trying...
16101MCID_676f0864756c8aefe8021314 38597682 Guochun Gong[author] Gong, Guochun[Full Author Name] gong, guochun[Author] trying2...
trying...
385976822024060920240611
1362-496252102024Jun10Nucleic acids researchNucleic Acids ResSomatic and intergenerational G4C2 hexanucleotide repeat instability in a human C9orf72 knock-in mouse model.573257555732-575510.1093/nar/gkae250Expansion of a G4C2 repeat in the C9orf72 gene is associated with familial Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). To investigate the underlying mechanisms of repeat instability, which occurs both somatically and intergenerationally, we created a novel mouse model of familial ALS/FTD that harbors 96 copies of G4C2 repeats at a humanized C9orf72 locus. In mouse embryonic stem cells, we observed two modes of repeat expansion. First, we noted minor increases in repeat length per expansion event, which was dependent on a mismatch repair pathway protein Msh2. Second, we found major increases in repeat length per event when a DNA double- or single-strand break (DSB/SSB) was artificially introduced proximal to the repeats, and which was dependent on the homology-directed repair (HDR) pathway. In mice, the first mode primarily drove somatic repeat expansion. Major changes in repeat length, including expansion, were observed when SSB was introduced in one-cell embryos, or intergenerationally without DSB/SSB introduction if G4C2 repeats exceeded 400 copies, although spontaneous HDR-mediated expansion has yet to be identified. These findings provide a novel strategy to model repeat expansion in a non-human genome and offer insights into the mechanism behind C9orf72 G4C2 repeat instability.© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.KojakNadaNRegeneron Pharmaceuticals, Tarrytown, NY 10591, USA.KunoJunkoJRegeneron Pharmaceuticals, Tarrytown, NY 10591, USA.FittipaldiKristina EKERegeneron Pharmaceuticals, Tarrytown, NY 10591, USA.KhanAmbereenARegeneron Pharmaceuticals, Tarrytown, NY 10591, USA.WengerDavidDRegeneron Pharmaceuticals, Tarrytown, NY 10591, USA.GlasserMichaelMRegeneron Pharmaceuticals, Tarrytown, NY 10591, USA.DonnianniRoberto ARARegeneron Pharmaceuticals, Tarrytown, NY 10591, USA.TangYajunYRegeneron Pharmaceuticals, Tarrytown, NY 10591, USA.ZhangJadeJRegeneron Pharmaceuticals, Tarrytown, NY 10591, USA.HulingKatieKRegeneron Pharmaceuticals, Tarrytown, NY 10591, USA.AllyRoxanneRRegeneron Pharmaceuticals, Tarrytown, NY 10591, USA.MujicaAlejandro OAORegeneron Pharmaceuticals, Tarrytown, NY 10591, USA.TurnerTerrenceTRegeneron Pharmaceuticals, Tarrytown, NY 10591, USA.MagardinoGinaGRegeneron Pharmaceuticals, Tarrytown, NY 10591, USA.HuangPei YiPYRegeneron Pharmaceuticals, Tarrytown, NY 10591, USA.KerkSze YenSYRegeneron Pharmaceuticals, Tarrytown, NY 10591, USA.DroguettGustavoGRegeneron Pharmaceuticals, Tarrytown, NY 10591, USA.PrissetteMarineMRegeneron Pharmaceuticals, Tarrytown, NY 10591, USA.RojasJoseJRegeneron Pharmaceuticals, Tarrytown, NY 10591, USA.GomezTeodoroTRegeneron Pharmaceuticals, Tarrytown, NY 10591, USA.GagliardiAnthonyARegeneron Pharmaceuticals, Tarrytown, NY 10591, USA.HuntCharleenCRegeneron Pharmaceuticals, Tarrytown, NY 10591, USA.RabinowitzJeremy SJSRegeneron Pharmaceuticals, Tarrytown, NY 10591, USA.GongGuochunGRegeneron Pharmaceuticals, Tarrytown, NY 10591, USA.PoueymirouWilliamWRegeneron Pharmaceuticals, Tarrytown, NY 10591, USA.ChiaoEricERegeneron Pharmaceuticals, Tarrytown, NY 10591, USA.ZambrowiczBrianBRegeneron Pharmaceuticals, Tarrytown, NY 10591, USA.SiaoChia-JenCJRegeneron Pharmaceuticals, Tarrytown, NY 10591, USA.KajimuraDaisukeD0009-0008-7399-0160Regeneron Pharmaceuticals, Tarrytown, NY 10591, USA.engJournal Article
EnglandNucleic Acids Res04110110305-10480C9orf72 Protein0C9orf72 protein, humanEC 3.6.1.3Msh2 protein, mouseEC 3.6.1.3MutS Homolog 2 ProteinIMAnimalsHumansMiceAmyotrophic Lateral SclerosisgeneticsC9orf72 ProteingeneticsDisease Models, AnimalDNA Breaks, Double-StrandedDNA Repeat ExpansiongeneticsFrontotemporal DementiageneticsGene Knock-In TechniquesGenomic InstabilitygeneticsMutS Homolog 2 Proteingenetics20243282024319202361220246105422024410124320244109152024410ppublish38597682PMC1116279810.1093/nar/gkae2507643286Lander  E.S., Linton  L.M., Birren  B., Nusbaum  C., Zody  M.C., Baldwin  J., Devon  K., Dewar  K., Doyle  M., FitzHugh  W.  et al. .  Initial sequencing and analysis of the human genome. Nature. 2001; 409:860–921.11237011Khristich  A.N., Mirkin  S.M.  On the wrong DNA track: molecular mechanisms of repeat-mediated genome instability. J. Biol. Chem.  2020; 295:4134–4170.PMC710531332060097Depienne  C., Mandel  J.L.  30 years of repeat expansion disorders: what have we learned and what are the remaining challenges?. Am. J. Hum. Genet.  2021; 108:764–785.PMC820599733811808McMurray  C.T.  Mechanisms of trinucleotide repeat instability during human development. Nat. Rev. Genet.  2010; 11:786–799.PMC317537620953213Dion  V.  Tissue specificity in DNA repair: lessons from trinucleotide repeat instability. Trends Genet.  2014; 30:220–229.24842550Usdin  K., House  N.C., Freudenreich  C.H.  Repeat instability during DNA repair: Insights from model systems. Crit. Rev. Biochem. Mol. Biol.  2015; 50:142–167.PMC445447125608779Heitz  D., Devys  D., Imbert  G., Kretz  C., Mandel  J.L.  Inheritance of the fragile X syndrome: size of the fragile X premutation is a major determinant of the transition to full mutation. J. Med. Genet.  1992; 29:794–801.PMC10161751453430Hagerman  R.J., Hagerman  P.J.  The fragile X premutation: into the phenotypic fold. Curr. Opin. Genet. Dev.  2002; 12:278–283.12076670Lozano  R., Rosero  C.A., Hagerman  R.J.  Fragile X spectrum disorders. Intractable Rare Dis. Res. 2014; 3:134–146.PMC429864325606363Wheeler  A.C., Bailey  D.B.  Jr, Berry-Kravis  E., Greenberg  J., Losh  M., Mailick  M., Mila  M., Olichney  J.M., Rodriguez-Revenga  L., Sherman  S  et al. .  Associated features in females with an FMR1 premutation. J. Neurodev. Disord.  2014; 6:30.PMC412143425097672Hayward  B.E., Usdin  K.  Mechanisms of genome instability in the fragile X-related disorders. Genes (Basel). 2021; 12:1633.PMC853610934681027Brook  J.D., McCurrach  M.E., Harley  H.G., Buckler  A.J., Church  D., Aburatani  H., Hunter  K., Stanton  V.P., Thirion  J.P., Hudson  T.  et al. .  Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3' end of a transcript encoding a protein kinase family member. Cell. 1992; 68:799–808.1310900Tome  S., Gourdon  G.  DM1 phenotype variability and triplet repeat instability: challenges in the development of new therapies. Int. J. Mol. Sci.  2020; 21:457.PMC701408731936870Morales  F., Vasquez  M., Cuenca  P., Campos  D., Santamaria  C., Del Valle  G., Brian  R., Sittenfeld  M., Monckton  D.G.  Parental age effects, but no evidence for an intrauterine effect in the transmission of myotonic dystrophy type 1. Eur. J. Hum. Genet.  2015; 23:646–653.PMC440261725052313Joosten  I.B.T., Hellebrekers  D., de Greef  B.T.A., Smeets  H.J.M., de Die-Smulders  C.E.M., Faber  C.G., Gerrits  M.M.  Parental repeat length instability in myotonic dystrophy type 1 pre- and protomutations. Eur. J. Hum. Genet.  2020; 28:956–962.PMC731698032203199Han  J.Y., Jang  W., Park  J.  Intergenerational influence of gender and the DM1 phenotype of the transmitting parent in Korean myotonic dystrophy type 1. Genes (Basel). 2022; 13:1465.PMC940846936011377Mankodi  A., Logigian  E., Callahan  L., McClain  C., White  R., Henderson  D., Krym  M., Thornton  C.A.  Myotonic dystrophy in transgenic mice expressing an expanded CUG repeat. Science. 2000; 289:1769–1773.10976074Seznec  H., Lia-Baldini  A.S., Duros  C., Fouquet  C., Lacroix  C., Hofmann-Radvanyi  H., Junien  C., Gourdon  G.  Transgenic mice carrying large human genomic sequences with expanded CTG repeat mimic closely the DM CTG repeat intergenerational and somatic instability. Hum. Mol. Genet.  2000; 9:1185–1194.10767343Gomes-Pereira  M., Foiry  L., Nicole  A., Huguet  A., Junien  C., Munnich  A., Gourdon  G.  CTG trinucleotide repeat “big jumps”: large expansions, small mice. PLoS Genet.  2007; 3:e52.PMC184769417411343Schmidt  M.H.M., Pearson  C.E.  Disease-associated repeat instability and mismatch repair. DNA Repair (Amst.). 2016; 38:117–126.26774442Wheeler  V.C., Dion  V.  Modifiers of CAG/CTG repeat instability: insights from mammalian models. J. Huntingtons Dis.  2021; 10:123–148.PMC799040833579861Zhao  X., Kumari  D., Miller  C.J., Kim  G.Y., Hayward  B., Vitalo  A.G., Pinto  R.M., Usdin  K.  Modifiers of somatic repeat instability in mouse models of Friedreich ataxia and the fragile X-related disorders: implications for the mechanism of somatic expansion in Huntington's disease. J Huntingtons Dis.  2021; 10:149–163.PMC799042833579860Manley  K., Shirley  T.L., Flaherty  L., Messer  A.  Msh2 deficiency prevents in vivo somatic instability of the CAG repeat in Huntington disease transgenic mice. Nat. Genet.  1999; 23:471–473.10581038van den Broek  W.J., Nelen  M.R., Wansink  D.G., Coerwinkel  M.M., te Riele  H., Groenen  P.J., Wieringa  B.  Somatic expansion behaviour of the (CTG)n repeat in myotonic dystrophy knock-in mice is differentially affected by Msh3 and Msh6 mismatch-repair proteins. Hum. Mol. Genet.  2002; 11:191–198.11809728Savouret  C., Brisson  E., Essers  J., Kanaar  R., Pastink  A., te Riele  H., Junien  C., Gourdon  G.  CTG repeat instability and size variation timing in DNA repair-deficient mice. EMBO J.  2003; 22:2264–2273.PMC15607412727892Wheeler  V.C., Lebel  L.A., Vrbanac  V., Teed  A., te Riele  H., MacDonald  M.E.  Mismatch repair gene Msh2 modifies the timing of early disease in Hdh(Q111) striatum. Hum. Mol. Genet.  2003; 12:273–281.12554681Dragileva  E., Hendricks  A., Teed  A., Gillis  T., Lopez  E.T., Friedberg  E.C., Kucherlapati  R., Edelmann  W., Lunetta  K.L., MacDonald  M.E.  et al. .  Intergenerational and striatal CAG repeat instability in Huntington's disease knock-in mice involve different DNA repair genes. Neurobiol. Dis.  2009; 33:37–47.PMC281128218930147Bourn  R.L., De Biase  I., Pinto  R.M., Sandi  C., Al-Mahdawi  S., Pook  M.A., Bidichandani  S.I.  Pms2 suppresses large expansions of the (GAA.TTC)n sequence in neuronal tissues. PLoS One. 2012; 7:e47085.PMC346949023071719Pinto  R.M., Dragileva  E., Kirby  A., Lloret  A., Lopez  E., St Claire  J., Panigrahi  G.B., Hou  C., Holloway  K., Gillis  T.  et al. .  Mismatch repair genes Mlh1 and Mlh3 modify CAG instability in Huntington's disease mice: genome-wide and candidate approaches. PLoS Genet.  2013; 9:e1003930.PMC381432024204323Tome  S., Manley  K., Simard  J.P., Clark  G.W., Slean  M.M., Swami  M., Shelbourne  P.F., Tillier  E.R., Monckton  D.G., Messer  A.  et al. .  MSH3 polymorphisms and protein levels affect CAG repeat instability in Huntington's disease mice. PLoS Genet.  2013; 9:e1003280.PMC358511723468640Ezzatizadeh  V., Sandi  C., Sandi  M., Anjomani-Virmouni  S., Al-Mahdawi  S., Pook  M.A.  MutLalpha heterodimers modify the molecular phenotype of Friedreich ataxia. PLoS One. 2014; 9:e100523.PMC407410424971578Lokanga  R.A., Zhao  X.N., Usdin  K.  The mismatch repair protein MSH2 is rate limiting for repeat expansion in a fragile X premutation mouse model. Hum. Mutat.  2014; 35:129–136.PMC395105424130133Zhao  X.N., Kumari  D., Gupta  S., Wu  D., Evanitsky  M., Yang  W., Usdin  K.  Mutsbeta generates both expansions and contractions in a mouse model of the fragile X-associated disorders. Hum. Mol. Genet.  2015; 24:7087–7096.PMC465405926420841Zhao  X.N., Lokanga  R., Allette  K., Gazy  I., Wu  D., Usdin  K.  A MutSbeta-dependent contribution of MutSalpha to repeat expansions in fragile X premutation mice?. PLoS Genet.  2016; 12:e1006190.PMC494885127427765Genetic Modifiers of Huntington's Disease, C.  Identification of genetic factors that modify clinical onset of Huntington's disease. Cell. 2015; 162:516–526.PMC452455126232222Genetic Modifiers of Huntington’s Disease (GeM-HD) Consortium  CAG repeat not polyglutamine length determines timing of Huntington's disease onset. Cell. 2019; 178:887–900.PMC670028131398342Flower  M., Lomeikaite  V., Ciosi  M., Cumming  S., Morales  F., Lo  K., Hensman Moss  D., Jones  L., Holmans  P., Investigators  T.-H.  et al. .  MSH3 modifies somatic instability and disease severity in Huntington's and myotonic dystrophy type 1. Brain. 2019; 142:1876–1886.PMC659862631216018Carvalho  C.M., Lupski  J.R.  Mechanisms underlying structural variant formation in genomic disorders. Nat. Rev. Genet.  2016; 17:224–238.PMC482762526924765Kim  J.C., Harris  S.T., Dinter  T., Shah  K.A., Mirkin  S.M.  The role of break-induced replication in large-scale expansions of (CAG)(n)/(CTG)(n) repeats. Nat. Struct. Mol. Biol.  2017; 24:55–60.PMC521597427918542Kononenko  A.V., Ebersole  T., Vasquez  K.M., Mirkin  S.M.  Mechanisms of genetic instability caused by (CGG)(n) repeats in an experimental mammalian system. Nat. Struct. Mol. Biol.  2018; 25:669–676.PMC608216230061600Garribba  L., Bjerregaard  V.A., Goncalves Dinis  M.M., Ozer  O., Wu  W., Sakellariou  D., Pena-Diaz  J., Hickson  I.D., Liu  Y.  Folate stress induces SLX1- and RAD51-dependent mitotic DNA synthesis at the fragile X locus in human cells. Proc. Natl. Acad. Sci. U.S.A.  2020; 117:16527–16536.PMC736827432601218DeJesus-Hernandez  M., Mackenzie  I.R., Boeve  B.F., Boxer  A.L., Baker  M., Rutherford  N.J., Nicholson  A.M., Finch  N.A., Flynn  H., Adamson  J.  et al. .  Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron. 2011; 72:245–256.PMC320298621944778Renton  A.E., Majounie  E., Waite  A., Simon-Sanchez  J., Rollinson  S., Gibbs  J.R., Schymick  J.C., Laaksovirta  H., van Swieten  J.C., Myllykangas  L.  et al. .  A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron. 2011; 72:257–268.PMC320043821944779Smeyers  J., Banchi  E.G., Latouche  M.  C9ORF72: what it is, what it does, and why it matters. Front. Cell Neurosci.  2021; 15:661447.PMC813152134025358van Blitterswijk  M., DeJesus-Hernandez  M., Niemantsverdriet  E., Murray  M.E., Heckman  M.G., Diehl  N.N., Brown  P.H., Baker  M.C., Finch  N.A., Bauer  P.O.  et al. .  Association between repeat sizes and clinical and pathological characteristics in carriers of C9ORF72 repeat expansions (Xpansize-72): a cross-sectional cohort study. Lancet Neurol.  2013; 12:978–988.PMC387978224011653Nordin  A., Akimoto  C., Wuolikainen  A., Alstermark  H., Jonsson  P., Birve  A., Marklund  S.L., Graffmo  K.S., Forsberg  K., Brannstrom  T.  et al. .  Extensive size variability of the GGGGCC expansion in C9orf72 in both neuronal and non-neuronal tissues in 18 patients with ALS or FTD. Hum. Mol. Genet.  2015; 24:3133–3142.25712133van der Ende  E.L., Jackson  J.L., White  A., Seelaar  H., van Blitterswijk  M., Van Swieten  J.C.  Unravelling the clinical spectrum and the role of repeat length in C9ORF72 repeat expansions. J. Neurol. Neurosurg. Psychiatry. 2021; 92:502–509.PMC805332833452054Van Mossevelde  S., van der Zee  J., Cruts  M., Van Broeckhoven  C.  Relationship between C9orf72 repeat size and clinical phenotype. Curr. Opin. Genet. Dev.  2017; 44:117–124.28319737O’Rourke  J.G., Bogdanik  L., Muhammad  A., Gendron  T.F., Kim  K.J., Austin  A., Cady  J., Liu  E.Y., Zarrow  J., Grant  S.  et al. .  C9orf72 BAC transgenic mice display typical pathologic features of ALS/FTD. Neuron. 2015; 88:892–901.PMC467238426637796Peters  O.M., Cabrera  G.T., Tran  H., Gendron  T.F., McKeon  J.E., Metterville  J., Weiss  A., Wightman  N., Salameh  J., Kim  J.  et al. .  Human C9ORF72 hexanucleotide expansion reproduces RNA foci and dipeptide repeat proteins but not neurodegeneration in BAC transgenic mice. Neuron. 2015; 88:902–909.PMC482834026637797Jiang  J., Zhu  Q., Gendron  T.F., Saberi  S., McAlonis-Downes  M., Seelman  A., Stauffer  J.E., Jafar-Nejad  P., Drenner  K., Schulte  D.  et al. .  Gain of toxicity from ALS/FTD-linked repeat expansions in C9ORF72 is alleviated by antisense oligonucleotides targeting GGGGCC-containing RNAs. Neuron. 2016; 90:535–550.PMC486007527112497Liu  Y., Pattamatta  A., Zu  T., Reid  T., Bardhi  O., Borchelt  D.R., Yachnis  A.T., Ranum  L.P.  C9orf72 BAC mouse model with motor deficits and neurodegenerative features of ALS/FTD. Neuron. 2016; 90:521–534.27112499Capecchi  M.R.  Altering the genome by homologous recombination. Science. 1989; 244:1288–1292.2660260Shimizu  M., Gellibolian  R., Oostra  B.A., Wells  R.D.  Cloning, characterization and properties of plasmids containing CGG triplet repeats from the FMR-1 gene. J. Mol. Biol.  1996; 258:614–626.8636996Ohshima  K., Montermini  L., Wells  R.D., Pandolfo  M.  Inhibitory effects of expanded GAA.TTC triplet repeats from intron I of the Friedreich ataxia gene on transcription and replication in vivo. J. Biol. Chem.  1998; 273:14588–14595.9603975Nair  R.R., Tibbit  C., Thompson  D., McLeod  R., Nakhuda  A., Simon  M.M., Baloh  R.H., Fisher  E.M.C., Isaacs  A.M., Cunningham  T.J.  Sizing, stabilising, and cloning repeat-expansions for gene targeting constructs. Methods. 2021; 191:15–22.PMC821568532721467Valenzuela  D.M., Murphy  A.J., Frendewey  D., Gale  N.W., Economides  A.N., Auerbach  W., Poueymirou  W.T., Adams  N.C., Rojas  J., Yasenchak  J.  et al. .  High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nat. Biotechnol.  2003; 21:652–659.12730667Osoegawa  K., Tateno  M., Woon  P.Y., Frengen  E., Mammoser  A.G., Catanese  J.J., Hayashizaki  Y., de Jong  P.J.  Bacterial artificial chromosome libraries for mouse sequencing and functional analysis. Genome Res.  2000; 10:116–128.PMC31049910645956Osoegawa  K., Mammoser  A.G., Wu  C., Frengen  E., Zeng  C., Catanese  J.J., de Jong  P.J.  A bacterial artificial chromosome library for sequencing the complete human genome. Genome Res.  2001; 11:483–496.PMC31104411230172Adams  D.J., Quail  M.A., Cox  T., van der Weyden  L., Gorick  B.D., Su  Q., Chan  W.I., Davies  R., Bonfield  J.K., Law  F.  et al. .  A genome-wide, end-sequenced 129Sv BAC library resource for targeting vector construction. Genomics. 2005; 86:753–758.16257172Zhang  Y., Buchholz  F., Muyrers  J.P., Stewart  A.F.  A new logic for DNA engineering using recombination in Escherichia coli. Nat. Genet.  1998; 20:123–128.9771703Montasser  M.E., Van Hout  C.V., Miloscio  L., Howard  A.D., Rosenberg  A., Callaway  M., Shen  B., Li  N., Locke  A.E., Verweij  N.  et al. .  Genetic and functional evidence links a missense variant in B4GALT1 to lower LDL and fibrinogen. Science. 2021; 374:1221–1227.34855475Poueymirou  W.T., Auerbach  W., Frendewey  D., Hickey  J.F., Escaravage  J.M., Esau  L., Dore  A.T., Stevens  S., Adams  N.C., Dominguez  M.G.  et al. .  F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses. Nat. Biotechnol.  2007; 25:91–99.17187059Bram  E., Javanmardi  K., Nicholson  K., Culp  K., Thibert  J.R., Kemppainen  J., Le  V., Schlageter  A., Hadd  A., Latham  G.J.  Comprehensive genotyping of the C9orf72 hexanucleotide repeat region in 2095 ALS samples from the NINDS collection using a two-mode, long-read PCR assay. Amyotroph. Lateral Scler. Frontotemporal Degener.  2019; 20:107–114.PMC651368030430876Cleary  E.M., Pal  S., Azam  T., Moore  D.J., Swingler  R., Gorrie  G., Stephenson  L., Colville  S., Chandran  S., Porteous  M.  et al. .  Improved PCR based methods for detecting C9orf72 hexanucleotide repeat expansions. Mol. Cell. Probes. 2016; 30:218–224.PMC497869927288208Long  A., Napierala  J.S., Polak  U., Hauser  L., Koeppen  A.H., Lynch  D.R., Napierala  M.  Somatic instability of the expanded GAA repeats in Friedreich's ataxia. PLoS One. 2017; 12:e0189990.PMC573621029261783Giesselmann  P., Brandl  B., Raimondeau  E., Bowen  R., Rohrandt  C., Tandon  R., Kretzmer  H., Assum  G., Galonska  C., Siebert  R.  et al. .  Analysis of short tandem repeat expansions and their methylation state with nanopore sequencing. Nat. Biotechnol.  2019; 37:1478–1481.31740840Wheeler  V.C., Auerbach  W., White  J.K., Srinidhi  J., Auerbach  A., Ryan  A., Duyao  M.P., Vrbanac  V., Weaver  M., Gusella  J.F.  et al. .  Length-dependent gametic CAG repeat instability in the Huntington's disease knock-in mouse. Hum. Mol. Genet.  1999; 8:115–122.9887339Duyao  M., Ambrose  C., Myers  R., Novelletto  A., Persichetti  F., Frontali  M., Folstein  S., Ross  C., Franz  M., Abbott  M.  et al. .  Trinucleotide repeat length instability and age of onset in Huntington's disease. Nat. Genet.  1993; 4:387–392.8401587Neto  J.L., Lee  J.M., Afridi  A., Gillis  T., Guide  J.R., Dempsey  S., Lager  B., Alonso  I., Wheeler  V.C., Pinto  R.M.  Genetic contributors to intergenerational CAG repeat instability in Huntington's disease knock-in mice. Genetics. 2017; 205:503–516.PMC528983227913616Jeggo  P.A., Pearl  L.H., Carr  A.M.  DNA repair, genome stability and cancer: a historical perspective. Nat. Rev. Cancer. 2016; 16:35–42.26667849Tubbs  A., Nussenzweig  A.  Endogenous DNA damage as a source of genomic instability in cancer. Cell. 2017; 168:644–656.PMC659173028187286Madabhushi  R., Pan  L., Tsai  L.H.  DNA damage and its links to neurodegeneration. Neuron. 2014; 83:266–282.PMC556444425033177Baratz  K.H., Tosakulwong  N., Ryu  E., Brown  W.L., Branham  K., Chen  W., Tran  K.D., Schmid-Kubista  K.E., Heckenlively  J.R., Swaroop  A.  et al. .  E2-2 protein and Fuchs's corneal dystrophy. N. Engl. J. Med.  2010; 363:1016–1024.20825314Fautsch  M.P., Wieben  E.D., Baratz  K.H., Bhattacharyya  N., Sadan  A.N., Hafford-Tear  N.J., Tuft  S.J., Davidson  A.E.  TCF4-mediated Fuchs endothelial corneal dystrophy: insights into a common trinucleotide repeat-associated disease. Prog. Retin. Eye Res.  2021; 81:100883.PMC798846432735996Masnovo  C., Lobo  A.F., Mirkin  S.M.  