CRISPR/Cas9技术,免疫缺陷小鼠 z-bio 2021-06-21 304

　　Purpose: Using CRISPR/Cas9 technology, targeted knockout of Rag2 gene encoding mouse T cells and B cells and IL2rg gene encoding NK cells to construct immunodeficient mice that combine T, B, and NK cells.

　　Method: According to the gene sequences of Rag2 and IL2rg reported by Genbank, sgRNAs of approximately 25bp were designed and synthesized for these exons, and then cloned into the pX330 vector after annealing. The ag2-sgRNA, IL2rg-sgRNA, and Cas9 recombinant plasmids were transcribed into mRNA in vitro, and the fertilized egg cells of BALB/c mice were microinjected, and the fertilized egg cells were transplanted into the recipient animal to establish the first mouse (F0). Wild-type F1 generation mice were obtained through mating, and F2 generation homozygous mice were selected after mating of mutant F1 generation mice. The genotype and phenotype of the offspring mice were detected by gene sequencing, flow cytometry and inoculation of human tumor cell lines.

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

　　Conclusion: The use of CRISPR/Cas9 technology can effectively realize the mutation of Rag2 and IL2rg genes in BABL/c mice, resulting in abnormal functions of mouse T, B, and NK cells.