Construction Of C57 BL/6 Mouse Immunodeficiency Animal Model By CRISPR/Cas9 Technology

With the development of molecular immunology,C57 BL/6 mouse has become a common experimental strain for studying immunology,oncology and genetics.The mouse is easy to reproduce,has a strong physique and has high accuracy of test results.Immunodeficient animals refer to animals with congenital genetic or artificial immunodeficiency.They are commonly used in tumor,pathology,anticancer drugs and gene therapy and are favorable tools for biomedical research.In this study,C57 BL/6 mice immune deficiency animal model was constructed by CRISPR/Cas9 technology,and its immune characteristics were studied to explore the expression rule of immune cells in the animal model.The main contents of this study are as follows:1.Construction of lyst gene deficient C57 BL/6 mouse animal model.lysosomal trafficking regulator gene(lyst)is a pathogenic gene of congenital granulocytic syndrome(Chediak-Higashi syndrome).The clinical manifestations of the diseased organism are including hypopigmentation,immune deficiency,photophobia,mild coagulation disorders,etc.In this study,CRISPR/Cas9 technology was used to design target primers(sgRNA)for exon 50 of lyst gene coding region in C57 BL/6 mice,and pUC57-lyst-sgRNA vector was recombinantly constructed.The recombinant plasmid and Cas9 plasmid were transcribed into mRNA in vitro by T7 RNA polymerase respectively,injected into mouse fertilized eggs,and transplanted into recipient mice.After the offspring mice are obtained,PCR and gene sequencing technologies are used to screen lyst gene edited animal individuals,and biological software is used to judge the deletion,mutation or insertion of C57 BL/6 mice,and flow cytometry is used to detect the immune characteristics of C57BL/6 mice.Mice edited with genomes are inbred in cages according to genotype homology.Finally,lyst gene deficient C57 BL/6 mice were obtained.The results showed that the number of NK cells in lyst gene-deficient C57 BL/6 mice constructed by CRISPR/Cas9 technology was reduced compared with that of wild mice of the same strain,and the color of some defective mice was lighter compared with that of wild mice of the same strain,which was consistent with the literature report.This study will lay a foundation for future humanized tumor research on NK cell immunodeficiency and C57 BL/6 mice model of severe combined immunodeficiency.2.Construction of prkdc gene deficient C57 BL/6 mouse animal model.The dysfunction of DNA-dependent protein kinas(prkdc)can directly affect the expression of T cells and B cells,causing the body to produce severe combined immunodeficiency.In this study,CRISPR/Cas9 technology was used to design target primers(sgRNA)for exon 85 of the coding region of prkdc gene in C57 BL/6 mice,and the pUC57-prkdc-sgRNA vector was recombinantly constructed.The recombinant plasmid and Cas9 plasmid were transcribed into mRNA in vitro by T7 RNA polymerase respectively,injected into mouse fertilized eggs,and transplanted into recipient mice.After the offspring mice were obtained,PCR and gene sequencing techniques were used to screen the animal individuals edited by prkdc gene,and the mice edited by genome were inbred according to genotype homology.Experimental results showed that C57 BL/6 mice with prkdc gene deficiency were finally constructed.Compared with wild mice of the same strain,the number of T and B cells was reduced,and even mice with almost zero T and B cells appeared.The slow growth of gene editing mice was consistent with the literature report.It can be used for humanized tumor research of T cell and B cell immune deficiency,and lays a foundation for gene therapy and C57 BL/6 mouse severe combined immune deficiency animal model.To sum up,the conclusion is as follows: immune deficiency animal models of lyst gene and prkdc gene of C57 BL/6 mice were respectively constructed by CRISPR/Cas9 technology.Finally,we found that the number of immune cells in the mice was reduced.This will lay a foundation for further research on humanized tumors,construction of C57 BL/6 mice model of severe combined immunodeficiency and research on molecular immunity.