Journal Article

Fluorescent fusion protein knockout mediated by anti-GFP nanobody

Affolter, Markus, Kanca, Oguz, Caussinus, Emmanuel

2012

Overview

The combination of an F-box domain with a single-domain antibody that recognizes green fluorescent protein (GFP) now allows controlled depletion of GFP fusions in mammalian cells and in flies. This system, called deGradFP, should be widely useful, as GFP fusions are available for many proteins in model organisms. The use of genetic mutations to study protein functions in vivo is a central paradigm of modern biology. Recent advances in reverse genetics such as RNA interference and morpholinos are widely used to further apply this paradigm. Nevertheless, such systems act upstream of the proteic level, and protein depletion depends on the turnover rate of the existing target proteins. Here we present deGradFP, a genetically encoded method for direct and fast depletion of target green fluorescent protein (GFP) fusions in any eukaryotic genetic system. This method is universal because it relies on an evolutionarily highly conserved eukaryotic function, the ubiquitin pathway. It is traceable, because the GFP tag can be used to monitor the protein knockout. In many cases, it is a ready-to-use solution, as GFP protein-trap stock collections are being generated in Drosophila melanogaster and in Danio rerio .

• Publisher: Nature Publishing Group US,Nature Publishing Group
• Subject: 631/1647/1888/2249 / 631/337/474/2085 / Amino Acid Sequence / Animals / Animals, Genetically Modified / Antibodies - immunology / Base Sequence / Biochemistry / Biological Microscopy / Biomedical and Life Sciences / Blotting, Western / Cytoskeletal Proteins - genetics / Cytoskeletal Proteins - metabolism / Drosophila melanogaster - embryology / Drosophila melanogaster - genetics / Drosophila melanogaster - metabolism / Drosophila Proteins - genetics / Drosophila Proteins - metabolism / Embryo, Nonmammalian - embryology / Embryo, Nonmammalian - metabolism / Eukaryotes / Gene Knockout Techniques / Genetics / Green fluorescent protein / Green Fluorescent Proteins - genetics / Green Fluorescent Proteins - immunology / Green Fluorescent Proteins - metabolism / HeLa Cells / Histones - genetics / Histones - metabolism / Homeodomain Proteins - genetics / Homeodomain Proteins - metabolism / Humans / Insects / Life Sciences / Luminescent Proteins - genetics / Luminescent Proteins - metabolism / Membrane Biology / Microscopy, Confocal / Models, Biological / Molecular biology / Molecular Sequence Data / Mutation / Physiological aspects / POU Domain Factors - genetics / POU Domain Factors - metabolism / Protein Structure / Proteins / Proteolysis / Recombinant Fusion Proteins - genetics / Recombinant Fusion Proteins - metabolism / Structure / technical-report / Ubiquitin / Wings, Animal - embryology / Wings, Animal - metabolism