Transmission experiments were carried out by intracerebral injection of patient brain homogenate into tg66 transgenic mice expressing human PrP. Homogenates were made from three-day formalin-fixed paraffin-embedded sections as described in the methods. At various times starting at 77dpi, injected mice were euthanized for examination of brain tissue by IHC. Mice studied at 77, 240 and 509 dpi were negative for PrPSc by IHC and showed no clinical signs suggestive of neurological disease (not shown). However, from 593 to 762 dpi, 4 of 8 mice tested had PrPSc detectable both by IHC using monoclonal antibody 3F4 and immunoblot by using the phosphotungstic acid (PTA) enrichment method with detection by 3F4 (Table 1).Table 1Transmission study of human, genetic mutant Y226X PrP isolate to tg66 transgenic mice expressing human PrPMouse numberDPIPrPSc IHCPrPSc western blotClinical TSE suspectClinical notes (reason for euth) & relevant necropsy findingsB321–377a–ntNonormalB325–277a–ntNonormalB326–1240a–ntNonormalB326–2240a–ntNonormalB326–3509a–ntNonormalB326–4509a–ntNonormalB323–1593++Noinjury necessitating euthanasiaB322–1601++Yesurine scalding, ataxic, poor nest, thin, dilated, thickened uterus, consolidated lung lobeB322–2609––Yesthin, circling, kyphosis, poor nesting, ataxic, tippy-toed gaitB323–2680––Yesthin, hunched, progressive paraparesis, bilaterally distended and inflamed uterusB324–1716++Notremor, mild ataxia, abnormal respirations, good body conditionB323–3718++YesWeight loss, poor coat quality, hunched postureB326–5720a––Noold age, thinB324–2762––Yesthin, hunched, wobblya- Indicates mice that were stereotactically microinjected into the striatum. As described in the methods, this technique was used to facilitate possible early detection of PrPSc replication at the site of the needle track, as was previously demonstrated . 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 or when they developed conditions requiring euthanasia for humane reasons. nt = not tested