Establishment of liver⁃specific Lcat knock⁃in mouse models by the CRISPER/Cas9 technique
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摘要:
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。
Abstract:
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.