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


CONCLUSIONS
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.
Qin
Xin-Tian
XT
State Key Laboratory of Oncology in South China, Guangzhou, Guangdong, PR China.
Lu
Yue
Y
Tan
Yin-Duo
YD
Chen
Xiao-Qin
XQ
Gen
Qi-Rong
QR
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism*; D002478:Cells, Cultured; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis* / Q000235:genetics; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D010957:Plasmids; D012333:RNA, Messenger / Q000378:metabolism; D013154:Spleen / Q000378:metabolism; D017154:Stromal Cells / Q000166:cytology / Q000378:metabolism*; D013752:Tetracycline / Q000494:pharmacology*; D014162:Transfection
D012333:RNA, Messenger; D007371:Interferon-gamma; D013752:Tetracycline