title: Efficient and accurate homologous recombination in hESCs and hiPSCs using helper-dependent adenoviral vectors.
abstract: Low efficiencies of gene targeting via homologous recombination (HR) have limited basic research and applications using human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Here, we show highly and equally efficient gene knockout and knock-in at both transcriptionally active (HPRT1, KU80, LIG1, LIG3) and inactive (HB9) loci in these cells using high-capacity helper-dependent adenoviral vectors (HDAdVs). Without the necessity of introducing artificial DNA double-strand breaks, 7-81% of drug-resistant colonies were gene-targeted by accurate HR, which were not accompanied with additional ectopic integrations. Even at the motor neuron-specific HB9 locus, the enhanced green fluorescent protein (EGFP) gene was accurately knocked in in 23-57% of drug-resistant colonies. In these clones, induced differentiation into the HB9-positive motor neuron correlated with EGFP expression. Furthermore, HDAdV infection had no detectable adverse effects on the undifferentiated state and pluripotency of hESCs and hiPSCs. These results suggest that HDAdV is one of the best methods for efficient and accurate gene targeting in hESCs and hiPSCs and might be especially useful for therapeutic applications.
authors.affiliation: Gene Therapy Division, Research Center for Genomic Medicine, Saitama Medical University, Saitama, Japan.
authors.forename: Emi
authors.initials: E
authors.lastname: Aizawa
authors.forename: Yuka
authors.initials: Y
authors.lastname: Hirabayashi
authors.forename: Yuzuru
authors.initials: Y
authors.lastname: Iwanaga
authors.forename: Keiichiro
authors.initials: K
authors.lastname: Suzuki
authors.forename: Kenji
authors.initials: K
authors.lastname: Sakurai
authors.forename: Miho
authors.initials: M
authors.lastname: Shimoji
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Aiba
authors.forename: Tamaki
authors.initials: T
authors.lastname: Wada
authors.forename: Norie
authors.initials: N
authors.lastname: Tooi
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: D000256:Adenoviridae / Q000235:genetics*; D034961:Antigens, Nuclear / Q000235:genetics; D002460:Cell Line; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D055879:Gene Knock-In Techniques; D055786:Gene Knockout Techniques; D023061:Gene Order; D018390:Gene Targeting; D005822:Genetic Vectors / Q000235:genetics*; D006579:Heterozygote; D059765:Homologous Recombination; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D000072200:Ku Autoantigen; D009154:Mutation; D000075223:Poly-ADP-Ribose Binding Proteins; D029867:Xenopus Proteins
chemical_list: D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; C000607892:LIG1 protein, human; D000075223:Poly-ADP-Ribose Binding Proteins; D029867:Xenopus Proteins; D007041:Hypoxanthine Phosphoribosyltransferase; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen; D011088:DNA Ligases; D000072481:DNA Ligase ATP; C000618816:DNA ligase III alpha protein, Xenopus; C000618817:LIG3 protein, human