Tetracycline-regulated highly inducible expression of the human prion protein in murine 3T3 cells

• Published in: Protein expression and purification 2005, Vol.39 (1), p.8-17
• Main Authors: Stuke, Andreas W., Strom, Alexander
• Format: Article
• Language: English
• Subjects: 3T3 Cells; 3T3 Tet-Off cells; Animals; Chromatography, Affinity; Gene Expression - drug effects; Gene Expression - physiology; Genetic Vectors; Glycosylation; Humans; IMAC; Mice; Prion protein; Prions - genetics; Prions - isolation & purification; Prions - metabolism; PRNP gene targeting; Protein Kinases - metabolism; Protein Synthesis Inhibitors - pharmacology; Tetracycline; Tetracycline - pharmacology; Transfection
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2004.09.013

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 P minCMV 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.