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 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.
Zou
Jizhong
J
Stem Cell Program, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Maeder
Morgan L
ML
Mali
Prashant
P
Pruett-Miller
Shondra M
SM
Thibodeau-Beganny
Stacey
S
Chou
Bin-Kuan
BK
Chen
Guibin
G
Ye
Zhaohui
Z
Park
In-Hyun
IH
Daley
George Q
GQ
Porteus
Matthew H
MH
Joung
J Keith
JK
Cheng
Linzhao
L
D002460:Cell Line; D003851:Deoxyribonucleases / Q000235:genetics; D053595:Embryonic Stem Cells / Q000378:metabolism*; D018390:Gene Targeting / Q000379:methods*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000378:metabolism*; D016335:Zinc Fingers
D008565:Membrane Proteins; C080086:phosphatidylinositol glycan-class A protein; D049452:Green Fluorescent Proteins; D003851:Deoxyribonucleases