A regulatory network for the efficient control of transgene expression.
BACKGROUND
Expression of heterologous genes in mammalian cells or organisms for therapeutic or experimental purposes often requires tight control of transgene expression. Specifically, the following criteria should be met: no background gene activity in the off-state, high gene expression in the on-state, regulated expression over an extended period, and multiple switching between on- and off-states.


METHODS
Here, we describe a genetic switch system for controlled transgene transcription using chimeric repressor and activator proteins functioning in a novel regulatory network. In the off-state, the target transgene is actively silenced by a chimeric protein consisting of multimerized eukaryotic transcriptional repression domains fused to the DNA-binding tetracycline repressor. In the on-state, the inducer drug doxycycline affects both the derepression of the target gene promoter and activation by the GAL4-VP16 transactivator, which in turn is under the control of an autoregulatory feedback loop.


RESULTS
The hallmark of this new system is the efficient transgene silencing in the off-state, as demonstrated by the tightly controlled expression of the highly cytotoxic diphtheria toxin A gene. Addition of the inducer drug allows robust activation of transgene expression. In stably transfected cells, this control is still observed after months of repeated cycling between the repressed and activated states of the target genes.


CONCLUSIONS
This system permits tight long-term regulation when stably introduced into cell lines. The underlying principles of this network system should have general applications in biotechnology and gene therapy.
Imhof
M O
MO
Laboratory of Molecular Biotechnology, Center for Biotechnology UNIL-EPFL, University of Lausanne, Switzerland.
Chatellard
P
P
Mermod
N
N
D000818:Animals; D002460:Cell Line; D004167:Diphtheria Toxin / Q000235:genetics / Q000378:metabolism*; D004318:Doxycycline / Q000494:pharmacology; D004921:Erythropoietin / Q000378:metabolism; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D051379:Mice; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D004167:Diphtheria Toxin; C070633:Gal-VP16; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D004921:Erythropoietin; D004318:Doxycycline