Versatile generation of precise gene edits in bovines using SEGCPN.

Background: Gene knockout and knock-in have been widely performed in large farm animals based on genome editing systems. However, many types of precise gene editing, including targeted deletion, gene tagging, and large gene fragment replacement, remain a challenge in large farm animals.

Results: Here, we established versatile self-excising gene-targeting technology in combination with programmable nucleases (SEGCPN) to efficiently generate various types of precise gene editing in bovine.

De novo genome assembly depicts the immune genomic characteristics of cattle.

Immunogenomic loci remain poorly understood because of their genetic complexity and size. Here, we report the de novo assembly of a cattle genome and provide a detailed annotation of the immunogenomic loci. The assembled genome contains 143 contigs (N50 ~ 74.

Efficient Editing of the ZBED6-Binding Site in Intron 3 of IGF2 in a Bovine Model Using the CRISPR/Cas9 System.

Background: Insulin-like growth factor 2 is a growth-promoting factor that plays an important role in the growth and development of mammals. A nucleotide substitution in intron 3 of -which disrupts the ZBED6-binding site-affects muscle mass, organ size, and fat deposition in pigs. The ZBED6-binding site is also conserved in cattle.

Efficient TALEN-mediated gene knockin at the bovine Y chromosome and generation of a sex-reversal bovine.

Functional elucidation of bovine Y-chromosome genes requires available genome editing technologies. Meanwhile, it has yet to be proven whether the bovine Sry gene is the main or single factor involved in the development of the male phenotype in bovine. Here, we efficiently knocked out four Y-linked genes (Sry, ZFY, DDX3Y, and EIF2S3Y) in bovine fetal fibroblasts (BFFs) with transcription activator-like effector nucleases (TALENs) individually.

Prolactin stimulation affects the stem cell-dependent mammary repopulating ability of embryonic mammary anlagen.

The function of prolactin in mammary gland development and lactation has been demonstrated in previous studies. However, the potential action of prolactin on mammary duct morphogenesis at mammary anlagen stage (E13.5˜15.

Production of hypoallergenic milk from DNA-free beta-lactoglobulin (BLG) gene knockout cow using zinc-finger nucleases mRNA.

The whey protein β-lactoglobulin (BLG) is a major milk allergen which is absent in human milk. Here, we for the first time generated DNA-free BLG bi-allelic knockout cow by zinc-finger nuclease (ZFNs) mRNA and produced BLG-free milk. According to the allergenicity evaluation of BLG-free milk, we found it can trigger lower allergic reaction of Balb/c mice including the rectal temperature drop and the allergen-specific immunoglobulin IgE production; BLG free-milk was easily digested by pepsin at 2 min, while BLG in control milk was still not completely digested after 60 min, and the binding of IgE from cow's milk allergy (CMA) patients to BLG free-milk was significantly lower than that to the control milk.

A novel glycosylated anti-CD20 monoclonal antibody from transgenic cattle.

The monoclonal antibody (mAb) against CD20 known as Rituxan is widely used to treat autoimmune diseases and lymphomas. However, further application of Rituxan faces challenges of high production cost, which limits its availability in developing countries. Here, we report a new approach for large production of a recombinant anti-CD20 mAb in the milk of transgenic cattle (at a yield of up to ~6.

Efficient targeted integration into the bovine Rosa26 locus using TALENs.

The genetic modification of cattle has many agricultural and biomedical applications. However, random integration often results in the unstable expression of transgenes and unpredictable phenotypes. Targeting genes to the "safe locus" and stably expressing foreign genes at a high level are desirable methods for overcoming these hurdles.

Effects of AANAT overexpression on the inflammatory responses and autophagy activity in the cellular and transgenic animal levels.

To explore the anti-inflammatory activity of endogenous produced melatonin, a melatonin-enriched animal model (goat) with AANAT transfer was successfully generated with somatic cell nuclear transfer (SCNT) technology. Basically, a pIRES2-EGFP-AANAT expression vector was constructed and was transferred into the female fetal fibroblast cells (FFCs) via electrotransfection and then the nuclear of the transgenic FFC was transferred to the eggs of the donor goats. The peripheral blood mononuclear cells (PBMCs) of the transgenic offspring expressed significantly higher levels of AANAT and melatonin synthetic function than those PBMCs from the wild-type (WT) animals.

CD24 and CD49f expressions of E14.5 mouse mammary anlagen cells define putative distribution of earlier embryonic mammary stem cell activities.

Stem cell biology offers promise for understanding the origins of the mammary gland. However, the distribution of mammary stem cell (MaSC) activities at earlier embryonic stages has not been fully identified. The markers for sorting adult MaSC, CD24, CD29, and CD49f have been applied to analyze fetal MaSCs.

Large-scale production of recombinant human lactoferrin from high-expression, marker-free transgenic cloned cows.

Human lactoferrin (hLF) is a valuable protein for pharmaceutical products and functional foods, and worldwide demand for this protein has steadily increased. However, large-scale recombinant human lactoferrin (rhLF) production using current animal bioreactor techniques is limited by the low expression of foreign proteins, the use of antibiotic resistance genes and the down-regulation of endogenous milk proteins. Here, we generated a herd of marker-free, hLF bacterial artificial chromosome (BAC) transgenic cloned cows, as confirmed by Polymerase chain reaction, Southern blot and Western blot analyses.

Purification and characterization of recombinant human bile salt-stimulated lipase expressed in milk of transgenic cloned cows.

