Tetracycline-regulated gene expression in epidermal keratinocytes.
The tetracycline-regulated expression system developed by Gossen and Bujard is a powerful genetic tool that permits the expression of any gene construct introduced into either cultured cells or transgenic animals to be precisely controlled. It involves two components, a regulatory component based on the prokaryotic tetracycline repressor (TetR) and a response plasmid that expresses the gene of interest under control of the tetracycline-response element. In this paper, we review the Tet system methodology, discuss the available vector systems, and describe how to prepare and characterize keratinocyte cell lines that express a gene under tetracycline control. The methodology involves the development of stable cell lines expressing the TetR protein (either tTA or rtTA, expressed as a fusion with the VP16 activation domain), and a second set of double-stable cell lines that contain both TetR and the response plasmid (tetracycline-response element-gene X) expressed under tetracycline control. As an example of this methodology, we discuss our recently developed keratinocyte cell lines that express human filaggrin in a tetracycline- regulated manner. This technique, now also available in retrovirus and adenovirus-based vectors, is applicable both to the study of genes that are toxic to cells and more generally to understand how genes regulate cell structure/function, growth, and differentiation.
Presland
Richard B
RB
Department of Oral Biology and Medicine, University of Seattle, WA, USA.
Fleckman
Philip
P
D002478:Cells, Cultured; D000091344:Filaggrin Proteins; D005786:Gene Expression Regulation / Q000187:drug effects*; D017930:Genes, Reporter; D006801:Humans; D007381:Intermediate Filament Proteins / Q000378:metabolism*; D015603:Keratinocytes / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D013752:Tetracycline / Q000494:pharmacology*; D014162:Transfection
C000717249:FLG protein, human; D000091344:Filaggrin Proteins; D007381:Intermediate Filament Proteins; D013752:Tetracycline