Methods for monitoring autophagy using GFP-LC3 transgenic mice

Methods Enzymol. 2009;452:13-23. doi: 10.1016/S0076-6879(08)03602-1.

Abstract

Several methods are now available for monitoring autophagy. Although biological methods are useful for cultured cells and homogenous tissues, these methods are not suitable for determining the autophagic activity of each cell type in heterogeneous tissues. Furthermore, intracellular localization of autophagosomes often provides valuable information. Thus, morphological assays are still important in many studies. Although electron microscopy has been the gold standard, recent studies of the molecular mechanism of autophagy have led to the development of several marker proteins for autophagosomes, the most widely used of which is LC3, a mammalian homolog of Atg8. These marker proteins allow identification of autophagic structures by fluorescence microscopy. This method has been applied to whole animals by generating green fluorescent protein (GFP)-LC3 transgenic mice. This chapter describes the background and practicality of, and possible precautions in the application of, this method using the GFP-LC3 transgenic mouse model.

MeSH terms

  • Animals
  • Autophagy / physiology*
  • Biological Assay / methods*
  • Genotype
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Mice
  • Mice, Transgenic
  • Microtubule-Associated Proteins / genetics
  • Microtubule-Associated Proteins / metabolism*
  • Polymerase Chain Reaction
  • Reproducibility of Results

Substances

  • Map1lc3b protein, mouse
  • Microtubule-Associated Proteins
  • Green Fluorescent Proteins