基于CRISPR/Cas9技术PCSK9基因敲除小鼠模型的构建及表型验证  

Construction and phenotypic verification of PCSK9 gene knockout mouse model based on CRISPR/Cas9 technology

作　　者：托罗娜依·米吉提 陈小翠 崔元峰 阿比旦·阿卜杜如苏力 陈邦党 马秀敏 Tuoluonayi Mijiti;CHEN Xiaocui;CUI Yuanfeng;Abidan Abudurusuli;CHEN Bangdang;MA Xiumin(Medical Laboratory Center,the Affiliated Tumor Hospital of Xinjiang Medical University,Urumqi 830000,China;School of Basic Medical Sciences,Xinjiang Medical University,Urumqi 830017,China;Clinical Medical Research Institute,the First Affiliated Hospital of Xinjiang Medical University,Urumqi 830011,China)

机构地区：[1]新疆医科大学附属肿瘤医院医学检验中心,乌鲁木齐830000 [2]新疆医科大学基础医学院,乌鲁木齐830017 [3]新疆医科大学第一附属医院临床医学研究院,乌鲁木齐830011

出　　处：《新疆医科大学学报》2024年第2期186-191,共6页Journal of Xinjiang Medical University

基　　金：新疆维吾尔自治区自然科学基金青年基金项目(2017D01C346)。

摘　　要：目的基于CRISPR/Cas9技术构建前枯草杆菌蛋白原转换酶9(Preprotein convertase subtilisin/kexin type9,PCSK9)基因敲除小鼠模型并对其表型进行初步验证。方法利用非同源末端连接(NHEJ)修复引入突变的方式,针对PCSK9基因靶序列设计单链向导RNA(sgRNA),将sgRNA和Cas9 mRNA显微注射到C57BL/6J小鼠受精卵,获得F0代敲除小鼠,通过繁育及基因型鉴定获得PCSK9基因敲除(PCSK9 KO)纯合子小鼠。采用PCR方法对PCSK9 KO小鼠进行基因鉴定,Western Blot检测肝组织PCSK9及低密度脂蛋白受体(LDL-R)蛋白的表达,同时检测心、脑、肾、脂肪组织中的PCSK9蛋白表达水平,q-PCR检测PCSK9的mRNA表达水平。采用酶法检测血清中总胆固醇(TC)及甘油三酯(TG)水平。结果PCR产物凝胶电泳以及PCSK9蛋白及mRNA表达结果表明成功获得全身性PCSK9基因敲除小鼠,肝组织LDL-R(3.42±0.56)蛋白表达显著增高。与WT组相比,PCSK9小鼠血清TC(90.75±17.81 mg/dL vs 54.19±2.42 mg/dL)、TG(81.26±11.98 mg/dL vs 50.92±11.93 mg/dL)显著降低(P<0.001)。结论基于CRISPR/Cas9技术成功构建获得PCSK9基因敲除小鼠,敲除PCSK9基因通过上调肝脏LDL-R蛋白显著降低血脂水平。Objective To establish a mouse model of preprotein convertase subtilisin/kexin type 9(PCSK9)gene knockout using CRISPR/Cas9 technology and to preliminarily validate its phenotype.Methods A single-stranded guide RNA(sgRNA)was designed for the target sequence of the PCSK9 gene through non-homologous end joining(NHEJ)repair and mutation.The sgRNA and Cas9 mRNA were microinjected into fertilized eggs of C57BL/6J mice to generate F0 generation positive knockout mice,followed by breeding and genotype identification to obtain homozygous PCSK9 knockout(PCSK9 KO)mice.PCR analysis was employed for genetic identification of PCSK9 KO mice,while western blotting was utilized to assess protein expression levels of PCSK9 and LDL-R in liver tissue,and the expression levels of PCSK9 protein in heart,brain,kidney and adipose tissues were detected.Additionally,q-PCR was performed to measure the mRNA expression level of PCSK9.Enzymatic methods were used for quantification of total cholesterol(TC)and triglyceride(TG)content in serum.Results The gel electrophoresis results of PCR products,along with the analysis of PCSK9 protein and mRNA expression,successfully demonstrated the generation of systemic PCSK9 knockout mice.The expression of LDL-R(3.42±0.56)protein in liver tissue was significantly increased.Compared with the WT group,the serum TC(90.75±17.81 mg/dL vs 54.19±2.42 mg/dL)and TG(81.26±11.98 mg/dL vs 50.92±11.93 mg/dL)of PCSK9 mice were significantly reduced(P<0.001).Conclusion The PCSK9 knockout mice were successfully generated using CRISPR/Cas9 technology.Knocking out the PCSK9 gene results in a substantial were decreased in lipid levels by upregulating liver LDL-R protein.

关 键 词：CRISPR/Cas9 PCSK9 基因敲除 基因型鉴定 血脂水平 

分 类 号：R575.1[医药卫生—消化系统]