NR AOTW
AU Heinig,L.; Strom,A.; Stuke,A.W.
TI Cell lines over-expressing PRNP genes of different species to characterise homo- and heterologous prions
QU International Conference - Prion diseases: from basic research to intervention concepts - TSE-Forum, 08.10.-10.10.2003, Gasteig, München - Poster session - DG-36
PT Konferenz-Poster
AB
During TSE pathogensis the cellular prion protein (PrPc) is converted by the pathogenic isoform (PrPsc) into insoluble PrP aggregates. This conversion requires PrPc as basic protein. Data with permanet PrPsc infected cells showed that over-expression of PrPc facilitates the conversion. In contrast PrP-knockout (PrP0/0) cell lines cannot be infected. In animal models a species-barrier was proposed since homologous PrPsc inoculums displayed shortend incubation period. The incubation times are explained by the different primery sequences and PrPsc conformations.
In order to provide an ex vivo system for the replication of PrPsc and to examine the species-barrier on a molecular level we have established a tetracycline (Tc)-regulated cell system over-expressing different prion protein (PRNP) genes. A vector cassette was constructed to over-express the human fatal familial insomnia (FFI) PRNP gene under the control of the Tc-responsive element. A stable integration was isolated from murine 3T3 Tet-Off fibroblast cells and characterised. The expression level was determined with anti-mouse and anti-human monoclonal antibodies in Western blots. In the absence of Tc the insert expression was induced twenty-fold and in the presence no expression was observed. This cell line produce membrane anchored and un-, mono- and di-glycosylated human PrP displaying proteinase K (PK) sensitivity. To create additional cell lines the PRNP gene in the vector was changed against the PRNP genes from mice, cow, sheep, elk, rhesus monkey, crab-eating macaque, gorilla, chimp, pygmy chimp and human. These constructs will be stably integrated into murine PrP0/0 cells with no endogenous PrPc and also Tet-Off cell lines form other species. The clones will be infected with murine, bovine and human prions and net amplification will be monitored by PK digestion and spectroscopy. These cells will be a new tool for PrP ex vivo propagation, its conversion and its interaction with cellular receptors.
AD Lars Heinig, Alexander Strom, Andreas W. Stuke, German Primte Centre, Germany
SP englisch
PO Deutschland