Bioassays in transgenic mice expressing human PrPC (129M)

The human PrP^N181Q/N197Q ORF, which contains 2 point mutations that change Asn to Gln in amino acid positions 181 and 197, thereby eliminating the two N-linked glycosylation sites on human PrP, was generated by PCR mutagenesis from the human PrP-129M transgene construct used for the generation of the Tg40 mice and described previously⁴⁰. The homozygous TgNN6h line expressing the human PrP^N181Q/N197Q ORF at about 60% of the PrP level found in wild type FVB mice was described previously¹². To assay for prion infectivity in the TgNN6h mice, 30 µl of the sample (brain homogenates or recombinanat human PrP preparations before or after QuIC) in PBS was injected into the mouse brain with a 26 gauge needle inserted to a depth of about 4 mm at the left parietal region of the cranium and the symptoms monitored as described previously⁴⁰. To prevent cross contamination, the intracerebral inoculations of synthetic prions were performed under sterile conditions in days when no other inoculations where carried out. The animals were housed in microisolator cages and stringent aseptic and biosafety techniques were followed for animal handling and husbandry as we described previously⁴⁰. Within three days after the appearance of symptoms or at death, the animal was euthanized and the brain was removed; one-half was frozen for biochemical studies, and the other half was stored in formalin for histology and immunohistochemistry analysis as described below.