Generation of porcine fetal fibroblasts expressing the tetracycline-inducible Cas9 gene by somatic cell nuclear transfer
| Autor: | Hengxi Wei, Guoqian Liu, Shouquan Zhang, Kai Liu, Li Li |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
pig
0301 basic medicine Nuclear Transfer Techniques Cancer Research Swine CRISPR-Associated Proteins Cell Gene Expression Mutagenesis (molecular biology technique) cell lines Biology somatic cell nuclear transfer Biochemistry 03 medical and health sciences 0302 clinical medicine Gene expression Genetics medicine Animals CRISPR Clustered Regularly Interspaced Short Palindromic Repeats Transgenes Molecular Biology Gene Cas9 Gene targeting Articles Sequence Analysis DNA Fibroblasts Molecular biology Mutagenesis Insertional 030104 developmental biology medicine.anatomical_structure Gene Expression Regulation Oncology Tetracyclines Gene Targeting Molecular Medicine Somatic cell nuclear transfer CRISPR-Cas Systems cas9 endonuclease 030217 neurology & neurosurgery |
| Zdroj: | Molecular Medicine Reports |
| ISSN: | 1791-3004 1791-2997 |
| DOI: | 10.3892/mmr.2016.5530 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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