载脂蛋白E背景E1A激活基因阻遏子转基因小鼠建立及鉴定

Establishment and identification of apolipoprotein E backgrouond cellular repressor of E1A⁃stimulated genes transgenic mice

目的建立并鉴定载脂蛋白E(ApoE)背景E1A激活基因阻遏子(CREG)转基因(Apo^E(⁃/⁃)CREG^(Tg))小鼠。方法将雄性Apo^E(⁃/⁃)小鼠与雌性CREG^(Tg)小鼠进行交配,获得雄性Apo^E(+/⁃)小鼠和雌性Apo^E(+/⁃)CREG^(Tg)小鼠,再将两者进行交配,以获得Apo^E(⁃/⁃)CREG^(Tg)小鼠。第4周时,提取鼠耳基因组DNA,采用聚合酶链式反应(PCR)和琼脂糖凝胶电泳进行基因型鉴定。提取小鼠脂肪组织和脾组织的RNA、蛋白,采用荧光定量PCR、蛋白免疫印迹法检测小鼠脂肪组织和脾组织CREG基因转录水平、蛋白表达水平。结果子代小鼠中,ApoE^(+/+)小鼠9只(12.33%),ApoE^(+/+)CREG^(Tg)小鼠10只(13.70%),Apo^E(+/⁃)小鼠19只(26.03%),Apo^E(+/⁃)CREG^(Tg)小鼠16只(21.92%),Apo^E(⁃/⁃)小鼠8只(10.95%),Apo^E(⁃/⁃)CREG^(Tg)小鼠11只(15.07%),成功构建Apo^E(⁃/⁃)CREG^(Tg)小鼠模型。Apo^E(⁃/⁃)CREG^(Tg)小鼠脂肪组织、脾组织的CREG基因转录水平、蛋白表达高于Apo^E(⁃/⁃)小鼠,差异均有统计学意义(P<0.05)。结论本研究成功建立了Apo^E(⁃/⁃)CREG^(Tg)小鼠模型,为深入阐明CREG在动脉粥样硬化等心血管疾病中的作用及机制提供了新的实验工具。

Objective Establishment and identification of apolipoprotein E(ApoE)background cellular repressor of E1A⁃stimulated genes(CREG)transgenic(Apo^E(⁃/⁃)CREG^(Tg))mice.Methods Male Apo^E(⁃/⁃)mice were mated with female CREG^(Tg) mice to obtain male Apo^E(+/⁃)mice and female Apo^E(+/⁃)CREG^(Tg) mice,then they were mated to obtain Apo^E(⁃/⁃)CREG^(Tg) mice.At the fourth week,genomic DNA was extracted,and genotypes were identified by polymerase chain reaction(PCR)and agarose gel electrophoresis.RNA and pro⁃tein were extracted from adipose tissue and spleen tissue of the mice,the transcription and protein expression of CREG gene in adipose tissue and spleen tissue were detected by real⁃time PCR and western blot.Results Among the offspring mice,9(12.33%)were ApoE^(+/+)type,10(13.70%)were ApoE^(+/+)CREG^(Tg) type,19(26.03%)were Apo^E(+/⁃)type,16(21.92%)were Apo^E(+/⁃)CREG^(Tg) type,8(10.95%)were Apo^E(⁃/⁃)type,and 11(15.07%)were Apo^E(⁃/⁃)CREG^(Tg) type,Apo^E(⁃/⁃)CREG^(Tg) mice were successfully constructed.CREG gene transcription level and protein expression in adipose tissue and spleen tissue of Apo^E(⁃/⁃)CREG^(Tg) mice were higher than those of Apo^E(⁃/⁃)type mice,with statistical significance(P<0.05).Conclusion Apo^E(⁃/⁃)CREG^(Tg) mice have been successfully established,which provides a tool for exploring the function and mechanism of CREG in atherosclerotic and other vascular disease.