Open AccessCre Recombination‐Mediated Cassette Exchange for Building Versatile Transgenic Human Embryonic Stem Cells Lines
Author(s) -
Du ZhongWei,
Hu BaoYang,
Ayala Melvin,
Sauer Brian,
Zhang SuChun
Publication year - 2009
Publication title -
stem cells
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.159
H-Index - 229
eISSN - 1549-4918
pISSN - 1066-5099
DOI - 10.1002/stem.38
Subject(s) - biology , transgene , embryonic stem cell , gene silencing , microbiology and biotechnology , stem cell , reporter gene , site specific recombination , transduction (biophysics) , cell culture , gene , gene expression , genetics , recombinase , recombination , biochemistry
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. S tem C ells 2009;27:1032–1041