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
Yale University School of Medicine, New Haven, Connecticut, USA.
Schatz
David
D
D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D003587:Cytomegalovirus / Q000235:genetics; D004274:DNA, Recombinant / Q000032:analysis / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects*; D005813:Genes, Synthetic; D005822:Genetic Vectors / Q000235:genetics; D017405:Herpes Simplex Virus Protein Vmw65 / Q000235:genetics; D051379:Mice; D041681:NIH 3T3 Cells / Q000187:drug effects / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D013752:Tetracycline / Q000494:pharmacology*; D015533:Transcriptional Activation; D014162:Transfection / Q000379:methods
D004274:DNA, Recombinant; D017405:Herpes Simplex Virus Protein Vmw65; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D013752:Tetracycline