Establishment of a Caco-2 cell line stably expressing Cas9 protein based on lentiviral packaging
10.13200/j.cnki.cjb.004252
- VernacularTitle:基于慢病毒包装构建稳定表达Cas9蛋白的Caco-2细胞系
- Author:
WANG Mingyue
- Publication Type:Journal Article
- Keywords:
Cas9 protein;
Caco-2 cell line;
Gene knockout;
Clustered regulatory interspaced short palindromic repeats(CRISPR)
- From:
Chinese Journal of Biologicals
2025;38(1):8-13+21
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a gene knockout cell library based on clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9(CRISPR/Cas9) system, and construct a cell line stably expressing Cas9 protein using human colon adenocarcinoma cells Caco-2. Methods Caco-2 cells were infected with packaged recombinant Cas9 lentivirus, and then Caco-2/Cas9 monoclonal cells were selected by medium containing blasticidin and twice limited dilution sorting,and identified by PCR and Western blot. Caco-2/Cas9 monoclonal cells were infected with lentivirus expressing both GFP gene and single guide RNA(sgRNA) targeting GFP gene. After puromycin screening, the knockout efficiency of Caco-2 cell line was calculated by flow cytometry, and the proliferation activity was detected by CCK-8 assay. Results Five Caco-2/Cas9 monoclonal cell lines, named Caco-2/Cas9-2, Caco-2/Cas9-3, Caco-2/Cas9-4, Caco-2/Cas9-5 and Caco-2/Cas9-6, were obtained, all of which were amplified for a 392 bp band of Cas9 gene. Cas9 protein was stably expressed in the cells and the knockout efficiency was 91. 27%, 20. 30%, 24. 13%, 11. 33%, 12. 27% and 8. 89%, respectively. The proliferative activity of Caco-2/Cas9-4, Caco-2/Cas9-5, Caco-2/Cas9-6 and uninfected Caco-2 cells was 2. 07, 1. 75, 1. 46 and 1. 40, respectively,with no significant difference between Caco-2/Cas9-5, Caco-2/Cas9-6 and uninfected Caco-2 cells(t = 1. 92 and 0. 37, each P > 0. 05). Conclusion A Caco-2/Cas9 monoclonal cell line, Caco-2/Cas9-6 monoclonal cells with high enzyme digestion activity of Cas9 and no significant change in proliferation activity compared with uninfected Caco-2 cells was screened out,which lays a foundation for further construction of high coverage knockout cell library and provides a platform for screening genes related to virus infection and genes with other specific functions.
