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Abstract / Description of output
Transcription activator-like effector nucleases (TALENs) are programmable nucleases that join FokI endonuclease with the modular DNA-binding domain of TALEs. Although zinc-finger nucleases enable a variety of genome modifications, their application to genetic engineering of livestock has been slowed by technical limitations of embryo-injection, culture of primary cells, and difficulty in producing reliable reagents with a limited budget. In contrast, we found that TALENs could easily be manufactured and that over half (23/36, 64%) demonstrate high activity in primary cells. Cytoplasmic injections of TALEN mRNAs into livestock zygotes were capable of inducing gene KO in up to 75% of embryos analyzed, a portion of which harbored biallelic modification. We also developed a simple transposon coselection strategy for TALEN-mediated gene modification in primary fibroblasts that enabled both enrichment for modified cells and efficient isolation of modified colonies. Coselection after treatment with a single TALEN-pair enabled isolation of colonies with mono- and biallelic modification in up to 54% and 17% of colonies, respectively. Coselection after treatment with two TALEN-pairs directed against the same chromosome enabled the isolation of colonies harboring large chromosomal deletions and inversions (10% and 4% of colonies, respectively). TALEN-modified Ossabaw swine fetal fibroblasts were effective nuclear donors for cloning, resulting in the creation of miniature swine containing mono- and biallelic mutations of the LDL receptor gene as models of familial hypercholesterolemia. TALENs thus appear to represent a highly facile platform for the modification of livestock genomes for both biomedical and agricultural applications.
Original language | English |
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Pages (from-to) | 17382-7 |
Number of pages | 6 |
Journal | Proceedings of the National Academy of Sciences (PNAS) |
Volume | 109 |
Issue number | 43 |
DOIs | |
Publication status | Published - 2012 |
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Dive into the research topics of 'Efficient TALEN-mediated gene knockout in livestock'. Together they form a unique fingerprint.Projects
- 2 Finished
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Refining genome editing in livestock
Non-EU industry, commerce and public corporations
30/04/14 → 29/04/16
Project: Research
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Evaluation of genome engineering strategies to target the porcine RELA gene
Non-EU industry, commerce and public corporations
1/01/13 → 31/07/16
Project: Research