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The standard scrapie cell assay: development, utility and prospects.

van der Merwe J, Aiken J, Westaway D, McKenzie D - Viruses (2015)

Bottom Line: The SSCA facilitates quantitative in vitro analysis of prion strains, titres and biological properties.Given its robust nature and potential for high throughput, the SSCA has substantial utility for in vitro characterization of prions and can be deployed in a number of settings.Here we provide an overview on establishing the SSCA, its use in studies of disease dissemination and pathogenesis, potential pitfalls and a number of remaining challenges.

View Article: PubMed Central - PubMed

Affiliation: Centre for Prions and Protein Folding Diseases, University of Alberta, Alberta, T6G2M8, Canada. jqvander@ualberta.ca.

ABSTRACT
Prion diseases are a family of fatal neurodegenerative diseases that involve the misfolding of a host protein, PrPC. Measuring prion infectivity is necessary for determining efficacy of a treatment or infectivity of a prion purification procedure; animal bioassays are, however, very expensive and time consuming. The Standard Scrapie Cell Assay (SSCA) provides an alternative approach. The SSCA facilitates quantitative in vitro analysis of prion strains, titres and biological properties. Given its robust nature and potential for high throughput, the SSCA has substantial utility for in vitro characterization of prions and can be deployed in a number of settings. Here we provide an overview on establishing the SSCA, its use in studies of disease dissemination and pathogenesis, potential pitfalls and a number of remaining challenges.

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Related in: MedlinePlus

CWD Isolate Titration in Elk21- Cells. Elk21- cells were exposed to serial dilutions of two different CWD isolates (elk, 132M, and white-tailed deer, 95Q/96G) and the generation of PrPSc determined by ELISPOT analysis following three passages of the cells. Elk21- cells demonstrated a dose-dependent PrPSc spot response to elk and white-tailed deer CWD. N = 6, mean ± SEM.
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viruses-07-00180-f002: CWD Isolate Titration in Elk21- Cells. Elk21- cells were exposed to serial dilutions of two different CWD isolates (elk, 132M, and white-tailed deer, 95Q/96G) and the generation of PrPSc determined by ELISPOT analysis following three passages of the cells. Elk21- cells demonstrated a dose-dependent PrPSc spot response to elk and white-tailed deer CWD. N = 6, mean ± SEM.

Mentions: Cell lines used in the SSCA exhibit distinct susceptibility to prion strains (Table 1). Similar to LD9 cells [14], L929 cells generate PrPSc in response to 22L, ME7 and RML prions (Figure 1). Importantly, these results demonstrate comparable responses to the already published LD9 subline [14,45]. In contrast, PK1 [14] and RK13moPrP cell lines generate PrPSc in response to RML and 22L prions but not ME7. While the SSCA was initially limited to murine cell lines infected with murine prions, a non-murine cell line compatible with the SSCA is the recently developed elk PrPC-expressing RK13 cell line (Elk21-) [48]. Based on a report demonstrating retroviral Gag-enhanced release of PrPSc from scrapie-infected cell lines [56], the elk PrPC RK13 line was further transfected with pcDNA3-gag, expressing the HIV-1 GAG precursor protein. This resulted in a cell line that, upon infection with elk CWD, exhibited an ~2-fold increase in PrPSc generation [48,56]. The Elk21- cell line generates PrPSc in response to both elk and white-tailed deer CWD [48], the latter generating significantly fewer PrPSc-positive cells compared to elk CWD [48] (Figure 2). The development of additional cervid cell lines will, therefore, help to further evaluate various CWD isolates from both deer and elk. To the best of our knowledge, there are no cell lines currently available that can be infected with primary isolates of BSE or CJD prions.


The standard scrapie cell assay: development, utility and prospects.

van der Merwe J, Aiken J, Westaway D, McKenzie D - Viruses (2015)

CWD Isolate Titration in Elk21- Cells. Elk21- cells were exposed to serial dilutions of two different CWD isolates (elk, 132M, and white-tailed deer, 95Q/96G) and the generation of PrPSc determined by ELISPOT analysis following three passages of the cells. Elk21- cells demonstrated a dose-dependent PrPSc spot response to elk and white-tailed deer CWD. N = 6, mean ± SEM.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4306833&req=5

viruses-07-00180-f002: CWD Isolate Titration in Elk21- Cells. Elk21- cells were exposed to serial dilutions of two different CWD isolates (elk, 132M, and white-tailed deer, 95Q/96G) and the generation of PrPSc determined by ELISPOT analysis following three passages of the cells. Elk21- cells demonstrated a dose-dependent PrPSc spot response to elk and white-tailed deer CWD. N = 6, mean ± SEM.
Mentions: Cell lines used in the SSCA exhibit distinct susceptibility to prion strains (Table 1). Similar to LD9 cells [14], L929 cells generate PrPSc in response to 22L, ME7 and RML prions (Figure 1). Importantly, these results demonstrate comparable responses to the already published LD9 subline [14,45]. In contrast, PK1 [14] and RK13moPrP cell lines generate PrPSc in response to RML and 22L prions but not ME7. While the SSCA was initially limited to murine cell lines infected with murine prions, a non-murine cell line compatible with the SSCA is the recently developed elk PrPC-expressing RK13 cell line (Elk21-) [48]. Based on a report demonstrating retroviral Gag-enhanced release of PrPSc from scrapie-infected cell lines [56], the elk PrPC RK13 line was further transfected with pcDNA3-gag, expressing the HIV-1 GAG precursor protein. This resulted in a cell line that, upon infection with elk CWD, exhibited an ~2-fold increase in PrPSc generation [48,56]. The Elk21- cell line generates PrPSc in response to both elk and white-tailed deer CWD [48], the latter generating significantly fewer PrPSc-positive cells compared to elk CWD [48] (Figure 2). The development of additional cervid cell lines will, therefore, help to further evaluate various CWD isolates from both deer and elk. To the best of our knowledge, there are no cell lines currently available that can be infected with primary isolates of BSE or CJD prions.

Bottom Line: The SSCA facilitates quantitative in vitro analysis of prion strains, titres and biological properties.Given its robust nature and potential for high throughput, the SSCA has substantial utility for in vitro characterization of prions and can be deployed in a number of settings.Here we provide an overview on establishing the SSCA, its use in studies of disease dissemination and pathogenesis, potential pitfalls and a number of remaining challenges.

View Article: PubMed Central - PubMed

Affiliation: Centre for Prions and Protein Folding Diseases, University of Alberta, Alberta, T6G2M8, Canada. jqvander@ualberta.ca.

ABSTRACT
Prion diseases are a family of fatal neurodegenerative diseases that involve the misfolding of a host protein, PrPC. Measuring prion infectivity is necessary for determining efficacy of a treatment or infectivity of a prion purification procedure; animal bioassays are, however, very expensive and time consuming. The Standard Scrapie Cell Assay (SSCA) provides an alternative approach. The SSCA facilitates quantitative in vitro analysis of prion strains, titres and biological properties. Given its robust nature and potential for high throughput, the SSCA has substantial utility for in vitro characterization of prions and can be deployed in a number of settings. Here we provide an overview on establishing the SSCA, its use in studies of disease dissemination and pathogenesis, potential pitfalls and a number of remaining challenges.

Show MeSH
Related in: MedlinePlus