[Construction and identification of the recombination adeno-associated virus carrying murine FoxP3 gene].
OBJECTIVE
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.


CONCLUSIONS
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.
Wang
Sheng-jun
SJ
Department of Immunology, Jiangsu University School of Medical Technology, Zhenjiang 212013, China. sjwjs@ujs.edu.cn
Ma
Jie
J
Ma
Bin
B
Mao
Chao-ming
CM
Tong
Jia
J
Yang
Min
M
Shao
Qi-xiang
QX
Xu
Hua-xi
HX
D000818:Animals; D002460:Cell Line; D000229:Dependovirus / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D051858:Forkhead Transcription Factors / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D010957:Plasmids; D016133:Polymerase Chain Reaction; D014161:Transduction, Genetic
D051858:Forkhead Transcription Factors; C498833:Foxp3 protein, mouse; D049452:Green Fluorescent Proteins