Related Experiment Videos
In vitro expression in eukaryotic cells of a prion protein gene cloned from scrapie-infected mouse brain
1Laboratory of Persistent Viral Diseases, National Institute of Allergy and Infectious Diseases, Hamilton, MT 59840.
Abstract:
It has been proposed that the causative agent of scrapie represents a class of infectious particle that is devoid of nucleic acid and that an altered form of the endogenous prion protein (PrP) is the agent. However, it has been difficult to exclude the possibility that PrP purified from scrapie tissues might be contaminated with a more conventional viral agent. To obtain PrP uncontaminated by scrapie-infected tissues, PrP cDNA cloned from a scrapie-infected mouse brain was expressed in mouse C127 cells in vitro. mRNA and protein encoded by the cloned PrP gene were identified. The expressed PrP polypeptides appeared to be glycosylated and were released from the cell surface into the medium. Homogenates of the cells expressing the cloned PrP gene were inoculated into susceptible mice but failed to induce clinical signs of scrapie. Thus, either PrP is not the transmissible agent of scrapie or the expressed PrP requires additional modification to be infectious.
Insights
Researchers explored if the prion protein (PrP) causes scrapie. Expressing PrP in cells and injecting it into mice did not cause scrapie, suggesting PrP alone may not be the infectious agent.
Area of Science:
- Neuroscience
- Molecular Biology
- Infectious Diseases
Background:
- Scrapie is a transmissible spongiform encephalopathy.
- The prion protein (PrP) is implicated as the infectious agent, but contamination with conventional viruses has been a concern.
Purpose of the Study:
- To investigate if the prion protein (PrP) is the causative agent of scrapie.
- To produce and test PrP free from scrapie-infected tissues.
Main Methods:
- Cloned PrP cDNA from a scrapie-infected mouse brain.
- Expressed PrP in mouse C127 cells in vitro.
- Inoculated susceptible mice with homogenates of cells expressing PrP.
Main Results:
- Identified mRNA and glycosylated PrP polypeptides expressed and released from cells.
- Mice inoculated with expressed PrP did not develop scrapie.
- The expressed PrP failed to induce clinical signs of scrapie.
Conclusions:
- The prion protein (PrP) expressed in vitro may not be the transmissible agent of scrapie.
- Alternatively, the expressed PrP might require further modification to become infectious.