Serial PMCA does not detect spontaneously formed PrP^Sc in Tg(FFI) and Tg(CJD) brains, but the mutant PrPs can be converted into PrP^Sc<i>in vitro</i>.

<p>(A and B) Homogenates of 3–4 pooled brains of C57BL/6J (Non-Tg), Tg(FFI-K5^+/-)/<i>Prnp</i>^0/0, Tg(FFI-26^+/-)/<i>Prnp</i>^0/0, Tg(CJD-A21^+/-)/<i>Prnp</i>^0/0 and Tg(CJD-66^+/-)/<i>Prnp</i>^0/0 mice were subjected to serial rounds of PMCA without (-) or with (+) a PrP^Sc inoculum (RML seed). Ten rounds of 48 PMCA cycles were done, and the samples were digested with 80 μg/ml PK before Western blot with anti-PrP antibody SAF84. (C and D) Brain lysates from Tg<i>a</i>20 mice inoculated with the reaction products of 17 rounds of PMCA (unseeded or seeded with RML, as indicated) were incubated with 0–20 μg of PK for 30 min at 37°C, and PrP was visualized by Western blotting using antibody 12B2. The undigested samples (0 μg/ml PK) represent 10 μg of protein, and the other samples 50 μg. Mice were killed at 86 (Non-Tg seeded), 558 (FFI-26 unseeded and CJD-66 seeded), 573 (FFI-26 seeded) and 611 (CJD-66 unseeded) days post-inoculation.