Establishment of Quaking Knockout Mouse Embryonic Fibroblast Cell Line Using CRISPR/Cas9 Technology
[Objective]CRISPR/Cas9 technology was used to generate a mouse embryonic fibroblast cell line(NIH3T3)with a knockout of the Quaking gene and to investigate its impact on NIH3T3 cell proliferation.[Method]Initially,two sgRNAs targeting Quaking exons were designed using an online platform,and two CRISPR/Cas9 recombinant lentiviral plasmids targeting the first and second exons of the Quaking gene were constructed successfully.These constructs with pcDNA3.1-Quaking overexpression plasmids were co-transfected into HEK293T cells,and the knockout efficiency of Quaking protein was assessed through Western blot analysis.Subsequently,the recombinant lentiviral plasmid(LentiCRISPRv2-sgRNA1)with high knockout efficiency was co-transfected with auxiliary packaging plasmids into HEK293T cells for lentivirus packaging.After lentiviral transduction of NIH3T3 cells,positive monoclonal cell lines were selected using puromycin.Finally,we confirmed the knockout effect through Western blot and immunofluorescence staining,demonstrating the absence of Quaking protein in these cells.[Result]Sequencing confirmed the occurrence of a targeted gene segment deletion.CCK8 assays revealed that Quaking gene knockout significantly inhibited NIH3T3 cell proliferation.[Conclusion]This study represents the first successful utilization of CRISPR/Cas9 technology to establish a Quaking gene knockout cell line in mouse embryonic fibroblast cells(NIH3T3),providing a valuable in vitro model for exploring the mechanistic role of the Quaking gene in the regulation of mouse physiological functions.
国家科技期刊平台
NSTL
万方数据
