Production of large, defined genome modifications in rats by targeting rat embryonic stem cells.

Rats were more frequently used than mice to model human disease before mouse embryonic stem cells (mESCs) revolutionized genetic engineering in mice. Rat ESCs (rESCs) were first reported over 10 years ago, yet they are not as frequently used as mESCs. CRISPR-based gene editing in zygotes is widely used in rats but is limited by the difficulty of inserting or replacing DNA sequences larger than about 10 kb.

Kappa-on-Heavy (KoH) bodies are a distinct class of fully-human antibody-like therapeutic agents with antigen-binding properties.

We describe a Kappa-on-Heavy (KoH) mouse that produces a class of highly diverse, fully human, antibody-like agents. This mouse was made by replacing the germline variable sequences of both the Ig heavy-chain (IgH) and Ig kappa (IgK) loci with the human IgK germline variable sequences, producing antibody-like molecules with an antigen binding site made up of 2 kappa variable domains. These molecules, named KoH bodies, structurally mimic naturally existing Bence-Jones light-chain dimers in their variable domains and remain wild-type in their antibody constant domains.

Deletion of Adam6 in Mus musculus leads to male subfertility and deficits in sperm ascent into the oviduct.

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.

Injecting Embryonic Stem Cells into Eight-Cell-Stage Mouse Embryos.

In this protocol, eight-cell-stage precompaction embryos from outbred mouse strains are used for the injection of hybrid or inbred embryonic stem (ES) cells. This process often leads to generation of fully ES cell-derived so-called F mice (VelociMice). Postinjection culture of embryos is necessary to achieve the highest ratio of fully ES cell-derived mice and high-degree chimeras.

Diverse Phenotypes and Specific Transcription Patterns in Twenty Mouse Lines with Ablated LincRNAs.

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.

Generation of fertile and fecund F0 XY female mice from XY ES cells.

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.

Precise and in situ genetic humanization of 6 Mb of mouse immunoglobulin genes.

Genetic humanization, which involves replacing mouse genes with their human counterparts, can create powerful animal models for the study of human genes and diseases. One important example of genetic humanization involves mice humanized for their Ig genes, allowing for human antibody responses within a mouse background (HumAb mice) and also providing a valuable platform for the generation of fully human antibodies as therapeutics. However, existing HumAb mice do not have fully functional immune systems, perhaps because of the manner in which they were genetically humanized.

Conditionals by inversion provide a universal method for the generation of conditional alleles.

Conditional mutagenesis is becoming a method of choice for studying gene function, but constructing conditional alleles is often laborious, limited by target gene structure, and at times, prone to incomplete conditional ablation. To address these issues, we developed a technology termed conditionals by inversion (COIN). Before activation, COINs contain an inverted module (COIN module) that lies inertly within the antisense strand of a resident gene.

Human thrombopoietin knockin mice efficiently support human hematopoiesis in vivo.

Hematopoietic stem cells (HSCs) both self-renew and give rise to all blood cells for the lifetime of an individual. Xenogeneic mouse models are broadly used to study human hematopoietic stem and progenitor cell biology in vivo. However, maintenance, differentiation, and function of human hematopoietic cells are suboptimal in these hosts.

Human IL-3/GM-CSF knock-in mice support human alveolar macrophage development and human immune responses in the lung.

Mice with a functional human immune system have the potential to allow in vivo studies of human infectious diseases and to enable vaccine testing. To this end, mice need to fully support the development of human immune cells, allow infection with human pathogens, and be capable of mounting effective human immune responses. A major limitation of humanized mice is the poor development and function of human myeloid cells and the absence of human immune responses at mucosal surfaces, such as the lung.

Producing fully ES cell-derived mice from eight-cell stage embryo injections.

In conventional methods for the generation of genetically modified mice, gene-targeted embryonic stem (ES) cells are injected into blastocyst-stage embryos or are aggregated with morula-stage embryos, which are then transferred to the uterus of a surrogate mother. F0 generation mice born from the embryos are chimeras composed of genetic contributions from both the modified ES cells and the recipient embryos. Obtaining a mouse strain that carries the gene-targeted mutation requires breeding the chimeras to transmit the ES cell genetic component through the germ line to the next (F1) generation (germ line transmission, GLT).

Embryonic stem cell tumor model reveals role of vascular endothelial receptor tyrosine phosphatase in regulating Tie2 pathway in tumor angiogenesis.

Inhibiting angiogenesis has become an effective approach for treating cancer and other diseases. However, our understanding of signaling pathways in tumor angiogenesis has been limited by the embryonic lethality of many gene knockouts. To overcome this limitation, we used the plasticity of embryonic stem (ES) cells to develop a unique approach to study tumor angiogenesis.

VelociMouse: fully ES cell-derived F0-generation mice obtained from the injection of ES cells into eight-cell-stage embryos.

With the completion of the human and mouse genome sequences and the development of high-throughput knockout mouse technologies, there is now a need for equally high-throughput methods for the production of mice for phenotypic studies. In response to this challenge, we recently developed a new method termed VelociMouse for the production of F0-generation mice that are fully derived from gene-targeted ES cells. In the version of the VelociMouse method described here, laser ablation of a portion of the zona pellucid (zp) of a normal eight-cell-stage embryo facilitates ES cell injection.

F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses.

