Gateway compatible vectors for analysis of gene function in the zebrafish

Authors

  • Jacques A. Villefranc,

    1. Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, Massachusetts
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    • J.A. Villefranc and Dr. Amigo contributed equally to this work.

  • Julio Amigo,

    1. Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, Massachusetts
    Current affiliation:
    1. Hematology Division, Department of Medicine, Brigham and Woman's Hospital and Harvard Medical School, Boston, MA 02115
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    • J.A. Villefranc and Dr. Amigo contributed equally to this work.

  • Nathan D. Lawson

    Corresponding author
    1. Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, Massachusetts
    • Program in Gene Function and Expression, University of Massachusetts Medical School, Lazare Research Building, Room 617, 364 Plantation Street, Worcester, MA 01605
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Abstract

The recent establishment of recombination-based cloning systems has greatly facilitated the analysis of gene function by allowing rapid and high-efficiency generation of plasmid constructs. However, the use of such an approach in zebrafish requires the availability of recombination-compatible plasmids that are appropriate for functional studies in zebrafish embryos. In this work, we describe the construction and validation of Gateway compatible vectors based on commonly used zebrafish plasmids. We have generated pCS-based plasmids that allow rapid generation of both N-terminal and C-terminal fusion proteins, and we demonstrate that mRNA synthesized from these plasmids encodes functional native or fusion proteins in injected zebrafish embryos. In parallel, we have established similar Gateway plasmids containing Tol2 cis elements that promote efficient integration into the zebrafish genome and allow expression of native or fusion proteins in a tissue-specific manner in the zebrafish embryo. Finally, we demonstrate the use of this system to rapidly identify tissue-specific cis elements to aid the establishment of blood vessel-specific transgenic constructs. Taken together, this work provides an important platform for the rapid functional analyses of open reading frames in zebrafish embryos. Developmental Dynamics 236:3077–3087, 2007. © 2007 Wiley-Liss, Inc.

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