Construction and application of high-throughput drug screening cell model based on Oct4 gene promoter activity
Objective A dual luciferase reporter gene vector was constructed by targeting the human Oct4 gene promoter,and a high-throughput drug screening cell model was established to screen marine-derived microbial secondary metabolites that could significantly upregulate the promoter activity of Oct4 gene.Methods The human Oct4 gene promoter sequence was cloned into the firefly luciferase reporter gene vector pGL6-TA,and the recombinant plasmid pGL6-TA-Oct4-luc was constructed and transiently co-transfected with the internal reference plasmid pRL-SV40-C into 293T cells,with OAC1 as the activator,a dual-luciferase reporter gene screening system was constructed,the activity of Oct4 gene promoter was reflected by detecting changes in relative luciferase expression level,and 309 samples of marine-derived microbial fermentation crude extracts were screened for activity;effect of crude extracts on Mesenchymal stem cells(MSCs)viability was measured using CCK-8 assay.Results The recombinant plasmid were successfully constructed by double enzyme digestion and gene sequencing.After the plasmids were transfected into 293T cells,the pGL6-TA-Oct4-luc recombinant plasmid had significant promoter activity compared with pGL6-TA vector(P＜0.05).Compared with 0 μmol/L,the activity of Oct4 gene promoter was significantly increased after 24 h of crude extract Z-179-1 treated cells(P＜0.05),and the effect is most significant at concentration of 10 μg/mL.CCK-8 assay showed that Z-179-1 significantly promoted the proliferation of Mesenchymal stem cells(MSCs)in a dose-dependent manner.Conclusion A high-throughput drug screening cell model targeting human Oct4 gene promoter was successfully constructed,and the marine-derived microbial fermentation crude extract Z-179-1 was screened to significantly activate the activity of Oct4 promoter,and its active components deserved further chemical research.
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