Generation of RNase L knockout cell lines by CRISPR/Cas system
10.7644/j.issn.1674-9960.2015.10.003
- VernacularTitle:利用CRISPR／Cas方法建立RNase L基因稳定敲除细胞株
- Author:
Ruihua LI
;
Hanjiang FU
;
Yiran ZHONG
;
Yuan SHEN
;
Jie ZHU
;
Xiaofei ZHENG
- Publication Type:Journal Article
- Keywords:
CRISPR/Cas system;
RNase L;
mice,gene knockout;
homologous recombination;
endoribonucleases;
transfection;
hygromycin B
- From:
Military Medical Sciences
2015;(10):742-746
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish RNase L gene knockout HEK 293 cell lines using CRISPR/Cas9 system.Methods Small guide RNA ( sgRNA) sequences of human RNase L were designed and sgRNAs were inserted into pCas-Guide and pCas-guide RNA(gRNA) vectors were obtained.The donor DNA sequences of the homologous arm were designed for RNase L knockout .In the presence of the right homologous arm , the resistance gene of hygromycin B and the left homologous arm as templates of homology-directed repair , the donor DNA template was amplified by overlopping PCR and cloned into the pBackZero-T expression vector and pBackZero-T-RNase LK vector was obtained .The pCas-gRNA vector and pBackZero-T-RNase LK vector were co-transfected into HEK293 cells to establish the stable expression cell line of RNase L gene knockout .Cells were cultured with hygromycin B , while Western blotting and DNA sequencing were used to analyze the gene of RNase L knockout from genome .Results and Conclusion The pCas-gRNA vector and pBackZero-T-RNase LK vector were successfully constructed.Five RNase L gene knockout HEK293 cell lines were generated,contributing to the study of the biological function and molecular mechanism of RNase L .
