Rapid retroviral delivery of tetracycline-inducible genes in a single autoregulatory cassette.
We describe a single autoregulatory cassette that allows reversible induction of transgene expression in response to tetracycline (tet). This cassette contains all of the necessary components previously described by others on two separate plasmids that are introduced sequentially over a period of months [Gossen, M. & Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89, 5547-5551]. The cassette is introduced using a retrovirus, allowing transfer into cell types that are difficult to transfect. Thus, populations of thousands of cells, rather than a few clones, can be isolated and characterized within weeks. To avoid potential interference of the strong retroviral long terminal repeat enhancer and promoter elements with the function of the tet-regulated cytomegalovirus minimal promoter, the vector is self-inactivating, eliminating transcription from the long terminal repeat after infection of target cells. Tandem tet operator sequences and the cytomegalovirus minimal promoter drive expression of a bicistronic mRNA, leading to transcription of the gene of interest (lacZ) and the internal ribosome entry site controlled transactivator (Tet repressor-VP16 fusion protein). In the absence of tet, there is a progressive increase in transactivator by means of an autoregulatory loop, whereas in the presence of tet, gene expression is prevented. Northern blot, biochemical, and single cell analyses have all shown that the construct yields low basal levels of gene expression and induction of one to two orders of magnitude. Thus, the current cassette of the retroviral construct (SIN-RetroTet vector) allows rapid delivery of inducible genes and should have broad applications to cultured cells, transgenic animals, and gene therapy.
Hofmann
A
A
Department of Molecular Pharmacology, Stanford University School of Medicine, CA 94305-5332, USA.
Nolan
G P
GP
Blau
H M
HM
D000818:Animals; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000187:drug effects / Q000235:genetics*; D004742:Enhancer Elements, Genetic; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000187:drug effects*; D017405:Herpes Simplex Virus Protein Vmw65 / Q000096:biosynthesis / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008979:Moloney murine leukemia virus; D009132:Muscles; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000096:biosynthesis / Q000378:metabolism*; D012190:Retroviridae; D013752:Tetracycline / Q000494:pharmacology*; D014158:Transcription, Genetic; D014162:Transfection; D001616:beta-Galactosidase / Q000096:biosynthesis*
D017405:Herpes Simplex Virus Protein Vmw65; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D001616:beta-Galactosidase; D013752:Tetracycline