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

Trihn, Le A. and Hochgreb, Tatiana and Graham, Matthew and Wu, David and Ruf-Zamojski, Frederique and Jayasena, Chathurani S. and Saxena, Ankur and Hawk, Rasheeda and Gonzales-Serricchio, Aidyl and Dixson, Alana and Chow, Elly and Gonzales, Constanza and Leung, Ho-Yin and Solomon, Ilana and Megason, Sean G. and Fraser, Scott E. and Bronner-Fraser, Marianne (2011) A versatile gene trap to visualize and interrogate the function of the vertebrate proteome. Genes and Development, 25 (21). pp. 2306-2320. ISSN 0890-9369. PMCID PMC3219234. https://resolver.caltech.edu/CaltechAUTHORS:20111129-075143013

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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.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1101/gad.174037.111DOIArticle
http://genesdev.cshlp.org/content/25/21/2306.abstractPublisherArticle
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3219234/PubMed CentralArticle
ORCID:
AuthorORCID
Graham, Matthew0000-0002-3168-0139
Saxena, Ankur0000-0001-8646-2887
Fraser, Scott E.0000-0002-5377-0223
Bronner-Fraser, Marianne0000-0003-4274-1862
Additional Information:© 2011 by Cold Spring Harbor Laboratory Press. The Authors acknowledge that six months after the full-issue publication date, the Article will be distributed under a Creative Commons CC-BY-NC License (Attribution-NonCommercial 4.0 International License, http://creativecommons.org/licenses/by-nc/4.0/). Received July 7, 2011; revised version accepted September 16, 2011. We thank P. Pantazis for comments on the manuscript, and members of the Fraser laboratory for helpful discussions. We are grateful to LeighAnn Fletcher for fish care. The T2KXIGd-in and pMDS-eGFP plasmids were provided by K. Kawakami and S. Parinov, respectively. This work was supported by NHGRI Center of Excellence in Genomic Science grant P50HG004071.
Funders:
Funding AgencyGrant Number
NIHP50HG004071
Subject Keywords:endogenous proteins; proteome; conditional mutagenesis; gene trap; genetic manipulation; gene knockdown
Issue or Number:21
PubMed Central ID:PMC3219234
Record Number:CaltechAUTHORS:20111129-075143013
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20111129-075143013
Official Citation:A versatile gene trap to visualize and interrogate the function of the vertebrate proteome Le A. Trinh, Tatiana Hochgreb, Matthew Graham, David Wu, Frederique Ruf-Zamojski, Chathurani S. Jayasena, Ankur Saxena, Rasheeda Hawk, Aidyl Gonzalez-Serricchio, Alana Dixson, Elly Chow, Constanza Gonzales, Ho-Yin Leung, Ilana Solomon, Marianne Bronner-Fraser, Sean G. Megason, and Scott E. Fraser Genes Dev. November 1, 2011 25: 2306-2320; doi:10.1101/gad.174037.111
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:27984
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:29 Nov 2011 17:44
Last Modified:09 Mar 2020 13:18

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