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
- Russell DeKelver1,
- Vivian Choi1,
- Erica Moehle1,
- David Paschon1,
- Dirk Hockemeyer2,
- Sebastiaan Meijsing3,
- Yasemin Sancak4,
- Victor Bartsevich1,
- Xiaoxia Cui5,
- Eveline Steine2,
- Jeffrey Miller1,
- Philip Tam1,
- David Sabatini4,
- Xiangdong Meng1,
- Igor Rupniewski1,
- Sunita Gopalan1,
- Helena Sun1,
- Kathleen Pitz1,
- Jeremy Rock1,
- Lei Zhang1,
- Gregory Davis5,
- Edward Rebar1,
- Rudolf Jaenisch4,
- Iain Cheeseman4,
- Keith Yamamoto6,
- Philip Gregory1 and
- Fyodor Urnov7,8
- * Corresponding author; email: furnov{at}sangamo.com
Abstract
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 placed in 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.
Footnotes
- Received February 18, 2010.
- Accepted May 11, 2010.
- Copyright © 2010, Cold Spring Harbor Laboratory Press
This manuscript is Open Access.
