Construction Of Human MPZL1 Gene Knockout SPC-A1 Stable Strain By CRISPR/Cas9 Technology

PZR(Protein zero related)is also named as protein related to myelin P0 protein,which was encoded by MPZL1 and located in human autosomal 1q24.1-24.2.It is a highly conserved protein and expressed in a lot of cell types.As a specific anchoring protein and physiological substract of SHP-2,PZR may play an important role in some SHP-2 signaling pathways.Research shows that PZR will affect cell migration and adhesion,and have found abnormal expression of PZR in NS/LS,HCC,BCa(HER2+)and other diseases.Reports on the biological function of PZR and its disease-related research are still limited,it is needed further work.In this study we constructed MPZL1(PZR)gene knockout stable cell lines in human lung adenocarcinoma cell SPC-A1 based on CRISPR/Cas9 gene editing technique.Firstly,according to the target design rules of CRISPR/Cas9 we designed two sgRNAs that specifically recognize the second exon-related sequence of MPZL1 gene.Next we constructed the recombinant eukaryotic expression plasmids by connected the two sgRNAs with PX458 and PX459.We have screend MPZL1 gene knockout SPC-A1 stable cells by Puromycin after co-transfected the two recombinant plasmids into SPC-A1 cells.Finally,the knockout result of MPZL1 gene in SPC-A1 cells was identified by PCR,gene sequencing and Western Blot.The results shows that we successfully constructed stable cell lines of SPC-A1 with MPZL1 gene knockout.In addition,CCK-8,cells in situ counting experiments,cell scratch test and suspension droplet aggregation experiment showed that migration ability of SPC-A1 cells was significantly reduced after the deletion of MPZL1 gene but MPZL1 gene didn’t have significantly effect on proliferation ability and cell-cell adhesion.In conclusion,we have successfully established a stable cell line SPC-A1 with MPZL1 gene knockout,which provides a gene knockout model for the future study of the biological function of MPZL1.And it laid a research foundation for the occurrence,development and treatment of lung cancer.