Cre Recombination-Mediated Cassette Exchange for Building Versatile Transgenic Human Embryonic Stem Cells Lines

被引:34
作者
Du, Zhong-Wei [1 ,2 ]
Hu, Bao-Yang [1 ,2 ]
Ayala, Melvin [1 ,2 ]
Sauer, Brian [3 ]
Zhang, Su-Chun [1 ,2 ]
机构
[1] Univ Wisconsin, Waisman Ctr, Dept Anat, Sch Med & Publ Hlth,WiCell Inst, Madison, WI 53705 USA
[2] Univ Wisconsin, Waisman Ctr, Dept Neurol, Sch Med & Publ Hlth,WiCell Inst, Madison, WI 53705 USA
[3] Stowers Inst Med Res, Kansas City, MO USA
关键词
Cre-loxP system; Human embryonic stem cells; Transgene expression; Differentiation; RMCE; GENE-EXPRESSION; DIFFERENTIATION; EFFICIENT; INTEGRATION; SYSTEM; MICE;
D O I
10.1002/stem.38
中图分类号
Q813 [细胞工程];
学科分类号
摘要
To circumvent the silencing effect of transgene expression in human embryonic stem cells (hESCs), we employed the Cre recombination-mediated cassette exchange strategy to target the silencing-resistant site in the genome. We have identified new loci that sustain transgene expression during stem cell expansion and differentiation to cells representing the three germ layers in vitro and in vivo. The built-in double loxP cassette in the established master hESC lines was specifically replaced by a targeting vector containing the same loxP sites, using the cell-permeable Cre protein transduction method, resulting in successful generation of new hESC lines with constitutive functional gene expression, inducible transgene expression, and lineage-specific reporter gene expression. This strategy and the master cell lines allow for rapid production of transgenic hESC lines in ordinary laboratories. STEM CELLS 2009; 27:1032-1041
引用
收藏
页码:1032 / 1041
页数:10
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