Protein knockouts in living eukaryotes using deGradFP and green fluorescent protein fusion targets

Curr Protoc Protein Sci. 2013 Sep 24;73:30.2.1-30.2.13. doi: 10.1002/0471140864.ps3002s73.

Abstract

This unit describes deGradFP (degrade Green Fluorescent Protein), an easy-to-implement protein knockout method applicable in any eukaryotic genetic system. Depleting a protein in order to study its function in a living organism is usually achieved at the gene level (genetic mutations) or at the RNA level (RNA interference and morpholinos). However, any system that acts upstream of the proteic level depends on the turnover rate of the existing target protein, which can be extremely slow. In contrast, deGradFP is a fast method that directly depletes GFP fusion proteins. In particular, deGradFP is able to counteract maternal effects in embryos and causes early and fast onset loss-of-function phenotypes of maternally contributed proteins.

Keywords: Drosophila; F-Box; GFP; nanobodies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Drosophila melanogaster / genetics
  • Gene Knockout Techniques / methods*
  • Green Fluorescent Proteins / chemistry
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism*
  • Protein Stability
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism*

Substances

  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins