Figure 1
In vitro seeded amplification of CWD prions from organotypic slice cultures using the RT-QuIC assay and PMCA. (A) RT-QuIC seeding curves showing that slice cultures from Tg12 mice (prnp+/−) efficiently seed the RT-QuIC assay producing strong positive peaks for the reactions seeded with 5 ng of CWD-infected slice culture homogenates at 42 dpi (red trace, labeled as Tg12 Slices CWD) or brain homogenates (BH) from a CWD-infected deer (blue trace, labeled as CWD BH). Slices cultured with NBH (grey trace) and prnp−/− slices cultured with CWD BH (black trace) or NBH (green trace) did not show seeding activity during the reaction period. Each trace is a combined average of 3 technical replicates and 2 biological replicates from a pool of 7–10 slices. (B) A representative full-length Western blot from the third of three serial rounds of PMCA performed on BH from the CWD slice culture model. All samples were treated with PK, except NBH from Tg5037 mice (NBH PK (−) in the first lane) used as a migration (positive) control. Only the positive control and Tg12 slices inoculated with CWD deer BH show immunoreactivity. (C) Representative PrPCWD immunohistochemistry (6H4 antibody) images showing a cross-section of 35-dpi slices. Slices from Tg12 mice inoculated with CWD deer BH showed immunoreactivity (top image), whereas NBH did not (bottom image). Scale bar, 10 µm. (D) Representative full-length Western blot showing the presence of PK-resistant CWD prions in Tg12, but not prnp−/−, slice culture homogenates, all probed with the anti-prnp mouse monoclonal antibody POM1. (E) DHE conversion assay showing the significantly higher generation of reactive oxygen species (ROS) for CWD BH-infected cerebellar slices when compared to NBH-treated or prnp−/− slice cultures. Data were analyzed using one-way ANOVA; n = 4 biological replicates. Data are mean ± SEM (*p ≤ 0.05, **p < 0.01, ***P < 0.001).
