摘要
目的：运用Cre/Loxp重组酶系统构建肝脏特异性CD36基因敲除小鼠并进行鉴定和验证,为研究CD36的生物学功能奠定基础。方法：构建CD36打靶载体,电转转染胚胎干细胞,通过长链PCR筛选出正确同源重组的阳性克隆,阳性胚胎干细胞克隆经扩增后,注射入C57BL/6J小鼠的囊胚中,获得嵌合小鼠,再与Flp小鼠交配筛选获得Flox杂合子小鼠,该小鼠与引进的Alb-Cre小鼠交配,在F3代获得CD36fl/fl：Alb-Cre＋基因型小鼠,即为肝脏特异性CD36敲除小鼠。采用PCR鉴定小鼠基因型,PCR、实时荧光定量PCR和Western blot验证小鼠肝脏CD36敲除效果,Western blot检测小鼠肾脏、脂肪和心肌组织CD36表达情况,HE染色观察小鼠肝脏形态学改变。结果：建立了CD36基因的Flox杂合子小鼠,与Alb-Cre小鼠交配后,在F3代筛选出CD36fl/fl：AlbCre-和CD36fl/fl：Alb-Cre＋基因型小鼠,DNA水平证实CD36fl/fl：Alb-Cre＋基因型小鼠肝脏CD36基因通过Cre/Loxp重组酶系统被敲除。与CD36fl/fl：Alb-Cre-基因型小鼠相比,CD36fl/fl：Alb-Cre＋基因型小鼠肝脏CD36mRNA和蛋白表达水平显著降低,肾脏、脂肪和心肌组织CD36蛋白表达无差别,肝脏形态学特征无明显差异。结论：通过Cre/Loxp重组酶系统成功构建了肝脏特异性CD36基因敲除小鼠,为研究CD36在肝脏代谢和肝脏疾病中的功能提供了动物模型。
Objective： To generate mice with a liver-specific knockout of CD36 gene using the Cre-Loxp system,which will lay a foundation for the study of the biological function of CD36. Methods： CD36 targeting vector was constructed and electroporated into embryonic stem cells. Positive clones with correct homologous recombination were screened by long-chain PCR. Positive embryonic stem cell clones were amplified and injected into the blastocysts of C57 BL/6 J mice to obtain chimeric mice. And then mated with Flp mice to obtain Flox heterozygous mice. The Flox mice were mated with the introduced Alb-Cre mice,and CD36 fl/fl： Alb-Cre＋mice were obtained in the F3 generation,which are liver-specific CD36 knockout mice. The genotypes of the mice were identified by PCR. PCR,real-time fluorescence quantitative PCR and Western blot were used to verify the knockout effects of CD36 gene in the liver. The expression of CD36 in kidney,adipose tissue and myocardial tissue was detected by Western blot. Morphological changes of the liver were observed by HE staining. Results：Flox heterozygous mice with CD36 gene were established. After mating with Alb-Cre mice,CD36 fl/fl： Alb-Cre-and CD36 fl/fl： Alb-Cre＋genotypes were screened in F3 generation. DNA levels confirmed that CD36 fl/fl： Alb-Cre＋genotype mouse liver CD36 gene was knocked out by the Cre/Loxp recombinase system. Compared with mice with CD36 fl/fl： Alb-Cre-mice,CD36 fl/fl： Alb-Cre＋mice had significantly reduced expression of CD36 mRNA and protein in the liver,and there was no difference in the expression of CD36 protein in kidney,adipose tissue and myocardial tissue. There was no significant difference in the morphological characteristics of liver. Conclusion：Liver-specific CD36 knockout mice were successfully generated by the Cre/Loxp recombinase system,providing an animal model for the study of CD36＇s function in hepatic metabolism and diseases.
作者
苏春晓
张晓玉
曾晗
陈压西
阮雄中
杨萍
SU Chun-xiao;ZHANG Xiao-yu;ZENG Han;CHEN Ya-xi;RUAN Xiong-zhong;YANG Ping(Centre for Lipid Research,Chongqing Key Laboratory of Lipid and Glucose Metabolism,Chongqing Medical University,Chongqing 400016,China)
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2018年第8期26-33,共8页
China Biotechnology
基金
国家自然科学基金(81400786)
重庆市教委科学技术研究项目(KJ1702029)资助项目
