Construction and characterization of a one-plasmid system for the controlled expression of genes in mammalian cells by tetracycline.
The two-plasmid system of Gossen and Bujard [Gossen and Bujard (1992) Proc. Natl. Acad. Sci. USA 89, 5547-5551] to express mammalian genes in a tetracycline-repressed fashion was combined into a single-plasmid system. Two variants of this single-plasmid system that differ in the multiple cloning site (MCS) region are described. These vectors were used to stably transfect raf kinase domain into the normal rat kidney epithelial cell line (NRKE) to obtain a conditionally transformed cell line. These vectors were also used to stably transfect wild-type and mutant human p53 into the human osteosarcoma cell line, SAOS-2. Tetracycline repressed gene expression in both cell lines; about 12-fold in NRKE and about 80-fold in SAOS-2 cell line.
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Cancer Research, Lilly Research Laboratories, A Division of Eli Lilly and Company, Indianapolis, IN 46285, USA. obrien_kathryn_a@Lilly.com
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D000818:Animals; D015153:Blotting, Western; D017931:DNA Primers / Q000737:chemistry; D005786:Gene Expression Regulation / Q000235:genetics*; D006801:Humans; D007668:Kidney / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf; D051381:Rats; D013752:Tetracycline / Q000494:pharmacology*; D014162:Transfection / Q000235:genetics; D014170:Transformation, Genetic / Q000235:genetics; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D011518:Proto-Oncogene Proteins; D016159:Tumor Suppressor Protein p53; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D013752:Tetracycline