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目的应用CRISPR/Cas9技术靶向敲除编码小鼠T、B细胞的Rag2基因及编码NK细胞的IL2rg基因,构建T、B细胞及NK细胞联合免疫缺陷小鼠。方法根据Genbank报道的Rag2及IL2rg基因序列,分别针对其外显子设计25 bp左右的sgRNA并进行合成,sgRNA退火后克隆入p X330载体。Rag2-sgRNA、IL2rg-sgRNA及Cas9重组质粒体外转录为mRNA后显微注射入BALB/c小鼠受精卵细胞,受精卵细胞移植到受体动物获得子代小鼠,首建鼠(F0)与野生型小鼠交配获得F1代小鼠,突变的F1代小鼠互交后筛选F2代纯合子小鼠。通过基因测序、流式细胞技术及接种人源性肿瘤细胞系方法检测子代小鼠基因型和表型。结果成功构建了Rag2-sgRNA、IL2rg-sgRNA重组质粒并对其进行了体外转录,mRNA显微注射并移植后获得57只F0小鼠。连续交配后,获得F2代纯合子小鼠。序列分析表明子代小鼠中IL2rg有两个基因型,分别是10 bp和11 bp的缺失突变;而Rag2只有一个基因型,为8 bp的缺失突变。与野生型BALB/c小鼠相比,小鼠外周血中CD3、B220及NKp46阳性细胞数量明显降低。接种人乳腺癌细胞系SKBR-2HL后,肿瘤生长良好,且随着时间延长肿瘤组织逐渐增大。结论利用CRISPR/Cas9技术可有效实现BABL/c小鼠体内Rag2、IL2rg基因突变,并导致小鼠T、B及NK细胞功能异常。
OBJECTIVE: To target knockout of Rag2 gene encoding mouse T and B cells and IL2rg gene encoding NK cells by CRISPR / Cas9 technology to construct T, B cells and NK cells combined immunodeficient mice. Methods According to the sequences of Rag2 and IL2rg reported in Genbank, sgRNAs of about 25 bp were designed and synthesized respectively. The sgRNA was annealed and cloned into pX330 vector. The Rag2-sgRNA, IL2rg-sgRNA and Cas9 recombinant plasmids were transcribed into mRNA in vitro and injected into the fertilized egg of BALB / c mice. The fertilized egg cells were transplanted to recipient animals to obtain offspring mice. The first mouse (F0) and wild type The mice of F1 generation were mated and the F1 generation of mutant mice were screened for F2 generation homozygous mice after reciprocal crossing. The offspring mouse genotypes and phenotypes were examined by gene sequencing, flow cytometry and inoculation of human tumor cell lines. Results The recombinant plasmids of Rag2-sgRNA and IL2rg-sgRNA were successfully constructed and transcribed in vitro. 57 F0 mice were obtained after microinjection and transplantation of mRNA. After continuous mating, F2 generation homozygous mice were obtained. Sequence analysis showed that there are two genotypes of IL2rg in offspring mice, which are 10 bp and 11 bp deletion mutations, while Rag2 has only one genotype and is an 8 bp deletion mutation. Compared with wild-type BALB / c mice, the number of CD3, B220 and NKp46-positive cells in peripheral blood of mice significantly decreased. After inoculating human breast cancer cell line SKBR-2HL, the tumor grew well, and the tumor tissue gradually increased with time. Conclusion The CRISPR / Cas9 technique can effectively induce the Rag2 and IL2rg gene mutations in BABL / c mice and lead to the dysfunction of T, B and NK cells in mice.