miR-26b慢病毒载体构建及其在人前体脂肪细胞中的表达验证

Construction of a recombinant lentiviral vector carrying miR-26band validation of its transduction efficiency in human pre-adipocytes

目的构建人miR-26b慢病毒表达载体,并检测其在人前体脂肪细胞中过表达效果。方法以人脂肪细胞基因组DNA为模板,PCR扩增包含miR-26b所在区域上下游100bp左右的基因组DNA,克隆慢病毒表达载体,进一步包装成慢病毒并感染人脂肪细胞,荧光定量PCR检测miR-26b表达。结果成功构建miR-26b慢病毒载体,病毒感染效率为75%左右,miR-26b过表达效率达2倍以上。结论成功构建了miR-26b慢病毒表达载体,可用于研究miR-26b在人脂肪细胞中的分子机制。

Objective To construct a lentiviral vector carrying miR-26b and validate its overexpression efficiency in human preadipocytes. Methods The minigene fragments of miR-26b were amplified by PCR from genomic DNA of human adipocytes and inserted into a lentiviral vector. The recombinant lentiviral vector was further packaged for generating lentiviral to infect human preadipocytes, miR-26b expression was detected by fluorescence quantitative-PCR. Results miR-26b lentiviral vector was successfully constructed and the transduction efficiency in human preadipocytes reached a level about 75%. The expression level of miR-26b increased more than two folds in the infected cell. Conclusion The molecular mechanism study of miR-26b in human adipocytes can be further carried out via successful construction of miR-26b lentiviral vector.