Fig 1.
Passage history of scrapie sources used in this study.
For the serial passage of scrapie prions in mice, the brain of a single mouse was chosen from the previous passage. The selected mice were named Mo1, Mo2, and Mo3 in Exp. 1 and Mo1′, Mo2′, and Mo3′ in Exp. 2. Mice that clinically and/or pathologically developed L-type disease are shown in grey, while those that developed S-type disease are shown in white. Survival days are indicated underneath each mouse. PrPSc-positive and -negative GT1-7 cells are shown in grey and white, respectively.
Table 1.
Infection of mice with L- and S-type prions with or without passaging in GT1-7 cells.
Fig 2.
Monitoring PrPSc accumulation in diseased mice throughout passaging.
(A) Representative western blot of PrPSc in mouse brains at different passages. Experiment number, survival days, passage number, disease phenotype, and lane number are indicated on the top of the panel. Each lane included 0.2 mg brain equivalents. (B) Immunoprecipitation assay with mAb 3H6. Lane number is indicated on the top of the panel. In lane 9, uninfected brain homogenate was probed with mAb 3H6. In all other lanes, PrPSc was detected with mAb T2-HRP. Molecular markers are shown on the left side of each panel.
Fig 3.
Representative images of PrPSc deposition types associated with each prion phenotype in ICR mice.
Sections of the hippocampus were subjected to immunostaining of PrPSc and H&E staining. Typical PrPSc distribution patterns and histopathology of the hippocampus affected with L-type (A and B) and S-type (C and D) prions are shown. Sections of the hippocampi of mice inoculated with cell homogenates prepared from GT/Mo1 (E and F), GT/Mo2 (G, H, I and J), GT/Mo3 (K and L), GT/Mo1 ′ (M and N), GT/Mo2′ (O and P), and GT/Mo3′ (Q and R) were also subjected to immunostaining of PrPSc and H&E staining. Immunohistochemical detection of PrPSc was performed using the mAb SAF84.
Fig 4.
Glycoform profiles of PrPSc in mice infected with GT1-7 cell culture-derived S-type and L-type prions.
PrPSc glycoform percentages of original mouse brains used for the infection of GT1-7 cells (Mo1, Mo2, Mo3, Mo1′, Mo2′, and Mo3′) were compared to those of mouse brains inoculated with GT1-7 cell homogenates (GT/Mo1, GT/Mo2, GT/Mo3, GT/Mo1′, GT/Mo2′, and GT/Mo3′). Results of GT1-7 cell homogenate-inoculated mice are shown as the mean ± standard deviation (n = 4). The PrPSc glycoprofile of GT/Mo2-inoculated mice in this graph shows the mean glycoform ratio of the mice that developed S-type disease. The inoculum (brain or GT1-7 cells) was intracerebrally injected into ICR mice. Experiment numbers are the same as in Fig 1.bar graph shows di-glycosylated (black columns), mono-glycosylated (gray columns), and unglycosylated (white columns) forms of PrPSc. L- and S-type at the bottom of each lane indicate the disease phenotypes. PrPSc was detected with mAb T2-HRP.
Table 2.
Comparison of the susceptibility of GT1-7 cells and ICR mice to L- and S-type prions.
Fig 5.
Illustration of breakdown during the transmission of L-type prions in mice and effective amplification in GT1-7 cells.
Grey mice and cells indicate the presence of L-type prions, while white mice and cells indicate S-type prions. Mice exhibiting the L-type disease still harbor S-type scrapie prions, resulting in the appearance of mice with the S-type disease at every passage from P2 to P4. GT1-7 cells accumulate PrPSc in response to L-type prions and can be used to re-propagate mice with L-type prions by injecting mice with cell homogenates. In contrast, GT1-7 cells challenged with S-type prions do not accumulate PrPSc, but they do harbor very low levels of S-type prion infectivity.