RT-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