Following intracerebral injection of tg66 mice with brain homogenate of this patient, mice were followed for clinical status weekly, but no mice with clinical signs suggestive of prion disease were noted at any time (Table 3). Individual mice were euthanized at various time-points between 77 and 798 dpi, but no PrPSc was detected in any mice by immunoblotting of brain tissue (Fig. 1b). IHC examination of brain for PrPSc deposition was also negative, and there was no gray matter vacuolation typical of prion diseases (Fig. 3). Thus, there was no evidence for transmission of prion infectivity to tg66 mice from the brain tissue of the Q227X patient.Table 3Transmission study of human, genetic mutant Q227X PrP to tg66 transgenic mice expressing human PrPMouse numberDPIPrPSc IHCPrPSc westernblotClinical TSE suspectClinical notes (reason for euth) & relevant necropsy findingsB302–277a–ntNonormalB299–177a–ntNonormalB302–3239a–ntNonormalB302–4239a–ntNonormalB304–1529a–ntNonormalB304–2529a–ntNonormalB630–1545––Nolung neoplasiaB296–1650––Noeye neoplasiaB295–1696––Noinjured, thin, ataxic, adequate nesting, lymphomaB296–2716––Noinjured, thin, barrel rolled twice, aware and responsiveB295–2743––Nodistended abdomen, liver neoplasiaB296–3756––NothinB306–3782a–ntNonormalB306–4782a–ntNonormalB340–1784––Nourine scaldingB295–3798––Nonormala- Indicates mice that were stereotactically microinjected into the striatum. Mice injected using this technique were euthanized electively and tissues were processed to directly screen the injection needle track and adjacent brain by IHC for any PrP replication. Mice without asterisks were intracerebrally inoculated with a 30ul volume of brain homogenate and euthanized when they developed neurologic signs consistent with prion infection (none) or conditions requiring euthanasia for humane reasons or at the termination of the experiment at 782 and 798 dpiFig. 3Histology and immunohistochemical staining of PrP in two brain regions of a tg66 mouse injected with Q227X brain homogenate at 743 dpi, and an uninfected aged control tg66 mouse (age 649 days). Panels a, c, e, g show PrP staining with biotinylated-3F4 antibody, and panels b, d, f, h show H&E staining. Uninfected cortex (a, b), Q227X injected cortex (c, d), uninfected pons (e, f), Q227X-injected pons (g, h). Panels a and c show darker tan staining than panels e and g due to a higher level of background PrPC in cortex compared to pons. No prion disease vacuoles or significant deposits suggestive of PrPSc were observed in uninfected or the Q227X-injected mice. Scale bar in panel a is 50 μm and is valid for all panels