β2m基因敲除小鼠构建  

Construction ofβ2m knockout mice

作　　者：李振 孙瑞林 刘雯 Zhen Li;Ruilin Sun;Wen Liu(Department of Cell and Genetic Medicine,School of Basic Medical Sciences,Fudan University,Shanghai 200300,China;Shanghai Model Organisms Center,Shanghai 201318,China)

机构地区：[1]复旦大学基础医学院细胞与遗传医学系,上海200300 [2]上海南方模式生物科技股份有限公司,上海201318

出　　处：《生物工程学报》2021年第8期2924-2935,共12页Chinese Journal of Biotechnology

摘　　要：2m(Beta-2-microglobulin)基因编码一个非糖基化蛋白,作为主要组织相容性复合体1类(MHCⅠ)的重要组分,发挥抗原递呈的作用。为了避免免疫介导的清除,人类肿瘤和病原体采取了不同的策略,其中包括MHCⅠ表达的丢失。合适的动物模型对于评估和开发肿瘤及其他疾病的临床治疗新方法,以及阐明目前临床有效治疗方法的机制至关重要。利用CRISPR/Cas9基因编辑、显微注射等方法构建了β2m基因敲除小鼠。随后,通过PCR鉴定、qPCR、流式分析等实验技术进行基因型和表型鉴定。基因型鉴定结果显示在该品系小鼠中,目的基因编码区目标区域缺失。qPCR检测发现,β2m的mRNA水平发生显著的下调。流式结果显示,在不同的免疫组织和器官中,CD8^(+)杀伤性T细胞显著减少。综上,成功构建β2m基因敲除小鼠,为后续体内研究β2m基因的功能奠定了基础。Theβ2m(Beta-2-microglobin)gene encodes a non-glycosylated protein that functions as an important component of major histocompatibility complexⅠ(MHCⅠ)for antigen presentation.To evade immune mediated clearance,human tumors and pathogens have adopted different strategies,including loss of MHCⅠexpression.Appropriate animal models are essential for understanding the mechanisms underpinning the clinical treatment of tumor and other human diseases.We constructedβ2m knockout mice using CRISPR/Cas9 gene editing tool through embryo microinjection.Subsequently,genotyping and phenotyping of knockout mice were performed by PCR,qPCR,and flow cytometry.Mice genotyping showed that the coding region of the target gene was absent in the knockout mice.Real time PCR showed that mRNA level ofβ2m was significantly downregulated.Flow cytometry showed that the proportions of CD8^(+)killer T cells was significantly reduced in a variety of tissues and organs of the immune system.Taken together,we have successfully constructed a strain ofβ2m knockout mice,which will facilitate subsequent in vivo study on the function and mechanism of theβ2m gene.

关 键 词：主要组织相容性复合物 Β2M 基因敲除小鼠 CRISPR/Cas9 

分 类 号：R-332[医药卫生]