title: [Construction of Rev-erbβ gene knockout HEK293 cell line with CRISPR/Cas9 system].
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.
authors: Gene Therapy Laboratory, College of Life Sciences, Shaanxi Normal University, Xi'an 710062, China; School of Modern Agriculture and Biotechnology, Ankang University, Ankang 725000, China.
authors: Fang
authors: F
authors: Chen
authors: Gene Therapy Laboratory, College of Life Sciences, Shaanxi Normal University, Xi'an 710062, China.
authors: Weifeng
authors: W
authors: Zhang
authors: Gene Therapy Laboratory, College of Life Sciences, Shaanxi Normal University, Xi'an 710062, China.
authors: Junli
authors: J
authors: Zhao
authors: Gene Therapy Laboratory, College of Life Sciences, Shaanxi Normal University, Xi'an 710062, China.
authors: Peiyan
authors: P
authors: Yang
authors: Gene Therapy Laboratory, College of Life Sciences, Shaanxi Normal University, Xi'an 710062, China.
authors: Rui
authors: R
authors: Ma
authors: Gene Therapy Laboratory, College of Life Sciences, Shaanxi Normal University, Xi'an 710062, China. *Corresponding author, E-mail: hbxia2001@163.com.
authors: Haibin
authors: H
authors: Xia
mesh_terms: D015153:Blotting, Western; D064113:CRISPR-Cas Systems / Q000235:genetics*; D055786:Gene Knockout Techniques / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D016133:Polymerase Chain Reaction; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: C090977:NR1D2 protein, human; D000094704:RNA, Guide, CRISPR-Cas Systems; D018160:Receptors, Cytoplasmic and Nuclear; D012097:Repressor Proteins