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

  1. Fyodor D. Urnov1,7
  1. 1 Sangamo BioSciences, Inc., Point Richmond Tech Center, Richmond, California 94804, USA;
  2. 2 The Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA;
  3. 3 Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94158, USA;
  4. 4 Sigma-Aldrich Research Biotechnology, St. Louis, Missouri 63103, USA;
  5. 5 Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    • ↵6 Present address: Max Planck Institute for Molecular Genetics, Ihnestrasse 63-73, 14195 Berlin, Germany.

    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 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.

    Footnotes

    Freely available online through the Genome Research Open Access option.

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    1. Published in Advance May 27, 2010, doi: 10.1101/gr.106773.110 Genome Res. 20: 1133-1142 Copyright © 2010 by Cold Spring Harbor Laboratory Press

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