title: Gene targeting in human pluripotent stem cells with adeno-associated virus vectors.
abstract: Human pluripotent stem cells, such as embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), have the ability to differentiate into various cell types, and will become a potential source of cellular materials for regenerative medicine. To make full use of hESCs or hiPSCs for both basic and clinical research, genetic modification, especially gene targeting via homologous recombination (HR), would be an essential technique. This report describes the successful gene targeting of the hypoxanthine phosphoribosyl transferase 1 (HPRT1) and the NANOG loci in human pluripotent stem cells with adeno-associated virus (AAV) vectors. At the HPRT1 locus, up to 1% of stable transformants were targeted via HR with an AAV-HPRT1 targeting vector, without loss of pluripotency. On the other hand, 20-87% of stable transformants were targeted using an AAV-NANOG-targeting vector designed for the promoter-trap strategy. In the KhES-3 cell line, which shows particularly high fragility to experimental manipulation, gene targeting was successful only by using an AAV vector but not by electroporation. In addition to hESC, gene targeting was achieved in hiPSC lines at similar frequencies. These data indicate that AAV vectors may therefore be a useful tool to introduce genetic modifications in hESCs and hiPSCs.
authors.affiliation: Gene Therapy Division, Research Center for Genomic Medicine, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1241, Japan.
authors.forename: Kaoru
authors.initials: K
authors.lastname: Mitsui
authors.forename: Keiichiro
authors.initials: K
authors.lastname: Suzuki
authors.forename: Emi
authors.initials: E
authors.lastname: Aizawa
authors.forename: Eihachiro
authors.initials: E
authors.lastname: Kawase
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Suemori
authors.forename: Norio
authors.initials: N
authors.lastname: Nakatsuji
authors.forename: Kohnosuke
authors.initials: K
authors.lastname: Mitani
mesh_terms: D000229:Dependovirus; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D000071317:Nanog Homeobox Protein; D039904:Pluripotent Stem Cells / Q000378:metabolism*
chemical_list: D018398:Homeodomain Proteins; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D007041:Hypoxanthine Phosphoribosyltransferase