利用CRISPER/Cas9技术构建肝脏特异性敲入Lcat基因小鼠

Establishment of liver⁃specific Lcat knock⁃in mouse models by the CRISPER/Cas9 technique

目的:通过CRISPR/Cas9介导的基因编辑技术构建卵磷脂胆固醇脂酰转移酶(lecithin⁃cholesterol acyltransferase,LCAT)基因敲入C57BL/6小鼠并与肝脏特异性表达Cre的转基因小鼠配繁得到肝脏特异性敲入Lcat基因C57BL/6小鼠模型。为Lcat基因在肝脏相关代谢疾病发生机制的研究提供动物模型。方法:利用CRISPR/Cas9技术构建Lcat基因敲入小鼠;利用肝脏特异性表达Cre的转基因小鼠与Lcat基因敲入小鼠交配得到肝脏特异性敲入Lcat基因小鼠;通过PCR法鉴定小鼠的基因型;利用实时荧光定量PCR(real⁃time quantitative PCR,qPCR)和Western blot技术验证Lcat基因的mRNA水平和蛋白水平。结果:PCR结果显示肝脏特异性敲入Lcat基因的小鼠模型构建成功;qPCR结果显示Lcat基因在肝脏中特异性高表达;WB结果显示,与对照组小鼠相比,LCAT蛋白在肝脏特异性敲入Lcat的小鼠肝脏中有明显更高的表达。结论:成功构建肝脏特异性敲入Lcat基因小鼠,为在动物水平探索Lcat基因在肝脏相关代谢疾病中的功能及相关发病机制提供研究平台。

Objective:To construct lecithin⁃cholesterolacyl transferase(LCAT)knock⁃in mice by CRISPR/Cas9⁃mediated gene editing and to obtain liver⁃specific overexpression of Lcat mice by mating with liver⁃specific Cre⁃expressing transgenic mice.Providing an animal model for the study of the mechanism of the Lcat gene in the development of liver⁃related metabolic diseases.Methods:Lcat knock⁃in mice were constructed by CRISPR/Cas9 technology;Liver⁃specific Cre⁃expressing transgenic mice were mated with Lcat knock⁃in mice to obtain liver⁃specific overexpressing Lcat mice;Genotyping mice by PCR;Quantitative real⁃time PCR(qPCR)and Western blot(WB)techniques were used to verify the expression of Lcat gene in C57BL/6 mice.Results:The PCR results showed that the liver⁃specific overexpression of Lcat gene in mice was successfully constructed;the qPCR results showed that the Lcat gene was specifically highly expressed in the liver,and the liver of knock⁃in mice showed higher Lcat expression;the WB results showed that LCAT protein was more highly expressed in the liver of liver⁃specific Lcat knock⁃in mice.Conclusion:Liver⁃specific overexpression of Lcat gene mice were successfully constructed,providing a platform for exploring the function of the Lcat gene at animal level in liver⁃related metabolic diseases and the associated pathogenesis.