Gene Targeting of a Disease-Related Gene in Human Induced Pluripotent Stem and Embryonic Stem Cells

We report here homologous recombination (HR)-mediated gene targeting of two different genes in human iPS 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 dono...

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Bibliographic Details
Published in:Cell stem cell 2009-07, Vol.5 (1), p.97-110
Main Authors: Zou, Jizhong, Maeder, Morgan L., Mali, Prashant, Pruett-Miller, Shondra M., Thibodeau-Beganny, Stacey, Chou, Bin-Kuan, Chen, Guibin, Ye, Zhaohui, Park, In-Hyun, Daley, George Q., Porteus, Matthew H., Joung, J. Keith, Cheng, Linzhao
Format: Article
Language:English
Subjects:
Biological and medical sciences
Cell differentiation, maturation, development, hematopoiesis
Cell Line
Cell physiology
Deoxyribonucleases - genetics
Embryonic Stem Cells - metabolism
Fundamental and applied biological sciences. Psychology
Gene Targeting - methods
Genes, Reporter
Green Fluorescent Proteins - genetics
Green Fluorescent Proteins - metabolism
Humans
HUMDISEASE
Membrane Proteins - genetics
Membrane Proteins - metabolism
Molecular and cellular biology
Pluripotent Stem Cells - metabolism
STEMCELL
Zinc Fingers
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Summary:We report here homologous recombination (HR)-mediated gene targeting of two different genes in human iPS 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.
ISSN:1934-5909
1875-9777
1875-9777
DOI:10.1016/j.stem.2009.05.023