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Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans
Published on: January 8, 2015
Prion protein gene expression in cultured cells
M R Scott1, D A Butler, D E Bredesen
1Department of Neurology, University of California, San Francisco 94143.
Abstract:
A single copy gene encodes both the scrapie (PrPSc) and cellular (PrPC) isoforms of the prion protein (PrP). Cultured cell lines were found to express the endogenous PrP mRNA at levels comparable to those observed in the brains of adult rodents; however, these cells were invariably found to express greatly reduced levels of PrP. In all the cell lines examined, PrP was undetectable by Western immunoblot analysis. These cells were also poor recipients for expression constructs linking the hamster PrP gene open reading frame to several strong eukaryotic promoters; stable clones derived by transfection of these expression vectors failed to show elevated expression of PrP. When extremely high levels of PrP mRNA were produced using either an insect baculovirus or a mammalian SV40 based vector, significant quantities of PrP were produced, although in both cases the proteins were apparently processed differently from the PrPC observed in brains. In an expression system using an SV40 late promoter vector in monkey COS-7 cells, a significant fraction of PrP was transported to the cell surface where PrPC is found in vivo. PrP synthesized by the baculovirus vector failed to induce scrapie in hamsters and did not possess the characteristics of the PrPSc isoform associated with infectivity. The SV40 late promoter vector system may permit experiments designed to elucidate the role of PrPSc during scrapie infection as well as the function of PrPC in normal metabolism.
Insights
Researchers investigated prion protein (PrP) expression in cell lines. They found that while PrP mRNA levels were normal, PrP protein levels were very low, suggesting post-transcriptional regulation is crucial for PrP expression.
Area of Science:
- Molecular Biology
- Neuroscience
- Biochemistry
Background:
- The prion protein (PrP) exists in two isoforms: cellular (PrPC) and scrapie (PrPSc).
- PrPSc is associated with transmissible spongiform encephalopathies like scrapie.
- Understanding PrP expression is key to understanding these diseases.
Purpose of the Study:
- To investigate the expression levels of PrP in cultured cell lines.
- To determine if cell lines can be used to study PrP processing and function.
- To explore methods for achieving higher PrP expression in vitro.
Main Methods:
- Cultured cell lines were analyzed for endogenous PrP mRNA and protein levels.
- Transfection experiments using expression vectors with strong eukaryotic promoters were performed.
- High-level PrP expression was induced using baculovirus and SV40-based vectors.
Main Results:
- Cultured cells showed PrP mRNA levels comparable to rodent brains but significantly reduced PrP protein levels, often undetectable by Western immunoblot.
- Transfection with standard expression vectors did not lead to elevated PrP expression.
- High-level expression using baculovirus or SV40 vectors produced PrP, but with altered processing compared to brain PrPC.
- SV40 vector-expressed PrP showed cell surface transport, while baculovirus-expressed PrP did not induce scrapie in hamsters.
Conclusions:
- Cultured cell lines exhibit post-transcriptional regulation of PrP expression.
- Mammalian expression systems like the SV40 late promoter vector show promise for studying PrPSc roles and PrPC function.
- Further research is needed to fully understand PrP processing and its implications in prion diseases.
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