title: deGradFP: A System to Knockdown GFP-Tagged Proteins.
abstract: Protein depletion by genetic means, in a very general sense including the use of RNA interference [1, 2] or CRISPR/Cas9-based methods, represents a central paradigm of modern biology to study protein functions in vivo. However, acting upstream the proteic level is a limiting factor if the turnover of the target protein is slow or the existing pool of the target protein is important (for instance, in insect embryos, as a consequence of a strong maternal contribution). In order to circumvent these problems, we developed deGradFP [3, 4]. deGradFP harnesses the ubiquitin-proteasome pathway to achieve direct depletion of GFP-tagged proteins. deGradFP is in essence a universal method because it relies on an evolutionarily conserved machinery for protein catabolism in eukaryotic cells; see refs. 5, 6 for review. deGradFP is particularly convenient in Drosophila melanogaster where it is implemented by a genetically encoded effector expressed under the control of the Gal4 system. deGradFP is a ready-to-use solution to perform knockdowns at the protein level if a fly line carrying a functional GFP-tagged version of the gene of interest is available. Many such lines have already been generated by the Drosophila community through different technologies allowing to make genomic rescue constructs or direct GFP knockins: protein-trap stock collections [7, 8] ( http://cooley.medicine.yale.edu/flytrap/ , http://www.flyprot.org/ ), P[acman] system [9], MiMIC lines [10, 11], and CRISPR/Cas9-driven homologous recombination.Two essential controls of a protein knockdown experiment are easily achieved using deGradFP. First, the removal of the target protein can be assessed by monitoring the disappearance of the GFP tag by fluorescence microscopy in parallel to the documentation of the phenotype of the protein knockdown (see Note 1 ). Second, the potential nonspecific effects of deGradFP can be assessed in control fly lacking a GFP-tagged target protein. So far, no nonspecific effects of the deGradFP effector have been reported [3].
authors.affiliation: Institute of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland.
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Caussinus
authors.affiliation: Growth & Development, Biozentrum, University of Basel, Room 200B, Klingelbergstrasse 50/70, 4056, Basel, Switzerland. markus.affolter@unibas.ch.
authors.forename: Markus
authors.initials: M
authors.lastname: Affolter
mesh_terms: D000818:Animals; D029721:Drosophila Proteins / Q000235:genetics* / Q000378:metabolism; D004331:Drosophila melanogaster / Q000235:genetics* / Q000378:metabolism; D004625:Embryo, Nonmammalian; D044783:F-Box Proteins / Q000235:genetics / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D008856:Microscopy, Fluorescence; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D059748:Proteolysis; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D019076:Transgenes; D000097573:Red Fluorescent Protein
chemical_list: D029721:Drosophila Proteins; D044783:F-Box Proteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D046988:Proteasome Endopeptidase Complex; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D000097573:Red Fluorescent Protein; C557617:GAL4 protein, Drosophila; C055002:GAL80 protein, S cerevisiae
keywords: F-Box; GFP; Gene expression; Nanobodies; Proteasome