A useful approach for exploring gene function involves generating mutant mice from genetically modified embryonic stem (ES) cells. Recent advances in genetic engineering of ES cells have shifted the bottleneck in this process to the generation of mice. Conventional injections of ES cells into blastocyst hosts produce F0 generation chimeras that are only partially derived from ES cells, requiring additional breeding to obtain mutant mice that can be phenotyped.

Recycling to the plasma membrane is delayed in EHD1 knockout mice.

EHD1 is a member of the EHD family that contains four mammalian homologs. Among the invertebrate orthologs are a single Drosophila and Caenorhabditis elegans proteins and two plant members. They all contain three modules, a N-terminal domain that contains nucleotide-binding motifs, a central coiled-coil domain involved in oligomerization and a C-terminal region that harbors the EH domain.

Genomic instability and aging-like phenotype in the absence of mammalian SIRT6.

The Sir2 histone deacetylase functions as a chromatin silencer to regulate recombination, genomic stability, and aging in budding yeast. Seven mammalian Sir2 homologs have been identified (SIRT1-SIRT7), and it has been speculated that some may have similar functions to Sir2. Here, we demonstrate that SIRT6 is a nuclear, chromatin-associated protein that promotes resistance to DNA damage and suppresses genomic instability in mouse cells, in association with a role in base excision repair (BER).

The exon 8-containing prosaposin gene splice variant is dispensable for mouse development, lysosomal function, and secretion.

Prosaposin is a multifunctional protein with diverse functions. Intracellularly, prosaposin is a precursor of four sphingolipid activator proteins, saposins A to D, which are required for hydrolysis of sphingolipids by several lysosomal exohydrolases. Secreted prosaposin has been implicated as a neurotrophic, myelinotrophic, and myotrophic factor as well as a spermatogenic factor.

Deletion of mouse rad9 causes abnormal cellular responses to DNA damage, genomic instability, and embryonic lethality.

The fission yeast Schizosaccharomyces pombe rad9 gene promotes cell survival through activation of cell cycle checkpoints induced by DNA damage. Mouse embryonic stem cells with a targeted deletion of Mrad9, the mouse ortholog of this gene, were created to evaluate its function in mammals. Mrad9(-/-) cells demonstrated a marked increase in spontaneous chromosome aberrations and HPRT mutations, indicating a role in the maintenance of genomic integrity.

High-throughput engineering of the mouse genome coupled with high-resolution expression analysis.

One of the most effective approaches for determining gene function involves engineering mice with mutations or deletions in endogenous genes of interest. Historically, this approach has been limited by the difficulty and time required to generate such mice. We describe the development of a high-throughput and largely automated process, termed VelociGene, that uses targeting vectors based on bacterial artificial chromosomes (BACs).

A novel gene, GliH1, with homology to the Gli zinc finger domain not required for mouse development.

The Sonic hedgehog (Shh)-Gli signaling pathway regulates development of many organs, including teeth. We cloned a novel gene encoding a transcription factor that contains a zinc finger domain with highest homology to the Gli family of proteins (61-64% amino acid sequence identity) from incisor pulp. Consistent with this sequence conservation, gel mobility shift assays demonstrated that this new Gli homologous protein, GliH1, could bind previously characterized Gli DNA binding sites.

Gli2, but not Gli1, is required for initial Shh signaling and ectopic activation of the Shh pathway.

The Shh signaling pathway is required in many mammalian tissues for embryonic patterning, cell proliferation and differentiation. In addition, inappropriate activation of the pathway has been implicated in many human tumors. Based on transfection assays and gain-of-function studies in frog and mouse, the transcription factor Gli1 has been proposed to be a major mediator of Shh signaling.

The upstream ectoderm enhancer in Pax6 has an important role in lens induction.

The Pax6 gene has a central role in development of the eye. We show, through targeted deletion in the mouse, that an ectoderm enhancer in the Pax6 gene is required for normal lens formation. Ectoderm enhancer-deficient embryos exhibit distinctive defects at every stage of lens development.

The HD mutation causes progressive lethal neurological disease in mice expressing reduced levels of huntingtin.

Huntingtin is an essential protein that with mutant polyglutamine tracts initiates dominant striatal neurodegeneration in Huntington's disease (HD). To assess the consequences of mutant protein when huntingtin is limiting, we have studied three lines of compound heterozygous mice in which both copies of the HD gene homolog (Hdh) were altered, resulting in greatly reduced levels of huntingtin with a normal human polyglutamine length (Q20) and/or an expanded disease-associated segment (Q111): Hdh(neoQ20)/Hdh(neoQ20), Hdh(neoQ20)/Hdh(null) and Hdh(neoQ20)/Hdh(neoQ111). All surviving mice in each of the three lines were small from birth, and had variable movement abnormalities.

Establishment and chimera analysis of 129/SvEv- and C57BL/6-derived mouse embryonic stem cell lines.

Hundreds of new mutant mouse lines are being produced annually using gene targeting and gene trap approaches in embryonic stem (ES) cells, and the number is expected to continue to grow as the human and mouse genome projects progress. The availability of robust ES cell lines and a simple technology for making chimeras is more attractive now than ever before. We established several new ES cell lines from 129/SvEv and C57BL/6 mice and tested their ability to contribute to the germline following blastocyst injections and/or the less expensive and easier method of morula-ES cell aggregation.