Pd-1基因敲除小鼠构建及初步表型验证

目的:细胞程序性死亡蛋白(programmed death ligand-1,PD-1)是机体T细胞的免疫检查点,也是肿瘤治疗的重要靶点。采用CRISPR/Cas9技术,利用非同源重组修复引入突变的方式,使基因蛋白读码框移码造成PD-1功能缺失,建立Pd-1基因敲除小鼠模型,为深入探究Pd-1基因功能及作用机制提供基础。方法:针对Pd-1基因2-4号外显子设计并合成2对sgRNA片段,与编码Cas9片段共同体外转录,通过受精卵显微注射方法将两者mRNA混合注射到C57BL/6小鼠受精卵中,经PCR产物测序鉴定获得F0代小鼠,之后与野生型C57BL/6小鼠交配获得F1代杂合子小鼠,F1代小鼠自交即获得F2代纯合子小鼠品系(Pd-1^-/-)。刀豆蛋白(concanavalin A,ConA)刺激Pd-1^-/-小鼠后,通过实时荧光定量PCR和流式细胞技术在mRNA和蛋白水平上分别检测Pd-1^-/-小鼠中Pd-1基因在转录和翻译过程中的表达情况,并通过ELISA方法检测Pd-1^-/-小鼠血清中IL-6、IFN-γ、IL12/IL23及TNF-α等因子的表达水平,初步分析Pd-1通路在T细胞反应调控中的作用机制及对免疫刺激的响应情况。结果:PCR及测序结果表明在小鼠基因组中Pd-1基因2-4号外显子被成功敲除;Real-Time PCR实验和流式检测结果显示:与野生型小鼠相比,Pd-1^-/-小鼠脾、肠系膜淋巴结、胸腺和血液各组织中Pd-1表达水平均显著降低;双抗夹心ELISA测定结果显示:Pd-1敲除后经ConA刺激,血清中IL-6和IFN-γ表达上调。结论:成功构建Pd-1基因敲除小鼠模型。Pd-1缺失能够上调IL-6和IFN-γ对ConA刺激的响应,增加ConA引起的炎症反应,为Pd-1的体内基因功能研究提供了新的小鼠模型和研究思路。

Pd-1 Gene Knockout Mouse Model Construction and Preliminary Phenotype Verification

Objective: Programmed cell death protein (PD-1) is a T cell immune checkpoint and an important target for tumor therapy. This article used CRISPR/Cas9 technology to repair the introduced mutations by non-homologous recombination, causing the frame shift of the gene protein reading frame and the loss of PD-1 function. Estabilishment of Pd-1 gene knockout mouse model provides the basis for in-depth exploration of Pd-1 gene function and mechanism. Methods: We designed and synthesized 2 pairs of sgRNA fragments for exons 2-4 of the Pd-1 gene, and transcribed them in vitro together with the Cas9 fragments encoding them. The two mRNAs were mixed into C57BL/6 mouse fertilized eggs by microinjection. F0 generation mice were obtained by PCR product sequencing and then mated with wild-type C57BL/6 mice to obtain F1 generation heterozygous mice. F1 generation mice were intercoursed to obtain F2 generation homozygous mouse strains (Pd-1^-/-). After it was stimulated with concanavalin (ConA), PD-1 in Pd-1^-/- mice was detected by Real-Time fluorescent quantitative PCR and flow cytometry at the mRNA and protein levels, respectively. The expression levels of IL-6, IFN-γ, IL12/IL23 and TNF-α in the serum of Pd-1^-/- mice were detected by the ELISA method, and the mechanism of Pd-1 pathway in the regulation of T cell response and its countermeasures were preliminarily analyzed. Results: PCR and sequencing results showed that exons 2-4 of the Pd-1 gene in the mouse genome were successfully knocked out; Real-Time PCR experiments and flow cytometry results showed that the expression of PD-1 was significantly reduced in Pd-1^-/- spleen, mesenteric lymph nodes, thymus and blood tissues compared with wild-type mice; the double-antibody sandwich ELISA test results showed that the expression of serum IL-6 and IFN-γ is up-regulated stimulated by ConA after Pd-1 gene was knocked out. Conclusion: The Pd-1 gene knockout mouse model has been successfully constructed. Preliminary analysis shows that Pd-1 deletion can upregulate the response of IL-6 and IFN-γ to ConA stimulation, increase the inflammatory response caused by ConA, and provide a new mouse model for the study of Pd-1 in vivo gene function and research ideas.