Autoregulated multicistronic expression vectors provide one-step cloning of regulated product gene expression in mammalian cells.
Regulated expression of a cloned gene often provides much higher final expression of the gene product. Also, regulated expression of an activity can enable optional metabolic engineering and simplify functional genomic research. We constructed di-, tri-, and quattrocistronic mammalian expression vectors which allow the simultaneous, coordinated, and adjustable expression of up to two product genes. A single, tetracycline-regulatable promoter, PhCMV*-1, drives high-level expression of a multicistronic expression unit, containing the product gene(s), the gene for tetracycline-responsive transactivator (tTA), and, in the case of pQuattro-tTA, also the neomycin resistance gene. This autoregulatory genetic configuration retains a very low basal transcription activity in the presence of tetracycline, thereby reducing or eliminating possible toxic effects of tTA expression. However, upon withdrawal of tetracycline, a positive feedback regulation loop is activated which leads to higher levels of tTA expression and consequently also to higher expression levels of all other cistrons encoded on the multicistronic expression unit. Since such multicistronic expression vectors combine all genetic elements necessary for high-level expression as well as regulation in a single multicistronic expression unit, they alleviate limitations of previously reported tetracycline-regulatable vector systems and allow straightforward, one-step genetic engineering of eucaryotic cells to give an adjustable phenotype under strict control of the external stimulus, here tetracycline. Because the expression vectors described here were used for the expression for several heterologous product genes such as the green fluorescent protein and the tumor suppressor gene p21 in several cell lines (CHO-K1, BHK-21, and HeLa), we expect these multicistronic, positive feedback regulation vectors to function in a wide variety of eucaryotic cells and to be useful for basic as well as for applied research applications. Other vectors based upon the same autoregulation and multicistronic expression concepts can be constructed using other regulator gene-regulated promoter elements.
Fussenegger
M
M
Institute of Biotechnology, ETH Zurich, Switzerland.
Moser
S
S
Mazur
X
X
Bailey
J E
JE
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D005246:Feedback; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D005796:Genes; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007668:Kidney; D008164:Luminescent Proteins / Q000235:genetics; D015183:Restriction Mapping; D013752:Tetracycline / Q000494:pharmacology; D015534:Trans-Activators / Q000235:genetics; D014162:Transfection
D008164:Luminescent Proteins; D015534:Trans-Activators; D049452:Green Fluorescent Proteins; D013752:Tetracycline