Bile salt-stimulated lipase (BSSL) is a lipolytic digestive enzyme with broad substrate specificity secreted from exocrine pancreas into the intestinal lumen in all species and from the lactating mammary gland into the milk of some species, notably humans but not cows. BSSL in breast milk facilitates digestion and absorption of milk fat and promotes growth of small for gestational age preterm infants. Thus, purified recombinant human BSSL (rhBSSL) can be used for treatment of patients with fat malabsorption and expressing rhBSSL in the milk of transgenic cloned cows would therefore be a mean to meet a medical need.

Large-scale production of functional human lysozyme from marker-free transgenic cloned cows.

Human lysozyme is an important natural non-specific immune protein that is highly expressed in breast milk and participates in the immune response of infants against bacterial and viral infections. Considering the medicinal value and market demand for human lysozyme, an animal model for large-scale production of recombinant human lysozyme (rhLZ) is needed. In this study, we generated transgenic cloned cows with the marker-free vector pBAC-hLF-hLZ, which was shown to efficiently express rhLZ in cow milk.

A mammary repopulating cell population characterized in mammary anlagen reveals essential mammary stroma for morphogenesis.

The cells with mammary repopulating capability can achieve mammary gland morphogenesis in a suitable cellular microenvironment. Using cell surface markers of CD24, CD29 and CD49f, mouse mammary repopulating unit (MRU) has been identified in adult mammary epithelium and late embryonic mammary bud epithelium. However, embryonic MRU remains to be fully characterized at earlier mammary anlagen stage.

Efficient generation of myostatin (MSTN) biallelic mutations in cattle using zinc finger nucleases.

Genetically engineered zinc-finger nucleases (ZFNs) are useful for marker-free gene targeting using a one-step approach. We used ZFNs to efficiently disrupt bovine myostatin (MSTN), which was identified previously as the gene responsible for double muscling in cattle. The mutation efficiency of bovine somatic cells was approximately 20%, and the biallelic mutation efficiency was 8.

A novel promoterless gene targeting vector to efficiently disrupt PRNP gene in cattle.

The PRNP gene encodes a cellular protein named prion, whose misfolded form has been implicated in a number of neuropathic diseases in mammals such as the Bovine Spongiform Encephalopathy (BSE) in cattle. BSE has brought devastating impact on the world economy and human health. Recently, several groups have performed the gene targeting strategy to disrupt the PRNP gene in bovine fibroblast cells and produce BSE-resistant cattle by somatic cell nuclear transfer (SCNT).

Identification of short hairpin RNA targeting foot-and-mouth disease virus with transgenic bovine fetal epithelium cells.

Background: Although it is known that RNA interference (RNAi) targeting viral genes protects experimental animals, such as mice, from the challenge of Foot-and-mouth disease virus (FMDV), it has not been previously investigated whether shRNAs targeting FMDV in transgenic dairy cattle or primary transgenic bovine epithelium cells will confer resistance against FMDV challenge.

Principal Finding: Here we constructed three recombinant lentiviral vectors containing shRNA against VP2 (RNAi-VP2), VP3 (RNAi-VP3), or VP4 (RNAi-VP4) of FMDV, and found that all of them strongly suppressed the transient expression of a FLAG-tagged viral gene fusion protein in 293T cells. In BHK-21 cells, RNAi-VP4 was found to be more potent in inhibition of viral replication than the others with over 98% inhibition of viral replication.

cDNA cloning of porcine PKD2 gene and RNA interference in LLC-PK1 cells.

Mutations in the PKD2 gene cause autosomal dominant polycystic kidney disease (ADPKD), a common, inherited disease that frequently leads to end-stage renal disease (ESRD). Swine show substantial similarity to humans physiologically and anatomically, and are therefore a good model system in which to decipher the structure and function of the PKD2 gene and to identify potential therapeutic targets. Here we report the cloning and characterization of the porcine PKD2 cDNA showing that the full-length gene (3370 bases) is highly expressed in kidney, with minimal expression in the liver.

Effect of microinjection of a single IVF-derived blastomere on the development of cloned embryos at the eight-cell stage in bovine.

This study was conducted to determine the effect of microinjection of a single blastomere from in vitro fertilization (IVF)-derived eight-cell embryo into eight-cell cloned embryos harboring the gene encoding recombinant human lactoferrin (rhLF), GFP, and NEO markers in bovine. The reconstructed chimeric embryos were assessed for their development to blastocyst, or to term after transfer, and tissues of offspring were evaluated by polymerase chain reaction (PCR) for the presence of nuclear transfer (NT)-derived transgenic cells, and the cloned embryos without microinjection were used as controls. The chimeric embryos showed slightly higher blastocyst rate than that for controls.

Repressive but not activating epigenetic modifications are aberrant on the inactive X chromosome in live cloned cattle.

X inactivation is the process of a chromosome-wide silencing of the majority of genes on the X chromosome during early mammalian development. This process may be aberrant in cloned animals. Here we show that repressive modifications, such as methylation of DNA, and the presence of methylated histones, H3K9me2 and H3K27me3, exhibit distinct aberrance on the inactive X chromosome in live clones.

Cattle mammary bioreactor generated by a novel procedure of transgenic cloning for large-scale production of functional human lactoferrin.

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.

High level expression of a functional human/mouse chimeric anti-CD20 monoclonal antibody in milk of transgenic mice.

Rituximab, a chimeric anti-CD20 monoclonal antibody, is one of the most successful biomedicines and has been used to treat at least 370,000 patients with indolent, aggressive non-Hodgkin's lymphoma and other malignant diseases. However, the global demand for rituximab and other therapeutic monoclonal antibodies is exponentially increasing and barely able to be met by current manufacturing capacities of mammalian cell culture. The mammary gland bioreactor has been regarded as an ideal substitute for mammalian cell culture to mass-produce recombinant monoclonal antibodies at the lowest possible cost.