Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome.
Isogenic settings are routine in model organisms, yet remain elusive for genetic experiments on human cells. We describe the use of designed zinc finger nucleases (ZFNs) for efficient transgenesis without drug selection into the PPP1R12C gene, a "safe harbor" locus known as AAVS1. ZFNs enable targeted transgenesis at a frequency of up to 15% following transient transfection of both transformed and primary human cells, including fibroblasts and hES cells. When added to this locus, transgenes such as expression cassettes for shRNAs, small-molecule-responsive cDNA expression cassettes, and reporter constructs, exhibit consistent expression and sustained function over 50 cell generations. By avoiding random integration and drug selection, this method allows bona fide isogenic settings for high-throughput functional genomics, proteomics, and regulatory DNA analysis in essentially any transformed human cell type and in primary cells.
DeKelver
Russell C
RC
Sangamo BioSciences, Inc., Point Richmond Tech Center, Richmond, California 94804, USA.
Choi
Vivian M
VM
Moehle
Erica A
EA
Paschon
David E
DE
Hockemeyer
Dirk
D
Meijsing
Sebastiaan H
SH
Sancak
Yasemin
Y
Cui
Xiaoxia
X
Steine
Eveline J
EJ
Miller
Jeffrey C
JC
Tam
Phillip
P
Bartsevich
Victor V
VV
Meng
Xiangdong
X
Rupniewski
Igor
I
Gopalan
Sunita M
SM
Sun
Helena C
HC
Pitz
Kathleen J
KJ
Rock
Jeremy M
JM
Zhang
Lei
L
Davis
Gregory D
GD
Rebar
Edward J
EJ
Cheeseman
Iain M
IM
Yamamoto
Keith R
KR
Sabatini
David M
DM
Jaenisch
Rudolf
R
Gregory
Philip D
PD
Urnov
Fyodor D
FD
D001483:Base Sequence; D002460:Cell Line; D004720:Endonucleases / Q000235:genetics; D018014:Gene Transfer Techniques; D056426:Genetic Loci; D015894:Genome, Human; D023281:Genomics / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D040901:Proteomics / Q000379:methods; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D016335:Zinc Fingers / Q000235:genetics*
D004720:Endonucleases