载脂蛋白E和巨噬细胞条件性PDHA1双基因敲除小鼠模型的构建和鉴定

Construction and Identification of Apolipoprotein E and Macrophage Monditional PDHA1 Double Gene Knockout Mice Model

目的构建并鉴定Cre-Loxp重组酶系统调控的PDHA1巨噬细胞条件性敲除小鼠和载脂蛋白E(ApoE)基因敲除小鼠杂交的双基因敲除小鼠模型。方法利用Cre-LoxP重组酶系统理论,将PDHA1f/w小鼠与Lyz2-Cre^(+)小鼠进行F1代自然交配,子代产生的PDHA1f/w-Lyz2-Cre^(+)小鼠进行F2代自然交配,产生F3代小鼠PDHA1^(f/f)-Lyz2-Cre^(+)即为PDHA1巨噬细胞条件性敲除小鼠模型,提取小鼠鼠尾组织基因组DNA,经PCR扩增及琼脂糖凝胶电泳后,以DNA水平判断小鼠基因型。取肝脏、脾脏组织利用q-PCR和Western blot技术检测PDHA1基因的敲除效果;将PDHA1^(f/f)-Lyz2-Cre^(+)与ApoE^(-/-)小鼠杂交,经数代杂交筛选得到PDHA1巨噬细胞条件性和ApoE双基因敲除小鼠(PDHA1^(f/f)-Lyz2-Cre^(+)-ApoE^(-/-))和对照小鼠(PDHA1^(f/f)-Lyz2-Cre--ApoE^(-/-))并进行基因型鉴定,取肝脏、脾脏组织利用q-PCR和Western blot技术检测巨噬细胞PDHA1基因和ApoE双基因的敲除效果。结果实时定量PCR和免疫印迹结果均显示,PDHA1的mRNA水平下调、蛋白表达降低(P均<0.05)。最终获得PDHA1巨噬细胞条件性敲除小鼠PDHA1^(f/f)-Lyz2-Cre^(+)以及PDHA1巨噬细胞条件性和ApoE双基因敲除小鼠(PDHA1^(f/f)-Lyz2-Cre^(+)-ApoE^(-/-)),两种类型基因敲除小鼠出生符合孟德尔遗传规律,存活并可育。结论本实验利用Cre-Loxp系统,成功构建并鉴定PDHA1巨噬细胞条件性敲除小鼠和ApoE基因敲除小鼠杂交的双基因敲除小鼠模型,为研究PDHA1在动脉粥样硬化疾病发病机制中的作用提供实验动物。

Objective To construct and identify the double gene knockout mouse model of PDHA1 macrophage conditional knockout mice and apolipoprotein E(ApoE)gene knockout mice regulated by Cre-LoxP recombinase system.Methods The method was based on the theory of Cre-LoxP recombinase system,PDHA1_(f/w) mice and Lyz2-Cre^(+)mice were naturally copulated by F1 generation.The PDHA1_(f/w)-Lyz2-Cre^(+)mice produced by the offspring were naturally mating with the F2 generation,and the F3 generation mice PDHA1^(f/f)-Lyz2-Cre^(+)was produced.It was PDHA1 macrophage conditional knockout mouse model,and the genomic DNA of mouse tail tissue was extracted,after the amplification of the PCR and agarose gel electrophoresis.The DNA level was used to determine the genotype of mice,and the knockout effect of PDHA1 gene was detected by sequencing and Western blot;Pdha1_(f/f)-lyz2 Cre^(+)was hybridized with ApoE^(-/-)mice.The conditioned and ApoE knockout mice(PDHA1^(f/f)-Lyz2-Cre^(+)-ApoE^(-/-))and control mice(PDHA1^(f/f)-Lyz2-Cre^(+)-ApoE^(-/-))were extracted and identified by genotyping,and then the liver was selected.The knockout effect of PDHA1 gene and ApoE gene in macrophages was detected by gene sequencing and Western blot in spleen.Results The results of real-time quantitative PCR and Western blot showed that the mRNA level of PDHA1 was significantly down regulated,and the protein expression was decreased(P all<0.05),finally obtain PDHA1^(f/f)-Lyz2-Cre^(+)and PDHA1 macrophage conditioned and ApoE gene knockout mice(PDHA1^(f/f)-Lyz2-Cre^(+)-ApoE^(-/-))were obtained.The two types of knockout mice were born in accordance with Mendel genetic law,survived and fertile.Conclusion In this experiment,we successfully constructed and identified the double gene knockout mouse model of PDHA1 macrophage conditional knockout mice and ApoE gene knockout mice by using Cre-LoxP system,so as to provide a research platform for studying the role of PDHA1 in the pathogenesis of atherosclerotic diseases.