In transmissible spongiform encephalopathy (TSE) pathogenesis the cellular prion protein (PrP(C)) is converted into its pathogenic PrP(Sc) isoform. Prion protein gene (Prnp) deficient mice (PrP(0/0)) are resistant to PrP(Sc) infection, but following reconstitution of Prnp they regain their susceptibility to infection. Therefore, it is challenging to simulate this natural situation in a cell culture model. We have previously reported the inducible stable expression of a human PrP(C) in murine 3T3 cells. In this study, we used murine PrP(0/0) cells stably expressing exemplarily the chimpanzee Prnp under the control of inducible tetracycline (Tet) system. The Prnp was integrated using a lentiviral vector. Its expression in the engineered PrP(0/0)Chimp1/Tet-Off cell line was analyzed by Western blot (Wb) and fluorescence activated cell sorting (FACS) analyses. PrP(C) was partially purified by using immobilized metal affinity chromatography (IMAC). Compared to all the other cell systems which possess an endogenous PrP(C) expression, here described cell line contains only an overexpressing species specific PrP(C) expression which is tightly regulated and can be turned-off at any time without showing any endogenous host PrP(C) expression. Consequently, a contamination of the isolated PrP(C) is impossible. This cell line potentially offers a new tool for simulation of mice bioassays widely used in TSE infection studies.