title: Genome editing in human pluripotent stem cells using site-specific nucleases.
abstract: Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) (Thomson, Science 282:1145-1147, 1998; Takahashi et al. Cell 131:861-872, 2007), collectively referred to as pluripotent stem cells (hPSCs), are currently used in disease modeling to address questions specific to humans and to complement our insight gained from model organisms (Soldner et al. Cell 146:318-331, 2011; Soldner and Jaenisch, Science 338:1155-1156, 2012). Recently, genetic engineering using site-specific nucleases has been established in hPSCs (Hockemeyer et al. Nat Biotechnol 27:851-857, 2009; Hockemeyer et al., Nat Biotechnol 29:731-734, 2011; Zou et al., Cell Stem Cell 5:97-110, 2011; Yusa et al., Nature 478:391-394, 2011; DeKelver et al., Genome Res 20:1133-1142, 2010), allowing a level of genetic control previously limited to model systems. Thus, we can now perform targeted gene knockouts, generate tissue-specific cell lineage reporters, overexpress genes from a defined locus, and introduce and repair single point mutations in hPSCs. This ability to genetically engineer pluripotent stem cells will significantly facilitate the study of human disease in a defined genetic context. Here we outline protocols for efficient gene targeting in hPSCs.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, 400B Li Ka Shing Center, Berkeley, CA, 94720-3370, USA.
authors.forename: Kunitoshi
authors.initials: K
authors.lastname: Chiba
authors.forename: Dirk
authors.initials: D
authors.lastname: Hockemeyer
mesh_terms: D003001:Cloning, Molecular / Q000379:methods; D059766:DNA End-Joining Repair; D004262:DNA Restriction Enzymes / Q000235:genetics* / Q000378:metabolism*; D018274:Electroporation; D053595:Embryonic Stem Cells / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D015870:Gene Expression; D018390:Gene Targeting; D005822:Genetic Vectors / Q000235:genetics; D060005:Genotyping Techniques; D059765:Homologous Recombination; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000378:metabolism*; D059010:Single-Cell Analysis / Q000379:methods
chemical_list: D004262:DNA Restriction Enzymes