Generation of porcine fetal fibroblasts expressing the tetracycline-inducible Cas9 gene by somatic cell nuclear transfer

Liu,Guoqian; Liu,Kai; Wei,Hengxi; Li,Li; Zhang,Shouquan

doi:10.3892/mmr.2016.5530

Generation of porcine fetal fibroblasts expressing the tetracycline-inducible Cas9 gene by somatic cell nuclear transfer

Authors:
- Guoqian Liu
- Kai Liu
- Hengxi Wei
- Li Li
- Shouquan Zhang
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Affiliations: Guangdong Provincial Key Lab of Agro‑Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong 510642, P.R. China
Published online on: July 19, 2016 https://doi.org/10.3892/mmr.2016.5530
Pages: 2527-2533
Copyright: © Liu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Cas9 endonuclease, from so-called clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems of Streptococcus pyogenes, type II functions as an RNA-guided endonuclease and edits the genomes of prokaryotic and eukaryotic organisms, including deletion and insertion by DNA double‑stranded break repair mechanisms. In previous studies, it was observed that Cas9, with a genome‑scale lentiviral single‑guide RNA library, could be applied to a loss‑of‑function genetic screen, although the loss‑of‑function genes have yet to be verified in vitro and this approach has not been used in porcine cells. Based on these observations, lentiviral Cas9 was used to infect porcine primary fibroblasts to achieve cell colonies carrying Cas9 endonuclease. Subsequently, porcine fetal fibroblasts expressing the tetracycline‑inducible Cas9 gene were generated by somatic cell nuclear transfer, and three 30 day transgenic porcine fetal fibroblasts (PFFs) were obtained. Polymerase chain reaction (PCR), reverse transcription‑PCR and western blot analysis indicated that the PFFs were Cas9‑positive. In addition, one of the three integrations was located near to known functional genes in the PFF1 cell line, whereas neither of the integrations was located in the PFF1 or PFF2 cell lines. It was hypothesized that these transgenic PFFs may be useful for conditional genomic editing in pigs, and for generating ideal modified porcine models.

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