Replication dependent and independent mechanisms of GAA repeat instability. DNA Repair (Amst.). 2022; 118:103385.PMC967532035952488Foiry  L., Dong  L., Savouret  C., Hubert  L., te Riele  H., Junien  C., Gourdon  G.  Msh3 is a limiting factor in the formation of intergenerational CTG expansions in DM1 transgenic mice. Hum. Genet.  2006; 119:520–526.16552576Liu  L., Malkova  A.  Break-induced replication: unraveling each step. Trends Genet.  2022; 38:752–765.PMC919787735459559Mayle  R., Campbell  I.M., Beck  C.R., Yu  Y., Wilson  M., Shaw  C.A., Bjergbaek  L., Lupski  J.R., Ira  G.  DNA REPAIR. Mus81 and converging forks limit the mutagenicity of replication fork breakage. Science. 2015; 349:742–747.PMC478262726273056Nickoloff  J.A., Sharma  N., Taylor  L., Allen  S.J., Hromas  R.  The safe path at the fork: ensuring replication-associated DNA double-strand breaks are repaired by homologous recombination. Front. Genet.  2021; 12:748033.PMC850286734646312Deem  A., Keszthelyi  A., Blackgrove  T., Vayl  A., Coffey  B., Mathur  R., Chabes  A., Malkova  A.  Break-induced replication is highly inaccurate. PLoS Biol.  2011; 9:e1000594.PMC303966721347245Beck  C.R., Carvalho  C.M.B., Akdemir  Z.C., Sedlazeck  F.J., Song  X., Meng  Q., Hu  J., Doddapaneni  H., Chong  Z., Chen  E.S.  et al. .  Megabase length hypermutation accompanies human structural variation at 17p11.2. Cell. 2019; 176:1310–1324.PMC643817830827684Costantino  L., Sotiriou  S.K., Rantala  J.K., Magin  S., Mladenov  E., Helleday  T., Haber  J.E., Iliakis  G., Kallioniemi  O.P., Halazonetis  T.D.  Break-induced replication repair of damaged forks induces genomic duplications in human cells. Science. 2014; 343:88–91.PMC404765524310611Bhowmick  R., Minocherhomji  S., Hickson  I.D.  RAD52 facilitates mitotic DNA synthesis following replication stress. Mol. Cell. 2016; 64:1117–1126.27984745Li  S., Wang  H., Jehi  S., Li  J., Liu  S., Wang  Z., Truong  L., Chiba  T., Wang  Z., Wu  X.  PIF1 helicase promotes break-induced replication in mammalian cells. EMBO J.  2021; 40:e104509.PMC804744033470420Tsuzuki  T., Fujii  Y., Sakumi  K., Tominaga  Y., Nakao  K., Sekiguchi  M., Matsushiro  A., Yoshimura  Y., Morita  T.  Targeted disruption of the Rad51 gene leads to lethality in embryonic mice. Proc. Natl. Acad. Sci. U.S.A.  1996; 93:6236–6240.PMC390058692798Pattamatta  A., Nguyen  L., Olafson  H.R., Scotti  M.M., Laboissonniere  L.A., Richardson  J., Berglund  J.A., Zu  T., Wang  E.T., Ranum  L.P.W.  Repeat length increases disease penetrance and severity in C9orf72 ALS/FTD BAC transgenic mice. Hum. Mol. Genet.  2021; 29:3900–3918.PMC790675633378537Kalef-Ezra  E., Edzeamey  F.J., Valle  A., Khonsari  H., Kleine  P., Oggianu  C., Al-Mahdawi  S., Pook  M.A., Anjomani Virmouni  S.  A new FRDA mouse model [Fxn (null):YG8s(GAA) >800] with more than 800 GAA repeats. Front. Neurosci.  2023; 17:930422.PMC990953836777637Freudenreich  C.H., Kantrow  S.M., Zakian  V.A.  Expansion and length-dependent fragility of CTG repeats in yeast. Science. 1998; 279:853–856.9452383Gadgil  R.Y., Romer  E.J., Goodman  C.C., Rider  S.D.  Jr, Damewood  F.J., Barthelemy  J.R., Shin-Ya  K., Hanenberg  H., Leffak  M  Replication stress at microsatellites causes DNA double-strand breaks and break-induced replication. J. Biol. Chem.  2020; 295:15378–15397.PMC765023932873711van den Broek  W.J., Wansink  D.G., Wieringa  B.  Somatic CTG*CAG repeat instability in a mouse model for myotonic dystrophy type 1 is associated with changes in cell nuclearity and DNA ploidy. BMC Mol. Biol.  2007; 8:61.PMC194026117645799Lee  J.M., Pinto  R.M., Gillis  T., St Claire  J.C., Wheeler  V.C.  Quantification of age-dependent somatic CAG repeat instability in Hdh CAG knock-in mice reveals different expansion dynamics in striatum and liver. PLoS One. 2011; 6:e23647.PMC316364121897851Dols-Icardo  O., Garcia-Redondo  A., Rojas-Garcia  R., Sanchez-Valle  R., Noguera  A., Gomez-Tortosa  E., Pastor  P., Hernandez  I., Esteban-Perez  J., Suarez-Calvet  M.  et al. .  Characterization of the repeat expansion size in C9orf72 in amyotrophic lateral sclerosis and frontotemporal dementia. Hum. Mol. Genet.  2014; 23:749–754.24057670Fratta  P., Polke  J.M., Newcombe  J., Mizielinska  S., Lashley  T., Poulter  M., Beck  J., Preza  E., Devoy  A., Sidle  K.  et al. .  Screening a UK amyotrophic lateral sclerosis cohort provides evidence of multiple origins of the C9orf72 expansion. Neurobiol. Aging. 2015; 36:546.e1–546.e7.PMC427044525179228Gijselinck  I., Van Mossevelde  S., van der Zee  J., Sieben  A., Engelborghs  S., De Bleecker  J., Ivanoiu  A., Deryck  O., Edbauer  D., Zhang  M.  et al. .  The C9orf72 repeat size correlates with onset age of disease, DNA methylation and transcriptional downregulation of the promoter. Mol. Psychiatry. 2016; 21:1112–1124.PMC496045126481318Gijselinck  I., Cruts  M., Van Broeckhoven  C.  The genetics of C9orf72 expansions. Cold Spring Harb. Perspect. Med.  2018; 8:a026757.PMC588016228130313Jackson  J.L., Finch  N.A., Baker  M.C., Kachergus  J.M., DeJesus-Hernandez  M., Pereira  K., Christopher  E., Prudencio  M., Heckman  M.G., Thompson  E.A.  et al. .  Elevated methylation levels, reduced expression levels, and frequent contractions in a clinical cohort of C9orf72 expansion carriers. Mol. Neurodegener.  2020; 15:7.PMC699339932000838Shastri  N., Tsai  Y.C., Hile  S., Jordan  D., Powell  B., Chen  J., Maloney  D., Dose  M., Lo  Y., Anastassiadis  T.  et al. .  Genome-wide identification of structure-forming repeats as principal sites of fork collapse upon ATR inhibition. Mol. Cell. 2018; 72:222–238.PMC640786430293786Tubbs  A., Sridharan  S., van Wietmarschen  N., Maman  Y., Callen  E., Stanlie  A., Wu  W., Wu  X., Day  A., Wong  N.  et al. .  Dual roles of poly(dA:dT) tracts in replication initiation and fork collapse. Cell. 2018; 174:1127–1142.PMC659173530078706Kaushal  S., Freudenreich  C.H.  The role of fork stalling and DNA structures in causing chromosome fragility. Genes Chromosomes Cancer. 2019; 58:270–283.PMC708308930536896Lokanga  R.A., Kumari  D., Usdin  K.  Common threads: aphidicolin-inducible and folate-sensitive fragile sites in the human genome. Front. Genet.  2021; 12:708860.PMC845601834567068Lopez-Gonzalez  R., Lu  Y., Gendron  T.F., Karydas  A., Tran  H., Yang  D., Petrucelli  L., Miller  B.L., Almeida  S., Gao  F.B.  Poly(GR) in C9ORF72-related ALS/FTD compromises mitochondrial function and increases oxidative stress and DNA damage in iPSC-derived motor neurons. Neuron. 2016; 92:383–391.PMC511136627720481Farg  M.A., Konopka  A., Soo  K.Y., Ito  D., Atkin  J.D.  The DNA damage response (DDR) is induced by the C9orf72 repeat expansion in amyotrophic lateral sclerosis. Hum. Mol. Genet.  2017; 26:2882–2896.28481984Pham  N., Yan  Z., Yu  Y., Faria Afreen  M., Malkova  A., Haber  J.E., Ira  G.  Mechanisms restraining break-induced replication at two-ended DNA double-strand breaks. EMBO J.  2021; 40:e104847.PMC812693333844333Symington  L.S.  End resection at double-strand breaks: mechanism and regulation. Cold Spring Harb. Perspect. Biol.  2014; 6:a016436.PMC410798925085909Caldecott  K.W.  Causes and consequences of DNA single-strand breaks. Trends Biochem. Sci.  2024; 49:68–78.38040599Whelan  D.R., Lee  W.T.C., Yin  Y., Ofri  D.M., Bermudez-Hernandez  K., Keegan  S., Fenyo  D., Rothenberg  E.  Spatiotemporal dynamics of homologous recombination repair at single collapsed replication forks. Nat. Commun.  2018; 9:3882.PMC615516430250272Gacy  A.M., Goellner  G., Juranic  N., Macura  S., McMurray  C.T.  Trinucleotide repeats that expand in human disease form hairpin structures in vitro. Cell. 1995; 81:533–540.7758107Poggi  L., Richard  G.F.  Alternative DNA structures in vivo: molecular evidence and remaining questions. Microbiol. Mol. Biol. Rev.  2021; 85:e00110-20.PMC854985133361270Lo Scrudato  M., Poulard  K., Sourd  C., Tome  S., Klein  A.F., Corre  G., Huguet  A., Furling  D., Gourdon  G., Buj-Bello  A.  Genome editing of expanded CTG repeats within the human DMPK gene reduces nuclear RNA foci in the muscle of DM1 mice. Mol. Ther.  2019; 27:1372–1388.PMC669745231253581Meijboom  K.E., Abdallah  A., Fordham  N.P., Nagase  H., Rodriguez  T., Kraus  C., Gendron  T.F., Krishnan  G., Esanov  R., Andrade  N.S.  et al. .  CRISPR/Cas9-mediated excision of ALS/FTD-causing hexanucleotide repeat expansion in C9ORF72 rescues major disease mechanisms in vivo and in vitro. Nat. Commun.  2022; 13:6286.PMC958724936271076Kosicki  M., Tomberg  K., Bradley  A.  Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements. Nat. Biotechnol.  2018; 36:765–771.PMC639093830010673Owens  D.D.G., Caulder  A., Frontera  V., Harman  J.R., Allan  A.J., Bucakci  A., Greder  L., Codner  G.F., Hublitz  P., McHugh  P.J.  et al. .  Microhomologies are prevalent at Cas9-induced larger deletions. Nucleic Acids Res.  2019; 47:7402–7417.PMC669865731127293348554752021121720220302
1095-920337465722021Dec03Science (New York, N.Y.)ScienceGenetic and functional evidence links a missense variant in B4GALT1 to lower LDL and fibrinogen.122112271221-122710.1126/science.abe0348Increased blood levels of low-density lipoprotein cholesterol (LDL-C) and fibrinogen are independent risk factors for cardiovascular disease. We identified associations between an Amish-enriched missense variant (p.Asn352Ser) in a functional domain of beta-1,4-galactosyltransferase 1 (B4GALT1) and 13.9 milligrams per deciliter lower LDL-C (P = 4.1 × 10–19) and 29 milligrams per deciliter lower plasma fibrinogen (P = 1.3 × 10–5). B4GALT1 gene–based analysis in 544,955 subjects showed an association with decreased coronary artery disease (odds ratio = 0.64, P = 0.006). The mutant protein had 50% lower galactosyltransferase activity compared with the wild-type protein. N-linked glycan profiling of human serum found serine 352 allele to be associated with decreased galactosylation and sialylation of apolipoprotein B100, fibrinogen, immunoglobulin G, and transferrin. B4galt1 353Ser knock-in mice showed decreases in LDL-C and fibrinogen. Our findings suggest that targeted modulation of protein galactosylation may represent a therapeutic approach to decreasing cardiovascular disease.MontasserMay EME0000-0002-9558-5456Division of Endocrinology, Diabetes and Nutrition and Program for Personalized and Genomic Medicine, Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201, USA.Van HoutCristopher VCV0000-0001-9689-5344Regeneron Genetics Center, LLC, Tarrytown, NY 10591, USA.Laboratorio Internacional de Investigatión sobre el Genoma Humano, Campus Juriquilla de la Universidad Nacional Autónoma de México, Querétaro, Querétaro 76230, México.MiloscioLawrenceLRegeneron Genetics Center, LLC, Tarrytown, NY 10591, USA.HowardAlicia DAD0000-0002-1616-5729Division of Endocrinology, Diabetes and Nutrition and Program for Personalized and Genomic Medicine, Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201, USA.Center for Biologics Evaluation and Research, US Food and Drug Administration, Silver Spring, MD 20993, USA.RosenbergAvrahamARegeneron Pharmaceuticals, Inc., Tarrytown, NY 10591, USA.CallawayMyrasolMRegeneron Pharmaceuticals, Inc., Tarrytown, NY 10591, USA.ShenBiaoB0000-0001-5791-1583Regeneron Pharmaceuticals, Inc., Tarrytown, NY 10591, USA.LiNingN0000-0001-6750-4308Regeneron Pharmaceuticals, Inc., Tarrytown, NY 10591, USA.LockeAdam EAE0000-0001-6227-198XRegeneron Genetics Center, LLC, Tarrytown, NY 10591, USA.VerweijNiekN0000-0002-4303-7685Regeneron Genetics Center, LLC, Tarrytown, NY 10591, USA.DeTanimaT0000-0001-9240-3744Regeneron Genetics Center, LLC, Tarrytown, NY 10591, USA.FerreiraManuel AMA0000-0001-9059-1825Regeneron Genetics Center, LLC, Tarrytown, NY 10591, USA.LottaLuca ALA0000-0002-2619-5956Regeneron Genetics Center, LLC, Tarrytown, NY 10591, USA.BarasArisARegeneron Genetics Center, LLC, Tarrytown, NY 10591, USA.DalyThomas JTJ0000-0002-0208-6827Regeneron Pharmaceuticals, Inc., Tarrytown, NY 10591, USA.HartfordSuzanne ASA0000-0001-8816-8585Regeneron Pharmaceuticals, Inc., Tarrytown, NY 10591, USA.LinWeiW0000-0002-1953-9765Regeneron Pharmaceuticals, Inc., Tarrytown, NY 10591, USA.MaoYuanYRegeneron Pharmaceuticals, Inc., Tarrytown, NY 10591, USA.YeBinB0000-0003-1083-8432Regeneron Genetics Center, LLC, Tarrytown, NY 10591, USA.WhiteDerekDRegeneron Pharmaceuticals, Inc., Tarrytown, NY 10591, USA.GongGuochunG0000-0003-4982-0491Regeneron Pharmaceuticals, Inc., Tarrytown, NY 10591, USA.PerryJames AJA0000-0001-5050-2074Division of Endocrinology, Diabetes and Nutrition and Program for Personalized and Genomic Medicine, Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201, USA.RyanKathleen AKA0000-0003-1158-8455Division of Endocrinology, Diabetes and Nutrition and Program for Personalized and Genomic Medicine, Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201, USA.FangQingQRegeneron Pharmaceuticals, Inc., Tarrytown, NY 10591, USA.TzonevaGannieG0000-0001-5784-7796Regeneron Genetics Center, LLC, Tarrytown, NY 10591, USA.PefanisEvangelosERegeneron Pharmaceuticals, Inc., Tarrytown, NY 10591, USA.HuntCharleenC0000-0003-0537-862XRegeneron Pharmaceuticals, Inc., Tarrytown, NY 10591, USA.TangYajunYRegeneron Pharmaceuticals, Inc., Tarrytown, NY 10591, USA.LeeLynnL0000-0001-8433-9367Regeneron Pharmaceuticals, Inc., Tarrytown, NY 10591, USA.Regeneron Genetics Center Collaboration‡Sztalryd-WoodleCaroleCDivision of Endocrinology, Diabetes and Nutrition and Program for Personalized and Genomic Medicine, Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201, USA.US Department of Veterans Affairs, Washington, DC 20420 USA.MitchellBraxton DBD0000-0003-4920-4744Division of Endocrinology, Diabetes and Nutrition and Program for Personalized and Genomic Medicine, Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201, USA.Geriatrics Research and Education Clinical Center, VA Medical Center, Baltimore, MD 21201, USA.HealyMatthewM0000-0001-6439-5038Enveda Biosciences, Boulder, CO 80301, USA.StreetenElizabeth AEA0000-0002-9771-6958Division of Endocrinology, Diabetes and Nutrition and Program for Personalized and Genomic Medicine, Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201, USA.Division of Genetics, Department of Pediatrics, University of Maryland School of Medicine, Baltimore, MD 21201, USA.TaylorSimeon ISI0000-0001-7500-7854Division of Endocrinology, Diabetes and Nutrition and Program for Personalized and Genomic Medicine, Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201, USA.O'ConnellJeffrey RJRDivision of Endocrinology, Diabetes and Nutrition and Program for Personalized and Genomic Medicine, Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201, USA.EconomidesAris NAN0000-0002-6508-8942Regeneron Genetics Center, LLC, Tarrytown, NY 10591, USA.Regeneron Pharmaceuticals, Inc., Tarrytown, NY 10591, USA.Della GattaGiusyG0000-0001-5888-6287Regeneron Genetics Center, LLC, Tarrytown, NY 10591, USA.ShuldinerAlan RAR0000-0001-9921-4305Regeneron Genetics Center, LLC, Tarrytown, NY 10591, USA.engU01 HL137181HLNHLBI NIH HHSUnited StatesU01 HL072515HLNHLBI NIH HHSUnited StatesR01 AG018728AGNIA NIH HHSUnited StatesR01 HL121007HLNHLBI NIH HHSUnited StatesR01 HL117626HLNHLBI NIH HHSUnited StatesR01 HL120393HLNHLBI NIH HHSUnited StatesP30 DK072488DKNIDDK NIH HHSUnited StatesJournal ArticleResearch Support, N.I.H., Extramural20211202
United StatesScience04045110036-80750Cholesterol, LDL0Glycoproteins0Polysaccharides9001-32-5FibrinogenEC 2.4.1.-GalactosyltransferasesEC 2.4.1.-beta-1,4-galactosyltransferase IGZP2782OP0N-Acetylneuraminic AcidX2RN3Q8DNEGalactoseIMAnimalsCholesterol, LDLbloodCoronary Artery Diseasegeneticsprevention & controlFemaleFibrinogenanalysisGalactosemetabolismGalactosyltransferasesgeneticsmetabolismGene Knock-In TechniquesGene Knockdown TechniquesGlycoproteinsbloodGlycosylationHumansLiverenzymologyMaleMiceMutation, MissenseN-Acetylneuraminic AcidmetabolismPolysaccharidesbloodWhole Genome Sequencing202112217192021123602021121860ppublish3485547510.1126/science.abe0348339862662021053120240104
2041-17231212021May13Nature communicationsNat CommunTissue-specific activation of gene expression by the Synergistic Activation Mediator (SAM) CRISPRa system in mice.27702770277010.1038/s41467-021-22932-4CRISPR-based transcriptional activation is a powerful tool for functional gene interrogation; however, delivery difficulties have limited its applications in vivo. Here, we created a mouse model expressing all components of the CRISPR-Cas9 guide RNA-directed Synergistic Activation Mediator (SAM) from a single transcript that is capable of activating target genes in a tissue-specific manner. We optimized Lipid Nanoparticles and Adeno-Associated Virus guide RNA delivery approaches to achieve expression modulation of one or more genes in vivo. We utilized the SAM mouse model to generate a hypercholesteremia disease state that we could bidirectionally modulate with various guide RNAs. Additionally, we applied SAM to optimize gene expression in a humanized Transthyretin mouse model to recapitulate human expression levels. These results demonstrate that the SAM gene activation platform can facilitate in vivo research and drug discovery.HuntCharleenC0000-0003-0537-862XRegeneron Pharmaceuticals, Inc., Tarrytown, NY, USA.HartfordSuzanne ASA0000-0001-8816-8585Regeneron Pharmaceuticals, Inc., Tarrytown, NY, USA.WhiteDerekDRegeneron Pharmaceuticals, Inc., Tarrytown, NY, USA.PefanisEvangelosERegeneron Pharmaceuticals, Inc., Tarrytown, NY, USA.HannaTimothyTRegeneron Pharmaceuticals, Inc., Tarrytown, NY, USA.HermanClarissaCRegeneron Pharmaceuticals, Inc., Tarrytown, NY, USA.WileyJarrellJRegeneron Pharmaceuticals, Inc., Tarrytown, NY, USA.BrownHeatherHRegeneron Pharmaceuticals, Inc., Tarrytown, NY, USA.SuQiQRegeneron Pharmaceuticals, Inc., Tarrytown, NY, USA.XinYurongYRegeneron Pharmaceuticals, Inc., Tarrytown, NY, USA.VoroninDennisDRegeneron Pharmaceuticals, Inc., Tarrytown, NY, USA.NguyenHienHRegeneron Pharmaceuticals, Inc., Tarrytown, NY, USA.AltarejosJudithJRegeneron Pharmaceuticals, Inc., Tarrytown, NY, USA.CrosbyKeithKRegeneron Pharmaceuticals, Inc., Tarrytown, NY, USA.HainesJefferyJRegeneron Pharmaceuticals, Inc., Tarrytown, NY, USA.CancelarichSarahSRegeneron Pharmaceuticals, Inc., Tarrytown, NY, USA.DrummondMeghanMRegeneron Pharmaceuticals, Inc., Tarrytown, NY, USA.Moller-TankSvenSRegeneron Pharmaceuticals, Inc., Tarrytown, NY, USA.MalpassRyanRRegeneron Pharmaceuticals, Inc., Tarrytown, NY, USA.BuckleyJacquelineJRegeneron Pharmaceuticals, Inc., Tarrytown, NY, USA.Del Pilar Molina-PortelaMariaMRegeneron Pharmaceuticals, Inc., Tarrytown, NY, USA.DroguettGustavoGRegeneron Pharmaceuticals, Inc., Tarrytown, NY, USA.FrendeweyDavidDRegeneron Pharmaceuticals, Inc., Tarrytown, NY, USA.ChiaoEricERegeneron Pharmaceuticals, Inc., Tarrytown, NY, USA.ZambrowiczBrianB0000-0001-8831-0406Regeneron Pharmaceuticals, Inc., Tarrytown, NY, USA.GongGuochunG0000-0003-4982-0491Regeneron Pharmaceuticals, Inc., Tarrytown, NY, USA. guochun.gong@regeneron.com.engJournal Article20210513
