title: Creation of a reversible on/off system for site-specific in vivo control of exogenous gene activity in the renal glomerulus.
abstract: Using genetically engineered glomerular mesangial cells, an in vivo gene transfer approach was developed that specifically targets the renal glomerulus. By combining this system with a tetracycline (Tc)-responsive promoter, the present study aimed to create a reversible on/off system for site-specific in vivo control of exogenous gene activity within the glomerulus. In the Tc regulatory system, a Tc-controlled transactivator (tTA) encoded by a regulator plasmid induces target gene transcription by binding to a tTA-responsive promoter located in a response plasmid. Tc inhibits this tTA-dependent transactivation via its affinity for tTA. In double-transfected cells, therefore, the activity of a transgene can be controlled by Tc. Cultured rat mesangial cells were cotransfected with a regulator plasmid and a response plasmid that introduces a beta-galactosidase gene. In vitro, stable double-transfectant MtTAG cells exhibited no beta-galactosidase activity in the presence of Tc. However, following withdrawal of Tc from culture media, expression of beta-galactosidase was induced within 24 h. When Tc was again added, the expression was rapidly resuppressed. Low concentrations of Tc were sufficient to maintain the silent state of tTA-dependent promoter. MtTAG cells were then transferred into the rat glomeruli via renal artery injection. In the isolated chimeric glomeruli, expression of beta-galactosidase was induced ex vivo in the absence of Tc, whereas it was repressed in its presence. When Tc-pretreated MtTAG cells were transferred into the glomeruli of untreated rats, beta-galactosidase expression was induced in vivo within 3 days. Oral administration of Tc dramatically suppressed this induction. These data demonstrate the feasibility of using mesangial cell vectors combined with the Tc regulatory system for site-specific in vivo control of exogenous gene expression in the glomerulus.
authors.affiliation: Glomerular Engineering Unit, Department of Medicne, University College London Medical School, United Kingdom.
authors.forename: M
authors.initials: M
authors.lastname: Kitamura
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D005786:Gene Expression Regulation / Q000187:drug effects; D018014:Gene Transfer Techniques; D005818:Genetic Engineering; D005920:Glomerular Mesangium / Q000166:cytology / Q000378:metabolism*; D008297:Male; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D013752:Tetracycline / Q000494:pharmacology; D015534:Trans-Activators / Q000378:metabolism; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000096:biosynthesis
chemical_list: D015534:Trans-Activators; D001616:beta-Galactosidase; D013752:Tetracycline