NR ATLX
AU Giri,R.K.; Carlson,G.A.
TI Mouse neurosphere cultures as bioassays for prion infectivity and for the study of prion replication and susceptibility
QU International Conference - Prion 2005: Between fundamentals and society's needs - 19.10.-21.10.2005, Congress Center Düsseldorf - Poster Session: Diagnosis DIA-61
PT Konferenz-Poster
AB
Few cell lines are susceptible to infection with prions, precluding in vitro analysis of the mechanisms underlying genetic differences in susceptibility to infection. Similarly, cell lines, such as mouse N2a, are resistant to some prion strains that are readily transmissible to mice. With one exception, sensitive bioassay of prions requires inoculation of mice with incubation times from months to over a year. Neurosphere lines grow as aggregates and contain CNS stem cell activity; we now report that these cultures can be infected with prions. Using a defined, serum-free medium, neurosphere lines were isolated from brains dissected from fetuses at embryonic day 12 to 15. In addition to expressing the stem cell-associated marker nestin, most cells from PrP transgenic or from wild-type mice express the normal isoform of PrP (PrPc), which is essential for prion replication. Scrapie brain homogenate (RML) was added to neurosphere cultures from FVB, FVB transgenic mice that overexpress mouse PrP (Tg4053), and FVB mice with a targeted null mutation in the PrP gene (Prnp). Presence of the proteinase K-resistant, misfolded PrPsc isoform was measured at each passage by Western, dot, or cell blots. A dramatic rise in PrPsc with time was observed in the Tg4053 cells while the level of PrPsc decayed with passage to undetectable levels in the cultures of cells lacking PrP; levels of PrPsc in FVB cultures persisted and then increased gradually over several passages. Prions produced in culture were transmissible to mice and produced typical scrapie pathology. Intracellular aggregates of PrP were seen in infected cultures. To date, infection of Tg4053 neurospheres by prion isolate diluted 1 to 50,000 has been achieved. Neurosphere lines from transgenic mice overexpressing PrP may provide a sensitive in vitro bioassay not only for mouse prions but also for prions from other species, including humans.
Supported by grants from the U.S. Department of Defense and NINDS, USPHS.
AD R.K.Giri, G.A.Carlson, McLaughlin Research Institute, Great Falls, MT 59405, USA
SP englisch
PO Deutschland