Construction of MAVS-/-HeLa cell line and its application in the interferon signaling pathway
[Objective]To construct a HeLa cell line with mitochondrial antiviral signaling(MAVS)gene knockout using CRISPR/Cas9 technology,and to preliminarily identify the growth characteristics of the cell line and its impact on the interferon signaling path-way.[Method]Guide RNA targeting the exons of the MAVS gene was designed and cloned into the LentiCRISPR v2 vector,and then packaged into lentiviruses.HeLa cells were infected with the lentiviruses and selected with puromycin to obtain HeLa cells with MAVS gene knockout.The knockout efficiency was validated using Western Blotting.Cell growth vitality was measured using the MTT assay.The changes of interferon-beta(IFN-β)mRNA levels in cells stimulated with poly(I∶C)were detected by RT-qPCR.[Result]Sequencing results indicated successful construction of the CRISPR-MAVS-gR plasmid,which was packaged into lentivirus and used to infect HeLa cells.Cells that were selected did not express MAVS mRNA and protein.The growth rate and activity of these cells were similar to wild-type HeLa cells.After stimulation with Poly(I∶C),there was no significant change in IFN-β levels within the cells.[Conclusion]The MAVS gene knockout MAVS-/-HeLa cell line was successfully obtained using CRISPR/Cas9 technology.The Poly(I∶C)agonists failed to activate the interferon signaling pathway within these cells.This study lays the foundation for further research on the MAVS-related antiviral innate immune signaling pathway.
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