EnglandNat Commun1015285552041-17230Lipid Nanoparticles0Liposomes0Prealbumin0RNA, Guide, CRISPR-Cas SystemsIMAnimalsCRISPR-Cas SystemsgeneticsCell LineGene ExpressiongeneticsGene Expression RegulationgeneticsGenetic EngineeringmethodsHEK293 CellsHumansHypercholesterolemiageneticspathologyLiposomespharmacologyMiceMice, Inbred C57BLMice, TransgenicNanoparticlesPrealbumingeneticsmetabolismRNA, Guide, CRISPR-Cas SystemsgeneticsmetabolismTranscriptional ActivationgeneticsC. Hunt, S.H., D.W., E.P., T.H., C. Herman, J. W., H.B., Q.S., D.V., J.A., K.C., J.H., S.C., M.D., S.M.-T., M.P.M.-P., G.D., D.F., E.C., B.Z., and G.G. are employees of Regeneron Pharmaceuticals Inc (“Regeneron”). Regeneron has filed patent applications around the described work. The remaining authors declare no competing interests.20201172021462021514613202151560202161602021513epublish33986266PMC811996210.1038/s41467-021-22932-410.1038/s41467-021-22932-4Prelich G. Gene overexpression: uses, mechanisms, and interpretation. Genetics. 2012;190:841–854. doi: 10.1534/genetics.111.136911.10.1534/genetics.111.136911PMC329625222419077Olalla Saad, S. T. et al. Comparison of different methods to overexpress large genes. J. Biol. Res.91, 10.4081/jbr.2018.7249 (2018).Guo Y, Zhang Y, Hu K. Sleeping Beauty transposon integrates into non-TA dinucleotides. Mob. DNA. 2018;9:8. doi: 10.1186/s13100-018-0113-8.10.1186/s13100-018-0113-8PMC580184029445422Liu C. Strategies for designing transgenic DNA constructs. Methods Mol. Biol. 2013;1027:183–201. doi: 10.1007/978-1-60327-369-5_8.10.1007/978-1-60327-369-5_8PMC381555123912987Groth AC, Olivares EC, Thyagarajan B, Calos MP. A phage integrase directs efficient site-specific integration in human cells. Proc. Natl Acad. Sci. USA. 2000;97:5995–6000. doi: 10.1073/pnas.090527097.10.1073/pnas.090527097PMC1854710801973Kolot M, Silberstein N, Yagil E. Site-specific recombination in mammalian cells expressing the Int recombinase of bacteriophage HK022–site-specific recombination in mammalian cells promoted by a phage integrase. Mol. Biol. Rep. 1999;26:207–213. doi: 10.1023/A:1007096701720.10.1023/A:100709670172010532317Tasic B, et al. Site-specific integrase-mediated transgenesis in mice via pronuclear injection. Proc. Natl Acad. Sci. USA. 2011;108:7902–7907. doi: 10.1073/pnas.1019507108.10.1073/pnas.1019507108PMC309348221464299Kim MJ, Ahituv N. The hydrodynamic tail vein assay as a tool for the study of liver promoters and enhancers. Methods Mol. Biol. 2013;1015:279–289. doi: 10.1007/978-1-62703-435-7_18.10.1007/978-1-62703-435-7_18PMC409602223824863Tschaharganeh DF, Lowe SW, Garippa RJ, Livshits G. Using CRISPR/Cas to study gene function and model disease in vivo. FEBS J. 2016;283:3194–3203. doi: 10.1111/febs.13750.10.1111/febs.13750PMC512036127149548Klug A. The discovery of zinc fingers and their development for practical applications in gene regulation and genome manipulation. Q. Rev. Biophys. 2010;43:1–21. doi: 10.1017/S0033583510000089.10.1017/S003358351000008920478078Konermann S, et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature. 2015;517:583–588. doi: 10.1038/nature14136.10.1038/nature14136PMC442063625494202Dominguez AA, Lim WA, Qi LS. Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation. Nat. Rev. Mol. Cell Biol. 2016;17:5–15. doi: 10.1038/nrm.2015.2.10.1038/nrm.2015.2PMC492251026670017Matharu, N. et al. CRISPR-mediated activation of a promoter or enhancer rescues obesity caused by haploinsufficiency. Science363, 10.1126/science.aau0629 (2019).PMC657048930545847Lundh M, Plucinska K, Isidor MS, Petersen PSS, Emanuelli B. Bidirectional manipulation of gene expression in adipocytes using CRISPRa and siRNA. Mol. Metab. 2017;6:1313–1320. doi: 10.1016/j.molmet.2017.07.001.10.1016/j.molmet.2017.07.001PMC564160129031730Dahlman JE, et al. Orthogonal gene knockout and activation with a catalytically active Cas9 nuclease. Nat. Biotechnol. 2015;33:1159–1161. doi: 10.1038/nbt.3390.10.1038/nbt.3390PMC474778926436575Liao HK, et al. In vivo target gene activation via CRISPR/Cas9-mediated trans-epigenetic modulation. Cell. 2017;171:1495–1507. doi: 10.1016/j.cell.2017.10.025.10.1016/j.cell.2017.10.025PMC573204529224783Ran FA, et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature. 2015;520:186–191. doi: 10.1038/nature14299.10.1038/nature14299PMC439336025830891Vora, S. et al. Rational design of a compact CRISPR-Cas9 activator for AAV- mediated delivery. Preprint at bioRxiv10.1101/298620 (2018).Lau CH, Ho JW, Lo PK, Tin C. Targeted transgene activation in the brain tissue by systemic delivery of engineered AAV1 expressing CRISPRa. Mol. Ther. Nucleic Acids. 2019;16:637–649. doi: 10.1016/j.omtn.2019.04.015.10.1016/j.omtn.2019.04.015PMC652623031108320Wangensteen KJ, et al. Combinatorial genetics in liver repopulation and carcinogenesis with a in vivo CRISPR activation platform. Hepatology. 2018;68:663–676. doi: 10.1002/hep.29626.10.1002/hep.29626PMC593014129091290Chavez A, et al. Comparison of Cas9 activators in multiple species. Nat. Methods. 2016;13:563–567. doi: 10.1038/nmeth.3871.10.1038/nmeth.3871PMC492735627214048Zambrowicz BP, et al. Disruption of overlapping transcripts in the ROSA beta geo 26 gene trap strain leads to widespread expression of beta-galactosidase in mouse embryos and hematopoietic cells. Proc. Natl Acad. Sci. USA. 1997;94:3789–3794. doi: 10.1073/pnas.94.8.3789.10.1073/pnas.94.8.3789PMC205199108056Friedrich G, Soriano P. Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice. Genes Dev. 1991;5:1513–1523. doi: 10.1101/gad.5.9.1513.10.1101/gad.5.9.15131653172Valenzuela DM, et al. High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nat. Biotechnol. 2003;21:652–659. doi: 10.1038/nbt822.10.1038/nbt82212730667Vihervaara A, Sistonen L. HSF1 at a glance. J. Cell Sci. 2014;127:261–266. doi: 10.1242/jcs.132605.10.1242/jcs.13260524421309Graslund T, Li X, Magnenat L, Popkov M, Barbas CF., 3rd Exploring strategies for the design of artificial transcription factors: targeting sites proximal to known regulatory regions for the induction of gamma-globin expression and the treatment of sickle cell disease. J. Biol. Chem. 2005;280:3707–3714. doi: 10.1074/jbc.M406809200.10.1074/jbc.M40680920015537646Tutucci E, et al. An improved MS2 system for accurate reporting of the mRNA life cycle. Nat. Methods. 2018;15:81–89. doi: 10.1038/nmeth.4502.10.1038/nmeth.4502PMC584357829131164Wu C, et al. BioGPS: an extensible and customizable portal for querying and organizing gene annotation resources. Genome Biol. 2009;10:R130. doi: 10.1186/gb-2009-10-11-r130.10.1186/gb-2009-10-11-r130PMC309132319919682Lai KM, et al. Diverse phenotypes and specific transcription patterns in twenty mouse lines with ablated LincRNAs. PLoS ONE. 2015;10:e0125522. doi: 10.1371/journal.pone.0125522.10.1371/journal.pone.0125522PMC440929325909911Su AI, et al. A gene atlas of the mouse and human protein-encoding transcriptomes. Proc. Natl Acad. Sci. USA. 2004;101:6062–6067. doi: 10.1073/pnas.0400782101.10.1073/pnas.0400782101PMC39592315075390Jensen, M. K. Design principles for nuclease-deficient CRISPR-based transcriptional regulators. FEMS Yeast Res. 18, 10.1093/femsyr/foy039 (2018).PMC593255529726937McGinn J, Marraffini LA. Molecular mechanisms of CRISPR-Cas spacer acquisition. Nat. Rev. Microbiol. 2019;17:7–12. doi: 10.1038/s41579-018-0071-7.10.1038/s41579-018-0071-730171202Kent WJ, et al. The human genome browser at UCSC. Genome Res. 2002;12:996–1006. doi: 10.1101/gr.229102.10.1101/gr.229102PMC18660412045153Consortium EP. An integrated encyclopedia of DNA elements in the human genome. Nature. 2012;489:57–74. doi: 10.1038/nature11247.10.1038/nature11247PMC343915322955616Davis CA, et al. The Encyclopedia of DNA elements (ENCODE): data portal update. Nucleic Acids Res. 2018;46:D794–D801. doi: 10.1093/nar/gkx1081.10.1093/nar/gkx1081PMC575327829126249Zhou H, et al. In vivo simultaneous transcriptional activation of multiple genes in the brain using CRISPR-dCas9-activator transgenic mice. Nat. Neurosci. 2018;21:440–446. doi: 10.1038/s41593-017-0060-6.10.1038/s41593-017-0060-629335603Kent WJ. BLAT-the BLAST-like alignment tool. Genome Res. 2002;12:656–664. doi: 10.1101/gr.229202.10.1101/gr.229202PMC18751811932250Gossler A, Joyner AL, Rossant J, Skarnes WC. Mouse embryonic stem cells and reporter constructs to detect developmentally regulated genes. Science. 1989;244:463–465. doi: 10.1126/science.2497519.10.1126/science.24975192497519Lau CH, Suh Y. In vivo genome editing in animals using AAV-CRISPR system: applications to translational research of human disease. F1000Res. 2017;6:2153. doi: 10.12688/f1000research.11243.1.10.12688/f1000research.11243.1PMC574912529333255Yin H, Kauffman KJ, Anderson DG. Delivery technologies for genome editing. Nat. Rev. Drug Discov. 2017;16:387–399. doi: 10.1038/nrd.2016.280.10.1038/nrd.2016.28028337020Senis E, et al. CRISPR/Cas9-mediated genome engineering: an adeno-associated viral (AAV) vector toolbox. Biotechnol. J. 2014;9:1402–1412. doi: 10.1002/biot.201400046.10.1002/biot.20140004625186301Magami Y, et al. Cell proliferation and renewal of normal hepatocytes and bile duct cells in adult mouse. Liver. 2002;22:419–425. doi: 10.1034/j.1600-0676.2002.01702.x.10.1034/j.1600-0676.2002.01702.x12390477Malato Y, et al. Fate tracing of mature hepatocytes in mouse liver homeostasis and regeneration. J. Clin. Invest. 2011;121:4850–4860. doi: 10.1172/JCI59261.10.1172/JCI59261PMC322600522105172Furuyama K, et al. Continuous cell supply from a Sox9-expressing progenitor zone in adult liver, exocrine pancreas and intestine. Nat. Genet. 2011;43:34–41. doi: 10.1038/ng.722.10.1038/ng.72221113154Lattin JE, et al. Expression analysis of G Protein-Coupled Receptors in mouse macrophages. Immunome Res. 2008;4:5. doi: 10.1186/1745-7580-4-5.10.1186/1745-7580-4-5PMC239451418442421Hordeaux J, et al. The GPI-linked protein LY6A drives AAV-PHP.B transport across the blood-brain barrier. Mol. Ther. 2019;27:912–921. doi: 10.1016/j.ymthe.2019.02.013.10.1016/j.ymthe.2019.02.013PMC652046330819613Chan KY, et al. Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems. Nat. Neurosci. 2017;20:1172–1179. doi: 10.1038/nn.4593.10.1038/nn.4593PMC552924528671695Wise PM, Scarbrough K, Weiland NG, Larson GH. Diurnal pattern of proopiomelanocortin gene expression in the arcuate nucleus of proestrous, ovariectomized, and steroid-treated rats: a possible role in cyclic luteinizing hormone secretion. Mol. Endocrinol. 1990;4:886–892. doi: 10.1210/mend-4-6-886.10.1210/mend-4-6-8862233745Goutman JD, Elgoyhen AB, Gomez-Casati ME. Cochlear hair cells: the sound-sensing machines. FEBS Lett. 2015;589:3354–3361. doi: 10.1016/j.febslet.2015.08.030.10.1016/j.febslet.2015.08.030PMC464102026335749Ogier JM, Burt RA, Drury HR, Lim R, Nayagam BA. Organotypic culture of neonatal murine inner ear explants. Front. Cell Neurosci. 2019;13:170. doi: 10.3389/fncel.2019.00170.10.3389/fncel.2019.00170PMC650923431130846Bas E, Gupta C, Van De Water TR. A novel organ of corti explant model for the study of cochlear implantation trauma. Anat. Rec. (Hoboken) 2012;295:1944–1956. doi: 10.1002/ar.22585.10.1002/ar.2258523044812Shapiro MD, Tavori H, Fazio S. PCSK9: from basic science discoveries to clinical trials. Circ. Res. 2018;122:1420–1438. doi: 10.1161/CIRCRESAHA.118.311227.10.1161/CIRCRESAHA.118.311227PMC597625529748367Melendez QM, Krishnaji ST, Wooten CJ, Lopez D. Hypercholesterolemia: the role of PCSK9. Arch. Biochem. Biophys. 2017;625-626:39–53. doi: 10.1016/j.abb.2017.06.001.10.1016/j.abb.2017.06.00128587771Hopkins PN, et al. Characterization of autosomal dominant hypercholesterolemia caused by PCSK9 gain of function mutations and its specific treatment with alirocumab, a PCSK9 monoclonal antibody. Circ. Cardiovasc. Genet. 2015;8:823–831. doi: 10.1161/CIRCGENETICS.115.001129.10.1161/CIRCGENETICS.115.001129PMC509846626374825Gertz MA, et al. Diagnosis, prognosis, and therapy of transthyretin amyloidosis. J. Am. Coll. Cardiol. 2015;66:2451–2466. doi: 10.1016/j.jacc.2015.09.075.10.1016/j.jacc.2015.09.07526610878Ueda M, Ando Y. Recent advances in transthyretin amyloidosis therapy. Transl. Neurodegener. 2014;3:19. doi: 10.1186/2047-9158-3-19.10.1186/2047-9158-3-19PMC416562225228988Fu L, Zhu X, Yi F, Liu GH, Izpisua Belmonte JC. Regenerative medicine: transdifferentiation in vivo. Cell Res. 2014;24:141–142. doi: 10.1038/cr.2013.165.10.1038/cr.2013.165PMC391590624343579Li YY, et al. Systematic analysis of head-to-head gene organization: evolutionary conservation and potential biological relevance. PLoS Comput. Biol. 2006;2:e74. doi: 10.1371/journal.pcbi.0020074.10.1371/journal.pcbi.0020074PMC148718016839196Kim HK, Pham MHC, Ko KS, Rhee BD, Han J. Alternative splicing isoforms in health and disease. Pflug. Arch. 2018;470:995–1016. doi: 10.1007/s00424-018-2136-x.10.1007/s00424-018-2136-x29536164Poueymirou WT, et al. F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses. Nat. Biotechnol. 2007;25:91–99. doi: 10.1038/nbt1263.10.1038/nbt126317187059Haeussler M, et al. Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol. 2016;17:148. doi: 10.1186/s13059-016-1012-2.10.1186/s13059-016-1012-2PMC493401427380939Arden, E. & Metzger, J. M. Inexpensive, serotype-independent protocol for native and bioengineered recombinant adeno-associated virus purification. J. Biol. Methods3, 10.14440/jbm.2016.102 (2016).PMC490228527294171Zolotukhin S, et al. Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther. 1999;6:973–985. doi: 10.1038/sj.gt.3300938.10.1038/sj.gt.330093810455399Liu F, Song Y, Liu D. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Ther. 1999;6:1258–1266. doi: 10.1038/sj.gt.3300947.10.1038/sj.gt.330094710455434Altarejos JY, et al. The Creb1 coactivator Crtc1 is required for energy balance and fertility. Nat. Med. 2008;14:1112–1117. doi: 10.1038/nm.1866.10.1038/nm.1866PMC266769818758446302894412020052620200825
1529-726810032019Mar01Biology of reproductionBiol ReprodDeletion of Adam6 in Mus musculus leads to male subfertility and deficits in sperm ascent into the oviduct.686696686-69610.1093/biolre/ioy210The Adisintegrin and metalloprotease domain-containing (ADAM) family of proteins is involved in cell adhesion, migration, proteolysis, and signaling. Many ADAMs are required for reproduction; however, the role of Adam6 has remained largely unknown. In the course of humanizing the mouse immunoglobulin heavy chain (IgH) locus, we generated Adam6-deficient mice that demonstrate severe subfertility. We decided to elucidate the role of ADAM6 in fertility and explore the underlying mechanisms. Despite normal sperm development and motility, Adam6-deficient mice display diminished male fertility, have abnormal sperm adhesion, and most importantly cannot transition from uterus to oviduct. To test whether ADAM6 is required for sperm's binding to extracellular matrix (ECM) components, we used a panel of ECM components and showed that unlike normal sperm, Adam6-deficient sperm cannot bind fibronectin, laminin, and tenascin. Reintroduction of Adam6 into these deficient mice repaired sperm interaction with ECM, restored male fertility, and corrected the sperm transport deficit. Together, our data suggest that ADAM6, either alone or in complex with other proteins, aids sperm transport through the female reproductive tract by providing a temporary site of attachment of sperm to ECM components prior to ascent into the oviduct.© The Author(s) 2018. Published by Oxford University Press on behalf of Society for the Study of Reproduction.VoroninaVera AVARegeneron Pharmaceuticals, Inc., Tarrytown, New York, USA.HarrisFaith MFMRegeneron Pharmaceuticals, Inc., Tarrytown, New York, USA.SchmahlJenniferJRegeneron Pharmaceuticals, Inc., Tarrytown, New York, USA.GalliganCarynCRegeneron Pharmaceuticals, Inc., Tarrytown, New York, USA.OristianDanielDRegeneron Pharmaceuticals, Inc., Tarrytown, New York, USA.ZamfirovaRalicaRRegeneron Pharmaceuticals, Inc., Tarrytown, New York, USA.GongGuochunGRegeneron Pharmaceuticals, Inc., Tarrytown, New York, USA.BaiYuYRegeneron Pharmaceuticals, Inc., Tarrytown, New York, USA.FuryWenWRegeneron Pharmaceuticals, Inc., Tarrytown, New York, USA.RajamaniSaathyakiSRegeneron Pharmaceuticals, Inc., Tarrytown, New York, USA.WallsJohnathon RJRRegeneron Pharmaceuticals, Inc., Tarrytown, New York, USA.PoueymirouWilliam TWTRegeneron Pharmaceuticals, Inc., Tarrytown, New York, USA.EsauLakeishaLRegeneron Pharmaceuticals, Inc., Tarrytown, New York, USA.GaleNicholas WNWRegeneron Pharmaceuticals, Inc., Tarrytown, New York, USA.AuerbachWojtekWRegeneron Pharmaceuticals, Inc., Tarrytown, New York, USA.MurphyAndrew JAJRegeneron Pharmaceuticals, Inc., Tarrytown, New York, USA.MacdonaldLynn ELERegeneron Pharmaceuticals, Inc., Tarrytown, New York, USA.engJournal Article
United StatesBiol Reprod02072240006-33630Adam6a protein, mouseEC 3.4.24.-ADAM ProteinsIMADAM ProteinsgeneticsmetabolismAnimalsFemaleInfertility, MalegeneticsMaleMiceMice, KnockoutOviductsSperm MotilitygeneticsphysiologySpermatozoaphysiologyextracellular matrixfemale reproductive tractmale subfertilityoviductsperm motility and transport20183302018882018103201810660202052760201810660ppublish3028944110.1093/biolre/ioy2105115557269799382017010620191210
2045-232262016Mar16Scientific reportsSci RepC9orf72 ablation causes immune dysregulation characterized by leukocyte expansion, autoantibody production, and glomerulonephropathy in mice.23204232042320410.1038/srep23204The expansion of a hexanucleotide (GGGGCC) repeat in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Both the function of C9ORF72 and the mechanism by which the repeat expansion drives neuropathology are unknown. To examine whether C9ORF72 haploinsufficiency induces neurological disease, we created a C9orf72-deficient mouse line. Null mice developed a robust immune phenotype characterized by myeloid expansion, T cell activation, and increased plasma cells. Mice also presented with elevated autoantibodies and evidence of immune-mediated glomerulonephropathy. Collectively, our data suggest that C9orf72 regulates immune homeostasis and an autoimmune response reminiscent of systemic lupus erythematosus (SLE) occurs in its absence. We further imply that haploinsufficiency is unlikely to be the causative factor in C9ALS/FTD pathology.AtanasioAmandaARegeneron Pharmaceuticals, Inc, Tarrytown, NY USA.DecmanVilmaVRegeneron Pharmaceuticals, Inc, Tarrytown, NY USA.WhiteDerekDRegeneron Pharmaceuticals, Inc, Tarrytown, NY USA.RamosMegMRegeneron Pharmaceuticals, Inc, Tarrytown, NY USA.IkizBurcinBRegeneron Pharmaceuticals, Inc, Tarrytown, NY USA.LeeHoi-ChingHCRegeneron Pharmaceuticals, Inc, Tarrytown, NY USA.SiaoChia-JenCJRegeneron Pharmaceuticals, Inc, Tarrytown, NY USA.BrydgesSusannahSRegeneron Pharmaceuticals, Inc, Tarrytown, NY USA.LaRosaElizabethERegeneron Pharmaceuticals, Inc, Tarrytown, NY USA.BaiYuYRegeneron Pharmaceuticals, Inc, Tarrytown, NY USA.FuryWenWRegeneron Pharmaceuticals, Inc, Tarrytown, NY USA.BurfeindPatriciaPRegeneron Pharmaceuticals, Inc, Tarrytown, NY USA.ZamfirovaRalicaRRegeneron Pharmaceuticals, Inc, Tarrytown, NY USA.WarshawGreggGRegeneron Pharmaceuticals, Inc, Tarrytown, NY USA.OrengoJamieJRegeneron Pharmaceuticals, Inc, Tarrytown, NY USA.OyejideAdelekanARegeneron Pharmaceuticals, Inc, Tarrytown, NY USA.FralishMichaelMRegeneron Pharmaceuticals, Inc, Tarrytown, NY USA.AuerbachWojtekWRegeneron Pharmaceuticals, Inc, Tarrytown, NY USA.PoueymirouWilliamWRegeneron Pharmaceuticals, Inc, Tarrytown, NY USA.FreudenbergJanJRegeneron Pharmaceuticals, Inc, Tarrytown, NY USA.GongGuochunGRegeneron Pharmaceuticals, Inc, Tarrytown, NY USA.ZambrowiczBrianBRegeneron Pharmaceuticals, Inc, Tarrytown, NY USA.ValenzuelaDavidDRegeneron Pharmaceuticals, Inc, Tarrytown, NY USA.YancopoulosGeorgeGRegeneron Pharmaceuticals, Inc, Tarrytown, NY USA.MurphyAndrewARegeneron Pharmaceuticals, Inc, Tarrytown, NY USA.ThurstonGavinGRegeneron Pharmaceuticals, Inc, Tarrytown, NY USA.LaiKa-Man VenusKMRegeneron Pharmaceuticals, Inc, Tarrytown, NY USA.engJournal Article20160316
EnglandSci Rep1015632882045-23220Autoantibodies0C9orf72 Protein0C9orf72 protein, mouse0Cytokines0Guanine Nucleotide Exchange FactorsIMActa Neuropathol. 2016 Jul;132(1):145-7. doi: 10.1007/s00401-016-1581-x27206760AnimalsAutoantibodiesbiosynthesisbloodAutoimmunityC9orf72 ProteinCytokinesbloodFemaleGlomerulonephritis, MembranoproliferativebloodgeneticsimmunologyGuanine Nucleotide Exchange FactorsgeneticsmetabolismLupus Erythematosus, SystemicgeneticsimmunologyLymphocyte ActivationLymphoid TissuepathologyMacrophagesimmunologyMaleMice, 129 StrainMice, Inbred C57BLMice, KnockoutPlasma CellsimmunologySequence Analysis, RNATranscriptome20151117201632201631760201631760201717602016316epublish26979938PMC479323610.1038/srep23204srep23204DeJesus-Hernandez M. et al. Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 72, 245–256 (2011).PMC320298621944778Renton A. E. et al. A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 72, 257–268 (2011).PMC320043821944779van Swieten J. C. & Grossman M. FTD/ALS families are no longer orphaned: the C9ORF72 story. Neurology 79, 962–964 (2012).22875094Donnelly C. J., Grima J. C. & Sattler R. Aberrant RNA homeostasis in amyotrophic lateral sclerosis: potential for new therapeutic targets? Neurodegener. Dis. Manag. 