应用Talen法制备apoE基因敲除大鼠模型

Establishment of apoE Gene Knockout Model on SD Rat Using Talen Method

目的应用转录激活因子样效应因子核酸酶（Talen）法制备载脂蛋白E（apoE）基因敲除大鼠模型，为进一步研究apoE基因功能，动脉粥样硬化（AS）疾病发病机制及治疗方法奠定基础。方法设计并合成针对大鼠apoE基因的Talen序列，应用原核显微注射方法制备子代鼠。通过PCR．测序及TA克隆-测序法检测仔鼠中apoE的突变，经传代及兄妹交配获得纯合型仔鼠，并对其基因型进行鉴定。应用Westemblot方法检测apoE-/-仔鼠各组织中ApoE蛋白表达水平；血清学分析血脂总胆固醇（CHO）及甘油三酯（TG）含量；应用RT—PCR方法检测脂质代谢相关基因三磷酸腺苷结合盒转运体A1（ABCA1）的表达。结果经鉴定选取在第二外显子处缺失1bp，造成开放阅读框移码突变的大鼠为阳性F0代仔鼠，该鼠经传代及筛选，最终获得apoE-/-大鼠。Westernblot检测显示apoE-/-鼠在心、肝、肾、脑、卵巢组织中未见ApoE蛋白的表达，表明在该模型中成功实现了apoE基因的敲除。apoE-/-鼠血脂CHO、TG均高于野生SD大鼠，其中CHO水平呈现显著性差异（P〈0．05）。在apoE-/-鼠肝组织中ABCA1的表达降低，可能起到促进AS形成的作用。结论应用Talen法成功制备apoE基因敲除SD大鼠；经筛选获得了纯合型apoE-/-大鼠，该模型实现了apoE基因敲除，并表现为高血脂及As性状。

Objective To establish the apoE gene knockout-rat model using Talen method, and provide a basis for further study on the function of the apoE gene, the pathogenesis and treatment of atherosclerotic disease. Methods The Talen sequences were designed and synthesized for the rat apoE gene, and then the linearized and purified fragments were injected into fertilized eggs of SD rat through microsopic injection. Positive rats were detected by PCR-sequencing and TA clone-sequencing. The homozygous type offspring were obtained by siblings mating, and the genotype was identified. Western blot was used to detect the expression level of ApoE protein in some tissues of apoE-/- rat. The plasma levels of total cholesterol and triglyceride levels have also been detected. The expression of ATP binding cassette transporter A1 （ABCA1） which was related to the lipid metabolism was detected by reverse transcriptase polymerase chain reaction （RT-PCR）. Results Sequencing analysis indicated that there had a deletion of 1 bp in the second exon, resulting in open reading frame shift mutation in the rat, and obtained the apoE-/- rats. Western blot test showed that no expression of ApoE protein in the heart, liver, kidney, brain and ovary of apoE-/- rats, and the apoE gene was successfully knocked out. The total cholesterol and triglyceride levels of apoE-/- rats were higher than that of wild type SD rats, and the total cholesterol levels have the significantly different （P〈0.05）. The expression of ABCA1 in the liver of the apoE-/- rats was decreased, which may play a role of AS promoting. Conclusions apoE-/- rats were obtained by Talen method. The apoE gene has been knocked out, and the rats can be considered as the model rats showed hyperlipemia and atherosclerosis.