长链非编码RNA-GAS5慢病毒表达载体构建及其转染效率鉴定

Construction of lentivirus expression vector with long non-coding RNA GAS5 and identification of its transfection efficiency

目的构建长链非编码RNA-GAS5(lncRNA-GAS5)过表达及干扰慢病毒载体,并在人滋养细胞中进行转染效率鉴定。方法获取lncRNA-GAS5基因序列,合成重组lncRNA-GAS5全基因序列,同时设计并合成3条lncRNA-GAS5 RNAi序列,构建过表达及干扰慢病毒载体,转染至293T细胞包装病毒并测定其滴度,转染人滋养细胞株HTR-8/SVneo后,使用荧光显微镜观察荧光表达情况,采用实时定量聚合酶链式反应法检测lncRNA-GAS5表达,并筛选出最佳干扰效率的表达载体。结果过表达载体感染细胞后,其表达量提高了2.12倍;干扰载体感染细胞后,其表达量降低了67.3%。结论本实验成功构建了lncRNA-GAS5慢病毒过表达和干扰载体,可为后续进一步研究提供基础。

Objective Long-chain non-coding RNA-GAS5(lncRNA-GAS5)over-expressed and interfered lentivirus vectors were constructed,and the transfection efficiency was identified in human trophoblast cells.Methods The lncRNA-GAS5 gene sequences were obtained and the full genetic sequences were synthesized.Meanwhile,three lncRNA-GAS5 RNAi sequences were designed.Over-expression and interference lentivirus vectors were built and transfected to 293 T cells to package the viruses and to measure their drop degree.Human trophoblastic cell line HTR-8/SVneo was transfected by the lentivirus vectors and the fluorescence expression can be detected by the fluorescence microscope.Real-time quantitative polymerase chain reaction(RT-qPCR)method was used to detect lncRNA-GAS5 expression and select the best expression vector of interference efficiency.Results After the over-expression vector infected cells,the expression level increased by 2.12 times,and the expression of interfering vector reduced by 67.3%.Conclusion The lncRNA-GAS5 over-expression and interference lentivirus vectors is successfully constructed,which provides experimental basis for further research.