RNAi screens of lysosomal trafficking

Methods Cell Biol. 2015:126:119-38. doi: 10.1016/bs.mcb.2014.10.030. Epub 2015 Jan 14.

Abstract

Here, we describe the general principles of RNA interference screens to study lysosomal functions in mammalian cells. Lysosomes occupy a central position in the biology of numerous processes such as degradation, microbial killing, and immunological antigen presentation to T cells. Selection of a screening system, conducting pooled versus arrayed screens, and appropriate steps in assay development, validation, and verification of novel gene candidates are all discussed. We focus on our experience in developing an arrayed short hairpin RNA screen to identify novel lysosomal trafficking proteins involved in vesicle and cargo trafficking and illustrate how such a trafficking library can be applied to screens involving lysosomes.

Keywords: Antigen presentation; CD1; Dendritic cell; Lysosome; Lysosome trafficking; MHC II; Macrophage; RNAi; RNAi screen; Vesicle trafficking; shRNA.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cells, Cultured
  • Gene Expression
  • Humans
  • Lysosomes / metabolism*
  • Protein Transport
  • Proteins / genetics*
  • Proteins / metabolism
  • RNA Interference*

Substances

  • Proteins
  • lysosomal proteins