4, 417–437 (2014).PMC430829225531686Haeusler A. R. et al. C9orf72 nucleotide repeat structures initiate molecular cascades of disease. Nature 507, 195–200 (2014).PMC404661824598541Lee Y. B. et al. Hexanucleotide repeats in ALS/FTD form length-dependent RNA foci, sequester RNA binding proteins, and are neurotoxic. Cell Rep 5, 1178–1186 (2013).PMC389846924290757Mori K. et al. hnRNP A3 binds to GGGGCC repeats and is a constituent of p62-positive/TDP43-negative inclusions in the hippocampus of patients with C9orf72 mutations. Acta Neuropathol 125, 413–423 (2013).23381195Porta S., Kwong L. K., Trojanowski J. Q. & Lee V. M. Drosha inclusions are new components of dipeptide-repeat protein aggregates in FTLD-TDP and ALS C9orf72 expansion cases. J Neuropathol Exp Neurol 74, 380–387 (2015).PMC436247825756586Sareen D. et al. Targeting RNA foci in iPSC-derived motor neurons from ALS patients with a C9ORF72 repeat expansion. Sci Transl Med 5, 208ra149 (2013).PMC409094524154603Xu Z. et al. Expanded GGGGCC repeat RNA associated with amyotrophic lateral sclerosis and frontotemporal dementia causes neurodegeneration. Proc Natl Acad Sci USA 110, 7778–7783 (2013).PMC365148523553836Ash P. E. et al. Unconventional translation of C9ORF72 GGGGCC expansion generates insoluble polypeptides specific to c9FTD/ALS. Neuron 77, 639–646 (2013).PMC359323323415312Mori K. et al. Bidirectional transcripts of the expanded C9orf72 hexanucleotide repeat are translated into aggregating dipeptide repeat proteins. Acta Neuropathol 126, 881–893 (2013).24132570Mori K. et al. The C9orf72 GGGGCC repeat is translated into aggregating dipeptide-repeat proteins in FTLD/ALS. Science 339, 1335–1338 (2013).23393093Zu T. et al. RAN proteins and RNA foci from antisense transcripts in C9ORF72 ALS and frontotemporal dementia. Proc Natl Acad Sci USA 110, E4968–4977 (2013).PMC387066524248382Stancu I. C., Vasconcelos B., Terwel D. & Dewachter I. Models of beta-amyloid induced Tau-pathology: the long and “folded” road to understand the mechanism. Mol Neurodegener 9, 51 (2014).PMC425565525407337Gijselinck I. et al. A C9orf72 promoter repeat expansion in a Flanders-Belgian cohort with disorders of the frontotemporal lobar degeneration-amyotrophic lateral sclerosis spectrum: a gene identification study. Lancet Neurol 11, 54–65 (2012).22154785Waite A. J. et al. Reduced C9orf72 protein levels in frontal cortex of amyotrophic lateral sclerosis and frontotemporal degeneration brain with the C9ORF72 hexanucleotide repeat expansion. Neurobiol Aging 35, 1779 e1775-1779 e1713 (2014).PMC398888224559645Fratta P. et al. C9orf72 hexanucleotide repeat associated with amyotrophic lateral sclerosis and frontotemporal dementia forms RNA G-quadruplexes. Sci Rep 2, 1016 (2012).PMC352782523264878Reddy K., Zamiri B., Stanley S. Y., Macgregor R. B. Jr. & Pearson C. E. The disease-associated r(GGGGCC)n repeat from the C9orf72 gene forms tract length-dependent uni- and multimolecular RNA G-quadruplex structures. J Biol Chem 288, 9860–9866 (2013).PMC361728623423380Sket P. et al. Characterization of DNA G-quadruplex species forming from C9ORF72 G4C2-expanded repeats associated with amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Neurobiol Aging 36, 1091–1096 (2015).25442110Ciura S. et al. Loss of function of C9orf72 causes motor deficits in a zebrafish model of amyotrophic lateral sclerosis. Ann Neurol 74, 180–187 (2013).23720273Therrien M., Rouleau G. A., Dion P. A. & Parker J. A. Deletion of C9ORF72 results in motor neuron degeneration and stress sensitivity in C. elegans. PLoS One 8, e83450 (2013).PMC386148424349511Koppers M. et al. C9orf72 ablation in mice does not cause motor neuron degeneration or motor deficits. Ann Neurol 78, 426–438 (2015).PMC474497926044557Lagier-Tourenne C. et al. Targeted degradation of sense and antisense C9orf72 RNA foci as therapy for ALS and frontotemporal degeneration. Proc Natl Acad Sci USA 110, E4530–4539 (2013).PMC383975224170860Suzuki N. et al. The mouse C9ORF72 ortholog is enriched in neurons known to degenerate in ALS and FTD. Nat Neurosci 16, 1725–1727 (2013).PMC439790224185425Gill A., Kidd J., Vieira F., Thompson K. & Perrin S. No benefit from chronic lithium dosing in a sibling-matched, gender balanced, investigator-blinded trial using a standard mouse model of familial ALS. PLoS One 4, e6489 (2009).PMC271446019649300Heng T. S., Painter M. W. & Immunological Genome Project, C. The Immunological Genome Project: networks of gene expression in immune cells. Nat Immunol 9, 1091–1094 (2008).18800157Vignali D. A. & Kuchroo V. K. IL-12 family cytokines: immunological playmakers. Nat Immunol 13, 722–728 (2012).PMC415881722814351Kilciksiz S., Karakoyun-Celik O., Agaoglu F. Y. & Haydaroglu A. A review for solitary plasmacytoma of bone and extramedullary plasmacytoma. Scientific World Journal 2012, 895765 (2012).PMC335466822654647Palumbo A. & Anderson K. Multiple myeloma. N Engl J Med 364, 1046–1060 (2011).21410373Tarlinton D. M. & Hodgkin P. D. Targeting plasma cells in autoimmune diseases. J Exp Med 199, 1451–1454 (2004).PMC221178015173204Browning J. L. B cells move to centre stage: novel opportunities for autoimmune disease treatment. Nat Rev Drug Discov 5, 564–576 (2006).16816838Ingegnoli F., Castelli R. & Gualtierotti R. Rheumatoid factors: clinical applications. Dis Markers 35, 727–734 (2013).PMC384543024324289Kurts C., Panzer U., Anders H. J. & Rees A. J. The immune system and kidney disease: basic concepts and clinical implications. Nat Rev Immunol 13, 738–753 (2013).24037418Apostolidis S. A., Lieberman L. A., Kis-Toth K., Crispin J. C. & Tsokos G. C. The dysregulation of cytokine networks in systemic lupus erythematosus. J Interferon Cytokine Res 31, 769–779 (2011).PMC318955321877904Perry D., Sang A., Yin Y., Zheng Y. Y. & Morel L. Murine models of systemic lupus erythematosus. J Biomed Biotechnol 2011, 271694 (2011).PMC304262821403825Sherer Y., Gorstein A., Fritzler M. J. & Shoenfeld Y. Autoantibody explosion in systemic lupus erythematosus: more than 100 different antibodies found in SLE patients. Semin Arthritis Rheum 34, 501–537 (2004).15505768Xu H. et al. Increased frequency of circulating follicular helper T cells in lupus patients is associated with autoantibody production in a CD40L-dependent manner. Cell Immunol 295, 46–51 (2015).25748125Migliorini P., Baldini C., Rocchi V. & Bombardieri S. Anti-Sm and anti-RNP antibodies. Autoimmunity 38, 47–54 (2005).15804705Kivity S., Agmon-Levin N., Zandman-Goddard G., Chapman J. & Shoenfeld Y. Neuropsychiatric lupus: a mosaic of clinical presentations. BMC Med 13, 43 (2015).PMC434974825858312Gulinello M. & Putterman C. The MRL/lpr mouse strain as a model for neuropsychiatric systemic lupus erythematosus. J Biomed Biotechnol 2011, 207504 (2011).PMC303842821331367Zandman-Goddard G., Chapman J. & Shoenfeld Y. Autoantibodies involved in neuropsychiatric SLE and antiphospholipid syndrome. Semin Arthritis Rheum 36, 297–315 (2007).17258299Sciascia S., Bertolaccini M. L., Roccatello D., Khamashta M. A. & Sanna G. Autoantibodies involved in neuropsychiatric manifestations associated with systemic lupus erythematosus: a systematic review. J Neurol 261, 1706–1714 (2014).24952022Zivkovic S. Autoimmune neurologic disorders. Curr Neuropharmacol 9, 399 (2011).PMC315159322379453Nataf S. & Pays L. Gene co-expression analysis unravels a link between C9orf72 and RNA metabolism in myeloid cells. Acta Neuropathol Commun 3, 64 (2015).PMC460829026472214Muller M., Emmendorffer A. & Lohmann-Matthes M. L. Expansion and high proliferative potential of the macrophage system throughout life time of lupus-prone NZB/W and MRL lpr/lpr mice. Lack of down-regulation of extramedullar macrophage proliferation in the postnatal period. Eur J Immunol 21, 2211–2217 (1991).1889463Herrmann M. et al. Impaired phagocytosis of apoptotic cell material by monocyte-derived macrophages from patients with systemic lupus erythematosus. Arthritis Rheum 41, 1241–1250 (1998).9663482Potter P. K., Cortes-Hernandez J., Quartier P., Botto M. & Walport M. J. Lupus-prone mice have an abnormal response to thioglycolate and an impaired clearance of apoptotic cells. J Immunol 170, 3223–3232 (2003).12626581Shao W. H. & Cohen P. L. Disturbances of apoptotic cell clearance in systemic lupus erythematosus. Arthritis Res Ther 13, 202 (2011).PMC315763621371352Zhou X. J. et al. Genetic association of PRDM1-ATG5 intergenic region and autophagy with systemic lupus erythematosus in a Chinese population. Ann Rheum Dis 70, 1330–1337 (2011).21622776Zhang Y. M. et al. Rare Variants of ATG5 Are Likely to Be Associated With Chinese Patients With Systemic Lupus Erythematosus. Medicine (Baltimore) 94, e939 (2015).PMC461636326039132Fernandez D. R. et al. Activation of mammalian target of rapamycin controls the loss of TCRzeta in lupus T cells through HRES-1/Rab4-regulated lysosomal degradation. J Immunol 182, 2063–2073 (2009).PMC267611219201859Clarke A. J. et al. Autophagy is activated in systemic lupus erythematosus and required for plasmablast development. Ann Rheum Dis 74, 912–920 (2015).PMC415219224419333Pierdominici M. et al. Role of autophagy in immunity and autoimmunity, with a special focus on systemic lupus erythematosus. FASEB J 26, 1400–1412 (2012).22247332Towns R. et al. Sera from patients with type 2 diabetes and neuropathy induce autophagy and colocalization with mitochondria in SY5Y cells. Autophagy 1, 163–170 (2005).16874076Farg M. A. et al. C9ORF72, implicated in amytrophic lateral sclerosis and frontotemporal dementia, regulates endosomal trafficking. Hum Mol Genet 23, 3579–3595 (2014).PMC404931024549040Xiao S. et al. Isoform-specific antibodies reveal distinct subcellular localizations of C9orf72 in amyotrophic lateral sclerosis. Ann Neurol 78, 568–583 (2015).26174152Freibaum B. D. et al. GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport. Nature 525, 129–133 (2015).PMC463139926308899Jovicic A. et al. Modifiers of C9orf72 dipeptide repeat toxicity connect nucleocytoplasmic transport defects to FTD/ALS. Nat Neurosci 18, 1226–1229 (2015).PMC455207726308983Zhang K. et al. The C9orf72 repeat expansion disrupts nucleocytoplasmic transport. Nature 525, 56–61 (2015).PMC480074226308891Dechiara T. M. et al. VelociMouse: fully ES cell-derived F0-generation mice obtained from the injection of ES cells into eight-cell-stage embryos. Methods Mol Biol 530, 311–324 (2009).19266341DeChiara T. M. et al. Producing fully ES cell-derived mice from eight-cell stage embryo injections. Methods Enzymol 476, 285–294 (2010).20691872Poueymirou W. T. et al. F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses. Nat Biotechnol 25, 91–99 (2007).17187059Valenzuela D. M. et al. High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nat Biotechnol 21, 652–659 (2003).12730667Frazier K. S. et al. Proliferative and nonproliferative lesions of the rat and mouse urinary system. Toxicol Pathol 40, 14S–86S (2012).22637735259099112016041120181113
1932-62031042015PloS onePLoS OneDiverse Phenotypes and Specific Transcription Patterns in Twenty Mouse Lines with Ablated LincRNAs.e0125522e0125522e012552210.1371/journal.pone.0125522In a survey of 20 knockout mouse lines designed to examine the biological functions of large intergenic non-coding RNAs (lincRNAs), we have found a variety of phenotypes, ranging from perinatal lethality to defects associated with premature aging and morphological and functional abnormalities in the lungs, skeleton, and muscle. Each mutant allele carried a lacZ reporter whose expression profile highlighted a wide spectrum of spatiotemporal and tissue-specific transcription patterns in embryos and adults that informed our phenotypic analyses and will serve as a guide for future investigations of these genes. Our study shows that lincRNAs are a new class of encoded molecules that, like proteins, serve essential and important functional roles in embryonic development, physiology, and homeostasis of a broad array of tissues and organs in mammals.LaiKa-Man VenusKMVelociGene, Regeneron Pharmaceuticals, Inc., Tarrytown, New York, United States of America.GongGuochunGVelociGene, Regeneron Pharmaceuticals, Inc., Tarrytown, New York, United States of America.AtanasioAmandaAVelociGene, Regeneron Pharmaceuticals, Inc., Tarrytown, New York, United States of America.RojasJoséJVelociGene, Regeneron Pharmaceuticals, Inc., Tarrytown, New York, United States of America.QuispeJosephJVelociGene, Regeneron Pharmaceuticals, Inc., Tarrytown, New York, United States of America.PoscaJulitaJVelociGene, Regeneron Pharmaceuticals, Inc., Tarrytown, New York, United States of America.WhiteDerekDVelociGene, Regeneron Pharmaceuticals, Inc., Tarrytown, New York, United States of America.HuangMeiMVelociGene, Regeneron Pharmaceuticals, Inc., Tarrytown, New York, United States of America.FedorovaDariaDVelociGene, Regeneron Pharmaceuticals, Inc., Tarrytown, New York, United States of America.GrantCraigCVelociGene, Regeneron Pharmaceuticals, Inc., Tarrytown, New York, United States of America.MiloscioLawrenceLVelociGene, Regeneron Pharmaceuticals, Inc., Tarrytown, New York, United States of America.DroguettGustavoGVelociGene, Regeneron Pharmaceuticals, Inc., Tarrytown, New York, United States of America.PoueymirouWilliam TWTVelociGene, Regeneron Pharmaceuticals, Inc., Tarrytown, New York, United States of America.AuerbachWojtekWVelociGene, Regeneron Pharmaceuticals, Inc., Tarrytown, New York, United States of America.YancopoulosGeorge DGDVelociGene, Regeneron Pharmaceuticals, Inc., Tarrytown, New York, United States of America.FrendeweyDavidDVelociGene, Regeneron Pharmaceuticals, Inc., Tarrytown, New York, United States of America.RinnJohnJDepartment of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, United States of America; Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America.ValenzuelaDavid MDMVelociGene, Regeneron Pharmaceuticals, Inc., Tarrytown, New York, United States of America.engJournal ArticleResearch Support, Non-U.S. Gov't20150424
United StatesPLoS One1012850811932-62030Membrane Transport Proteins0RNA, Long Noncoding9068-45-5lactose permeaseIMAllelesAnimalsEmbryonic DevelopmentgeneticsFemaleGenes, ReportergeneticsMaleMammalsgeneticsMembrane Transport ProteinsgeneticsMiceMice, Inbred C57BLMice, KnockoutPhenotypeRNA, Long NoncodinggeneticsTranscription, GeneticgeneticsTranscriptomegeneticsCompeting Interests: K-MVL, GG, AA, J. Rojas, JQ, JP, DW, MH, D. Fedorova, CG, LM, GD, WTP, WA, GDY, DF, DMV are employees of VelociGene, Regeneron Pharmaceuticals, Inc. A patent application has been filed relating to this work, title: LincRNA-deficient non-human animals and application No: US 14/454,464 and PCT/US2014/050178. There is no product in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.201411220153142015425602015425602016412602015424epublish25909911PMC440929310.1371/journal.pone.0125522PONE-D-14-49290Mattick JS. The genetic signatures of noncoding RNAs. PLoS Genet 2009;5: e1000459 10.1371/journal.pgen.100045910.1371/journal.pgen.1000459PMC266726319390609Carninci P, Kasukawa T, Katayama S, Gough J, Frith MC, Maeda N, et al. The transcriptional landscape of the mammalian genome. Science. 2005;309: 1559–1563.16141072Djebali S, Davis CA, Merkel A, Dobin A, Lassmann T, Mortazavi A, et al. Landscape of transcription in human cells. Nature. 2012;489: 101–108. 10.1038/nature1123310.1038/nature11233PMC368427622955620Kapranov P, Cheng J, Dike S, Nix DA, Duttagupta R, Willingham AT, et al. RNA maps reveal new RNA classes and a possible function for pervasive transcription. Science. 2007;316: 1484–1488.17510325Derrien T, Johnson R, Bussotti G, Tanzer A, Djebali S, Tilgner H, et al. The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution, and expression. Genome Res. 2012;22: 1775–1789. 10.1101/gr.132159.11110.1101/gr.132159.111PMC343149322955988Guttman M, Amit I, Garber M, French C, Lin MF, Feldser D, et al. Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals. Nature. 2009;458: 223–227. 10.1038/nature0767210.1038/nature07672PMC275484919182780Khalil AM, Guttman M, Huarte M, Garber M, Raj A, Rivea Morales D, et al. Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression. Proc Natl Acad Sci U S A. 2009;106: 11667–11672. 10.1073/pnas.090471510610.1073/pnas.0904715106PMC270485719571010Capecchi MR. Generating mice with targeted mutations. Nat Med. 2001;7: 1086–1090.11590420Evans MJ. The cultural mouse. Nat Med. 2001;7: 1081–1083.11590418Smithies O. Forty years with homologous recombination. Nat Med. 2001;7: 1083–1086.11590419Prosser HM, Koike-Yusa H, Cooper JD, Law FC, Bradley A. A resource of vectors and ES cells for targeted deletion of microRNAs in mice. Nat Biotechnol. 2011;29: 840–845. 10.1038/nbt.192910.1038/nbt.1929PMC324203221822254Marahrens Y, Panning B, Dausman J, Strauss W, Jaenisch R. Xist-deficient mice are defective in dosage compensation but not spermatogenesis. Genes Dev. 1997;11: 156–166.9009199Sado T, Wang Z, Sasaki H, Li E. Regulation of imprinted X-chromosome inactivation in mice by Tsix. Development. 2001;128: 1275–1286.11262229Leighton PA, Ingram RS, Eggenschwiler J, Efstratiadis A, Tilghman SM. Disruption of imprinting caused by deletion of the H19 gene region in mice. Nature. 1995;375: 34–39.7536897Mohammad F, Mondal T, Guseva N, Pandey GK, Kanduri C. Kcnq1ot1 noncoding RNA mediates transcriptional gene silencing by interacting with Dnmt1. Development. 2010;137: 2493–2499. 10.1242/dev.04818110.1242/dev.04818120573698Sleutels F, Zwart R, Barlow DP. The non-coding Air RNA is required for silencing autosomal imprinted genes. Nature. 2002;415: 810–813.11845212Takahashi N, Okamoto A, Kobayashi R, Shirai M, Obata Y, Ogawa H, et al. Deletion of Gtl2, imprinted non-coding RNA, with its differentially methylated region induces lethal parent-origin-dependent defects in mice. Hum Mol Genet 2009;18: 1879–1888. 10.1093/hmg/ddp10810.1093/hmg/ddp10819264764Grote P, Wittler L, Hendrix D, Koch F, Wahrisch S, Beisaw A, et al. The tissue-specific lncRNA Fendrr is an essential regulator of heart and body wall development in the mouse. Dev Cell. 2013;24: 206–214. 10.1016/j.devcel.2012.12.01210.1016/j.devcel.2012.12.012PMC414917523369715Zambrowicz BP, Imamoto A, Fiering S, Herzenberg LA, Kerr WG, Soriano P. Disruption of overlapping transcripts in the ROSA beta geo 26 gene trap strain leads to widespread expression of beta-galactosidase in mouse embryos and hematopoietic cells. Proc Natl Acad Sci U S A 1997;94: 3789–3794.PMC205199108056Zhang B, Arun G, Mao YS, Lazar Z, Hung G, Bhattacharjee G, et al. The lncRNA Malat1 is dispensable for mouse development but its transcription plays a cis-regulatory role in the adult. Cell Rep. 2012;2: 111–123. 10.1016/j.celrep.2012.06.00310.1016/j.celrep.2012.06.003PMC340858722840402Thai TH, Calado DP, Casola S, Ansel KM, Xiao C, Xue Y, et al. Regulation of the germinal center response by microRNA-155. Science. 2007;316: 604–608.17463289Valenzuela DM, Murphy AJ, Frendewey D, Gale NW, Economides AN, Auerbach W, et al. High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nat Biotechnol. 2003;21: 652–659.12730667Adams DJ, Quail MA, Cox T, van der Weyden L, Gorick BD, Su Q, et al. A genome-wide, end-sequenced 129Sv BAC library resource for targeting vector construction. Genomics. 2005;86: 753–758.16257172Frendewey D, Chernomorsky R, Esau L, Om J, Xue Y, Murphy AJ, et al. The loss-of-allele assay for ES cell screening and mouse genotyping. Methods Enzymol. 2010;476: 295–307. 10.1016/S0076-6879(10)76017-110.1016/S0076-6879(10)76017-120691873Dechiara TM, Poueymirou WT, Auerbach W, Frendewey D, Yancopoulos GD, Valenzuela DM. VelociMouse: fully ES cell-derived F0-generation mice obtained from the injection of ES cells into eight-cell-stage embryos. Methods Mol Biol. 2009;530: 311–324. 10.1007/978-1-59745-471-1_1610.1007/978-1-59745-471-1_1619266341DeChiara TM, Poueymirou WT, Auerbach W, Frendewey D, Yancopoulos GD, Valenzuela DM. Producing fully ES cell-derived mice from eight-cell stage embryo injections. Methods Enzymol. 2010;476: 285–294. 10.1016/S0076-6879(10)76016-X10.1016/S0076-6879(10)76016-X20691872Poueymirou WT, Auerbach W, Frendewey D, Hickey JF, Escaravage JM, Esau L, et al. F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses. Nat Biotechnol. 2007;25: 91–99.17187059Cabili MN, Trapnell C, Goff L, Koziol M, Tazon-Vega B, Regev A, et al. Integrative annotation of human large intergenic noncoding RNAs reveals global properties and specific subclasses. Genes Dev. 2011;25: 1915–1927. 10.1101/gad.1744661110.1101/gad.17446611PMC318596421890647Sauvageau M, Goff LA, Lodato S, Bonev B, Groff AF, Gerhardinger C, et al. Multiple knockout mouse models reveal lincRNAs are required for life and brain development. Elife (Cambridge). 2013;2: e01749 10.7554/eLife.0174910.7554/eLife.01749PMC387410424381249Tsai MC, Manor O, Wan Y, Mosammaparast N, Wang JK, Lan F, et al. Long noncoding RNA as modular scaffold of histone modification complexes. Science. 2010;329: 689–693. 10.1126/science.119200210.1126/science.1192002PMC296777720616235Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987;15: 8125–8148.PMC3063493313277Hostikka SL, Capecchi MR. The mouse Hoxc11 gene: genomic structure and expression pattern. Mech Dev. 1998;70: 133–145.9510030Lu P, Yu Y, Perdue Y, Werb Z. The apical ectodermal ridge is a timer for generating distal limb progenitors. Development. 2008;135: 1395–1405. 10.1242/dev.01894510.1242/dev.018945PMC257450918359901Schorderet P, Duboule D. Structural and functional differences in the long non-coding RNA hotair in mouse and human. PLoS Genet. 2011;7: e1002071 10.1371/journal.pgen.100207110.1371/journal.pgen.1002071PMC310275021637793Nagy A. Manipulating the mouse embryo: a laboratory manual Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 2003.Dimri G, Lee X, Basile G, Acosta M, Scott G, Roskelley C, et al. A biomarker that identifies sensescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A. 1995;92: 9363–9367.PMC409857568133Carpenter S, Aiello D, Atianand MK, Ricci EP, Gandhi P, Hall LL, et al. A long noncoding RNA mediates both activation and repression of immune response genes. Science. 2013;341: 789–792. 10.1126/science.124092510.1126/science.1240925PMC437666823907535Li L, Liu B, Wapinski OL, Tsai MC, Qu K, Zhang J, et al. Targeted disruption of Hotair leads to homeotic transformation and gene derepression. Cell Rep. 2013;5: 3–12. 10.1016/j.celrep.2013.09.00310.1016/j.celrep.2013.09.003PMC403829524075995Bishop NB, Stankiewicz P, Steinhorn RH. Alveolar capillary dysplasia. Am J Respir Crit Care Med. 2011;184: 172–179. 10.1164/rccm.201010-1697CI10.1164/rccm.201010-1697CIPMC317288721471096Szafranski P, Dharmadhikari AV, Brosens E, Gurha P, Kolodziejska KE, Zhishuo O, et al. Small noncoding differentially methylated copy-number variants, including lncRNA genes, cause a lethal lung developmental disorder. Genome Res. 2013;23: 23–33. 10.1101/gr.141887.11210.1101/gr.141887.112PMC353068123034409Marin-Bejar O, Marchese FP, Athie A, Sanchez Y, Gonzalez J, Segura V, et al. Pint lincRNA connects the p53 pathway with epigenetic silencing by the Polycomb repressive complex 2. Genome Biol. 2013;14: R104PMC405382224070194Rufini A, Tucci P, Celardo I, Melino G. Senescence and aging: the critical roles of p53. Oncogene. 2013;32: 5129–5143. 10.1038/onc.2012.64010.1038/onc.2012.64023416979250871742015082120211021
1573-93682412015FebTransgenic researchTransgenic ResGeneration of fertile and fecund F0 XY female mice from XY ES cells.192919-2910.1007/s11248-014-9815-yKnown examples of male to female sex reversal in mice are caused by either strain incompatibilities or mutations in genes required for male sex determination. The resultant XY females are often sterile or exhibit very poor fertility. We describe here embryonic stem (ES) cell growth conditions that promote the production of healthy, anatomically normal fertile and fecund female F0 generation mice completely derived from gene-targeted XY male ES cells. The sex reversal is a transient trait that is not transmitted to the F1 progeny. Growth media with low osmolality and reduced sodium bicarbonate, maintained throughout the gene targeting process, enhance the yield of XY females. As a practical application of the induced sex reversal, we demonstrate the generation of homozygous mutant mice ready for phenotypic studies by the breeding of F0 XY females with their isogenic XY male clonal siblings, thereby eliminating one generation of breeding and the associated costs.KunoJunkoJRegeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, NY, 10591, USA.PoueymirouWilliam TWTGongGuochunGSiaoChia-JenCJClarkeGeorgiaGEsauLakeishaLKojakNadaNPoscaJulitaJAtanasioAmandaAStreinJohnJYancopoulosGeorge DGDLaiKa-Man VenusKMDeChiaraThomas MTMFrendeweyDavidDAuerbachWojtekWValenzuelaDavid MDMengJournal Article20140803
