Inducible and irreversible control of gene expression using a single transgene.
Experimental or therapeutic designs involving the conditional expression of genes often require the use of two different transgenes; this can represent a major undertaking. One of these systems takes advantage of inducible recombinases. Here we show a novel use of such enzymes, in that an inducible recombinase-encoding sequence can function to both block the transcription of a gene placed downstream and, subsequently, irreversibly activate transcription of this very same gene. This double function, which circumvents the need for two transgenes, can be achieved by flanking the inducible recombinase gene by two of its target sequences. In our design we used as the inducible recombinase gene the Cre-ER(T) gene flanked by two loxP sites. This cassette was placed between a mouse phosphoglycerate kinase promoter and the enhanced green fluorescent protein (EGFP) coding sequence. Massive EGFP gene expression in BHK cells bearing this transgene was observed upon administration of 4-hydroxytamoxifen (4-OHT), the inducer of the recombinant activity of Cre-ER(T). In the absence of 4-OHT EGFP production was prevented. Because of its simplicity (only a single transgene needs to be used) this strategy is of obvious interest in certain protocols of gene or cell therapy and in a variety of experimental designs in which conditional expression of genes is required.
Fuhrmann-Benzakein
E
E
Department of Morphology and Department of Pathology, University of Geneva Medical School, 1 Rue Michel-Servet, CH-1211 Geneva 4, Switzerland.
García-Gabay
I
I
Pepper
M S
MS
Vassalli
J D
JD
Herrera
P L
PL
D000818:Animals; D002460:Cell Line; D004247:DNA / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D049452:Green Fluorescent Proteins; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D016133:Polymerase Chain Reaction; D011960:Receptors, Estrogen / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013629:Tamoxifen / Q000031:analogs & derivatives* / Q000494:pharmacology; D014162:Transfection; D019076:Transgenes / Q000235:genetics*; D014764:Viral Proteins
D008164:Luminescent Proteins; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D013629:Tamoxifen; D049452:Green Fluorescent Proteins; C016601:afimoxifene; D004247:DNA; C045073:Cre recombinase; D019426:Integrases