The DOI matches its Crossref record; 5 of the 5 references it cites were found in a registry or an index.
Efficient TALEN-mediated gene knockout in livestock
Open accessAbstract
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.
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Citations
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“Compared with long-range deletions, inversion events are rare and have only been reported in vivo at rates of 0.2– 1% and 4% in zebrafish (Gupta et al. 2013; Xiao et al. 2013) and pigs (Carlson et al. 2012), respectively.”
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Self-citationTrait-associated noncoding variant regions affect TBX3 regulation and cardiac conduction
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Metadata
- Authors
- Carlson, D. F., Tan, W., Lillico, S. G., Stverakova, D., Proudfoot, C., Christian, M., Voytas, D. F., Long, C. R., Whitelaw, C. B. A., & Fahrenkrug, S. C.
- Venue
- Proceedings of the National Academy of Sciences
- Published
- 2012-10
- Volume
- 109
- Issue
- 43
- Pages
- 17382-17387
- ISSN
- 0027-8424 (ISSN-L)
- 1091-6490
- OpenAlex
- W1976508887
- Cited by
- 580
- References
- 5
- FWCI
- 31.02
- Type
- journal article
- Language
- en
- Open access
- green
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