NetherlandsTransgenic Res92091200962-8819IMAnimalsDisorders of Sex DevelopmentgeneticsEmbryonic Stem CellscytologyFemaleFertilitygeneticsGene TargetingGonadal Dysgenesis, 46,XYgeneticsMaleMiceMicroinjectionsMutationSex Determination Processes20143312014722014846020148560201582260ppublish2508717410.1007/s11248-014-9815-yScience. 1994 Aug 5;265(5173):808-117914033Dev Cell. 2012 Nov 13;23(5):1020-3123102580Cell. 2001 Aug 10;106(3):319-2911509181Nat Biotechnol. 2007 Jan;25(1):59-6017211398Nat Biotechnol. 2003 Jun;21(6):652-912730667Development. 1989 Sep;107(1):95-1052534072Am J Physiol Renal Physiol. 2010 Jan;298(1):F24-3419759267Methods Enzymol. 2010;476:295-30720691873Sci Rep. 2013 Nov 05;3:313624190364Science. 1993 Mar 19;259(5102):1760-37681220Development. 2004 May;131(9):1891-90115056615Nat Rev Rheumatol. 2011 Jun 14;7(8):447-5621670768Proc Natl Acad Sci U S A. 1998 Nov 10;95(23):13453-89811821Development. 1990 Jul;109(3):635-462401216Science. 1982 Aug 6;217(4559):535-77089579Dev Biol. 2008 Feb 1;314(1):71-8318155190Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3120-37724525Nat Biotechnol. 2007 Jan;25(1):91-917187059Nat Biotechnol. 2013 Jun;31(6):530-223666012Nat Genet. 2003 May;34(1):32-312679814Dev Cell. 2012 Nov 13;23(5):1032-4223102581PLoS Biol. 2009 Sep;7(9):e100019619753101Development. 2002 Oct;129(19):4627-3412223418Bone. 2009 Feb;44(2):199-20718845279Cell. 2001 Mar 23;104(6):875-8911290325Nature. 2003 Nov 20;426(6964):291-514628051Nature. 1998 Jun 18;393(6686):688-929641679Biol Reprod. 2006 Jan;74(1):195-20116207837Am J Physiol Renal Physiol. 2008 Oct;295(4):F867-7618480174243812492015033020240104
2050-084X22013Dec31eLifeElifeMultiple knockout mouse models reveal lincRNAs are required for life and brain development.e01749e01749e0174910.7554/eLife.01749Many studies are uncovering functional roles for long noncoding RNAs (lncRNAs), yet few have been tested for in vivo relevance through genetic ablation in animal models. To investigate the functional relevance of lncRNAs in various physiological conditions, we have developed a collection of 18 lncRNA knockout strains in which the locus is maintained transcriptionally active. Initial characterization revealed peri- and postnatal lethal phenotypes in three mutant strains (Fendrr, Peril, and Mdgt), the latter two exhibiting incomplete penetrance and growth defects in survivors. We also report growth defects for two additional mutant strains (linc-Brn1b and linc-Pint). Further analysis revealed defects in lung, gastrointestinal tract, and heart in Fendrr(-/-) neonates, whereas linc-Brn1b(-/-) mutants displayed distinct abnormalities in the generation of upper layer II-IV neurons in the neocortex. This study demonstrates that lncRNAs play critical roles in vivo and provides a framework and impetus for future larger-scale functional investigation into the roles of lncRNA molecules. DOI: http://dx.doi.org/10.7554/eLife.01749.001.SauvageauMartinMDepartment of Stem Cell and Regenerative Biology, Harvard University, Cambridge, United States.GoffLoyal ALALodatoSimonaSBonevBoyanBGroffAbigail FAFGerhardingerChiaraCSanchez-GomezDiana BDBHacisuleymanEzgiELiEricESpenceMatthewMLiapisStephen CSCMallardWilliamWMorseMichaelMSwerdelMavis RMRD'EcclessisMichael FMFMooreJennifer CJCLaiVenusVGongGuochunGYancopoulosGeorge DGDFrendeweyDavidDKellisManolisMHartRonald PRPValenzuelaDavid MDMArlottaPaolaPRinnJohn LJLengRC1 CA147187CANCI NIH HHSUnited StatesR01 NS078164NSNINDS NIH HHSUnited StatesP01 GM099117GMNIGMS NIH HHSUnited StatesF30 NS062489NSNINDS NIH HHSUnited StatesNS073124NSNINDS NIH HHSUnited StatesNS062489NSNINDS NIH HHSUnited StatesDP2 OD006670ODNIH HHSUnited StatesR01 HG004037HGNHGRI NIH HHSUnited StatesR01 NS062849NSNINDS NIH HHSUnited StatesR01 NS073124NSNINDS NIH HHSUnited StatesP50 HG006193HGNHGRI NIH HHSUnited StatesDP2OD006670ODNIH HHSUnited StatesNS078164NSNINDS NIH HHSUnited StatesJournal ArticleResearch Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov't20131231
EnglandElife1015796142050-084X0RNA, Long NoncodingIMElife. 2013 Dec 31;2:e01968. doi: 10.7554/eLife.0196824381251AnimalsBraingrowth & developmentMiceMice, KnockoutRNA, Long Noncodinggeneticsphysiologybrain developmentdevelopmental defectknockout mouse modelslethalitylong noncoding RNAsVL: Employee of Regeneron Pharmaceuticals. GG: Employee of Regeneron Pharmaceuticals. GDY: Employee of Regeneron Pharmaceuticals. DF: Employee of Regeneron Pharmaceuticals. DMV: Employee of Regeneron Pharmaceuticals. The other authors declare that no competing interests exist.201412602014126020141261201411epublish24381249PMC387410410.7554/eLife.017492/0/e01749Almada AE, Wu X, Kriz AJ, Burge CB, Sharp PA. 2013. Promoter directionality is controlled by U1 snRNP and polyadenylation signals. Nature 499:360–363. 10.1038/nature1234910.1038/nature12349PMC372071923792564Anguera MC, Ma W, Clift D, Namekawa S, Kelleher RJ, Lee JT. 2011. Tsx produces a long noncoding RNA and has general functions in the germline, stem cells, and brain. PLOS Genetics 7:e1002248. 10.1371/journal.pgen.100224810.1371/journal.pgen.1002248PMC316469121912526Arlotta P, Molyneaux BJ, Chen J, Inoue J, Kominami R, Macklis JD. 2005. Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo. Neuron 45:207–221. 10.1016/j.neuron.2004.12.03610.1016/j.neuron.2004.12.03615664173Arnold SJ, Huang G-J, Cheung AFP, Era T, Nishikawa S-I, Bikoff EK, Molnár Z, Robertson EJ, Groszer M. 2008. The T-box transcription factor Eomes/Tbr2 regulates neurogenesis in the cortical subventricular zone. Genes and Development 22:2479–2484. 10.1101/gad.47540810.1101/gad.475408PMC254669718794345Berger UV, Hediger MA. 2001. Differential distribution of the glutamate transporters GLT-1 and GLAST in tanycytes of the third ventricle. The Journal of Comparative Neurology 433:101–114. 10.1002/cne.112810.1002/cne.112811283952Brunner AL, Beck AH, Edris B, Sweeney RT, Zhu SX, Li R, Montgomery K, Varma S, Gilks T, Guo X, Foley JW, Witten DM, Giacomini CP, Flynn RA, Pollack JR, Tibshirani R, Chang HY, van de Rijn M, West RB. 2012. Transcriptional profiling of long non-coding RNAs and novel transcribed regions across a diverse panel of archived human cancers. Genome Biology 13:R75. 10.1186/gb-2012-13-8-r7510.1186/gb-2012-13-8-r75PMC405374322929540Cabili MN, Trapnell C, Goff L, Koziol M, Tazon-Vega B, Regev A, Rinn JL. 2011. Integrative annotation of human large intergenic noncoding RNAs reveals global properties and specific subclasses. Genes and Development 25:1915–1927. 10.1101/gad.1744661110.1101/gad.17446611PMC318596421890647Chen B, Schaevitz LR, McConnell SK. 2005. Fezl regulates the differentiation and axon targeting of layer 5 subcortical projection neurons in cerebral cortex. Proceedings of the National Academy of Sciences of the United States of America 102:17184–17189. 10.1073/pnas.050873210210.1073/pnas.0508732102PMC128256916284245Condie BG, Capecchi MR. 1993. Mice homozygous for a targeted disruption of Hoxd-3 (Hox-4.1) exhibit anterior transformations of the first and second cervical vertebrae, the atlas and the axis. Development 119:579–5957910549Cubelos B, Sebastian-Serrano A, Kim S, Moreno-Ortiz C, Redondo JM, Walsh CA, Nieto M. 2008. Cux-2 controls the proliferation of neuronal intermediate precursors of the cortical subventricular zone. Cerebral Cortex 18:1758–1770. 10.1093/cercor/bhm19910.1093/cercor/bhm19918033766Dominguez MH, Ayoub AE, Rakic P. 2012. Pou-iii transcription factors (Brn1, Brn2, and Oct6) influence neurogenesis, molecular identity, and migratory destination of upper-layer cells of the cerebral cortex. Cerebral Cortex 23:2632–2643. 10.1093/cercor/bhs25210.1093/cercor/bhs252PMC379274122892427Edwards AM, Isserlin R, Bader GD, Frye SV, Willson TM, Yu FH. 2011. Too many roads not taken. Nature 470:163–165. 10.1038/470163a10.1038/470163a21307913Eggan KK, Akutsu HH, Loring JJ, Jackson-Grusby LL, Klemm MM, Rideout WMW, Yanagimachi RR, Jaenisch RR. 2001. Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation. Proceedings of the National Academy of Sciences of the United States of America 98:6209–6214. 10.1073/pnas.10111889810.1073/pnas.101118898PMC3344711331774Englund C, Fink A, Lau C, Pham D, Daza RAM, Bulfone A, Kowalczyk T, Hevner RF. 2005. Pax6, Tbr2, and Tbr1 are expressed sequentially by radial glia, intermediate progenitor cells, and postmitotic neurons in developing neocortex. Journal of Neuroscience 25:247–251. 10.1523/JNEUROSCI.2899-04.200510.1523/JNEUROSCI.2899-04.2005PMC672518915634788Gascoigne DK, Cheetham SW, Cattenoz PB, Clark MB, Amaral PP, Taft RJ, Wilhelm D, Dinger ME, Mattick J. 2012. Pinstripe: a suite of programs for integrating transcriptomic and proteomic datasets identifies novel proteins and improves differentiation of protein-coding and non-coding genes. Bioinformatics 28:3042–3050. 10.1093/bioinformatics/bts58210.1093/bioinformatics/bts58223044541Goff LA, Davila J, Swerdel MR, Moore JC, Cohen RI, Wu H, Sun YE, Hart RP. 2009. Ago2 Immunoprecipitation identifies predicted MicroRNAs in human embryonic stem cells and neural Precursors. PLOS ONE 4:e7192. 10.1371/journal.pone.000719210.1371/journal.pone.0007192PMC274566019784364Gomez JA, Wapinski OL, Yang YW, Bureau J-F, Gopinath S, Monack DM, Chang HY, Brahic M, Kirkegaard K. 2013. The NeST long ncRNA controls microbial susceptibility and epigenetic activation of the interferon-γ locus. Behavioural Brain Research 152:743–754. 10.1016/j.cell.2013.01.01510.1016/j.cell.2013.01.015PMC357709823415224Gordon FE, Nutt CL, Cheunsuchon P, Nakayama Y, Provencher KA, Rice KA, Zhou Y, Zhang X, Klibanski A. 2010. Increased expression of angiogenic genes in the brains of mouse Meg3-null embryos. Endocrinology 151:2443–2452. 10.1210/en.2009-115110.1210/en.2009-1151PMC287581520392836Grote P, Wittler L, Hendrix D, Koch F, Währisch S, Beisaw A, Macura K, Bläss G, Kellis M, Werber M, Herrmann BG. 2013. The tissue-specific lncRNA Fendrr is an essential regulator of heart and body wall development in the mouse. Developmental Cell 24:206–214. 10.1016/j.devcel.2012.12.01210.1016/j.devcel.2012.12.012PMC414917523369715Guo T, Mandai K, Condie BG, Wickramasinghe SR, Capecchi MR, Ginty DD. 2010. An evolving NGF-Hoxd1 signaling pathway mediates development of divergent neural circuits in vertebrates. Nature Neuroscience 14:31–36. 10.1038/nn.271010.1038/nn.2710PMC318091821151121Guttman M, Rinn JL. 2012. Modular regulatory principles of large non-coding RNAs. Nature 482:339–346. 10.1038/nature1088710.1038/nature10887PMC419700322337053Guttman M, Amit I, Garber M, French C, Lin MF, Feldser D, Huarte M, Zuk O, Carey BW, Cassady JP, Cabili MN, Jaenisch R, Mikkelsen TS, Jacks T, Hacohen N, Bernstein BE, Kellis M, Regev A, Rinn JL, Lander ES. 2009. Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals. Nature 458:223–227. 10.1038/nature0767210.1038/nature07672PMC275484919182780Guttman M, Donaghey J, Carey BW, Garber M, Grenier JK, Munson G, Young G, Lucas AB, Ach R, Bruhn L, Yang X, Amit I, Meissner A, Regev A, Rinn JL, Root DE, Lander ES. 2011. lincRNAs act in the circuitry controlling pluripotency and differentiation. Nature 477:295–300. 10.1038/nature1039810.1038/nature10398PMC317532721874018Guttman M, Garber M, Levin JZ, Donaghey J, Robinson J, Adiconis X, Fan L, Koziol MJ, Gnirke A, Nusbaum C, Rinn JL, Lander ES, Regev A. 2010. Ab initio reconstruction of cell type-specific transcriptomes in mouse reveals the conserved multi-exonic structure of lincRNAs. Nature Biotechnology 28:503–510. 10.1038/nbt.163310.1038/nbt.1633PMC286810020436462Guttman M, Russell P, Ingolia NT, Weissman JS, Lander ES. 2013. Ribosome profiling provides evidence that large noncoding RNAs do not encode proteins. Behavioural Brain Research 154:240–251. 10.1016/j.cell.2013.06.00910.1016/j.cell.2013.06.009PMC375656323810193Jabaudon D, Shnider JS, Tischfield DJ, Galazo MJ, Macklis JD. 2012. RORβ induces barrel-like neuronal clusters in the developing neocortex. Cerebral Cortex 22:996–1006. 10.1093/cercor/bhr18210.1093/cercor/bhr182PMC332834321799210Khalil AM, Guttman M, Huarte M, Garber M, Raj A, Rivea Morales D, Thomas K, Presser A, Bernstein BE, van Oudenaarden A, Regev A, Lander ES, Rinn JL. 2009. Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression. Proceedings of the National Academy of Sciences of the United States of America 106:11667–11672. 10.1073/pnas.090471510610.1073/pnas.0904715106PMC270485719571010Koop KE, MacDonald LM, Lobe CG. 1996. Transcripts of Grg4, a murine groucho-related gene, are detected in adjacent tissues to other murine neurogenic gene homologues during embryonic development. Mechanisms of Development 59:73–87. 10.1016/0925-4773(96)00582-510.1016/0925-4773(96)00582-58892234Kowalczyk MS, Higgs DR, Gingeras TR. 2012. Molecular biology: RNA discrimination. Nature 482:310–311. 10.1038/482310a10.1038/482310a22337043Land PW, Simons DJ. 1985. Cytochrome oxidase staining in the rat SmI barrel cortex. The Journal of Comparative Neurology 238:225–235. 10.1002/cne.90238020910.1002/cne.9023802092413086Lin MF, Jungreis I, Kellis M. 2011. PhyloCSF: a comparative genomics method to distinguish protein coding and non-coding regions. Bioinformatics 27:i275–i282. 10.1093/bioinformatics/btr20910.1093/bioinformatics/btr209PMC311734121685081Mahlapuu M, Enerbäck S, Carlsson P. 2001. Haploinsufficiency of the forkhead gene Foxf1, a target for sonic hedgehog signaling, causes lung and foregut malformations. Development 128:2397–240611493558McEvilly RJ. 2002. Transcriptional regulation of cortical neuron Migration by POU domain factors. Science 295:1528–1532. 10.1126/science.106713210.1126/science.106713211859196Mercer TR, Dinger ME, Sunkin SM, Mehler MF, Mattick JS. 2008. Specific expression of long noncoding RNAs in the mouse brain. Proceedings of the National Academy of Sciences of the United States of America 105:716–721. 10.1073/pnas.070672910510.1073/pnas.0706729105PMC220660218184812Mercer TR, Mattick JS. 2013. Structure and function of long noncoding RNAs in epigenetic regulation. Nature Structural and Molecular Biology 20:300–307. 10.1038/nsmb.248010.1038/nsmb.248023463315Merkin J, Russell C, Chen P, Burge CB. 2012. Evolutionary dynamics of gene and isoform regulation in mammalian tissues. Science 338:1593–1599. 10.1126/science.122818610.1126/science.1228186PMC356849923258891Molyneaux BJ, Arlotta P, Hirata T, Hibi M, Macklis JD. 2005. Fezl is required for the birth and specification of corticospinal motor neurons. Neuron 47:817–831. 10.1016/j.neuron.2005.08.03010.1016/j.neuron.2005.08.03016157277Molyneaux BJ, Arlotta P, Menezes JRL, Macklis JD. 2007. Neuronal subtype specification in the cerebral cortex. Nature Reviews Neuroscience 8:427–437. 10.1038/nrn215110.1038/nrn215117514196Moseley ML, Zu T, Ikeda Y, Gao W, Mosemiller AK, Daughters RS, Chen G, Weatherspoon MR, Clark HB, Ebner TJ, Day JW, Ranum LP. 2006. Bidirectional expression of CUG and CAG expansion transcripts and intranuclear polyglutamine inclusions in spinocerebellar ataxia type 8. Nature Genetics 38:758–769. 10.1038/ng182710.1038/ng182716804541Nakagawa S, Naganuma T, Shioi G, Hirose T. 2011. Paraspeckles are subpopulation-specific nuclear bodies that are not essential in mice. The Journal of Cell Biology 193:31–39. 10.1083/jcb.20101111010.1083/jcb.201011110PMC308219821444682Ørom UA, Derrien T, Beringer M, Gumireddy K, Gardini A, Bussotti G, Lai F, Zytnicki M, Notredame C, Huang Q, Guigo R, Shiekhattar R. 2010. Long noncoding RNAs with enhancer-like function in human cells. Cell 143:46–58. 10.1016/j.cell.2010.09.00110.1016/j.cell.2010.09.001PMC410808020887892Pauli A, Valen E, Lin MF, Garber M, Vastenhouw NL, Levin JZ, Fan L, Sandelin A, Rinn JL, Regev A, Schier AF. 2012. Systematic identification of long noncoding RNAs expressed during zebrafish embryogenesis. Genome Research 22:577–591. 10.1101/gr.133009.11110.1101/gr.133009.111PMC329079322110045Petersen CCH. 2007. The functional organization of the barrel cortex. Neuron 56:339–355. 10.1016/j.neuron.2007.09.01710.1016/j.neuron.2007.09.01717964250Ponting CP, Oliver PL, Reik W. 2009. Evolution and functions of long noncoding RNAs. Behavioural Brain Research 136:629–641. 10.1016/j.cell.2009.02.00610.1016/j.cell.2009.02.00619239885Poueymirou WT, Auerbach W, Frendewey D, Hickey JF, Escaravage JM, Esau L, Doré AT, Stevens S, Adams NC, Dominguez MG, Gale NW, Yancopoulos GD, DeChiara TM, Valenzuela DM. 2006. F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses. Nature Biotechnology 25:91–99. 10.1038/nbt126310.1038/nbt126317187059Qureshi IA, Mattick JS, Mehler MF. 2010. Long non-coding RNAs in nervous system function and disease. Brain Research 1338:20–35. 10.1016/j.brainres.2010.03.11010.1016/j.brainres.2010.03.110PMC288365920380817Raj A, van den Bogaard P, Rifkin SA, van Oudenaarden A, Tyagi S. 2008. Imaging individual mRNA molecules using multiple singly labeled probes. Nature Methods 5:877–879. 10.1038/nmeth.125310.1038/nmeth.1253PMC312665318806792Ramos AD, Diaz A, Nellore A, Delgado RN, Park K-Y, Gonzales-Roybal G, Oldham MC, Song JS, Lim DA. 2013. Integration of genome-wide approaches identifies lncRNAs of adult neural stem cells and their progeny in vivo. Cell Stem Cell 12:616–628. 10.1016/j.stem.2013.03.00310.1016/j.stem.2013.03.003PMC366280523583100Rinn JL, Chang HY. 2012. Genome regulation by long noncoding RNAs. Annual Review of Biochemistry 81:145–166. 10.1146/annurev-biochem-051410-09290210.1146/annurev-biochem-051410-092902PMC385839722663078Ripoche MA, Kress C, Poirier F, Dandolo L. 1997. Deletion of the H19 transcription unit reveals the existence of a putative imprinting control element. Genes and Development 11:1596–1604. 10.1101/gad.11.12.159610.1101/gad.11.12.15969203585Sessa A, Mao C-A, Hadjantonakis A-K, Klein WH, Broccoli V. 2008. Tbr2 directs conversion of radial glia into basal precursors and guides neuronal amplification by indirect neurogenesis in the developing neocortex. Neuron 60:56–69. 10.1016/j.neuron.2008.09.02810.1016/j.neuron.2008.09.028PMC288776218940588Shen Y, Yue F, McCleary DF, Ye Z, Edsall L, Kuan S, Wagner U, Dixon J, Lee L, Lobanenkov VV, Ren B. 2012. A map of the cis-regulatory sequences in the mouse genome. Nature 488:116–120. 10.1038/nature1124310.1038/nature11243PMC404162222763441Sigova AA, Mullen AC, Molinie B, Gupta S, Orlando DA, Guenther MG, Almada AE, Lin C, Sharp PA, Giallourakis CC, Young RA. 2013. Divergent transcription of long noncoding RNA/mRNA gene pairs in embryonic stem cells. Proceedings of the National Academy of Sciences of the United States of America 110:2876–2881. 10.1073/pnas.122190411010.1073/pnas.1221904110PMC358194823382218Struhl K. 2007. Transcriptional noise and the fidelity of initiation by RNA polymerase II. Nature Structural and Molecular Biology 14:103–105. 10.1038/nsmb0207-10310.1038/nsmb0207-10317277804Sugitani Y, Nakai S, Minowa O, Nishi M, Jishage K-I, Kawano H, Mori K, Ogawa M, Noda T. 2002. Brn-1 and Brn-2 share crucial roles in the production and positioning of mouse neocortical neurons. Genes and Development 16:1760–1765. 10.1101/gad.97800210.1101/gad.978002PMC18640112130536Sun L, Goff LA, Trapnell C, Alexander R, Lo KA, Hacisuleyman E, Sauvageau M, Tazon-Vega B, Kelley DR, Hendrickson DG, Yuan B, Kellis M, Lodish HF, Rinn JL. 2013. Long noncoding RNAs regulate adipogenesis. Proceedings of the National Academy of Sciences of the United States of America 110:3387–3392. 10.1073/pnas.122264311010.1073/pnas.1222643110PMC358721523401553Szafranski P, Dharmadhikari AV, Brosens E, Gurha P, Kolodziejska KE, Zhishuo O, Dittwald P, Majewski T, Mohan KN, Chen B, Person RE, Tibboel D, de Klein A, Pinner J, Chopra M, Malcolm G, Peters G, Arbuckle S, Guiang SF, III, Hustead VA, Jessurun J, Hirsch R, Witte DP, Maystadt I, Sebire N, Fisher R, Langston C, Sen P, Stankiewicz P. 2013. Small noncoding differentially methylated copy-number variants, including lncRNA genes, cause a lethal lung developmental disorder. Genome Research 23:23–33. 10.1101/gr.141887.11210.1101/gr.141887.112PMC353068123034409Tarabykin V, Stoykova A, Usman N, Gruss P. 2001. Cortical upper layer neurons derive from the subventricular zone as indicated by Svet1 gene expression. Development 128:1983–199311493521Trapnell C, Hendrickson DG, Sauvageau M, Goff L, Rinn JL, Pachter L. 2012a. Differential analysis of gene regulation at transcript resolution with RNA-seq. Nature Biotechnology 31:46–53. 10.1038/nbt.245010.1038/nbt.2450PMC386939223222703Trapnell C, Pachter L, Salzberg SL. 2009. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25:1105–1111. 10.1093/bioinformatics/btp12010.1093/bioinformatics/btp120PMC267262819289445Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, Pimentel H, Salzberg SL, Rinn JL, Pachter L. 2012b. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nature Protocols 7:562–578. 10.1038/nprot.2012.01610.1038/nprot.2012.016PMC333432122383036Tsai M-C, Manor O, Wan Y, Mosammaparast N, Wang JK, Lan F, Shi Y, Segal E, Chang HY. 2010. Long noncoding RNA as modular scaffold of histone modification complexes. Science 329:689–693. 10.1126/science.119200210.1126/science.1192002PMC296777720616235Ulitsky I, Bartel DP. 2013. lincRNAs: genomics, evolution, and mechanisms. Cell 154:26–46. 10.1016/j.cell.2013.06.02010.1016/j.cell.2013.06.020PMC392478723827673Ulitsky I, Shkumatava A, Jan CH, Sive H, Bartel DP. 2011. Conserved function of lincRNAs in vertebrate embryonic development despite rapid sequence evolution. Cell 147:1537–1550. 