Journal
CELL STEM CELL
Volume 5, Issue 1, Pages 97-110Publisher
CELL PRESS
DOI: 10.1016/j.stem.2009.05.023
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Funding
- Stem Cell Research Foundation [S2005-026]
- Johns Hopkins Institute for Cell Engineering
- NIH [R01 HL073781, R01GM069906, R21RR024189, R21HL091808, R01 HL079295]
- Maryland Stem Cell Research Postdoctoral Fellowship Grant
- Cystic Fibrosis Foundation
- Burroughs-Wellcome Fund
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We report here homologous recombination (HR)-mediated gene targeting of two different genes in human PS cells (hiPSCs) and human ES cells (hESCs). HR-mediated correction of a chromosomally integrated mutant GFP reporter gene reaches efficiencies of 0.14%-0.24% in both cell types transfected by donor DNA with plasmids expressing zinc finger nucleases (ZFNs). Engineered ZFNs that induce a sequence-specific double-strand break in the GFP gene enhanced HR-mediated correction by > 1400-fold without detectable alterations in stem cell karyotypes or pluripotency. Efficient HR-mediated insertional mutagenesis was also achieved at the endogenous PIG-A locus, with a > 200-fold enhancement by ZFNs targeted to that gene. Clonal PIG-A null hESCs and iPSCs with normal karyotypes were readily obtained. The phenotypic and biological defects were rescued by PIG-A transgene expression. Our study provides the first demonstration of HR-mediated gene targeting in hiPSCs and shows the power of ZFNs for inducing specific genetic modifications in hiPSCs, as well as hESCs.
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