鸡gga-miR-199a慢病毒表达系统的构建

Construction of Chicken gga-miR-199a Lentiviral Expression System

为建立一种稳定持续表达鸡gga-miR-199a的系统,将从鸡DF-1细胞扩增到的gga-miR-199a前体序列克隆入pLL3.7慢病毒载体质粒中用以包装慢病毒。同时构建了更换pLL3.7质粒中鼠源U6启动子更换为鸡源及人源U6启动子的pLL3.7慢病毒载体质粒;将其与慢病毒包装辅助质粒共转至HEK293T细胞中,收集浓缩病毒颗粒并侵染细胞并获得过表达gga-miR-199a的单细胞克隆。提取单细胞克隆总RNA进行gga-miR-199a-5P的定量检测。结果表明,经慢病毒侵染后的细胞中鸡gga-miR-199a表达量显著提高,且人源U6启动子的转录效率最高,该研究成功建立了一种稳定持续表达鸡gga-miR-199a的慢病毒系统,说明该系统有利于在鸡活体或原代细胞中开展miRNA功能的研究。

In order to establish a stable and continuous expression system for chicken gga-miR-199a,the gga-miR-199a precursor sequence amplified from chicken DF-1 cells was cloned into the pLL3.7 lentiviral vector plasmid to package the lentivirus.For the U6 promoters from different species may possess variant transcription efficiency,pLL3.7 plasmid that replaced the mouse origin U6 promoter with the chicken and human origin was also constructed.The HEK293T cells were infected with the concentrated lentivirus particles that packaged by co-transfecting pLL3.7 with helper plasmid into HEK293T cells.Ther a single cell clone overexpressing gga-miR-199a was obtained.Extract the total RNA of single-cell cloned cells after infection for quantitative detection of gga-miR-199a-5P.The results showed that the expression of gga-miR-199a in the cells infected was significantly increased,and the human origin U6 promoter had the highest transcription efficiency.In summary,this study has successfully established a lentiviral system that can stably and continuously express gga-miR-199a,which is conducive to the study of miRNA functions in live chicken or primary cells.