期刊
STEM CELLS
卷 27, 期 5, 页码 1032-1041出版社
WILEY
DOI: 10.1002/stem.38
关键词
Cre-loxP system; Human embryonic stem cells; Transgene expression; Differentiation; RMCE
资金
- NIH [NS061243]
- National MS Society
- EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT [P30HD003352] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE [R21NS061243, P01NS057778, R01NS045926] Funding Source: NIH RePORTER
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
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