小鼠miR367逆转录病毒载体的构建与检测

为了探明小鼠体细胞重编程过程中miR367的作用机制,试验构建小鼠miR367逆转录病毒载体。根据NCBI上小鼠miR367的相关序列,从小鼠基因组中扩增miR367并将其连接到pMD18-T载体,然后对重组质粒进行双酶切并回收目的片段。将目的片段与pMXs逆转录病毒载体连接,然后依次经过酶切、PCR筛选阳性克隆和测序,最终构建逆转录病毒载体miR367-pMXs;采用磷酸钙法将miR367-pMXs转染plat-E细胞,以包装逆转录病毒颗粒;用逆转录病毒颗粒侵染小鼠胚胎成纤维细胞,在侵染后的第5天,提取被侵染细胞的总RNA,逆转录后获得cDNA,并采用Q-PCR方法检测侵染细胞中的miR367表达量。结果表明,被逆转录病毒侵染后,小鼠胚胎成纤维细胞中miR367的相对表达量比侵染前约提高了170倍。上述结果表明,成功构建小鼠miR367的逆转录病毒载体,为开展体细胞重编程机制的相关研究提供依据。

Construction and Detection of Retrovirus Vector with miR367 in Mouse

The aim of this study was to construct the retroviral vector with mouse miR367 and construction its mechanism of action in cell reprogramming. miR367 was cloned from mouse genome, and then linked with vector pMD18-T.The recombinant plasmid was digested and the segment with miR367 was purified, then subcloned into retroviral vector pMXs. Plat-E cells were transfected by the vector. Viral suspension was infected MEF cells in order to detect the expression of miR367. PCR and restriction enzyme digestion revealed that miR367-pMXs plasmid was constructed successfully. Q-PCR revealed that the expression of miR367 was enhanced in the infected-MEF.Result showed that mouse miR367 retroviral vector had been successfully constructed, and it had laid a good foundation for further research of cell reprogramming.