NR AWFQ
AU Giri,R.K.; Young,R.; Orr,M.; Pitstick,R.; Carlson,G.A.
TI Genetic differences in susceptibility of neurosphere cultures to prion infection
QU International Conference - Prion 2006: Strategies, advances and trends towards protection of society - 3.10.-6.10.2006, Torino, Italy, Lingotto Conference Centre - Poster sessions GEN-12
PT Konferenz-Poster
AB Prion protein genotype (Prnp in mice) is the most important determinant of susceptibility and disease phenotype in every known prion disorder. Background genes other than Prnp also significantly influence incubation time in mice and chromosomal locations have been determined for several of these modifier loci. Due to the limited genetic diversity and aneuploidy of prion infectible cell lines and the consequent reliance on expensive and time-consuming bioassays in mice, there is only limited understanding of the cellular mechanisms underlying genetic differences in susceptibility. CNS-stem cell-containing neurosphere cultures were produced to test their suitability for studying the genetics of susceptibility to prions. Efficiency of infection, spread from cell to cell, and rate of prion replication can be discriminated in neurosphere cultures using filter-based immmunostaining for PK-resistant PrPsc or conformation-dependent immunocytochemistry (Giri et al., Proc Natl Acad Sci USA 2006,103:3875). The RML strain of prions produces short incubation times in mice expressing the a allele of Prnp and long incubation times in Prnpb mice. Neurosphere lines were isolated from C57Bl/6J (B6), B6.I-Prnpb, Tg(MoPrP-A)B4053, and Tg(MoPrP-B)C2091 mice; the transgenic lines express comparable levels of the alternative PrP allotypes. Infection could be established at higher dilutions of RML prions and spread of infection through the cultures was more rapid in neurosphere lines expressing PrP-A than those expressing PrP-B, reflecting the short and long incubation times of the mice from which they were derived. For example, by passage 3, most neurosphere colonies were PrPsc-positive in TgB4053 cells incubated with isolate diluted 10 (exp-8). The PrP-B producing neurospheres were far less sensitive to infection, with de novo PrPsc production achieved only by dilutions of 10 (exp-4) to 10 (exp-5). To our knowledge, this is the first demonstration of allelic differences in prion susceptibility in mice modeled in tissue culture.
AD McLaughlin Research Institute, Great Falls, Montana USA. E-mail: gac@po.mri.montana.edu
SP englisch
PO Italien