10.1016/j.cell.2011.11.05510.1016/j.cell.2011.11.055PMC337635622196729Valenzuela DM, Murphy AJ, Frendewey D, Gale NW, Economides AN, Auerbach W, Poueymirou WT, Adams NC, Rojas J, Yasenchak J, Chernomorsky R, Boucher M, Elsasser AL, Esau L, Zheng J, Griffiths JA, Wang X, Su H, Xue Y, Dominguez MG, Noguera I, Torres R, Macdonald LE, Stewart AF, DeChiara TM, Yancopoulos GD. 2003. High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nature Biotechnology 21:652–659. 10.1038/nbt82210.1038/nbt82212730667Visel A, Zhu Y, May D, Afzal V, Gong E, Attanasio C, Blow MJ, Cohen JC, Rubin EM, Pennacchio LA. 2010. Targeted deletion of the 9p21 non-coding coronary artery disease risk interval in mice. Nature 464:409–412. 10.1038/nature0880110.1038/nature08801PMC293807620173736Wang KC, Yang YW, Liu B, Sanyal A, Corces-Zimmerman R, Chen Y, Lajoie BR, Protacio A, Flynn RA, Gupta RA, Wysocka J, Lei M, Dekker J, Helms JA, Chang HY. 2011. A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression. Nature 472:120–124. 10.1038/nature0981910.1038/nature09819PMC367075821423168Watson CM, Trainor PA, Radziewic T, Pelka GJ, Zhou SX, Parameswaran M, Quinlan GA, Gordon M, Sturm K, Tam PPL. 2008. Application of lacZ transgenic mice to cell lineage studies. Methods in Molecular Biology 461:149–164. 10.1007/978-1-60327-483-8_1010.1007/978-1-60327-483-8_10PMC309376719030795Welker C, Woolsey TA. 1974. Structure of layer IV in the somatosensory neocortex of the rat: description and comparison with the mouse. The Journal of Comparative Neurology 158:437–453. 10.1002/cne.90158040510.1002/cne.9015804054141363White JK Gerdin A-K Karp NA Ryder E Buljan M Bussell JN Salisbury J Clare S Ingham NJ Podrini C Houghton R Estabel J Bottomley JR Melvin DG Sunter D Adams NC, Sanger Institute Mouse Genetics Project, Tannahill D Logan DW Macarthur DG Flint J Mahajan VB Tsang SH Smyth I Watt FM Skarnes WC Dougan G Adams DJ Ramirez-Solis R Bradley A Steel KP. 2013. Genome-wide generation and systematic phenotyping of knockout mice reveals new roles for many genes. Behavioural Brain Research 154:452–464. 10.1016/j.cell.2013.06.02210.1016/j.cell.2013.06.022PMC371720723870131Wong-Riley M. 1979. Changes in the visual system of monocularly sutured or enucleated cats demonstrable with cytochrome oxidase histochemistry. Brain Research 171:11–28. 10.1016/0006-8993(79)90728-510.1016/0006-8993(79)90728-5223730Yang L, Lin C, Jin C, Yang JC, Tanasa B, Li W, Merkurjev D, Ohgi KA, Meng D, Zhang J, Evans CP, Rosenfeld MG. 2013. lncRNA-dependent mechanisms of androgen-receptor-regulated gene activation programs. Nature 500:598–602. 10.1038/nature1245110.1038/nature12451PMC403438623945587Yoon J-H, Abdelmohsen K, Srikantan S, Yang X, Martindale JL, De S, Huarte M, Zhan M, Becker KG, Gorospe M. 2012. LincRNA-p21 suppresses target mRNA translation. Molecular Cell 47:648–655. 10.1016/j.molcel.2012.06.02710.1016/j.molcel.2012.06.027PMC350934322841487Zhang B, Arun G, Mao YS, Lazar Z, Hung G, Bhattacharjee G, Xiao X, Booth CJ, Wu J, Zhang C, Spector DL. 2012. The lncRNA Malat1 is dispensable for mouse development but its transcription plays a cis-regulatory role in the adult. Cell Reports 2:111–123. 10.1016/j.celrep.2012.06.00310.1016/j.celrep.2012.06.003PMC340858722840402Zhu J, Sanborn JZ, Diekhans M, Lowe CB, Pringle TH, Haussler D. 2007. Comparative genomics search for losses of long-established genes on the human lineage. PLOS Computational Biology 3:e247–e247. 10.1371/journal.pcbi.003024710.1371/journal.pcbi.0030247PMC213496318085818Zimmer C. 2004. Dynamics of Cux2 expression suggests that an early pool of SVZ precursors is fated to become upper cortical layer neurons. Cerebral Cortex 14:1408–1420. 10.1093/cercor/bhh10210.1093/cercor/bhh10215238450208859282011071420211020
2042-004820112010Sep05Veterinary medicine internationalVet Med IntOocyte source and hormonal stimulation for in vitro fertilization using sexed spermatozoa in cattle.14562610.4061/2011/145626The aim of this study was to investigate the efficiency of in vitro embryo production in cattle utilizing sexed sperm from two bulls and oocytes recovered by OPU. Twenty donor animals were employed in eight OPU replicates: the first four OPU trials were conducted on animals without hormone treatment, and the last four were run on the same animals, following FSH subcutaneous and intramuscular administration. A higher rate of blastocyst development was recorded in stimulated, as compared to nonstimulated animals, (25.2% versus 12.8%, P = .001). Ocytes derived from slaughterhouse (SH) ovaries were also fertilized with sperm from the same bulls. Overall, non-sexed sperm used with oocytes derived from SH ovaries was significantly more efficient for blastocyst development than was sexed sperm with these same SH derived oocytes and sexed sperm with stimulated donor oocytes (39.8% versus 25.0% and 25.2%, P = .001). In conclusion, the use of sexed sperm with OPU-derived oocytes resulted in a significantly higher blastocyst development when donors were hormonally stimulated; furthermore, the level of efficiency achieved was comparable to that attained when the same sexed sperm was tested on oocytes derived from SH ovaries.PresicceGiorgio AGAIstituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy.XuJieJGongGuochunGMorenoJuan FJFChaubalSanjeevSXueFeiFBellaAntoninoASenatoreElena MEMYangXiangzhongXTianX CindyXCDuFuliangFengJournal Article20100905
United StatesVet Med Int1015242032042-00482010542010611201078201010260201010560201010561201095epublish20885928PMC294659410.4061/2011/145626145626Cran DG. XY sperm separation and use in artificial insemination and other ARTs. Society of Reproduction and Fertility Supplement. 2007;65:475–491.17644986De Vries A, Overton M, Fetrow J, Leslie K, Eicker S, Rogers G. Exploring the impact of sexed semen on the structure of the dairy industry. Journal of Dairy Science. 2008;91(2):847–856.18218773Garner DL, Seidel GE., Jr. History of commercializing sexed semen for cattle. Theriogenology. 2008;69(7):886–895.18343491Wilson RD, Weigel KA, Fricke PM, et al. In vitro production of Holstein embryos using sex-sorted sperm and oocytes from selected cull cows. Journal of Dairy Science. 2005;88(2):776–782.15653544Wilson RD, Fricke PM, Leibfried-Rutledge ML, Rutledge JJ, Penfield CMS, Weigel KA. In vitro production of bovine embryos using sex-sorted sperm. Theriogenology. 2006;65(6):1007–1015.16122781Xu J, Guo Z, Su L, et al. Developmental potential of vitrified Holstein cattle embryos fertilized in vitro with sex-sorted sperm. Journal of Dairy Science. 2006;89(7):2510–2518.16772569Maxwell WMC, Evans G, Hollinshead FK, et al. Integration of sperm sexing technology into the ART toolbox. Animal Reproduction Science. 2004;82-83:79–95.15271445Presicce GA. Reproduction in the water buffalo. Reproduction in Domestic Animals. 2007;42, supplement 2:24–32.17688599Liang XW, Lu YQ, Chen MT, et al. In vitro embryo production in buffalo (Bubalus bubalis) using sexed sperm and oocytes from ovum pick up. Theriogenology. 2008;69(7):822–826.18336893Wheeler MB, Rutledge JJ, Fischer-Brown A, VanEtten T, Malusky S, Beebe DJ. Application of sexed semen technology to in vitro embryo production in cattle. Theriogenology. 2006;65(1):219–227.16263159Lu KH, Seidel GE., Jr. Effects of heparin and sperm concentration on cleavage and blastocyst development rates of bovine oocytes inseminated with flow cytometrically-sorted sperm. Theriogenology. 2004;62(5):819–830.15251233Palma GA, Olivier NS, Neumüller CH, Sinowatz F. Effects of sex-sorted spermatozoa on the efficiency of in vitro fertilization and ultrastructure of in vitro produced bovine blastocysts. Anatomia, Histologia, Embryologia. 2008;37(1):67–73.18197903Zhang M, Lu KH, Seidel GE., Jr. Development of bovine embryos after in vitro fertilization of oocytes with flow cytometrically sorted, stained and unsorted sperm from different bulls. Theriogenology. 2003;60(9):1657–1663.14580648Kruip TA, Boni R, Wurth YA, Roelofsen MWM, Pieterse MC. Potential use of ovum pick-up for embryo production and breeding in cattle. Theriogenology. 1994;42(4):675–684.16727573De Roover R, Genicot G, Leonard S, Bols P, Dessy F. Ovum pick up and in vitro embryo production in cows superstimulated with an individually adapted superstimulation protocol. Animal Reproduction Science. 2005;86(1-2):13–25.15721656Humblot P, Holm P, Lonergan P, et al. Effect of stage of follicular growth during superovulation on developmental competence of bovine oocytes. Theriogenology. 2005;63(4):1149–1166.15710200Chaubal SA, Ferre LB, Molina JA, et al. Hormonal treatments for increasing the oocyte and embryo production in an OPU-IVP system. Theriogenology. 2007;67(4):719–728.17140652Presicce GA, Senatore EM, Santis GD, et al. Hormonal stimulation and oocyte maturational competence in prepuberal Mediterranean Italian buffaloes (Bubalus bubalis) Theriogenology. 2002;57(7):1877–1884.12041691De Roover R, Feugang JMN, Bols PEJ, Genicot G, Hanzen CH. Effects of ovum pick-up frequency and fsh stimulation: a retrospective study on seven years of beef cattle in vitro embryo production. Reproduction in Domestic Animals. 2008;43(2):239–245.18226024Tamassia M, Heyman Y, Lavergne Y, et al. Evidence of oocyte donor cow effect over oocyte production and embryo development in vitro. Reproduction. 2003;126(5):629–637.14611636Fujita T, Umeki H, Shimura H, Kugumiya K, Shiga K. Effect of group culture and embryo-culture conditioned medium on development of bovine embryos. Journal of Reproduction and Development. 2006;52(1):137–142.16293943Johnson LA, Flook JP, Hawk HW. Sex preselection in rabbits: live births from X and Y sperm separated by DNA and cell sorting. Biology of Reproduction. 1989;41(2):199–203.2804212Pieterse MC, Vos PLAM, Kruip THAM, et al. Transvaginal ultrasound guided follicular aspiration of bovine oocytes. Theriogenology. 1991;35(4):857–862.16726954Brackett BG, Bousquet D, Boice ML, Donawick WJ, Evans JF, Dressel MA. Normal development following in vitro fertilization in the cow. Biology of Reproduction. 1982;27(1):147–158.6896830Lonergan P, Fair T, Corcoran D, Evans ACO. Effect of culture environment on gene expression and developmental characteristics in IVF-derived embryos. Theriogenology. 2006;65(1):137–152.16289260Nagao Y, Iijima R, Saeki K. Interaction between embryos and culture conditions during in vitro development of bovine early embryos. Zygote. 2008;16(2):127–133.18405433Lopes AS, Martinussen T, Greve T, Callesen H. Effect of days post-partum, breed and ovum pick-up scheme on bovine oocyte recovery and embryo development. Reproduction in Domestic Animals. 2006;41(3):196–203.16689881Su L, Yang S, He X, et al. Effect of donor age on the developmental competence of bovine oocytes retrieved by Ovum Pick Up. Reproduction in Domestic Animals. In press.19281593Bols PE, Van Soom A, Ysebaert MT, Vandenheede JMM, De Kruif A. Effects of aspiration vacuum and needle diameter on cumulus oocyte complex morphology and developmental capacity of bovine oocytes. Theriogenology. 1996;45(5):1001–1014.16727859Bungartz L, Lucas-Hahn A, Rath D, Niemann H. Collection of oocytes from cattle via follicular aspiration aided by ultrasound with or without gonadotropin pretreatment and in different reproductive stages. Theriogenology. 1995;43(3):667–675.16727658Merton JS, De Roos APW, Mullaart E, et al. Factors affecting oocyte quality and quantity in commercial application of embryo technologies in the cattle breeding industry. Theriogenology. 2003;59(2):651–674.12499010Hasler JF, McCauley AD, Schermerhorn EC, Foote RH. Superovulatory responses of Holstein cows. Theriogenology. 1983;19(1):83–99.Jensen AM, Greve T, Madej A, Edqvist L-E. Endocrine profiles and embryo quality in the PMSG-PGF2α treated cow. Theriogenology. 1982;18(1):33–44.16725723Bo GA, Hockley DK, Nasser LF, Mapletoft RJ. Superovulatory response to a single subcutaneous injection of Folltropin-V in beef cattle. Theriogenology. 1994;42(6):963–975.16727601Yamamoto M, Ooe M, Kawaguchi M, Suzuki T. Superovulation in the cow with a single intramuscular injection of FSH dissolved in polyvinylpyrrolidone. Theriogenology. 1994;41(3):747–755.16727429Chaubal SA, Molina JA, Ohlrichs CL, et al. Comparison of different transvaginal ovum pick-up protocols to optimise oocyte retrieval and embryo production over a 10-week period in cows. Theriogenology. 2006;65(8):1631–1648.16243385Gibbons JR, Beal WE, Krisher RL, Faber EG, Pearson RE, Gwazdauskas FC. Effects of once- versus twice-weekly transvaginal follicular aspiration on bovine oocyte recovery and embryo development. Theriogenology. 1994;42(3):405–419.16727548Rick G, Hadeler DG, Lemme E, et al. Long term OPU in heifers from 6 to 15 months of age. Theriogenology. 1996;45(1):p. 356.Blondin P, Coenen K, Guilbault LA, Sirard M-A. Superovulation can reduce the developmental competence of bovine embryos. Theriogenology. 1996;46(7):1191–1203.16727982Gibbons JR, Wiltbank MC, Ginther OJ. Functional interrelationships between follicles greater than 4 mm and the follicle stimulating hormone surge in heifers. Biology of Reproduction. 1997;42:405–419.9369172Blondin P, Sirard M-A. Oocyte and follicular morphology as determining characteristics for developmental competence in bovine oocytes. Molecular Reproduction and Development. 1995;41(1):54–62.7619506Carolan C, Lonergan P, Khatir H, Mermillod P. In vitro production of bovine embryos using individual oocytes. Molecular Reproduction and Development. 1996;45(2):145–150.8914071Keefer CL, Stice SL, Paprocki AM, Golueke P. In vitro culture of bovine IVM-IVF embryos: cooperative interaction among embryos and the role of growth factors. Theriogenology. 1994;41(6):1323–1331.16727487Donnay I, Van Langendonckt A, Auquier P, et al. Effects of co-culture and embryo number on the in vitro development of bovine embryos. Theriogenology. 1997;47(8):1549–1561.16728097Palma GA, Clement-Sengewald A, Berg U, Brem G. Role of embryo number in the development of in vitro produced bovine embryos. Theriogenology. 1992;37:p. 271.Ward FA, Lonergan P, Enright BP, Boland MP. Factors affecting recovery and quality of oocytes for bovine embryo production in vitro using ovum pick-up technology. Theriogenology. 2000;54(3):433–446.11051326Ferry L, Mermillod P, Massip A, Dessy F. Bovine embryos cultured in serum-poor oviduct-conditioned medium need cooperation to reach the blastocyst stage. Theriogenology. 1994;42(3):445–453.16727551Senatore EM, Mannino ME, Suarez Novoa MV, et al. Synergistic effect on embryo development by inclusion of supplemental embryos embedded in agar chips. Reproduction, Fertility and Development. 2009;21(1):p. 208.206779302010111620160511
2152-49981222010AprCellular reprogrammingCell ReprogramCulture conditions and enzymatic passaging of bovine ESC-like cells.151160151-6010.1089/cell.2009.0049The goals of the current study were to (1) improve culture conditions and (2) chemical passaging of bovine embryonic stem cell-like (bESC-like) cells. Specifically, the effects of human leukemia inhibitory factor (hLIF), two types of feeders, mouse embryonic fibroblast (MEF) and bovine embryonic fibroblast (BEF), as well as three different enzymatic treatments including Trypsin-EDTA, TrypLE, and Liberase Blendzymes 3 were investigated. The addition of hLIF at 1000 U/mL to the culture medium (41.2 and 36.9%), and the use of either MEF or BEF feeders (40.3 and 38.1%) had no significant effect on the ability of inner cell masses (ICMs) to form primary cell colonies compared to controls. All bESC-like cells were first dissociated mechanically for three passages followed by enzymatic dissociation. The ability to maintain ESC morphology to passage 10 was compared among the three enzymes above. More bESC-like cell lines survived beyond passage 10 when treated with TrypLE compared to Trypson-EDTA (28.8 and 12.6%; p < 0.05), and bESC-like cells differentiated quickly when treated with Liberase Blendzyme 3. The bESC-like cells generated in our study displayed typical stem cell morphology and expressed specific markers such as SSEA-1, AP, OCT-4, and Nanog. When removed from feeders, these bESC-like cells formed embryoid bodies (EBs) in a suspension culture. When EBs were cultured on tissue culture plates, they differentiated into various cell types. In summary, we were able to culture bESC-like cells more than 10 passages by enzymatic dissociation, which is important in gene targeting, maintenance, and banking of bESC lines.GongGuochunGDepartment of Animal Science and Center for Regenerative Biology, University of Connecticut , Storrs, CT 06269-4243, USA.RoachMarsha LMLJiangLeLYangXiangzhongXTianXiuchun CindyXCengJournal Article
United StatesCell Reprogram1015281762152-49710Culture Media0Leukemia Inhibitory FactorIMAnimalsCattleCell Culture TechniquesCell DifferentiationCells, CulturedcytologyCulture MediapharmacologyEmbryo Culture TechniquesmethodsEmbryonic Stem CellscytologyFemaleFertilization in VitroHumansLeukemia Inhibitory FactormetabolismMiceSignal TransductionTime Factors20108460201084602010111760ppublish2067793010.1089/cell.2009.0049204324622010081620151119
1097-465222432010SepJournal of cellular physiologyJ Cell PhysiolDirect and progressive differentiation of human embryonic stem cells into the chondrogenic lineage.664671664-7110.1002/jcp.22166Treatment of common and debilitating degenerative cartilage diseases particularly osteoarthritis is a clinical challenge because of the limited capacity of the tissue for self-repair. Because of their unlimited capacity for self-renewal and ability to differentiate into multiple lineages, human embryonic stem cells (hESCs) are a potentially powerful tool for repair of cartilage defects. The primary objective of the present study was to develop culture systems and conditions that enable hESCs to directly and uniformly differentiate into the chondrogenic lineage without prior embryoid body (EB) formation, since the inherent cellular heterogeneity of EBs hinders obtaining homogeneous populations of chondrogenic cells that can be used for cartilage repair. To this end, we have subjected undifferentiated pluripotent hESCs to the high density micromass culture conditions we have extensively used to direct the differentiation of embryonic limb bud mesenchymal cells into chondrocytes. We report that micromass cultures of pluripotent hESCs undergo direct, rapid, progressive, and substantially uniform chondrogenic differentiation in the presence of BMP2 or a combination of BMP2 and TGF-beta1, signaling molecules that act in concert to regulate chondrogenesis in the developing limb. The gene expression profiles of hESC-derived cultures harvested at various times during the progression of their differentiation has enabled us to identify cultures comprising cells in different phases of the chondrogenic lineage ranging from cultures just entering the lineage to well differentiated chondrocytes. Thus, we are poised to compare the abilities of hESC-derived progenitors in different phases of the chondrogenic lineage for cartilage repair.(c) 2010 Wiley-Liss, Inc.GongGuochunGDepartment of Reconstructive Sciences, Center for Regenerative Medicine and Skeletal Development, University of Connecticut Health Center, Farmington, Connecticut 06030, USA.FerrariDeborahDDealyCaroline NCNKosherRobert ARAengJournal ArticleResearch Support, Non-U.S. Gov't
United StatesJ Cell Physiol00502220021-95410Biomarkers0Bone Morphogenetic Protein 20Transforming Growth Factor beta1IMAnimalsBiomarkersmetabolismBone Morphogenetic Protein 2metabolismCell Culture TechniquesCell DifferentiationphysiologyCell LineageCells, CulturedChondrocytescytologyphysiologyChondrogenesisphysiologyEmbryo, MammaliancytologyphysiologyEmbryonic Stem CellscytologyphysiologyGene Expression ProfilingGene Expression Regulation, DevelopmentalHumansMicePluripotent Stem CellscytologyphysiologyTransforming Growth Factor beta1metabolism2010516020105160201081760ppublish2043246210.1002/jcp.22166189416332009011320181113
1932-62033102008PloS onePLoS OneCattle mammary bioreactor generated by a novel procedure of transgenic cloning for large-scale production of functional human lactoferrin.e3453e3453e345310.1371/journal.pone.0003453Large-scale production of biopharmaceuticals by current bioreactor techniques is limited by low transgenic efficiency and low expression of foreign proteins. In general, a bacterial artificial chromosome (BAC) harboring most regulatory elements is capable of overcoming the limitations, but transferring BAC into donor cells is difficult. We describe here the use of cattle mammary bioreactor to produce functional recombinant human lactoferrin (rhLF) by a novel procedure of transgenic cloning, which employs microinjection to generate transgenic somatic cells as donor cells. Bovine fibroblast cells were co-microinjected for the first time with a 150-kb BAC carrying the human lactoferrin gene and a marker gene. The resulting transfection efficiency of up to 15.79 x 10(-2) percent was notably higher than that of electroporation and lipofection. Following somatic cell nuclear transfer, we obtained two transgenic cows that secreted rhLF at high levels, 2.5 g/l and 3.4 g/l, respectively. The rhLF had a similar pattern of glycosylation and proteolytic susceptibility as the natural human counterpart. Biochemical analysis revealed that the iron-binding and releasing properties of rhLF were identical to that of native hLF. Importantly, an antibacterial experiment further demonstrated that rhLF was functional. Our results indicate that co-microinjection with a BAC and a marker gene into donor cells for somatic cell cloning indeed improves transgenic efficiency. Moreover, the cattle mammary bioreactors generated with this novel procedure produce functional rhLF on an industrial scale.YangPenghuaPState Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing, People's Republic of China.WangJianwuJGongGuochunGSunXiuzhuXZhangRanRDuZhuoZLiuYingYLiRongRDingFangrongFTangBoBDaiYunpingYLiNingNengJournal ArticleResearch Support, Non-U.S. Gov't20081020
