NR ATHV
AU Barenco,M.G.; Valori,C.; Schubert,A.; Hammann,J.; Crugnola,A.; Löwer,J.; Weissmann,C.; Montrasio,F.; Rossi,D.
TI Generation and Characterization of a Novel PrP Knockout Cell Line
QU International Conference - Prion 2005: Between fundamentals and society's needs - 19.10.-21.10.2005, Congress Center Düsseldorf - Poster Session: Cell Biology of PrPc and PrPsc CELL-27
PT Konferenz-Poster
AB
The cellular prion protein (PrPc) has been proposed to play a role in several cellular mechanisms, such as apoptosis, copper homeostasis, oxidative stress, cell adhesion, cell differentiation and cell signalling. In order to develop in vitro models for studying prion protein-dependent pathways, we have first generated a novel PrP knockout cell line by transducing cerebellar primary cell cultures isolated from PrP knockout mice (Zürich I) with a retroviral vector carrying an expression cassette for a temperature-sensitive form of the SV40 Large T Antigen. After isolation of a clonal cell line (PrP0/0 ML), we investigated by immunofluorescence microscopy the expression of lineage-specific cell markers at permissive and non-permissive temperatures. We further investigated the effects of serum deprivation as well as that of the addition of growth factors on the differentiation potential of this cell line. Here we show that at the permissive temperature the PrP0/0 ML cell line express nestin, an intermediate filament protein that is abundantly expressed in neuroepithelial stem cells. However, the expression of this marker is strongly reduced upon non-permissive conditions. In addition, we demonstrate that the PrP0/0 ML cells are unipotent neuronal precursors which can specifically differentiate into neurons under non-permissive temperature, low serum concentration and BDNF-treatment, as shown by the expression of several neuronal markers.
In a second step, we reconstituted PrPc expression in PrP0/0 ML cell line by introducing a CMV promoter-driven murine PrP-encoding expression cassette. Clones expressing low, average, and high PrPc levels were generated. The role of PrP in neuronal differentiation, cell survival, and signalling is currently being investigated by studies analyzing PrP-knockout and PrP-expressing cells.
AD M.G.Barenco, A.Schubert, Joanna Hammann (hamjo@pei.de), J.Löwer, F.Montrasio, Prion Research Group, Paul-Ehrlich-Institut, Langen, Germany; C.Valori, A.Crugnola, D.Rossi, Center of Excellence on Neurodegenerative Diseases, Department of Pharmacological Sciences, University of Milan, Milan, Italy; C.Weissmann, Department of Infectology, Scripps Florida Research Institute, Schmidt Building, 777 Glades Road, Boca Raton FL 33431, USA
SP englisch
PO Deutschland