Efficient integration of transgenes into a defined locus in human embryonic stem cells.
Random integration is one of the more straightforward methods to introduce a transgene into human embryonic stem (ES) cells. However, random integration may result in transgene silencing and altered cell phenotype due to insertional mutagenesis in undefined gene regions. Moreover, reliability of data may be compromised by differences in transgene integration sites when comparing multiple transgenic cell lines. To address these issues, we developed a genetic manipulation strategy based on homologous recombination and Cre recombinase-mediated site-specific integration. First, we performed gene targeting of the hypoxanthine phosphoribosyltransferase 1 (HPRT) locus of the human ES cell line KhES-1. Next, a gene-replacement system was created so that a circular vector specifically integrates into the targeted HPRT locus via Cre recombinase activity. We demonstrate the application of this strategy through the creation of a tetracycline-inducible reporter system at the HPRT locus. We show that reporter gene expression was responsive to doxycycline and that the resulting transgenic human ES cells retain their self-renewal capacity and pluripotency.
Sakurai
Kenji
K
Institute for Frontier Medical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan. sakurai@scdi.or.jp
Shimoji
Miho
M
Tahimic
Candice G T
CG
Aiba
Kazuhiro
K
Kawase
Eihachiro
E
Hasegawa
Kouichi
K
Amagai
Yuji
Y
Suemori
Hirofumi
H
Nakatsuji
Norio
N
D002460:Cell Line; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D005260:Female; D005786:Gene Expression Regulation; D018390:Gene Targeting / Q000379:methods*; D056426:Genetic Loci; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D011995:Recombination, Genetic; D019076:Transgenes
D007041:Hypoxanthine Phosphoribosyltransferase