[Construction of Rev-erbβ gene knockout HEK293 cell line with CRISPR/Cas9 system]

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2016 Nov;32(11):1446-1452.
[Article in Chinese]

Abstract

Objective 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.

MeSH terms

  • Blotting, Western
  • CRISPR-Cas Systems / genetics*
  • Gene Knockout Techniques / methods*
  • HEK293 Cells
  • Humans
  • Polymerase Chain Reaction
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • Receptors, Cytoplasmic and Nuclear / genetics
  • Receptors, Cytoplasmic and Nuclear / metabolism
  • Repressor Proteins / genetics
  • Repressor Proteins / metabolism

Substances

  • NR1D2 protein, human
  • RNA, Guide, CRISPR-Cas Systems
  • Receptors, Cytoplasmic and Nuclear
  • Repressor Proteins