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deGradFP: A System to Knockdown GFP-Tagged Proteins.
JournalArticle (Originalarbeit in einer wissenschaftlichen Zeitschrift)
ID 3644116
Author(s) Caussinus, Emmanuel; Affolter, Markus
Author(s) at UniBasel Affolter, Markus;
Year 2016
Title deGradFP: A System to Knockdown GFP-Tagged Proteins.
Journal Methods Mol Biol
Volume 1478
Pages / Article-Number 177-187
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].
ISSN/ISBN 1940-6029
Full Text on edoc
Digital Object Identifier DOI 10.1007/978-1-4939-6371-3_9
PubMed ID http://www.ncbi.nlm.nih.gov/pubmed/27730581