Inducible gene expression using an autoregulatory, tetracycline-controlled system.
The protocols in this unit describe the transfection of adherent cells and the testing of resultant clones for inducible transactivator or target gene protein expression. Stably transfected fibroblast cell lines expressing transactivator and target gene(s) can be derived by first cotransfecting pTet-tTAk and a plasmid encoding a selectable marker and obtaining stable lines with inducible transactivator expression. These lines are subsequently stably cotransfected with plasmids encoding the target gene(s) and a second selectable marker. The procedure may also be used to cotransfect pTet-tTAk with the target gene-encoding plasmid(s) and a single selectable marker plasmid. A support protocol describes methods to test stably transfected cell lines for inducible gene expression, for transient transfection and induction of tet-regulated plasmids, and for detection of the tTAk gene in cells (or transgenic mice).
Shockett
Penny
P
Southeastern Louisiana University, Hammond, Louisiana.
Schatz
David
D
Howard Hughes Medical Institute and Yale University School of Medicine, New Haven, Connecticut.
D000818:Animals; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods*; D005819:Genetic Markers / Q000187:drug effects / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics; D008967:Molecular Biology / Q000379:methods*; D041681:NIH 3T3 Cells; D010957:Plasmids / Q000235:genetics; D013752:Tetracycline / Q000494:pharmacology; D015534:Trans-Activators / Q000235:genetics*; D014162:Transfection / Q000379:methods*
D005819:Genetic Markers; D015534:Trans-Activators; D013752:Tetracycline