Lac/Tet dual-inducible system functions in mammalian cell lines.
The Escherichia coli Lac repressor (Lac system) and tetracycline responsive promoter (Tet system) systems have been used individually to regulate gene expression at the cellular as well as the organismal levels. In this study, these two systems were combined (designated Lac/Tet dual-inducible system) to regulate two inducible genes simultaneously in a single cell. The isopropyl-beta-D-thiogalactopyranoside (IPTG) and tetracycline (used for the operation of the Lac and the Tet systems) were non-cytotoxic to the cells when added together into the cells at around the optimal concentrations (IPTG: < or = 5 mM; tetracycline: < 1.5 micrograms). The rate and efficiency of induction and repression of two inducible genes regulated by the Lac/Tet dual-inducible system were similar to the results obtained when one inducible gene is regulated by one inducible system in a single cell. The Lac/Tet dual-inducible system could function in many cell lines, which was demonstrated by regulating the expression of beta-galactosidase and luciferase reporter genes in five tumor cell lines by transient transfection analysis. The feasibility of introducing a second inducible system into an already established inducible cell line was confirmed. Finally, we showed that the Lac/Tet dual-inducible system functions at translational and at functional levels in a stable cell line named 7-4-b, which contains the Ha-ras and bc1-2 inducible genes. In conclusion, this study extends the application of prokaryotic inducible systems from the regulation of a single gene to two genes and helps clarify the relationship between two genes and the effects of two genes on the cells.
Liu
H S
HS
Department of Microbiology & Immunology, Medical College, National Cheng Kung University, Tainan, Taiwan, ROC.
Lee
C H
CH
Lee
C F
CF
Su
I J
IJ
Chang
T Y
TY
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D002460:Cell Line; D053938:DNA Fragmentation / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005786:Gene Expression Regulation / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D019254:Genes, bcl-2 / Q000235:genetics; D011905:Genes, ras / Q000235:genetics; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D056968:Lac Repressors; D051379:Mice; D010957:Plasmids / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics*; D013752:Tetracycline / Q000633:toxicity; D014162:Transfection / Q000235:genetics
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D007544:Isopropyl Thiogalactoside; D013752:Tetracycline