Constructing PLC?1(S345F)Gene Point Mutation Mice And Sik1 Gene Knockout Mice With CRISPR/Cas9 Gene-editing Technology

Objective:In the study of patients with Cutaneous T cell lymphoma?CTCL?,Plcg1was found to be mutated in the genomic DNA of three patients,two of which?c.1034T>C,S345F?had a redundant mutation in exon eleven that further affected the catalytic domain of PLC?protein.To correlation between further investigate the relation of the point mutation of PLC?1 and the pathogenesis of CTCL,we generate PLC?1?S345F?gene point mutant mice and preliminarily analyzed the phenotype of the immune cells in these mice.Further characterization of its role in the pathogenesis of CTCL will be carried out.We found enhanced expression of Sik1 gene in the analysis of gene differential expression of germinal center B cells compared with na?ve B cells.However,the role of Sik1 gene in the development of immune cells was not yet clear,so we planned to prepare Sik1 gene knockout mice to promote the study on the role of Sik1 gene in immune system.Methods:CRISPR/Cas9 gene-editing technology,with its advantages of simple design and high efficiency,has been widely used.We designed sgRNA targeting mouse Plcg1 gene and Sik1 for pronuclear microinjections.Then we sequenced the regions of genes of interest to verify the mutation/deletion.We then carried out flow cytometry an analyze the development of each lineage of lymphocytes and their subpopulations from PLC?1?S345F?point mutation mice and wild-type mice.Results:PLC?1?S345F?heterozygous mice were confirmed by PCR sequencing in F0 generation of mice after microinjection.After breeding of two generations,we successfully established PLC?1^S345F/+and PLC?1^S345F/S345F mouse.Flow cytometry analysis showed that there was no significant change in the proportion of the lymphocytes and their subpopulations in PLC?1?S345F?point mutation mice compared with wild-type mice.Sik1gene knockout mice were also successfully verified in vitro,currently we have microinjected the gRNA with the most efficiency into murine embryonic cells.Conclusion:PLC?1?S345F?gene point mutation has no significant effect on B or T lymphocyte development in mice,Further investigation of its role in the pathogenesis of CTCL is needed in an appropriate mouse model.This study aims to elucidate the molecular mechanisms of PLC?1?S345F?in the normal development of T cells and its potential role in the carcinogenesis of T cell lymphoma.