Efficient gene targeting by homologous recombination in rat embryonic stem cells

The rat is the preferred experimental animal in many biological studies. With the recent derivation of authentic rat embryonic stem (ES) cells it is now feasible to apply state-of-the art genetic engineering in this species using homologous recombination. To establish whether rat ES cells are amenable to in vivo recombination, we tested targeted disruption of the hypoxanthine phosphoribosyltransferase (hprt) locus in ES cells derived from both inbred and outbred strains of rats. Targeting vectors that replace exons 7 and 8 of the hprt gene with neomycinR/thymidine kinase selection cassettes were electroporated into male Fisher F344 and Sprague Dawley rat ES cells. Approximately 2% of the G418 resistant colonies also tolerated selection with 6-thioguanine, indicating inactivation of the hprt gene. PCR and Southern blot analysis confirmed correct site-specific targeting of the hprt locus in these clones. Embryoid body and monolayer differentiation of targeted cell lines established that they retained differentiation potential following targeting and selection. This report demonstrates that gene modification via homologous recombination in rat ES cells is efficient, and should facilitate implementation of targeted, genetic manipulation in the rat.     

Generating gene knockout rats by homologous recombination in embryonic stem cells

We describe here a detailed protocol for generating gene knockout rats by homologous recombination in embryonic stem (ES) cells. This protocol comprises the following procedures: derivation and expansion of rat ES cells, construction of gene-targeting vectors, generation of gene-targeted rat ES cells and, finally, production of gene-targeted rats. The major differences between this protocol and the classical mouse gene-targeting protocol include ES cell culture methods, drug selection scheme, colony picking and screening strategies. This ES cell-based gene-targeting technique allows sophisticated genetic modifications to be performed in the rat, as many laboratories have been doing in the mouse for the past two decades. Recently we used this protocol to generate Tp53 (also known as p53) gene knockout rats. The entire process requires ～1 year to complete, from derivation of ES cells to generation of knockout rats.     

Use of genetically modified embryonic stem cells for creating transgenic animals

Targeted genetic modification of embryonic stem cells (ESC) was used to obtain nondifferentiated cell clones containing the foreign genetic material in the genome. It was demonstrated that transgenic animals may be obtained by ESC injection in preimplantation embryos and subsequent transplantation of the embryos into a recipient female. Using this method, we constructed chimeric animals with a modified genome.     

Generation of genetically modified mice by oocyte injection of androgenetic haploid embryonic stem cells

Haploid cells are amenable for genetic analysis. Recent success in the derivation of mouse haploid embryonic stem cells (haESCs) via parthenogenesis has enabled genetic screening in mammalian cells. However, successful generation of live animals from these haESCs, which is needed to extend the genetic analysis to the organism level, has not been achieved. Here, we report the derivation of haESCs from androgenetic blastocysts. These cells, designated as AG-haESCs, partially maintain paternal imprints, express classical ESC pluripotency markers, and contribute to various tissues, including the germline, upon injection into diploid blastocysts. Strikingly, live mice can be obtained upon injection of AG-haESCs into MII oocytes, and these mice bear haESC-carried genetic traits and develop into fertile adults. Furthermore, gene targeting via homologous recombination is feasible in the AG-haESCs. Our results demonstrate that AG-haESCs can be used as a genetically tractable fertilization agent for the production of live animals via injection into oocytes.     

The role and fate of DNA ends for homologous recombination in embryonic stem cells.

We have analyzed the gene-targeting frequencies and recombination products generated by a series of vectors which target the hprt locus in embryonic stem cells and found the existence of alternative pathways that depend on the location of the double-strand break within the vector. A double-strand break in the targeting homology was found to increase the targeting frequency compared with a double-strand break at the edge of or outside the target homology; this finding agrees with the double-strand break repair model proposed for Saccharomyces cerevisiae. Although a double-strand break in the homology is important for efficient targeting, observations reported here suggest that the terminal ends are not always directly involved in the initial recombination event. Short terminal heterologous sequences which block the homologous ends of the vector may be incorporated into the target locus. A modification of the double-strand break repair model is described to account for this observation.     

Site-specific recombination in human embryonic stem cells induced by cell-permeant Cre recombinase

The biomedical application of human embryonic stem (hES) cells will increasingly depend on the availability of technologies for highly controlled genetic modification. In mouse genetics, conditional mutagenesis using site-specific recombinases has become an invaluable tool for gain- and loss-of-function studies. Here we report highly efficient Cre-mediated recombination of a chromosomally integrated loxP-modified allele in hES cells and hES cell-derived neural precursors by protein transduction. Recombinant modified Cre recombinase protein translocates into the cytoplasm and nucleus of hES cells and subsequently induces recombination in virtually 100% of the cells. Cre-transduced hES cells maintain the expression of pluripotency markers as well as the capability of differentiating into derivatives of all three germ layers in vitro and in vivo. We expect this technology to provide an important technical basis for analyzing complex genetic networks underlying human development as well as generating highly purified, transplantable hES cell-derived cells for regenerative medicine.     

