Cao Jiang, Chen Chong, Xu Kai-lin, Pan Xiu-ying, Li Zhen-yu, Zeng Ling-yu
Department of Hematology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2009 Mar;40(2):199-202.
To construct the self-inactivating lentiviral vector containing murine Foxp3 gene and detect its expression in the CD4+ CD25- T cells.
The bicistronic lentiviral transfer plasmid containing Foxp3 gene and internal ribosomal entry site-green fluorescent protein gene (IRES-GFP) was constructed. Human embryonic kidney 293T cells were co-transfected by lentiviral packing system, which including the packaging plasmid deltaNRF, the transfer plasmid and the envelope plasmid VSVG using liposome. Then manifested the efficiency of gene transduction with Flow Cytometry (FCM) and the expressions of Foxp3 mRNA and protein were assayed by RT-PCR and Western blotting.
The lentiviral transfer plasmid pXZ208-Foxp3-IRES-GFP was constructed, the virus titres were above 10(6) IU/mL in the supernatant. After coculture with thus cells, the CD4+ CD25- T cells in experimental group expressed significantly higher amounts of Foxp3 mRNA and protein than control group, and the efficiency of gene transduction was about 55.95%.
The self-inactivating lentiviral vector containing murine Foxp3 gene are successfully constructed. This gene delivery system can transduce Foxp3 gene into CD4+ CD25- T cells with highly efficient expression.
构建含小鼠Foxp3基因的自失活慢病毒载体，并检测其在CD4+CD25-T细胞中的表达。
构建含Foxp3基因和内部核糖体进入位点-绿色荧光蛋白基因（IRES-GFP）的双顺反子慢病毒转移质粒。采用脂质体将慢病毒包装系统（包括包装质粒deltaNRF、转移质粒和包膜质粒VSVG）共转染人胚肾293T细胞。然后用流式细胞术（FCM）检测基因转导效率，用RT-PCR和Western印迹法检测Foxp3 mRNA和蛋白的表达。
构建了慢病毒转移质粒pXZ208-Foxp3-IRES-GFP，上清中病毒滴度高于10(6)IU/mL。与这些细胞共培养后，实验组CD4+CD25-T细胞中Foxp3 mRNA和蛋白的表达量明显高于对照组，基因转导效率约为55.95%。
成功构建了含小鼠Foxp3基因的自失活慢病毒载体。该基因传递系统可将Foxp3基因高效转导至CD4+CD25-T细胞中并表达。
