To screen for PrP amyloid seeding activity in tg66 brains we performed RT-QuIC analysis on brain homogenates from tg66 mice inoculated with each GSS mutant. For the RT-QuIC assay we used bank vole as a substrate and followed established protocols . Brains derived from 2 tg66 mice infected with vCJD were run as positive controls (Fig. 6). Brain tissue from the 4 Y226X-inoculated mice that previously had detectable PrPres deposition showed strong seeding activity following a short lag phase (less than 30 h) in 4/4 wells tested for each mouse (Fig. 6). In contrast, brains from Y226X-inoculated mice that tested negative for prion deposition by WB and IHC did not show significant seeding activity by RT-QuIC prior to 45 h of testing. Interestingly, despite the presence of PrPres deposition by IHC, none of the 7 G131V-inoculated tg66 mice tested by RT-QuIC had detectable seeding activity. Six Q227X-inoculated tg66 mice were also screened by RT-QuIC and did not have detectable seeding activity. Thus, transmission of the G131V and Q227X diseases to tg66 mice was not supported by these RT-QuIC data (Fig. 6).Fig. 6RT-QuIC analysis of tg66 mouse brain following inoculation with human brain expressing GSS mutations Y226X, Q227X and G131V. RT-QuIC was performed on tg66 mouse brain homogenates using bank vole recPrP substrate. Curves show the average of the fluorescence values for 4 replicate wells at each time-point. In each panel, two tg66 mice inoculated with vCJD were run as positive controls (▲ and ▼). a Y226X-inoculated tg66 mice. Open symbols show the four Y226X inoculated tg66 mice which were PrPSc-positive by immunoblot and IHC (B322–1, B323–1, B323–3, B324–1). The asterisk and black circle depict two Y226X mice (B322–2, B324–2) which gave low level fluorescence in one well out of 4 after 30 h and did not meet criteria for a positive reaction (see Methods). Two other Y226X mice (B323–2, B326–5), which were negative for PrPSc by immunoblot and IHC, and two uninoculated tg66 mice are collectively shown with the solid black diamond symbol, and all four had no fluorescence above background. b Q227X-inoculated tg66 mice. Two mice (B295–2, B296–2) had low level fluorescence between 40 and 50 h but did not meet criteria for a positive reaction. The remaining four Q227X mice tested (B295–1, B295–3, B296–3, B340–1) and two uninoculated tg66 mice were negative and are shown with the solid black diamond. c G131V-inoculated tg66 mice. The seven G131V-inoculated tg66 mice tested (B297–2, B297–3, B297–4, B298–1, B298–2, B303–1, B632–1) and two uninoculated tg66 mice all showed no seeding activity, indicated by the solid black diamond