title: A versatile gene trap to visualize and interrogate the function of the vertebrate proteome.
abstract: 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.
authors: Beckman Institute, Division of Biology, California Institute of Technology, Pasadena, USA. ltrinh@caltech.edu
authors: Le A
authors: le A
authors: Trinh
authors: Tatiana
authors: T
authors: Hochgreb
authors: Matthew
authors: M
authors: Graham
authors: David
authors: D
authors: Wu
authors: Frederique
authors: F
authors: Ruf-Zamojski
authors: Chathurani S
authors: CS
authors: Jayasena
authors: Ankur
authors: A
authors: Saxena
authors: Rasheeda
authors: R
authors: Hawk
authors: Aidyl
authors: A
authors: Gonzalez-Serricchio
authors: Alana
authors: A
authors: Dixson
authors: Elly
authors: E
authors: Chow
authors: Constanza
authors: C
authors: Gonzales
authors: Ho-Yin
authors: HY
authors: Leung
authors: Ilana
authors: I
authors: Solomon
authors: Marianne
authors: M
authors: Bronner-Fraser
authors: Sean G
authors: SG
authors: Megason
authors: Scott E
authors: SE
authors: Fraser
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D030562:Databases, Protein; D004625:Embryo, Nonmammalian; D005822:Genetic Vectors; D020407:Internet; D008164:Luminescent Proteins / Q000235:genetics; D058977:Molecular Sequence Annotation; D009154:Mutation; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015027:Zebrafish
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D020543:Proteome; D011993:Recombinant Fusion Proteins; C434479:citrine protein, bacteria