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, Scott E. Fraser

Research output: Contribution to journalArticlepeer-review

90 Scopus citations

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.

Original languageEnglish
Pages (from-to)2306-2320
Number of pages15
JournalGenes and Development
Volume25
Issue number21
DOIs
StatePublished - 1 Nov 2011
Externally publishedYes

Keywords

  • Conditional mutagenesis
  • Endogenous proteins
  • Gene knockdown
  • Gene trap
  • Genetic manipulation
  • Proteome

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