Aim: To construct the recombination adeno-associated virus (rAAV) carrying murine forkhead box P3 (FoxP3) gene, and then detect the expression in NIH3T3 cells.
Methods: The recombination adeno-associated virus (rAAV) vector containing internal ribosome entry site which could coordinate the expression of FoxP3 gene and enhance green fluorescent protein gene was constructed. HEK293 cells were transfected by the transfection cocktail containing the constructed rAAV vector, trans plasmid and helper plasmid by the calcium phosphate method. The infectious rAAV carrying FoxP3 gene (rAAV/FoxP3) suspensions were harvested and purified by heparin affinity chromatography. The purity and titre of rAAV were detected by SDS-PAGE electrophoresis and real-time PCR, respectively. NIH3T3 cells were transfected by rAAV, the transfection efficiency was analyzed by flow cytometry and FoxP3 mRNA expression was detected by real-time PCR.
Results: The titre of the infectious rAAV was 5x 10(12) vg/mL by real-time PCR analysis. After NIH3T3 cells were transfected, the transfection efficiency of the infectious rAAV/FoxP3 was up to 92.88% and high level of FoxP3 mRNA was detected.
Conclusion: rAAV carrying FoxP3 gene was successfully constructed and expressed in NIH3T3 cells, which will be of benefit to further researches on the function of FoxP3.