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目的 :建立携带突变位点R258C的平滑肌肌动蛋白α(ACTA2)基因的转基因小鼠模型。方法:利用基因重组技术,将人工合成的小鼠ACTA2基因第七外显子258位精氨酸(R)突变为半胱氨酸(C),再克隆到质粒p Bluescript KS的多克隆位点,通过显微注射法,把线性化、纯化后含有鼠ACTA2启动子/外显子1/内含子1/外显子2~9(含R258C)/3′端非翻译区的质粒注射入C57BL/6J小鼠受精卵中,将存活的胚胎移植入同期发情的假孕受体母鼠内,获得子代小鼠。用聚合酶链反应(polymerase chain reaction,PCR)和Southern印迹杂交方法检测子代鼠尾DNA,鉴定基因型。结果:移植注射胚胎后得到32只新生仔鼠,经PCR和Southern印迹杂交检测得到3只阳性小鼠。结论:携带突变位点R258C的ACTA2基因的转基因鼠模型构建成功,为进一步探索ACTA2基因突变在胸主动脉瘤发病中的作用和机制提供了重要的前期工作基础。
OBJECTIVE: To establish a transgenic mouse model of ACTA2 gene carrying the mutation R258C. Methods: The arginine (R) at position 7 of exon 7 of ACTA2 gene was mutated to cysteine (C) by gene recombination technique and cloned into the multi-cloning site of plasmid p Bluescript KS , The plasmid containing the mouse ACTA2 promoter / exon 1 / intron 1 / exon 2 to 9 (including R258C) / 3 ’untranslated region after linearization and purification was injected by microinjection C57BL / 6J mouse zygotes, the surviving embryos were transplanted into pregnant estrus females in the same period of estrus to obtain offspring mice. The offspring mouse tail DNA was detected by polymerase chain reaction (PCR) and Southern blot hybridization to identify the genotypes. RESULTS: Thirty-two newborn pups were obtained after transplantation of embryos. Three positive mice were obtained by PCR and Southern blotting. CONCLUSION: The transgenic mouse model of ACTA2 gene carrying the mutation locus R258C was successfully constructed, which provided important pre-operative basis for further exploring the role and mechanism of ACTA2 gene mutation in the pathogenesis of thoracic aortic aneurysm.