PD-L1基因敲除小鼠构建及初步表型验证

目的 程序性死亡配体-1(PD-L1)是免疫调节途径的重要因子,是抗肿瘤免疫疗法中重要的靶标之一。利用CRISPR/Cas9技术成功构建PD-L1基因敲除小鼠模型,并初步分析其表型。方法 构建Cas9和sgRNA载体,并转录获得RNA,通过显微注射方式将RNA注射到C57BL/6小鼠受精卵中,经过鉴定获得F₀代阳性小鼠。F₀代小鼠与野生型C57BL/6小鼠交配获得F₁代杂合子小鼠,再通过F₁代小鼠自交获得F₂代纯合子小鼠品系。随后通过Real-Time PCR和流式实验分别检测PD-L1基因在mRNA和蛋白质水平上的表达情况。结果 Real-Time PCR和流式实验检测结果显示与野生型C57小鼠相比,PD-L1纯合子小鼠的PD-L1 mRNA相对表达水平和细胞上的蛋白质表达均有显著性下降,仅测定到本底的信号,证实已成功构建PD-L1基因敲除小鼠品系,为PD-L1体内基因功能研究提供了新的小鼠模型。

Construction and Preliminary Phenotypic Verification of PD-L1 Knockout Mice

Objective: Programmed death ligand-1 (PD-L1) is an important factor in the immunoregulatory pathway and is one of the important targets in anti-tumor immunotherapy. The PD-L1 knockout mouse model was successfully constructed using CRISPR/Cas9 technology, and its phenotype was initially analyzed.Methods: Cas9 and sgRNA vectors were constructed and transcribed to obtain RNA. RNA was injected into C57BL/6 mouse fertilized eggs by microinjection, and F₀ generation positive mice were obtained. F₀ generation mice were mated with wild type C57BL/6 mice to obtain F₁ generation heterozygous mice, and F₂ generation homozygous mouse strains were obtained by self-crossing of F₁ generation mice. Subsequently, the expression of PD-L1 gene at the mRNA and protein levels was detected by Real-Time PCR and flow cytometry, respectively.Results: Real-Time PCR and flow cytometry showed that the PD-L1 mRNA expression level and protein expression on PD-L1 homozygous mice with only background signals were significantly decreased, compared with wild-type C57 mice. It was confirmed that the PD-L1 knockout mouse strain was successfully constructed, which provided a new mouse model for PD-L1 gene function research in vivo.