A versatile gene trap to visualize and interrogate the function of the vertebrate proteome
- Le A. Trinh1,5,
- Tatiana Hochgreb1,
- Matthew Graham2,
- David Wu1,
- Frederique Ruf-Zamojski1,3,
- Chathurani S. Jayasena1,
- Ankur Saxena1,
- Rasheeda Hawk1,
- Aidyl Gonzalez-Serricchio1,
- Alana Dixson1,
- Elly Chow1,
- Constanza Gonzales1,
- Ho-Yin Leung1,
- Ilana Solomon1,
- Marianne Bronner-Fraser1,
- Sean G. Megason1,4 and
- Scott E. Fraser1
- 1Beckman Institute, Division of Biology, and
- 2Center for Advanced Computing Research, California Institute of Technology, Pasadena, California 91125, USA
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.
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Footnotes
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↵Corresponding author.
5E-mail ltrinh{at}caltech.edu.
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Supplemental material is available for this article.
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Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.174037.111.
- Received July 7, 2011.
- Accepted September 16, 2011.
- Copyright © 2011 by Cold Spring Harbor Laboratory Press