For each PrP mutant human brain sample, three first passage tg66 mice were selected for second passage into tg66 mice. Tg66 mouse brains were homogenized to a 20% homogenate as described in the immunoblotting section. For injection, the 20% brain homogenates were further diluted to a 1% homogenate in a phosphate buffered balanced salt solution with 2% fetal bovine serum. Each recipient mouse was anesthetized with isoflurane and intracerebrally injected with 30 μL of 1% homogenate. Nine to 12 recipient mice were injected per donor mouse. Following inoculation, mice are being monitored for onset of prion disease signs as described above.