Germ line transmission of an inactive N-myc allele generated by homologous recombination in mouse embryonic stem cells.

We have disrupted one allele of the N-myc locus in mouse embryonic stem (ES) cells by using homologous recombination techniques and have obtained germ line transmission of null N-myc ES cell lines with transmission of the null N-myc allele to the offspring. The creation of mice with a deficient N-myc allele will allow the generation of offspring bearing null N-myc alleles in both chromosomes and permit study of the role that this proto-oncogene plays in embryonic development.     

Genetic recombination pathways and their application for genome modification of human embryonic stem cells

Human embryonic stem cells are pluripotent cells derived from early human embryo and retain a potential to differentiate into all adult cell types. They provide vast opportunities in cell replacement therapies and are expected to become significant tools in drug discovery as well as in the studies of cellular and developmental functions of human genes. The progress in applying different types of DNA recombination reactions for genome modification in a variety of eukaryotic cell types has provided means to utilize recombination-based strategies also in human embryonic stem cells. Homologous recombination-based methods, particularly those utilizing extended homologous regions and those employing zinc finger nucleases to boost genomic integration, have shown their usefulness in efficient genome modification. Site-specific recombination systems are potent genome modifiers, and they can be used to integrate DNA into loci that contain an appropriate recombination signal sequence, either naturally occurring or suitably pre-engineered. Non-homologous recombination can be used to generate random integrations in genomes relatively effortlessly, albeit with a moderate efficiency and precision. DNA transposition-based strategies offer substantially more efficient random strategies and provide means to generate single-copy insertions, thus potentiating the generation of genome-wide insertion libraries applicable in genetic screens.     

A simple protocol for transfecting human mesenchymal stem cells

Objectives and results

Mesenchymal stromal cells (MSCs) are potential targets for cell and gene therapy-based approaches against a variety of different diseases. The MSCs from bone marrow are a promising target population as they are capable of differentiating along multiple lineages and have significant expansion capability. These characteristics make them strong candidates for delivering genes and restoring organ systems function. However, as other primary cells, MSCs are difficult to transfect. In order to standardize a simple protocol for transfection of MSCs, we conducted a series of experiments and achieved a protocol that does not require the use of viral particles or specific expensive equipment.

Conclusion

MSCs transfection at early passages using a ratio lipid/DNA of 3.0 µL/µg with Lipofectamine 3000^® yields good transfection efficiencies for human MSCs (up to 26%) and is rapid, simple, and safe.

     

Specification of motoneurons from human embryonic stem cells

An understanding of how mammalian stem cells produce specific neuronal subtypes remains elusive. Here we show that human embryonic stem cells generated early neuroectodermal cells, which organized into rosettes and expressed Pax6 but not Sox1, and then late neuroectodermal cells, which formed neural tube-like structures and expressed both Pax6 and Sox1. Only the early, but not the late, neuroectodermal cells were efficiently posteriorized by retinoic acid and, in the presence of sonic hedgehog, differentiated into spinal motoneurons. The in vitro-generated motoneurons expressed HB9, HoxC8, choline acetyltransferase and vesicular acetylcholine transporter, induced clustering of acetylcholine receptors in myotubes, and were electrophysiologically active. These findings indicate that retinoic acid action is required during neuroectoderm induction for motoneuron specification and suggest that stem cells have restricted capacity to generate region-specific projection neurons even at an early developmental stage.     

Developmental study of fragile X syndrome using human embryonic stem cells derived from preimplantation genetically diagnosed embryos

We report on the establishment of a human embryonic stem cell (HESC) line from a preimplantation fragile X-affected embryo and demonstrate its value as an appropriate model to study developmentally regulated events that are involved in the pathogenesis of this disorder. Fragile X syndrome results from FMR1 gene inactivation due to a CGG expansion at the 5'UTR region of the gene. Early events in FMR1 silencing have not been fully characterized due to the lack of appropriate animal or cellular models. Here we show that, despite the presence of a full mutation, affected undifferentiated HESCs express FMR1 and are DNA unmethylated. However, epigenetic silencing by DNA methylation and histone modification occurs upon differentiation. Our unique cell system allows the dissection of the sequence by which these epigenetic changes are acquired and illustrates the importance of HESCs in unraveling developmentally regulated mechanisms associated with human genetic disorders.     

Derivation of human embryonic stem cells by immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of the body suggests that they hold great promise for both medical applications and as a research tool for addressing fundamental questions in development and disease. Here, we provide a concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos by immunosurgical isolation of the inner cell mass.     

