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Homologous recombination contributes to the repair of zinc-finger-nuclease induced double strand breaks in pig primary cells and facilitates recombination with exogenous DNA

Paper ID Volume ID Publish Year Pages File Format Full-Text
23224 43421 2014 8 PDF Available
Title
Homologous recombination contributes to the repair of zinc-finger-nuclease induced double strand breaks in pig primary cells and facilitates recombination with exogenous DNA
Abstract

•Nucleases support homologous recombination in somatic cells.•Homologous recombination contributes to DNA repair in somatic cells.•Nuclease treated primary cells develop normally in vitro after nuclear transfer.

Site-specific nucleases have become powerful tools for genome editing by the introduction of end-joining-mediated mutations, but it is unclear to which extent induced double strand breaks will also facilitate homologous recombination with exogenous DNA. This question is, however, of particular importance for somatic cells, which have to be modified for the generation of large animal models, but, on the other hand, have also been described to be reluctant to recombination-based DNA repair.Here, we examined zinc-finger nucleases for their potential to introduce modifications in pig somatic cells via end-joining or recombination. We found that co-transfection with nuclease-encoding plasmids resulted in a dramatic boost of recombination with different targeting vectors, suggesting a much more prominent role of this repair pathway in somatic cells than was previously thought. Although recombination with any of the vectors even occurred on both alleles of the target gene, we found also evidence for distinct properties of the used vectors regarding their preference for mono-allelic or bi-allelic modification.Thus, we show that the combined usage of site-specific nucleases and targeting vectors does not only promote homologous recombination in somatic cells but might also resemble a promising tool for detailed examination of DNA repair pathways.

Keywords
BAC, bacterial artificial chromosome; DSB, double strand break; HR, homologous recombination; PKC, porcine kidney cells; SCNT, somatic cell nuclear transfer; ZFN, zinc-finger-nucleaseSite-specific nucleases; Double strand break repair; Homologous recombin
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Homologous recombination contributes to the repair of zinc-finger-nuclease induced double strand breaks in pig primary cells and facilitates recombination with exogenous DNA
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 177, 10 May 2014, Pages 74–81
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us