Construction of HEK293 cell line with apoptotic protease activating factor 1 knockout and its effect on recombinant protein expression
Objective To construct an HEK293 stable cell line with apoptotic protease activating factor 1(Apaf1)knockout and detect its effects on the expression of recombinant proteins in the cells,so as to provide an effective cell model for further exploring the function and mechanism of Apaf1 gene.Methods The recombinant plasmid with Apaf1 gene knockout was constructed using Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated systems(CRISPR/Cas)gene editing system and transfected into HEK293 cells with liposomes.Apaf1 gene deficient cells were screened by puromycin,and then the monoclonal cells were obtained by limited dilution method and sequenced.The relative expression levels of mRNA and protein of Apaf1 in the HEK293 monoclonal cell line(HEK293-Apaf1-KO group)and wild type HEK293 cell line(HEK293-WT group)were detected by RT-qPCR and Western blot respectively.The effects of Apaf1 knockout on the prolifera-tion and apoptosis of HEK293 cells were measured by CCK-8 and flow cytometry respectively.Moreover,the effects of Apaf1 knockout on transient transfection of secreted alkaline phosphatase(SEAP)and stable transfection of vitronectin(VN)plasmids into HEK293 cells were detected by flow cytometry and Western blot respectively.Results A HEK293 monoclonal cell line with Apaf1 knockout,1-38,was obtained by screening and identification.Compared with HEK293-WT group,the relative expression levels of mRNA and protein of Apaf1 in HEK293-Apaf1-KO group decreased significantly(t=73.98 and 75.57,respectively,each P＜0.000 1),the cell proliferation increased significantly(t=3.634,P＜0.01),the apoptosis level showed no significant difference(t=0.162 1,P＞0.05),and the expression of SEAP and VN proteins increased by 2.5 and 1.8 folds(t=-51.199 and 2.47,respectively,each P＜0.01).Conclusion The constructed Apaf1 knockout HEK293 cell line can increase the expression levels of SEAP and VN proteins,and is expected to be applied to the production of recombinant protein drugs.
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