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 target...
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| Format: | Article |
| Language: | en_US |
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Cold Spring Harbor Laboratory Press
2012
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| Online Access: | http://hdl.handle.net/1721.1/72607 https://orcid.org/0000-0002-3829-5612 https://orcid.org/0000-0002-1446-7256 |
| _version_ | 1811068630197075968 |
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| author | DeKelver, Russell C. Choi, Vivian M. Moehle, Erica A. Paschon, David E. Hockemeyer, Dirk Meijsing, Sebastiaan H. Sancak, Yasemin Cui, Xiaoxia Steine, Eveline J. Miller, Jeffrey C. Tam, Phillip Bartsevich, Victor V. Meng, Xiangdong Rupniewski, Igor Gopalan, Sunita M. Sun, Helena C. Pitz, Kathleen J. Rock, Jeremy M. Davis, Gregory D. Rebar, Edward J. Yamamoto, Keith R. Jaenisch, Rudolf Gregory, Philip D. Urnov, Fyodor D. Zhang, Lei, Ph. D Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, fl. 2014. Cheeseman, Iain M Sabatini, David |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology DeKelver, Russell C. Choi, Vivian M. Moehle, Erica A. Paschon, David E. Hockemeyer, Dirk Meijsing, Sebastiaan H. Sancak, Yasemin Cui, Xiaoxia Steine, Eveline J. Miller, Jeffrey C. Tam, Phillip Bartsevich, Victor V. Meng, Xiangdong Rupniewski, Igor Gopalan, Sunita M. Sun, Helena C. Pitz, Kathleen J. Rock, Jeremy M. Davis, Gregory D. Rebar, Edward J. Yamamoto, Keith R. Jaenisch, Rudolf Gregory, Philip D. Urnov, Fyodor D. Zhang, Lei, Ph. D Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, fl. 2014. Cheeseman, Iain M Sabatini, David |
| author_sort | DeKelver, Russell C. |
| collection | MIT |
| description | 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. |
| first_indexed | 2024-09-23T07:58:48Z |
| format | Article |
| id | mit-1721.1/72607 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T07:58:48Z |
| publishDate | 2012 |
| publisher | Cold Spring Harbor Laboratory Press |
| record_format | dspace |
| spelling | mit-1721.1/726072022-09-30T01:26:54Z 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 DeKelver, Russell C. Choi, Vivian M. Moehle, Erica A. Paschon, David E. Hockemeyer, Dirk Meijsing, Sebastiaan H. Sancak, Yasemin Cui, Xiaoxia Steine, Eveline J. Miller, Jeffrey C. Tam, Phillip Bartsevich, Victor V. Meng, Xiangdong Rupniewski, Igor Gopalan, Sunita M. Sun, Helena C. Pitz, Kathleen J. Rock, Jeremy M. Davis, Gregory D. Rebar, Edward J. Yamamoto, Keith R. Jaenisch, Rudolf Gregory, Philip D. Urnov, Fyodor D. Zhang, Lei, Ph. D Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, fl. 2014. Cheeseman, Iain M Sabatini, David Massachusetts Institute of Technology. Department of Biology Cheeseman, Iain M. Sancak, Yasemin Cheeseman, Iain McPherson Sabatini, David M. Jaenisch, Rudolf 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. National Institutes of Health (U.S.) (Grant number R37-CA084198) National Institutes of Health (U.S.) (Grant number RO1-CA087869) National Institutes of Health (U.S.) (Grant number RO1-HD045022) Howard Hughes Medical Institute. National Institutes of Health (U.S.) (Grant number R01 CA103866) National Institutes of Health (U.S.) (Grant number AI47389) United States. Dept. of Defense (Grant number W81XWH-07-0448) W. M. Keck Foundation 2012-09-11T13:33:20Z 2012-09-11T13:33:20Z 2010-05 Article http://purl.org/eprint/type/JournalArticle 1088-9051 http://hdl.handle.net/1721.1/72607 DeKelver, R. C. et al. “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.” Genome Research 20.8 (2010): 1133–1142. Copyright © 2010 by Cold Spring Harbor Laboratory Press https://orcid.org/0000-0002-3829-5612 https://orcid.org/0000-0002-1446-7256 en_US http://dx.doi.org/10.1101/gr.106773.110 Genome Research Creative Commons Attribution Non-Commercial http://creativecommons.org/licenses/by-nc/3.0/ application/pdf Cold Spring Harbor Laboratory Press PMC |
| spellingShingle | DeKelver, Russell C. Choi, Vivian M. Moehle, Erica A. Paschon, David E. Hockemeyer, Dirk Meijsing, Sebastiaan H. Sancak, Yasemin Cui, Xiaoxia Steine, Eveline J. Miller, Jeffrey C. Tam, Phillip Bartsevich, Victor V. Meng, Xiangdong Rupniewski, Igor Gopalan, Sunita M. Sun, Helena C. Pitz, Kathleen J. Rock, Jeremy M. Davis, Gregory D. Rebar, Edward J. Yamamoto, Keith R. Jaenisch, Rudolf Gregory, Philip D. Urnov, Fyodor D. Zhang, Lei, Ph. D Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, fl. 2014. Cheeseman, Iain M Sabatini, David 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 |
| title | 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 |
| title_full | 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 |
| title_fullStr | 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 |
| title_full_unstemmed | 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 |
| title_short | 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 |
| title_sort | 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 |
| url | http://hdl.handle.net/1721.1/72607 https://orcid.org/0000-0002-3829-5612 https://orcid.org/0000-0002-1446-7256 |
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