Fang Chen, Weifeng Zhang, Junli Zhao, Peiyan Yang, Rui Ma, Haibin Xia
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 2016 NovObjective To prepare Rev-erbβ knockout HEK293 cells using clustered regularly interspaced short palindromic repeats/Cas 9 nuclease (CRISPR/Cas9) gene editing technology. Methods The knock-in or knockout of Rev-erbβ gene could be realized by single-guide RNA (sgRNA)-mediated Cas9 cutting of target DNA, and followed by DNA homologous recombination or non-homologous end joining-mediated DNA repair. Firstly, four sgRNAs were designed for Rev-erbβ gene. The sgRNA1 and sgRNA2 with the higher activity were respectively used to construct pCMV-hCas9-U6-Rev-erbβ sgRNA1 and pCMV-hCas9-U6-Rev-erbβ sgRNA2. Then, pCMV-hCas9-U6-Rev-erbβ sgRNA1, pCMV-hCas9-U6-Rev-erbβ sgRNA2 and pAd5-E1/hRev-erbβ donor plasmid vectors were co-transfected into HEK293 cells. Through drug screening, cloning and sequencing, the Rev-erbβ gene-knockout HEK293 (Rev-erbβ-/-) cell lines were obtained with one chain integrated with exogenous gene fragment and the other chain for deletion mutants. Finally, the HEK293 (Rev-erbβ-/-) cell lines (C3-6) was detected with real-time quantitative PCR and Western blotting. Results Expression of Rev-erbβ mRNA and protein was undetectable in HEK293 Rev-erbβ-/- cell line. Conclusion Using CRISPR/Cas9 technology, the HEK293 Rev-erbβ-/- cell line has been successfully constructed, which would provide an effective tool for the study on the function of Rev-erbβ.
Fang Chen, Weifeng Zhang, Junli Zhao, Peiyan Yang, Rui Ma, Haibin Xia. Construction of Rev-erbβ gene knockout HEK293 cell line with CRISPR/Cas9 system]. Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology. 2016 Nov;32(11):1446-1452
PMID: 27774932
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