动物造模,CRISPR/Cas9技术 z-bio 2022-06-08 350

　　Objective: To use CRISPR/Cas9 technology to target knockout of Rag2 gene encoding mouse T and B cells and IL2rg gene encoding NK cells, and to construct T, B cells and NK cells combined immunodeficiency mice.

　　Methods: According to the Rag2 and IL2rg gene sequences reported by Genbank, sgRNAs of about 25 bp were designed and synthesized for their exons respectively. The sgRNAs were annealed and cloned into the pX330 vector. Rag2-sgRNA, IL2rg-sgRNA and Cas9 recombinant plasmids were transcribed into mRNA in vitro, then microinjected into fertilized egg cells of BALB/c mice, and the fertilized egg cells were transplanted into recipient animals to obtain offspring mice. Mice were bred to obtain F1 generation mice, and the mutant F1 generation mice were crossed to screen F2 generation homozygous mice. Genotype and phenotype of offspring mice were detected by gene sequencing, flow cytometry and inoculation with human tumor cell lines.

　　RESULTS: Rag2-sgRNA and IL2rg-sgRNA recombinant plasmids were successfully constructed and transcribed in vitro. 57 F0 mice were obtained after mRNA microinjection and transplantation. After serial mating, F2 generation homozygous mice were obtained. Sequence analysis showed that there were two genotypes of IL2rg in offspring mice, which were 10 bp and 11 bp deletion mutations, respectively; while Rag2 had only one genotype, which was 8 bp deletion mutations. Compared with wild-type BALB/c mice, the numbers of CD3, B220 and NKp46 positive cells in the peripheral blood of mice were significantly reduced. After inoculation with human breast cancer cell line SKBR-2HL, the tumor grew well, and the tumor tissue gradually increased with time.

　　Conclusion: The use of CRISPR/Cas9 technology can effectively achieve Rag2 and IL2rg gene mutations in BABL/c mice, and lead to abnormal T, B and NK cell functions in mice.