Construction of lentiviral vector carrying mouse RORγt and expression of RORγt in 293FT cells / 中国实验血液学杂志

ABSTRACT

This study was aimed to construct a lentiviral vector carrying mouse RORγt and glp gene, and to detect the expression of RORγt in the 293FT cells. The RORγt fragment was amplified by RT-PCR from mouse thymus and cloned into PCR 2.1 vector. The RORγt DNA fragment was prepared by digestion and inserted into MigR1 plasmid, then the RORγt-IRES-GFP was directionally linked with lentiviral transfer plasmid pTK208 to generate a lentiviral vector pXZ9-RORγt. The recombinant lentivirus were produced by co-transfected three plasmids into 293FT packing cells using lipofectamine 2000. After transfection, the lentiviral supernatant was collected and concentrated via ultracentrifugation. The 293FT cells were infected by the concentrated lentivirus, GFP expression was examined under a fluorescent microscope and the expression of RORγt protein was detected by Western blot. The results showed that the RORγt fragment was amplified from cDNA of mouse thymus and recombinant lentiviral vector pXZ9-RORγt was constructed successfully. High titer lentivirus were prepared after one round ultracentrifugation. RORγt expression could be detected in 293FT cells after virus infection. It is concluded that the lentiviral vector pXZ9- RORγt containing mouse RORγt-IRES-GFP is successfully constructed; RORγt can express in 293FT cells via lentiviral vector transduction, which provides an optional tool for further research on the mechanism of RORγt controlling Th17 cell differentiation.