[Regulatory expression of human interferon-gamma gene in murine bone marrow stromal cells by Tet-off system].
OBJECTIVE
The Tet-off system is a gene expression regulating system, which has high efficiency, low toxicity and strict turning-off function. This study was to investigate the regulatory expression of human interferon-gamma (IFNgamma) gene in murine bone marrow stromal cells (BMSCs) by Tet-off system.


METHODS
Plasmid pTre and human IFNgamma gene were digested using Sac II and Xba I, purified, and ligated by T4 ligase. The recombinant pTre-IFNgamma was identified by restriction analysis and sequencing. pTet-off and pTre-IFNgamma were co-transfected into murine BMSCs. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect IFNgamma mRNA 48 h after transfection. ELISA was used to detect IFNgamma protein in the cell culture medium everyday to analyze the secretary mode of IFNgamma. Then, Tetracycline was added to the culture medium immediately after co-transfection in gradient concentration and 48 h after co-transfection to observe its effect on IFNgamma secretion.


RESULTS
Restriction analysis and sequencing confirmed the orientation and sequence of the recombinant plasmid pTre-IFNgamma were correct. RT-PCR detected IFNgamma mRNA in BMSCs 48 h after co-transfection. ELISA showed the secretion of IFNgamma lasted 6 days and a peak appeared in the third 24 h, which was (720.09+/-241.51) pg per 1 x10(6) cells. Increasing tetracycline decreased the secretion of IFNgamma in the first 48 h: 10-100 ng/ml tetracycline decreased the secretion to nearly 0. When tetracycline was added 48 h after co-transfection, the secretion was obviously suppressed 8 h later, and the suppression was strengthened as time went by.


CONCLUSIONS
The Tet-off system can efficiently and rapidly down-regulate the expression of human IFNgamma gene in murine BMSCs.
Tan
Yin-Duo
YD
State Key Laboratory of Oncology in South China, Guangzhou, Guangdong, 510060, P. R. China.
Lu
Yue
Y
Xu
Hai-Peng
HP
Chen
Xiao-Qin
XQ
Li
Xiao-Qing
XQ
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism*; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D007371:Interferon-gamma / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids; D012333:RNA, Messenger / Q000378:metabolism; D017154:Stromal Cells / Q000166:cytology / Q000378:metabolism*; D013752:Tetracycline / Q000008:administration & dosage / Q000494:pharmacology*; D014162:Transfection
D012333:RNA, Messenger; D007371:Interferon-gamma; D013752:Tetracycline