A versatile gene trap to visualize and interrogate the function of the vertebrate proteome

We report a multifunctional gene-trapping approach, which generates full-length Citrine fusions with endogenous proteins and conditional mutants from a single integration event of the FlipTrap vector. We identified 170 FlipTrap zebrafish lines with diverse tissue-specific expression patterns and dis...

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Published inGenes & development Vol. 25; no. 21; pp. 2306 - 2320
Main Authors Trinh, Le A, Hochgreb, Tatiana, Graham, Matthew, Wu, David, Ruf-Zamojski, Frederique, Jayasena, Chathurani S, Saxena, Ankur, Hawk, Rasheeda, Gonzalez-Serricchio, Aidyl, Dixson, Alana, Chow, Elly, Gonzales, Constanza, Leung, Ho-Yin, Solomon, Ilana, Bronner-Fraser, Marianne, Megason, Sean G, Fraser, Scott E
Format Journal Article
LanguageEnglish
Published United States Cold Spring Harbor Laboratory Press 01.11.2011
Subjects
Animals
Bacterial Proteins - genetics
Danio rerio
Databases, Protein
Embryo, Nonmammalian
Genetic Vectors
Internet
Luminescent Proteins - genetics
Molecular Sequence Annotation
Mutation
Proteome
Proteomics - methods
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - metabolism
Resource/Methodology
Zebrafish
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Summary:We report a multifunctional gene-trapping approach, which generates full-length Citrine fusions with endogenous proteins and conditional mutants from a single integration event of the FlipTrap vector. We identified 170 FlipTrap zebrafish lines with diverse tissue-specific expression patterns and distinct subcellular localizations of fusion proteins generated by the integration of an internal citrine exon. Cre-mediated conditional mutagenesis is enabled by heterotypic lox sites that delete Citrine and "flip" in its place mCherry with a polyadenylation signal, resulting in a truncated fusion protein. Inducing recombination with Cerulean-Cre results in fusion proteins that often mislocalize, exhibit mutant phenotypes, and dramatically knock down wild-type transcript levels. FRT sites in the vector enable targeted genetic manipulation of the trapped loci in the presence of Flp recombinase. Thus, the FlipTrap captures the functional proteome, enabling the visualization of full-length fluorescent fusion proteins and interrogation of function by conditional mutagenesis and targeted genetic manipulation.
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Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.
Present addresses: 3Department of Basic Neurosciences, University of Geneva Medical Center, CH-1211 Geneva 4, Switzerland
ISSN:0890-9369
1549-5477
DOI:10.1101/gad.174037.111