摘要
利用ＰＣＲ、Ｗｅｓｔｅｒｎ ｂｌｏｔ、免疫组化、免疫金标电镜、Ｓｏｕｔｈｅｒｎ ｂｌｏｔ从ＤＮＡ水平、蛋白水平分析干扰素诱导后Ｍｘ－Ｃｒｅ转基因小鼠肝组织中Ｃｒｅ重组酶的表达及其表达产物的活性。在对Ｍｘ－ｃｒｅ转基因小鼠基因组中整合有ｃｒｅ基因进行确定后，通过干扰素诱导Ｍｘ－ｃｒｅ转基因小鼠表达Ｃｒｅ重组酶，结果表明转基因小鼠肝细胞核和细胞质中均有Ｃｒｅ重组酶的表达，并在超微水平进一步证实。将含表达的Ｃｒｅ重组酶的肝细胞核抽提液加入到带有ｌｏｘＰ位点的ＤＮＡ中进行重组，分析证明Ｍｘ－Ｃｒｅ转基因小鼠表达的Ｃｒｅ重组酶具有重组活性，从而建立了体外检测Ｍｘ－Ｃｒｅ转基因小鼠表达的Ｃｒｅ重组酶活性的方法。
The Cre recombinase and its activity in C57-TgN(Mx-Cre) transgenic mice is studied by polymerase chain reaction (PCR), Western blot, immunohistochemistry, immunogold electron microscopy and Southern blot. C57-TgN(Mx-Cre) transgenic mice harbouring cre gene in genomic DNA is demonstrated by PCR, and these mice which are induced by INF- αlb could express Cre recombinase, which is confirmed by Western blot. With immunohistochemistry, we find that the Cre recombinase expresses in hepatocyte cytoplasm and nuclear of C57-TgN(Mx-Cre) transgenic mice. Cre recombinase expressed in hepatocyte cytoplasm and nuclear is further confirmed by immunogold electon microscopy. And it is supported that the Cre recombinase which is created from C57-TgN(Mx-Cre) transgenic mice induced by INF-αld b can direct DNA recombination reaction in vitro. All evidence leads us supporting the view that the Cre recombinase expressed in C57- TgN(Mx-Cre) transgenic mice has activity. Thus we find a method to detect the activity of Cre recombinase in vitro.
基金
国家自然科学基金重点(399993420
39430210)
国家人类基因组南方中心(CNCS99M11)项目~~
