title: Tetracycline-regulated highly inducible expression of the human prion protein in murine 3T3 cells.
abstract: To provide an in vitro system that allows inducible or conditional overexpression of human prion protein (PrP), we have established a tetracycline (Tc)-regulated system in murine 3T3 L1 fibroblast cells. A replacement-type gene targeting vector cassette was constructed to express the human fatal familial insomnia (FFI) prion protein gene (PRNP) under control of a Tc-responsive element. Following stable integration of the vector into 3T3 Tet-Off cells, we have isolated and characterised six 3T3 L1 pTet-Off FFI clones. These clones were analysed by PCR and their expression level was determined by Western blot using species specific monoclonal antibodies (anti-mouse and human 3B5, 4F2, 12F10, 11C6, 8G8, and 14D3; anti-mouse l3). Addition of the antibiotic Tc to the culture medium turned off expression of human PrP. This supression was repeatedly reversible. However, no significant transcriptional leakiness of repressed PminCMV promoter was observed. In the absence of Tc, expression of human PrP was induced 10- to 20-fold as estimated from densitometric analyses. PrP was analysed by Proteinase K (PK) digestions and found to be PK sensitive. Subcellular fractionation revealed that PrP was located mainly in the cytoplasmic membrane fraction. Furthermore, we partially purified PrP by PrP-specific copper-binding. After immobilised metal affinity chromatography, majority of PrP showed a molecular weight consistent with non-glycosylated PrP. These clones offer a new tool to facilitate the investigation of PrP interaction with potential cellular ligands and PrP ex vivo propagation.
authors.affiliation: German Primate Centre (DPZ), Department of Virology and Immunology, Kellnerweg 4, D-37077 Göttingen, Germany. astuke@dpz.gwdg.de
authors.forename: Andreas W
authors.initials: AW
authors.lastname: Stuke
authors.forename: Alexander
authors.initials: A
authors.lastname: Strom
mesh_terms: D016475:3T3 Cells; D000818:Animals; D002846:Chromatography, Affinity; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology*; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D051379:Mice; D011328:Prions / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D013752:Tetracycline / Q000494:pharmacology; D014162:Transfection
chemical_list: D011328:Prions; D011500:Protein Synthesis Inhibitors; D011494:Protein Kinases; D013752:Tetracycline