[Regulating human interferon-gamma gene expression in marrow stromal cells in mice by Tet-off system].

BACKGROUND & OBJECTIVE: We have constructed plasmid "pTre-IFN-gamma" and proved that the Tet-off system could regulate the expression of human interferon-gamma (IFN-gamma) gene in murine marrow stromal cells in vitro. This study was to investigate the regulatory reversibility of Tet-off system and its effect on the expression of human IFN-gamma gene in murine marrow stromal cells in mice. METHODS: Plasmids pTet-off and pTre-IFN-gamma were co-transfected into murine marrow stromal cells. The expression of IFN-gamma in marrow stromal cells was detected with ELISA. The marrow stromal cells were transfused into BABL/c naked mice after co-transfection. The expression of IFN-gamma mRNA in the spleen was detected by real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: IFN-gamma protein was detected in the culture solution of marrow stromal cells after co-transfection. The secretion peak appeared within the first 72 h. The protein level of IFN-gamma was significantly lower in 300 ng/ml tetracycline hydrochloride-treated marrow stroma cells than in untreated cells [(67.11+/-22.14) pg/1 x 10(7) cells vs. (319.96+/-29.04) pg/1 x 10(7) cells, P<0.001]; its expression was increased when removed tetracycline hydrochloride (P=0.032). The expression of human IFN-gamma mRNA was detected in the spleen. The mRNA level of IFN-gamma was significantly higher in untreated group than in continuous tetracycline hydrochloride-treated group [(1.5+/-0.7)x10(5) copies . (100 mg)(-1) vs. (6.9+/-5.3)x10(2) copies . (100 mg)(-1), P<0.001]; its expression in the mice received tetracycline hydrochloride for one single time lay between the above two groups with significant difference. CONCLUSION: In mice, Tet-off system could rapidly, efficiently and reversibly regulate the expression of human IFN-gamma gene in marrow stromal cells in vitro and in vivo.

[Regulatory expression of human interferon-gamma gene in murine bone marrow stromal cells by Tet-off system].

BACKGROUND & 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. CONCLUSION: The Tet-off system can efficiently and rapidly down-regulate the expression of human IFNgamma gene in murine BMSCs.