Inducible expression based on regulated recombination: a single vector strategy for stable expression in cultured cells.
When fused to the ligand binding domain (LBD) of steroid hormone nuclear receptors, site-specific recombinases (SSRs) acquire a ligand-dependent activity. Here, we describe the use of SSR-LBD fusion proteins in an inducible expression system, introduced into cells in a single step. A single transgene contains a constitutively active, bi-directional enhancer/promoter, which directs expression, on one side, of an SSR-LBD fusion protein gene and, on the other, a selectable marker/inducible gene cassette. The selectable marker, the puromycin acetyltransferase (pac) gene, is used for stable genomic integration of the transgene and is flanked by recombination target sites. The inducible gene is not expressed because the pac gene lies between it and the promoter. Activation of the SSR-LBD by a ligand induces recombination and the pac gene is excised. The inducible gene is thus positioned next to the promoter and so is expressed. This describes a ligand-inducible expression strategy that relies on regulated recombination rather than regulated transcription. By inducible expression of diptheria toxin, evidence that this system permits inducible expression of very toxic proteins is presented. The combination of the complete regulatory circuit and inducible gene in one transgene relates expression of the selectable marker gene to expression from the bi-directional enhancer/promoter. We exploit this relationship to show that graded increases in selection pressure can be used to select for clones with different induction properties.
Angrand
P O
PO
Gene Expression Program, EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.
Woodroofe
C P
CP
Buchholz
F
F
Stewart
A F
AF
D002460:Cell Line; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D007700:Kinetics; D011691:Puromycin / Q000494:pharmacology; D011995:Recombination, Genetic
D011691:Puromycin; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase