A tetracycline controlled activation/repression system with increased potential for gene transfer into mammalian cells

Background Tight control of gene activity has been achieved in cells and tr ansgenic organisms using the Tet regulatory systems. Unregulated basal tran scription can, however, be observed whenever integration of target genes dr iven by promoters responsive to tetracycline controlled transcriptional act ivators (tTA, rtTA) does not occur at suitable chromosomal sites. Moreover, in viral vectors containing both the tTA coding sequence and the regulated target gene, proximity of the enhancer element driving tTA/rtTA expression to the responsive unit will lead to elevated background levels. Similarly when tTA/rtTA responsive transcription units are in a nonintegrated state a s eg., during transient expression, intrinsic residual transcription persis ts in their 'off' state, which can differ in intensity among different cell types. Methods To efficiently repress such background activities we generated tetr acycline controlled transcriptional silencers (tTS) that bind promoters res ponsive for rtTA in absence of the effector doxycycline (Dox). Addition of Dox prevents binding of tTS thus relieving repression, promotes binding of rtTA and thereby switches the promoter from an actively repressed to an act ivated state. Results Of several tTS - fusions between a modified Tet repressor and trans criptional silencing domains - tTS(Kid) was found to be most effective in r educing the activity of two target promoters. Ten to 200 fold repression is seen in transient expression whereas in stably transfected HeLa cells the regulatory range of the rtTA system was increased by three orders of magnit ude. Conclusions The new system appears particularly suited for the transfer of toxic genes into appropriate chromosomal sites as well as for tight regulat ion of genes carried by viral or episomal vectors. Copyright (C) 1999 John Wiley & Sons Ltd.