Gene Targeting in Human Pluripotent Stem Cells
Department of Reproductive Medicine, UCSD Medical Center, University of California, San Diego, San Diego, CA, USA. Methods in molecular biology (Clifton, N.J.)
(Impact Factor: 1.29).
01/2011; 767:355-67. DOI: 10.1007/978-1-61779-201-4_26
Targeted homologous recombination (HR) is an essential tool in stem cell biology. It can be used to study gene function and is a highly developed technology in the mouse where precise genetic modifications are introduced into the genome via HR in mouse embryonic stem cells (mESCs). However, gene targeting has not been widely applied to the study of human pluripotent stem cells (hPSCs) due to its relatively low efficiency in human cell lines. To overcome this technical hurdle, we have developed and established a protocol that allows efficient gene targeting in hPSC lines. This chapter provides a detailed protocol for efficiently performing gene targeting in hPSCs by electroporation. The protocol describes methods for cell preparation, antibiotic selection, and excision of the selectable marker following gene targeting. While we can only target one allele at a time, HR covers a broad range of important applications including making knock-in reporter lines and knock-in lineage tracers, generating disease models that are caused by dominant mutants, repair of patient-derived induced PSCs that only involve a single allele mutation, and knocking out genes that are located on the X chromosome in male lines. When targeting to both alleles is needed, such as generation of a knockout cell line, the cells can be electroporated twice with targeting vectors designed to target each of the alleles. This protocol will find broad applications in generating lineage-specific reporter lines and point mutations in genetic repair in disease models using hPSCs.
Available from: Claes Andréasson
- "Although knockout and conditional ablation of genes are the first steps in understanding gene function in vivo, allelic variants such as disease-mimicking point mutations or whole gene replacement (e.g., with human disease variants) are quickly emerging as necessary tools to phenocopy human genetic disease in model organisms. With regard to species relevance or for clinical applications, these mutations are increasingly created in human ES or iPS cells –, a process that is still somewhat tedious due to its use in niche applications . Nonetheless, paired with new technologies to increase homologous recombination such as zinc-finger nucleases, TALens, and CRISPR –, targeting vectors are employed widely for genome modification. "
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ABSTRACT: Efficient gene targeting in embryonic stem cells requires that modifying DNA sequences are identical to those in the targeted chromosomal locus. Yet, there is a paucity of isogenic genomic clones for human cell lines and PCR amplification cannot be used in many mutation-sensitive applications. Here, we describe a novel method for the direct cloning of genomic DNA into a targeting vector, pRTVIR, using oligonucleotide-directed homologous recombination in yeast. We demonstrate the applicability of the method by constructing functional targeting vectors for mammalian genes Uhrf1 and Gfap. Whereas the isogenic targeting of the gene Uhrf1 showed a substantial increase in targeting efficiency compared to non-isogenic DNA in mouse E14 cells, E14-derived DNA performed better than the isogenic DNA in JM8 cells for both Uhrf1 and Gfap. Analysis of 70 C57BL/6-derived targeting vectors electroporated in JM8 and E14 cell lines in parallel showed a clear dependence on isogenicity for targeting, but for three genes isogenic DNA was found to be inhibitory. In summary, this study provides a straightforward methodological approach for the direct generation of isogenic gene targeting vectors.
Available from: Xiangyang Miao
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ABSTRACT: Transgenic animal technology is one of the fastest growing biotechnology areas. It is used to integrate exogenous genes into the animal genome by genetic engineering technology so that these genes can be inherited and expressed by offspring. The transgenic efficiency and precise control of gene expression are the key limiting factors in the production of transgenic animals. A variety of transgenic technologies are available. Each has its own advantages and disadvantages and needs further study because of unresolved technical and safety issues. Further studies will allow transgenic technology to explore gene function, animal genetic improvement, bioreactors, animal disease models, and organ transplantation. This article reviews the recently developed animal transgenic technologies, including the germ line stem cell-mediated method to improve efficiency, gene targeting to improve accuracy, RNA interference-mediated gene silencing technology, zinc-finger nuclease gene targeting technology and induced pluripotent stem cell technology. These new transgenic techniques can provide a better platform to develop transgenic animals for breeding new animal varieties and promote the development of medical sciences, livestock production, and other fields.
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To generate rat induced pluripotent stem cells (iPSCs) using the PiggyBac (PB) transposon system, and to explore whether these iPSCs would be amenable to genetic manipulation.
Materials and methods:
The PB transposon system was used to reprogramme rat embryonic fibroblasts (EF) to become iPSCs. Cells were identified with regard to pluripotency and differentiation capacity in vitro and in vivo, population growth characteristics and gene expression; furthermore, targeting vector was electroporated into them. Correct recombination colonies were acquired by positive and negative selection, and then phenotype confirmed by Southern blotting.
The rat EF cells were reprogrammed into iPSCs successfully, using the PB transposon system. Cell morphology was found to display characteristics of rat embryonic stem cells (ESCs) and results of immunofluorescence staining and PCR indicated that they expressed pluripotency markers. In vivo and in vitro differentiation experiments proved that the cells could differentiate into all phenotypes from three germ layers, and to form chimaeras with high rat iPSC contribution. After electroporation with p53 targeting vector, approximately (5.44 ± 0.74) × 10(-6) colonies tolerated selection. Southern blotting confirmed that p53 gene was targeted successfully in the colonies.
The PB transposon system proved to be an effective method for reprogramming of rat EF cells into iPSCs. The rat iPSCs were amenable to gene targeting mediated by routine homologous recombination.
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