title: Fluorescent fusion protein knockout mediated by anti-GFP nanobody.
abstract: 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.
authors.affiliation: Biozentrum, University of Basel, Basel, Switzerland. emmanuel.caussinus@unibas.ch
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Caussinus
authors.forename: Oguz
authors.initials: O
authors.lastname: Kanca
authors.forename: Markus
authors.initials: M
authors.lastname: Affolter
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D000906:Antibodies / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster / Q000196:embryology / Q000235:genetics / Q000378:metabolism*; D004625:Embryo, Nonmammalian / Q000196:embryology / Q000378:metabolism; D055786:Gene Knockout Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006367:HeLa Cells; D006657:Histones / Q000235:genetics / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008954:Models, Biological; D008969:Molecular Sequence Data; D050809:POU Domain Factors / Q000235:genetics / Q000378:metabolism; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014921:Wings, Animal / Q000196:embryology / Q000378:metabolism*
chemical_list: D000906:Antibodies; D003598:Cytoskeletal Proteins; D029721:Drosophila Proteins; D006657:Histones; D018398:Homeodomain Proteins; D008164:Luminescent Proteins; D050809:POU Domain Factors; D011993:Recombinant Fusion Proteins; C104121:nub protein, Drosophila; D049452:Green Fluorescent Proteins; C075578:nullo protein, Drosophila