Objective: To construct the self-inactivating lentiviral vector containing murine Foxp3 gene and detect its expression in the CD4+ CD25- T cells.
Methods: 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.
Results: 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%.
Conclusions: 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.