United StatesPLoS One1012850811932-62030Recombinant ProteinsEC 3.4.21.-LactoferrinIMAnimalsAnimals, Genetically ModifiedgeneticsBioreactorsCattleCell TransplantationFibroblastsmetabolismHumansLactoferrinbiosynthesisgeneticsMammary Glands, AnimalmetabolismNuclear Transfer TechniquesRecombinant ProteinsTransfectionmethodsCompeting Interests: The authors have declared that no competing interests exist.20086720089172008102390200911490200810239020081020ppublish18941633PMC256548710.1371/journal.pone.0003453Lonnerdal B, Iyer S. Lactoferrin: Molecular Structure and Biological Function. Annu Rev Nutr. 1995;15:93–110.8527233Lönnerdal B, Bryantthe A. Absorption of iron from recombinant human lactoferrin in young US women. Am J Clin Nutr. 2006;83:305–309.16469988Zimecki M, Artym J, Chodaczek G, Kocieba M, Kruzel ML. Protective effects of lactoferrin in Escherichia coli-induced bacteremia in mice: Relationship to reduced serum TNF alpha level and increased turnover of neutrophils. Inflam Res. 2004;53:292–296.15241563Wakabayashi H, Takakura N, Yamauchi K, Teraguchi S, Uchida K, et al. Effect of lactoferrin feeding on the host antifungal response in guinea-pigs infected or immunised with Trichophyton mentagrophytes. J Med Microbiol. 2002;51:844–850.12435063Isamida T, Tanaka T, Omata Y, Yamauchi K, Shimazachi K, et al. Protective effect of lactoferrin against Toxoplasma gondii infection in mice. J Vet Med Sci. 1998;60:241–244.9524950Strate BWA, Beljaars L, Molema G, Harmsen MC, Meijer DKF. Antiviral activities of lactoferrin. Antivir Res. 2001;52:225–239.11675140Baveye S, Elass E, Mazurier J, Spik G, Legrand D. Lactoferrin: a multifunctional glycoprotein involved in the modulation of the inflammatory process. Clin Chem Lab Med. 1999;37:281–286.10353473Vorland LH. Lactoferrin: a multifunctional glycoprotein. APMIS. 1999;107:971–981.10598868Naot D, Grey A, Reid IR, Cornish J. Lactoferrin-a novel bone growth factor. Clin Med Res. 2005;3:93–101.PMC118343916012127Ward PP, May GS, Headon DR, Conneely OM. An inducible expression system for the production of human lactoferrin in Aspergillus nidulans. Gene. 1992;122:219–223.1452033Ward PP, Piddington CS, Cunningham GA, Zhou X, Wyatt RD, et al. A system for production of commercial quantities of human lactoferrin: a broad spectrum natural antibiotic. Biotechnology (N Y) 1995;13:498–503.9634791Liang QW, Richardson T. Expression and characterization of human lactoferrin in yeast Saccharomyces cerevisiae. J Agric Food Chem. 1993;41:1800–1807.Stowell KM, Rodo TA, Funk WD, Tweeid JW. Expression of cloned human lactoferrin in baby-hamster kidney cells. Biochem J. 1991;276:349–355.PMC11510982049066Legrand D, Salmon V, Coddeville B, Benaissa M, Plancke Y, et al. Structural determination of two N-linked glycans isolated from recombinant human lactoferrin expressed in BHK cells. FBES letter. 1995;365:57–60.7774715Salmon V, Legrand D, Slomianny MC, el Yazidi I, Spik G, et al. Production of human lactoferrin in transgenic tobacco plants. Protein Expr Purif. 1998;13:127–135.9631525Chong DK, Langridge WH. Expression of full-length bioactive antimicrobial human lactoferrin in potato plants. Transgenic Res. 2000;9:71–78.10853271Platenburg GJ, Kootwijk EP, Kooiman PM, Woloshuk SL, Nuijens JH, et al. Expression of human lactoferrin in milk of transgenic mice. Transgenic Res. 1994;3:99–108.8193642Nuijens JH, van Berkel PH, Geerts ME, Hartevelt PP, de Boer HA, et al. Characterization of recombinant human lactoferrin secreted in milk of transgenic mice. J Biol Chem. 1997;272:8802–8807.9079716Wall RJ. Biotechnology for the production of modified and innovative animal products: transgenic livestock bioreactors. Livest Prod Sci. 1999;59:243–255.Wall RJ, Powell AM, Paape MJ, Kerr DE, Bannerman DD, et al. Genetically enhanced cows resist intramammary Staphylococcus aureus infection. Nat Biotechnol. 2005;23:445–451.15806099Brophy B, Smolenski G, Wheeler T, Wells D, L'Huillier P, et al. Cloned transgenic cattle produce milk with higher levels of beta-casein and kappa-casein. Nat Biotechnol. 2003;21:157–162.12548290van Berkel HC, Welling MW, Geerts M, van Veen HA, Ravensbergen B, et al. Large scale production of recombinant human lactoferrin in the milk of transgenic cows. Nat Biotech. 2002;20:484–487.11981562Krimpenfort P, Rademakers A, Eyestone W, van der Schans A, van den Broek S, et al. Generation of transgenic dairy cattle using ‘in vitro’ embryo production. Biotechnology (N Y) 1991;9:844–847.1367358Liu Z, Zhao C, Fan B, Dai Y, Zhao Z, et al. Variable expression of human lactoferrin gene in mice milk driven by its 90 KB upstream flanking sequences. Anim Biotechnol. 2004;15:21–31.15248598Dean W, Santos F, Stojkovic M, Zakhartchenko V, Walter J, et al. Conservation of methylation reprogramming in mammalian development: Aberrant reprogramming in cloned embryos. Proc Natl Acad Sci U S A. 2001;98:13734–13738.PMC6111011717434Wee G, Koo D-B, Song B-S, Kim J-S, Kang M-J, et al. Inheritable Histone H4 Acetylation of Somatic Chromatins in Cloned Embryos. J Biol Chem. 2006;281:6048–6057.16371357van Berkel PHC, Geerts MDJ, Van Veen HA, KooIman PM, Pieper FR, et al. Glycosylated and unglycosylated human lactoferrins both bind iron and show identical affinities towards human lysozyme and bacterial lipopolysaccharide, but differ in their susceptibilities towards tryptic proteolysis. Biochem J. 1995;312:107–114.PMC11362337492299Giraldo P, Montoliu L. Size matters: use of YACs, BACs and PACs in transgenic animals. Transgenic Res. 2001;10:83–103.11305364Hejna JA, Johnstone PL, Kohler SL, Bruun DA, Reifsteck CA, et al. Functional complementation by electroporation of human BACs into mammalian fibroblast cells. Nucleic Acids Res. 1998;26:1124–1125.PMC1473389461477Magin-Lachmann C, Kotzamanis G, D'Aiuto L, Cooke H, Huxley C, et al. In vitro and in vivo delivery of intact BAC DNA – comparison of different methods. J Gene Med. 2004;6:195–209.14978773Kittler R, Pelletier L, Ma C, Poser I, Fischer S, et al. RNA interference rescue by bacterial artificial chromosome transgenesis in mammalian tissue culture cells. Proc Natl Acad Sci U S A. 2005;102:2396–2401.PMC54899215695330Xie HX. Differences in the efficiency and stability of gene expression after transfection and nuclear injection: a study with a chick delta-crystallin gene. Cell Struct Funct. 1983;8:315–325.6673837Zhang X-F, Wu G-X, Chen J-Q, Zhang A-M, Liu S-G, et al. Transfer of an expression YAC into goat fetal fibroblasts by cell fusion for mammary gland bioreactor. Biochemical and Biophysical Research Communications. 2005;333:58.15936717Yang XW, Model P, Heintz N. Homologous recombination based modification in Esherichia coli and germline transmission in transgenic mice of a bacterial artificial chromsome. Nat Biotechnol. 1997;15:859–865.9306400Eyestone WH. Production and breeding of transgenic cattle using in vitro embryo production technology. Theriogenology. 1999;51:509–517.10729109Takahashi S, Ito Y. Evaluation of meat products from cloned cattle: biological and biochemical properties. Cloning Stem Cells. 2004;6:165–171.15268791Tian XC, Kubota C, Sakashita K, Izaike Y, Okano R, et al. Meat and milk compositions of bovine clones. Proc Natl Acad Sci U S A. 2005;102:6261–6266.PMC108836715829585Walsh MK, Lucey JA, Govindasamy-Lucey S, Pace MM, Bishop MD. Comparison of milk produced by cows cloned by nuclear transfer with milk from non-cloned cows. Cloning Stem Cells. 2003;5:213.14588139van Veen HA, Geerts MEJ, van Berkel PHC, Nuijens JH. Analytical cation-exchange chromatography to assess the identity, purity, and N-terminal integrity of human lactoferrin. Anal Biochem. 2002;309:60–66.12381362Foley AA, Bates GW. The purification of lactoferrin from human whey by batch extraction. Anal Biochem. 1987;162:296–300.3496808Palmano KP, Elgar DF. Detection and quantitation of lactoferrin in bovine whey samples by reversed phase high-performance liquid chromatography on polystyrene-divinylbenzene. J Chromatogr A. 2002;947:307–311.11883664Zhao C, Liu Z, Fan B, Dai Y, Wang L, et al. Differential glycosylation of rhLf expressed in the mammary gland of transgenic mice. Anim Biotechnol. 2006;17:13–20.16621756Spik G, Strecker G, Fournet B, Bouquelet S, Montreuil J, et al. Primary structure of the glycans from human lactotransferrin. Eur J Biochem. 1982;121:413–419.7060557Petit BS, Dubos JW, Chirat F, Coddeville B, Demaizieres G, et al. Comparative analysis of the site-specific N-glycosylation of human lactoferrin produced in maize and tobacco plants. Eur J Biochem. 2003;270:3235–3242.12869199Lopez M, Coddeville B, Langridge J, Plancke Y, Sautiere P, et al. Microheterogeneity of the oligosaccharides carried by the recombinant bovine lactoferrin expressed in Mamestra brassicae cells. Glycoblology. 1997;7:635–651.9254046Davidson LA, Lonnerdal B. Persistence of human milk proteins in the breast-fed infant. Acta Paediatr Scand. 1987;76:733–740.3661174van Veen HA, Geerts MEJ, van Berkel PHC, Nuijens JH. The role of N-linked glycosylation in the protection of human and bovine lactoferrin against tryptic proteolysis. Eur J Biochem. 2004;271:678–684.14764083Crichton RR, Charloteaux-Wauters M. Iron transport and storage. Eur J Biochem. 1987;164:485–506.3032619Iyer S, Lonnerdal B. Lactoferrin, lactoferrin receptors and iron metabolism. Eur J Clin Nutr. 1993;47:232–241.8491159Levay PF, Viljoen M. Lactoferrin: ageneral review. Haematologica. 1995;80:252–267.7672721Brock JH. Lactoferrin in human milk: its role in iron absorption and protection against enteric infection in the newborn. infant. Arch Dis Child. 1980;55:417–421.PMC16269337002055Hyvonen P, Suojala L, Orro T, Haaranen J, Simola O, et al. Transgenic cows that produce recombinant human lactoferrin in milk are not protected from experimental Escherichia coli intramammary infection. Infect Immun. 2006;74:6206–6212.PMC169553516954396Gong G, Dai Y, Fan B, Zhu H, Zhu S, et al. Birth of Calves Expressing the Enhanced Green Fluorescent Protein After Transfer of Fresh or Vitrified/Thawed Blastocysts Produced by Somatic Cell Nuclear Transfer. Mol Reprod Dev. 2004;69:278–288.15349839Rosenkrans JCF, First NL. Culture of bovine zygotes to the blastocyst stage: Effects of amino acids and vitamins. Theriogenology. 1991;35:266.Candiano G, Bruschi M, Musante L, Santucci L, Ghiggeri GM, et al. Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis. 2004;25:1327–1333.15174055161396142006012620061115
0093-691X6462005Oct01TheriogenologyTheriogenologyBovine oocytes vitrified by the open pulled straw method and used for somatic cell cloning supported development to term.138113911381-91The objective of the present study was to determine if oocytes vitrified by the open pulled straw (OPS) method could subsequently be used to produce somatic cell cloned cattle. Post-thaw survival rates were 77.0, 79.1, 97.2 and 97.5% for oocytes vitrified with EDFS30 (15% ethylene glycol, 15% dimethyl sulfoxide, ficoll and sucrose), EDFS40 (20% ethylene glycol, 20% dimethyl sulfoxide, ficoll and sucrose), EDFSF30 (15% ethylene glycol, 15% dimethyl sulfoxide, ficoll, sucrose and FBS) and EDFSF40 (20% ethylene glycol, 20% dimethyl sulfoxide, ficoll, sucrose and FBS), respectively. The parthenogenetic blastocyst rates of the vitrified-thawed oocytes activated with 5 microM of the calcium ionophore A23187 for 5 min and 2 microM of 6-dimethylaminopurin (6-DMAP) for 4h ranged from 10.3 to 23.0%, with the highest group not significantly differing from that of the controls (33.2%). In total, 722 vitrified-thawed oocytes were used as recipients for nuclear transfer, of which 343 fused (47.6%). Fifty-six (16.3%) of the reconstructed embryos reached the blastocyst stage after 7d of in vitro culture. Twenty-four blastocysts derived from vitrified-thawed oocytes were transferred to six Luxi yellow cattle recipients. Two recipients (33%) were diagnosed pregnant; one aborted 97 d after transfer, whereas the other delivered a cloned calf after 263 d. As a control, 28 synchronous Luxi yellow cattle recipients each received a single blastocyst produced using a fresh oocyte as a nuclear recipient; 10 recipients were diagnosed pregnant, of which 6 (21.4% of the original 28) delivered cloned calves. In conclusion, bovine oocytes vitrified by the OPS method and subsequently thawed supported development (to term) of somatic cell cloned embryos.HouYun-PengYPCollege of Animal Science and Technology, China Agricultural University, Beijing 100094, China.DaiYun-PingYPZhuShi-EnSEZhuHua-BinHBWuTong-YiTYGongGuo-ChunGCWangHai-PingHPWangLi-LiLLLiuYingYLiRongRWanRongRLiNingNengJournal ArticleResearch Support, Non-U.S. Gov't20050425
United StatesTheriogenology04215100093-691XIMAnimalsAnimals, NewbornBlastocystphysiologyCattleembryologyphysiologyCloning, OrganismCryopreservationmethodsveterinaryFemaleFertilization in VitroveterinaryNuclear Transfer TechniquesOocytesphysiologyParthenogenesisPregnancyPregnancy Outcome20047252005969020061279020059690ppublish1613961410.1016/j.theriogenology.2005.03.012S0093-691X(05)00083-X156231602005062820191109
1006-93054752004OctScience in China. Series C, Life sciencesSci China C Life SciGeneration of cloned calves from different types of somatic cells.470476470-6Six types of bovine somatic cell lines, including a granulosa cell line of Chinese red-breed yellow cattle (YGR), a granulosa cell line of Holstein cow (HGR), two skin fibroblast cell lines of two adult Holstein cows respectively (AFB1 and AFB2), a skin fibroblast cell line (FFB) and an oviduct epithelial cell line (FOV) of a Holstein fetus, were established. Somatic cell nuclear transfer (SCNT) was carried out using these cells as nuclei donor, and a total of 12 healthy calves were cloned. The effects of different types of donor cells on developmental potential of bovine SCNT embryos were investigated. (i) There was no significant difference in development rates to the blastocyst stage for SCNT embryos from YGR and HGR (33.2% and 35.1%, respectively). Pregnancy rates of them were 33.3% and 30.2%, respectively; and birth rates were 16.7% and 11.6%, respectively. (ii) Development rates to the blastocyst stage for SCNT embryos from different individuals (AFB1 and AFB2) differed significantly (27.9% and 39.4%, respectively, P < 0.05). Pregnancy rates of them were 36.2% and 36.4%, respectively; and birth rates were 14.9 % and 27.3%, respectively. (iii) There was significant difference in development rates to the blastocyst stage for SCNT embryos from FFB and FOV of the same fetus (37.9% and 41.5%, respectively, P < 0.05). Pregnancy rates of them were 45.7% and 24.1%, respectively; and birth rates were 22.9% and 10.3%, respectively. Finally, developmental potential of bovine SCNT embryos from all four types of somatic cells from Holstein cows (HGR, AFB, FFB and FOV) were compared. For in vitro development stage, development rates to the blastocyst stage for SCNT embryos from HGR, AFB, FFB and FOV were 35.1%A, 29.4%B, 37.9%A and 41.5%c, respectively (pABC < 0.05); for in vivo development stage, pregnancy rates of them were 30.2%, 36.2%, 45.7% and 24.1%, respectively; and birth rates of them were 11.6%, 17.2%, 22.9% and 10.3% respectively.GongGuochunGState Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, China.DaiYunpingYZhuHuabingHWangHaipingHWangLiliLLiRongRWanRongRLiuYingYLiNingNengJournal ArticleResearch Support, Non-U.S. Gov't
ChinaSci China C Life Sci96118091006-9305IMAnimalsBlastomeresCattleCell LineCell NucleusmetabolismCells, CulturedCloning, MolecularCloning, OrganismmethodsEmbryo TransferEmbryo, MammaliancytologyFemaleFibroblastsmetabolismMicrosatellite RepeatsOocytesmetabolismOvarymetabolismPregnancyPregnancy, Animal20041230902005629902004123090ppublish1562316010.1360/03yc0224153792512004121420191108
1006-93054722004AprScience in China. Series C, Life sciencesSci China C Life SciProduction of transgenic blastocyst by nuclear transfer from different types of somatic cells in cattle.183189183-9The present study examined the effects of genetic manipulation to the donor cell and different types of transgenic donor cells on developmental potential of bovine nuclear transfer (NT) embryos. Four types of bovine somatic cells, including granulosa cells, fetal fibroblasts, fetal oviduct epithelial cells and fetal ovary epithelial cells, were transfected with a plasmid (pCE-EGFP-Ires-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neor) genes by electroporation. After 14 days selection with 800 microg/mL G418, transgenic cell lines from each type of somatic cells were obtained. Nontransgenic granulosa cells and all 4 types of transgenic somatic cells were used as nuclear donor to produce transgenic embryos by NT. There was no significant difference in development rates to the blastocyst stage for NT embryos from transgenic and nontransgenic granulosa cells (44.6% and 42.8%, respectively), and transfer of NT embryos derived from transgenic and nontransgenic granulosa cells to recipients resulted in similar pregnancy rates on day 90 (19% and 25%, respectively). The development rates to the blastocyst stage of NT embryos were significantly different among different types of transgenic donor cells (P<0.05). Blastocyst rates from fetal oviduct epithelial cell and granulosa cell (49.1% and 44.6%, respectively) were higher than those from fetal fibroblast (32.7%) and fetal ovary epithelial cell (22.5%). These results suggest that (i) genetic manipulation to donor cells has no negative effect on in vitro and early in vivo developmental competence of bovine NT embryos and (ii) granulosa and fetal oviduct epithelial cells can be used to produce transgenic bovine NT embryos more efficiently. In addition, GFP can be used to select transgenic NT embryos as a non-invasive selective marker.GongGuochunGState Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, China.DaiYunpingYFanBaoliangBZhuHuabingHWangHaipingHWangLiliLFangChanggeCWanRongRLiuYingYLiRongRLiNingNengJournal ArticleResearch Support, Non-U.S. Gov't
ChinaSci China C Life Sci96118091006-9305IMAnimalsAnimals, Genetically ModifiedBlastocystphysiologyCattleFemaleGenes, ReporterGranulosa CellscytologyNuclear Transfer TechniquesOocytescytologyphysiologyRestriction Mapping2004924502004121690200492450ppublish1537925110.1360/03yc0015153498392005031420061115
1040-452X6932004NovMolecular reproduction and developmentMol Reprod DevBirth of calves expressing the enhanced green fluorescent protein after transfer of fresh or vitrified/thawed blastocysts produced by somatic cell nuclear transfer.278288278-88The present study examined effects of genetic manipulation and serum starvation on in vitro developmental potential of bovine somatic cell nuclear transfer (SCNT) embryos and vitrification on in vivo developmental competence of transgenic SCNT blastocysts. Fetal oviduct epithelial cells (FOECs) were isolated from the oviduct of a Day 147 bovine fetus and transfected with a plasmid (pCE-EGFP-IRES-NEO) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neor) genes. There were no significant differences (P > 0.05) in cleavage rates or development rates to the blastocyst stage for SCNT embryos derived from FOECs (72.5 and 47.8%, respectively) or transfected FOECs (TFOECs, 73.8 and 47.7%, respectively); nor from serum-fed (73.6 and 47.2%, respectively) or serum-starved (72.7 and 48.3%, respectively) cells. Seventeen of Day 7 GFP-embryos (eight fresh blastocysts and nine vitrified/thawed blastocysts ) were transferred to recipients with one embryo per recipient. Two (25%) recipients were confirmed pregnant at Day 60 in fresh blastocysts group, and three recipients (33%) were confirmed pregnant at Day 60 in vitrified/thawed blastocysts group. Two healthy calves (25%) were obtained from fresh blastocysts and one (11%) from vitrified/thawed blastocysts. Microsatellite analysis confirmed that the three clones were genetically identical to the donor cells. Moreover, PCR and Southern blot demonstrated integration of transgene in genomic DNA of all three cloned calves. Expression of GFP in skin biopsies isolated from transgenic cloned calves and fibroblasts derived from the skin biopsies revealed the activity of EGFP gene, and G418 resistance in vitro of these fibroblasts confirmed the activity of Neor gene. Our results show that genetic manipulation and serum starvation of donor cells (FOECs) do not affect in vitro developmental competence of bovine SCNT embryos, and vitrified transgenic SCNT blastocysts can develop to term successfully.GongGuochunGState Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing, China.DaiYunpingYFanBaoliangBZhuHuabingHZhuShienSWangHaipingHWangLiliLTangBoBLiRongRWanRongRLiuYingYHuangYinhuaYZhangLeiLSunXiuzhuXLiNingNengJournal ArticleResearch Support, Non-U.S. Gov't
United StatesMol Reprod Dev89033331040-452XIMAnimalsAnimals, Genetically ModifiedBlastocystmetabolismBlotting, SouthernCattleCloning, OrganismCryopreservationGenes, ReporterMicrosatellite RepeatsNuclear Transfer TechniquesPolymerase Chain Reaction2004975020053159020049750ppublish1534983910.1002/mrd.20130trying2...
Publications by Guochun Gong | LitMetric
Browse Categories

