Generation of Infectious Prions and Detection With the Prion-Infected Cell Assay

Methods Mol Biol. 2017;1658:105-118. doi: 10.1007/978-1-4939-7244-9_9.

Abstract

Cell lines propagating prions are an efficient and useful means for studying the cellular and molecular mechanisms implicated in prion disease. Utilization of cell-based models has led to the finding that PrPC and PrPSc are released from cells in association with extracellular vesicles known as exosomes. Exosomes have been shown to act as vehicles for infectivity, transferring infectivity between cell lines and providing a mechanism for prion spread between tissues. Here, we describe the methods for generating a prion-propagating cell line with prion-infected brain homogenate, cell lysate, conditioned media, and exosomes and also detection of protease-resistant PrP with the prion-infected cell assay.

Keywords: Cell culture; Exosomes; Extracellular vesicles; GT1-7; Infection; PICA; PrP; Prion; Prion-infected cell assay; RK13.

MeSH terms

  • Animals
  • Cell Line
  • Cloning, Molecular
  • Culture Media, Conditioned / chemistry
  • Endopeptidase K / chemistry
  • Exosomes / chemistry*
  • Exosomes / pathology
  • Gene Expression
  • High-Throughput Screening Assays*
  • Humans
  • Hypothalamus / metabolism
  • Hypothalamus / pathology
  • Immunoblotting / methods*
  • Mice
  • Neurons / metabolism*
  • Neurons / pathology
  • Plasmids / chemistry
  • Plasmids / metabolism
  • PrPC Proteins / chemistry
  • PrPC Proteins / genetics*
  • PrPC Proteins / metabolism
  • PrPSc Proteins / chemistry
  • PrPSc Proteins / genetics*
  • PrPSc Proteins / metabolism
  • Protein Folding
  • Rabbits
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism

Substances

  • Culture Media, Conditioned
  • PrPC Proteins
  • PrPSc Proteins
  • Recombinant Proteins
  • Endopeptidase K