平滑肌细胞特异表达Cre重组酶转基因小鼠的建立

Generation of smooth muscle cell specific Cre transgenic mice

目的:建立平滑肌细胞特异表达Cre重组酶的转基因小鼠。方法:用分子克隆的方法构建含有α平滑肌肌动蛋白(α-SMA)启动子、Cre重组酶基因和polyA的转基因载体α-SMA-Cre。以显微注射的方法将5.3kb的转基因片段引入小鼠基因组。通过PCR和LacZ染色以检测Cre重组酶在体内介导重组的功能。结果:共注射282枚受精卵,移植至10只假孕母小鼠的输卵管中发育,获得子代小鼠19只,经PCR鉴定有4只小鼠在基因组上整合有Cre基因,整合率为21%。将该小鼠与基因组上携带LoxP位点的Smad4条件基因打靶小鼠交配,通过PCR在所有含有平滑肌细胞的组织基因组DNA中检测到重组后的234bp特异条带。与“报告”小鼠—ROSA26交配,LacZ染色后小肠壁平滑肌细胞中特异地检测到Cre重组酶活性。结论:成功构建了平滑肌细胞特异表达Cre重组酶的转基因小鼠,该小鼠在平滑肌细胞中特异表达Cre重组酶,并能在体内成功地介导LoxP位点间的重组。

Objective:To establish transgenic mice that express Cre recombinase in smooth muscle cells specifically. Methods: A smooth muscle cell specific transgenic construct (α-SMA-Cre) containing the smooth muscle cell specific α-SMA promoter, the Cre recombinase gene and polyA was constructed. The 5.3 kb DNA fragments were microinjected into fertilized eggs. PCR analysis and LacZ staining were used to examine the function of Cre recombinase in the transgenic mice. Results: The injected 282 eggs were implanted into the oviducts of 10 female mice. In the 19 offspring, there were 4 mice carrying the transgene identified by PCR, and the efficiency of integration was 21%. The SMA-Cre transgenic mice were mated with Smad4 conditional knockout mice and ROSA26 mice to check the Cre mediated recombination in multiple tissues. After the Cre mediated recombination, primers amplified a fragment of 234 bp from the genomic DNA of all the tissues containing smooth muscle cells. LacZ staining showed that smooth muscles of intestine were positive. Conclusion:A kind of transgenic mouse that expresses Cre recombinase in smooth muscle cells specifically was established. The Cre recombinase was expressed in smooth muscle cells and could mediate the recombination between the LoxP sites in vivo.