人miR-17-92 cluster慢病毒载体构建及其在人脂肪细胞的表达验证

Construction of a recombinant lentiviral vector carrying miR-17-92 cluster and validation of its transduction efficiency in human adipocytes

目的构建人miR-17-92cluster慢病毒表达载体,包被病毒并检测其在人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-17-92cluster所在区域上下游100bp左右的基因组DNA,克隆入慢病毒表达载体,与Pcgvp、Rev、Vsvg包装质粒共转染HEK-293T细胞包装病毒,收取病毒上清液感染人脂肪细胞。36h开始观察荧光标记,72h收取细胞,抽提RNA,RT-PCR检测miR-17-92cluster各miRNA及miR-103、let-7e的表达。结果成功构建人miR-17-92cluster慢病毒表达载体。包装获得的病毒感染人脂肪细胞的效率可达到75%以上,miR-17-92cluster各miRNA的过表达效率均达到2倍以上,且没有干扰miRNA内源性成熟机制。结论成功构建了人miR-17-92cluster慢病毒表达载体。包被的慢病毒可以在人脂肪细胞中实现了该cluster的过表达效果,为研究人miR-17-92cluster在人脂肪细胞中的功能提供参考。

Objective To construct a recombinant lentiviral vector carrying miR-17-92 cluster and validate of its transduction efficiency in human adipocytes. Methods The minigene fragments of miR-17-92 cluster were cloned from genomic DNA of human adipocytes and inserted in a lentivirus expression vector. The sequencing validated recombinant lentiviral expression vector and packaging plasmids Pcgvp,Rev,Vsvg were co-transfected into HEK-293T cells to generate lentivirus.The supernatant containing lentiviral particles was transducted into human adipocytes,and its transduction efficiency was validated by fluorescent observation and miRNA expression. Results miR-17-92 cluster lentiviral vector was constructed successfully and transduction efficiency in human adipocytes reached a level over 75%.The expression level of six members belonging to miR-17-92 cluster increased more than two folds in the infected cells and ectopic expresion did not interrupt the endogenous microRNA pathway. Conclusion miR-17-92 cluster lentiviral vector has been constructed successfully.The transduction efficiency in human adipocytes is confermed,which is helpful in the further study of the function of miR-17-92 cluster in human adipocytes.