【摘要】 实验于2005-10/2006-06在江苏省干细胞重点实验室进行。体外分离培养新生SD大鼠心室肌细胞2周后,Trizol法抽提细胞总RNA,RT-PCR获得心肌营养素1基因,与克隆载体PMD18-T-Vector连接,转化TOP10感受态细菌,阳性克隆经鉴定正确后,PCR扩增心肌营养素1基因,EcoRI和BamHI双酶切,T4DNA连接酶连接,构建pEGZ-CT-1的反转录病毒载体。经PCR及酶切鉴定,测序表明心肌营养素1基因成功插入pEGZ-Term载体。pEGZ-CT-1载体的构建,为转基因细胞的制备和移植治疗心肌梗死奠定了基础。更多还原

【Abstract】 The experiment was performed at the Key Laboratory for Stem Cell of Jiangsu Province from October 2005 to June 2006. With Trizol method, the total RNA was obtained from the ventricular cells isolated and cultured for two weeks from the neonatal SD rat. Cardiotrophin-1 (CT-1) was obtained by using RT-PCR reaction and connected with PMD18-T-Vector, which were transformed into the competence bacteria TOP10 in which positive clones were identified. The CT-1 gene after amplified with PCR was digested with restriction endonuclease EcoR I and BamH I, and the products and pEGZ-Term were ligated with T4DNA ligase to construct the retrovirus vector of pEGZ-CT-1. After identification of PCR and digestion, the sequencing showed that the full-length of CT-1 was successfully inserted into pEGZ-Term. Establishment of pEGZ-CT-1 retroviral expressing vector lays a foundation for the further preparation of transgenic cells and therapy of myocardial infarction with transplantation.更多还原