UNIT 30.2 Protein Knockouts in Living Eukaryotes Using deGradFP and Green Fluorescent Protein Fusion Targets

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. Curr. Protoc. Protein Sci. 73:30.2.1-30.2.13. - 2013 by John Wiley & Sons, Inc.