Mouse embryonic stem cells exhibit high levels of extrachromosomal homologous recombination in a chloramphenicol acetyltransferase assay system.

Mouse embryonic stem (ES) cells were compared to COS1 and CV1 cells for their ability to perform extrachromosomal homologous recombination. RSVCAT plasmid substrates consisting of overlapping chloramphenicol acetyltransferase (CAT) gene fragments were transiently transfected into cells and extracts were assayed for CAT activity. Approximately 10% activity, relative to transfection with a complete CAT gene, was recovered for the recombination substrates in each of the cell lines tested. ES cells, therefore, as other cell lines, are capable of high levels of extrachromosomal recombination.     

A role for RAD54B in homologous recombination in human cells.

In human somatic cells, homologous recombination is a rare event. To facilitate the targeted modification of the genome for research and gene therapy applications, efforts should be directed toward understanding the molecular mechanisms of homologous recombination in human cells. Although human genes homologous to members of the RAD52 epistasis group in yeast have been identified, no genes have been demonstrated to play a role in homologous recombination in human cells. Here, we report that RAD54B plays a critical role in targeted integration in human cells. Inactivation of RAD54B in a colon cancer cell line resulted in severe reduction of targeted integration frequency. Sensitivity to DNA-damaging agents and sister-chromatid exchange were not affected in RAD54B-deficient cells. Parts of these phenotypes were similar to those of Saccharomyces cerevisiae tid1/rdh54 mutants, suggesting that RAD54B may be a human homolog of TID1/RDH54. In yeast, TID1/RDH54 acts in the recombinational repair pathway via roles partially overlapping those of RAD54. Our findings provide the first genetic evidence that the mitotic recombination pathway is functionally conserved from yeast to humans.     

A scalable system for production of functional pancreatic progenitors from human embryonic stem cells

Development of a human embryonic stem cell (hESC)-based therapy for type 1 diabetes will require the translation of proof-of-principle concepts into a scalable, controlled, and regulated cell manufacturing process. We have previously demonstrated that hESC can be directed to differentiate into pancreatic progenitors that mature into functional glucose-responsive, insulin-secreting cells in vivo. In this study we describe hESC expansion and banking methods and a suspension-based differentiation system, which together underpin an integrated scalable manufacturing process for producing pancreatic progenitors. This system has been optimized for the CyT49 cell line. Accordingly, qualified large-scale single-cell master and working cGMP cell banks of CyT49 have been generated to provide a virtually unlimited starting resource for manufacturing. Upon thaw from these banks, we expanded CyT49 for two weeks in an adherent culture format that achieves 50-100 fold expansion per week. Undifferentiated CyT49 were then aggregated into clusters in dynamic rotational suspension culture, followed by differentiation en masse for two weeks with a four-stage protocol. Numerous scaled differentiation runs generated reproducible and defined population compositions highly enriched for pancreatic cell lineages, as shown by examining mRNA expression at each stage of differentiation and flow cytometry of the final population. Islet-like tissue containing glucose-responsive, insulin-secreting cells was generated upon implantation into mice. By four- to five-months post-engraftment, mature neo-pancreatic tissue was sufficient to protect against streptozotocin (STZ)-induced hyperglycemia. In summary, we have developed a tractable manufacturing process for the generation of functional pancreatic progenitors from hESC on a scale amenable to clinical entry.     

Generation of functional hemangioblasts from human embryonic stem cells

Recent evidence suggests the existence of progenitor cells in adult tissues that are capable of differentiating into vascular structures as well as into all hematopoietic cell lineages. Here we describe an efficient and reproducible method for generating large numbers of these bipotential progenitors-known as hemangioblasts-from human embryonic stem (hES) cells using an in vitro differentiation system. Blast cells expressed gene signatures characteristic of hemangioblasts, and could be expanded, cryopreserved and differentiated into multiple hematopoietic lineages as well as into endothelial cells. When we injected these cells into rats with diabetes or into mice with ischemia-reperfusion injury of the retina, they localized to the site of injury in the damaged vasculature and appeared to participate in repair. Injection of the cells also reduced the mortality rate after myocardial infarction and restored blood flow in hind limb ischemia in mouse models. Our data suggest that hES-derived blast cells (hES-BCs) could be important in vascular repair.     

Derivation of human embryonic stem cells from single blastomeres

This protocol details a method to derive human embryonic stem (hES) cells from single blastomeres. Blastomeres are removed from morula (eight-cell)-stage embryos and cultured until they form multicell aggregates. These blastomere-derived cell aggregates are plated into microdrops seeded with mitotically inactivated feeder cells, and then connected with neighboring microdrops seeded with green fluorescent protein-positive hES cells. The resulting blastomere-derived outgrowths are cultured in the same manner as blastocyst-derived hES cells. The whole process takes about 3-4 months.