Publications by authors named "Guochun Gong"

  • Page 1 of 1
Somatic and intergenerational G4C2 hexanucleotide repeat instability in a human C9orf72 knock-in mouse model.
Nada Kojak Junko Kuno Kristina E Fittipaldi Ambereen Khan David Wenger Guochun Gong

Nucleic Acids Res

June 2024

Article Synopsis
  • The study explores the link between a repeated genetic sequence (G4C2) in the C9orf72 gene and conditions like familial ALS and FTD.
  • Researchers created a new mouse model with 96 copies of the G4C2 repeat to better understand how these repeats can become unstable over generations.
  • Two main mechanisms were identified for repeat expansion: minor increases due to a mismatch repair pathway and larger expansions triggered by DNA breaks, with implications for understanding the genetic instability seen in human cases.
View Article and Find Full Text PDF
Genetic and functional evidence links a missense variant in to lower LDL and fibrinogen.
May E Montasser Cristopher V Van Hout Lawrence Miloscio Alicia D Howard Avraham Rosenberg Guochun Gong

Science

December 2021

Increased blood levels of low-density lipoprotein cholesterol (LDL-C) and fibrinogen are independent risk factors for cardiovascular disease. We identified associations between an Amish-enriched missense variant (p.Asn352Ser) in a functional domain of beta-1,4-galactosyltransferase 1 () and 13.

View Article and Find Full Text PDF
Tissue-specific activation of gene expression by the Synergistic Activation Mediator (SAM) CRISPRa system in mice.
Charleen Hunt Suzanne A Hartford Derek White Evangelos Pefanis Timothy Hanna Guochun Gong

Nat Commun

May 2021

CRISPR-based transcriptional activation is a powerful tool for functional gene interrogation; however, delivery difficulties have limited its applications in vivo. Here, we created a mouse model expressing all components of the CRISPR-Cas9 guide RNA-directed Synergistic Activation Mediator (SAM) from a single transcript that is capable of activating target genes in a tissue-specific manner. We optimized Lipid Nanoparticles and Adeno-Associated Virus guide RNA delivery approaches to achieve expression modulation of one or more genes in vivo.

View Article and Find Full Text PDF
Deletion of Adam6 in Mus musculus leads to male subfertility and deficits in sperm ascent into the oviduct.
Vera A Voronina Faith M Harris Jennifer Schmahl Caryn Galligan Daniel Oristian Guochun Gong

Biol Reprod

March 2019

The Adisintegrin and metalloprotease domain-containing (ADAM) family of proteins is involved in cell adhesion, migration, proteolysis, and signaling. Many ADAMs are required for reproduction; however, the role of Adam6 has remained largely unknown. In the course of humanizing the mouse immunoglobulin heavy chain (IgH) locus, we generated Adam6-deficient mice that demonstrate severe subfertility.

View Article and Find Full Text PDF
C9orf72 ablation causes immune dysregulation characterized by leukocyte expansion, autoantibody production, and glomerulonephropathy in mice.
Amanda Atanasio Vilma Decman Derek White Meg Ramos Burcin Ikiz Guochun Gong

Sci Rep

March 2016

Article Synopsis
  • The hexanucleotide repeat expansion in the C9ORF72 gene is the leading cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but its functional impact and disease mechanism are still unclear.
  • Research using a mouse model lacking C9orf72 revealed immune system alterations, such as increased myeloid cells, activated T cells, and elevated autoantibodies.
  • The findings suggest that C9orf72 is important for maintaining immune balance and may indicate that deficiency in this gene isn't the main cause of ALS/FTD pathology.
View Article and Find Full Text PDF
Diverse Phenotypes and Specific Transcription Patterns in Twenty Mouse Lines with Ablated LincRNAs.
Ka-Man Venus Lai Guochun Gong Amanda Atanasio José Rojas Joseph Quispe

PLoS One

April 2016

In a survey of 20 knockout mouse lines designed to examine the biological functions of large intergenic non-coding RNAs (lincRNAs), we have found a variety of phenotypes, ranging from perinatal lethality to defects associated with premature aging and morphological and functional abnormalities in the lungs, skeleton, and muscle. Each mutant allele carried a lacZ reporter whose expression profile highlighted a wide spectrum of spatiotemporal and tissue-specific transcription patterns in embryos and adults that informed our phenotypic analyses and will serve as a guide for future investigations of these genes. Our study shows that lincRNAs are a new class of encoded molecules that, like proteins, serve essential and important functional roles in embryonic development, physiology, and homeostasis of a broad array of tissues and organs in mammals.

View Article and Find Full Text PDF
Generation of fertile and fecund F0 XY female mice from XY ES cells.
Junko Kuno William T Poueymirou Guochun Gong Chia-Jen Siao Georgia Clarke

Transgenic Res

February 2015

Known examples of male to female sex reversal in mice are caused by either strain incompatibilities or mutations in genes required for male sex determination. The resultant XY females are often sterile or exhibit very poor fertility. We describe here embryonic stem (ES) cell growth conditions that promote the production of healthy, anatomically normal fertile and fecund female F0 generation mice completely derived from gene-targeted XY male ES cells.

View Article and Find Full Text PDF
Multiple knockout mouse models reveal lincRNAs are required for life and brain development.
Martin Sauvageau Loyal A Goff Simona Lodato Boyan Bonev Abigail F Groff Guochun Gong

Elife

December 2013

Many studies are uncovering functional roles for long noncoding RNAs (lncRNAs), yet few have been tested for in vivo relevance through genetic ablation in animal models. To investigate the functional relevance of lncRNAs in various physiological conditions, we have developed a collection of 18 lncRNA knockout strains in which the locus is maintained transcriptionally active. Initial characterization revealed peri- and postnatal lethal phenotypes in three mutant strains (Fendrr, Peril, and Mdgt), the latter two exhibiting incomplete penetrance and growth defects in survivors.

View Article and Find Full Text PDF
Oocyte source and hormonal stimulation for in vitro fertilization using sexed spermatozoa in cattle.
Giorgio A Presicce Jie Xu Guochun Gong Juan F Moreno Sanjeev Chaubal

Vet Med Int

September 2010

The aim of this study was to investigate the efficiency of in vitro embryo production in cattle utilizing sexed sperm from two bulls and oocytes recovered by OPU. Twenty donor animals were employed in eight OPU replicates: the first four OPU trials were conducted on animals without hormone treatment, and the last four were run on the same animals, following FSH subcutaneous and intramuscular administration. A higher rate of blastocyst development was recorded in stimulated, as compared to nonstimulated animals, (25.

View Article and Find Full Text PDF
Culture conditions and enzymatic passaging of bovine ESC-like cells.
Guochun Gong Marsha L Roach Le Jiang Xiangzhong Yang Xiuchun Cindy Tian

Cell Reprogram

April 2010

The goals of the current study were to (1) improve culture conditions and (2) chemical passaging of bovine embryonic stem cell-like (bESC-like) cells. Specifically, the effects of human leukemia inhibitory factor (hLIF), two types of feeders, mouse embryonic fibroblast (MEF) and bovine embryonic fibroblast (BEF), as well as three different enzymatic treatments including Trypsin-EDTA, TrypLE, and Liberase Blendzymes 3 were investigated. The addition of hLIF at 1000 U/mL to the culture medium (41.

View Article and Find Full Text PDF
Direct and progressive differentiation of human embryonic stem cells into the chondrogenic lineage.
Guochun Gong Deborah Ferrari Caroline N Dealy Robert A Kosher

J Cell Physiol

September 2010

Treatment of common and debilitating degenerative cartilage diseases particularly osteoarthritis is a clinical challenge because of the limited capacity of the tissue for self-repair. Because of their unlimited capacity for self-renewal and ability to differentiate into multiple lineages, human embryonic stem cells (hESCs) are a potentially powerful tool for repair of cartilage defects. The primary objective of the present study was to develop culture systems and conditions that enable hESCs to directly and uniformly differentiate into the chondrogenic lineage without prior embryoid body (EB) formation, since the inherent cellular heterogeneity of EBs hinders obtaining homogeneous populations of chondrogenic cells that can be used for cartilage repair.

View Article and Find Full Text PDF
Cattle mammary bioreactor generated by a novel procedure of transgenic cloning for large-scale production of functional human lactoferrin.
Penghua Yang Jianwu Wang Guochun Gong Xiuzhu Sun Ran Zhang

PLoS One

January 2009

Large-scale production of biopharmaceuticals by current bioreactor techniques is limited by low transgenic efficiency and low expression of foreign proteins. In general, a bacterial artificial chromosome (BAC) harboring most regulatory elements is capable of overcoming the limitations, but transferring BAC into donor cells is difficult. We describe here the use of cattle mammary bioreactor to produce functional recombinant human lactoferrin (rhLF) by a novel procedure of transgenic cloning, which employs microinjection to generate transgenic somatic cells as donor cells.

View Article and Find Full Text PDF
Bovine oocytes vitrified by the open pulled straw method and used for somatic cell cloning supported development to term.
Yun-Peng Hou Yun-Ping Dai Shi-En Zhu Hua-Bin Zhu Tong-Yi Wu Guo-Chun Gong

Theriogenology

October 2005

The objective of the present study was to determine if oocytes vitrified by the open pulled straw (OPS) method could subsequently be used to produce somatic cell cloned cattle. Post-thaw survival rates were 77.0, 79.

View Article and Find Full Text PDF
Generation of cloned calves from different types of somatic cells.
Guochun Gong Yunping Dai Huabing Zhu Haiping Wang Lili Wang

Sci China C Life Sci

October 2004

Six types of bovine somatic cell lines, including a granulosa cell line of Chinese red-breed yellow cattle (YGR), a granulosa cell line of Holstein cow (HGR), two skin fibroblast cell lines of two adult Holstein cows respectively (AFB1 and AFB2), a skin fibroblast cell line (FFB) and an oviduct epithelial cell line (FOV) of a Holstein fetus, were established. Somatic cell nuclear transfer (SCNT) was carried out using these cells as nuclei donor, and a total of 12 healthy calves were cloned. The effects of different types of donor cells on developmental potential of bovine SCNT embryos were investigated.

View Article and Find Full Text PDF
Production of transgenic blastocyst by nuclear transfer from different types of somatic cells in cattle.
Guochun Gong Yunping Dai Baoliang Fan Huabing Zhu Haiping Wang

Sci China C Life Sci

April 2004

The present study examined the effects of genetic manipulation to the donor cell and different types of transgenic donor cells on developmental potential of bovine nuclear transfer (NT) embryos. Four types of bovine somatic cells, including granulosa cells, fetal fibroblasts, fetal oviduct epithelial cells and fetal ovary epithelial cells, were transfected with a plasmid (pCE-EGFP-Ires-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neor) genes by electroporation. After 14 days selection with 800 microg/mL G418, transgenic cell lines from each type of somatic cells were obtained.

View Article and Find Full Text PDF
Birth of calves expressing the enhanced green fluorescent protein after transfer of fresh or vitrified/thawed blastocysts produced by somatic cell nuclear transfer.
Guochun Gong Yunping Dai Baoliang Fan Huabing Zhu Shien Zhu

Mol Reprod Dev

November 2004

The present study examined effects of genetic manipulation and serum starvation on in vitro developmental potential of bovine somatic cell nuclear transfer (SCNT) embryos and vitrification on in vivo developmental competence of transgenic SCNT blastocysts. Fetal oviduct epithelial cells (FOECs) were isolated from the oviduct of a Day 147 bovine fetus and transfected with a plasmid (pCE-EGFP-IRES-NEO) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neor) genes. There were no significant differences (P > 0.

View Article and Find Full Text PDF
Privacy Policy Site Map

© LitMetric 2024. All rights reserved.