Cre recombination-mediated cassette exchange for building versatile transgenic human embryonic stem cells lines.
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.
Du
Zhong-Wei
ZW
Department of Anatomy and Neurology, School of Medicine and Public Health, Waisman Center, WiCell Institute, University of Wisconsin, Madison, WI 53705, USA.
Hu
Bao-Yang
BY
Ayala
Melvin
M
Sauer
Brian
B
Zhang
Su-Chun
SC
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D002463:Cell Membrane Permeability; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D005786:Gene Expression Regulation; D020868:Gene Silencing; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D019426:Integrases / Q000378:metabolism*; D051379:Mice; D016254:Mutagenesis, Insertional; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D000076104:Oligodendrocyte Transcription Factor 2; D009928:Organ Specificity; D011995:Recombination, Genetic / Q000235:genetics*; D014162:Transfection; D019076:Transgenes / Q000235:genetics*
D051792:Basic Helix-Loop-Helix Proteins; D049452:Green Fluorescent Proteins; D019426:Integrases; D009419:Nerve Tissue Proteins; D000076104:Oligodendrocyte Transcription Factor 2; C405708:OLIG2 protein, human; C045073:Cre recombinase