小鼠miR-29c过表达慢病毒载体的构建与鉴定

Construction and identification of mouse miR-29c overexpression lentiviral vector

目的构建小鼠miR-29c过表达重组慢病毒载体,并对表达产物进行鉴定。方法应用PCR法从小鼠MLE-12细胞中提取pre-miR-29c基因,测序后将其克隆到pLenti-CMV-EGFP慢病毒载体,通过PCR筛选及测序对阳性克隆进行鉴定。将miR-29c过表达慢病毒载体转染至293T细胞,进行慢病毒的包装和滴度测定。以RT-PCR检测慢病毒转染MLE-12细胞中miR-29c的表达量。结果构建miR-29c慢病毒表达载体Lv-miR-29c经PCR和测序鉴定正确,并获得滴度为5.65×108 IU/ml的病毒浓缩液,转染MLE-12细胞后miR-29c基因成功过表达。结论成功构建miR-29c过表达慢病毒载体,为进一步研究miR-29c的功能奠定实验基础。

Objective To construct mouse miR-29c overexpression recombinant lentiviral vector and to identify the expressed product. Methods The pre-miR-29c gene was amplified from the mouse genome by PCR. The amplified product was cloned into p Lenti-CMV-EGFP lentiviral vector. The positive clones were identified by PCR screening and sequencing. The miR-29c overexpression lentiviral vector was transfected into 293 T cells for lentivirus packaging and titer determination. The lentivirus was infected with MLE-12 cells and the expression of miR-29c was detected by real-time PCR. Results PCR and DNA sequencing assays demonstrated that the miR-29c lentiviral expression vector Lv-miR-29c was successfully constructed and the titer of virus solution was 5.65 ×10^8 IU/ml. The miR-29c gene was overexpressed in MLE-12 cells. Conclusion The construction of miR-29c overexpression lentiviral vector is successful, which will lay the groundwork for the further study of the function of miR-29c.