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The Zebrafish Tol2 System: A Modular and Flexible Gateway-Based Transgenesis Approach
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FLPe functions in zebrafish embryos.

Andrew C Wong1, Bruce W Draper, Alison L Van Eenennaam

  • 1Department of Animal Science, University of California, Davis, CA 95616, USA.

Transgenic Research
|June 17, 2010
PubMed
Summary
This summary is machine-generated.

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The FLP/FRT recombination system efficiently excises transgenes in zebrafish (Danio rerio) at 28.5°C. This study demonstrates FLPe recombinase effectiveness for genetic manipulation in zebrafish, with germline excision observed in most cases.

Area of Science:

  • Molecular Biology
  • Genetics
  • Developmental Biology

Background:

  • The FLP/FRT site-specific recombination system is a powerful tool for genetic manipulation.
  • Assessing its efficiency in zebrafish (Danio rerio) is crucial for advancing genetic studies in this model organism.

Purpose of the Study:

  • To evaluate the efficiency of the FLP/FRT site-specific recombination system in zebrafish.
  • To determine the efficacy of FLPe recombinase for transgene excision in zebrafish embryos and germline.

Main Methods:

  • Development of a transgenic zebrafish construct with a muscle-specific promoter driving EGFP flanked by FRT sites.
  • Microinjection of FLPe capped RNA into single-cell stage zebrafish embryos.
  • Analysis of EGFP expression, PCR, and DNA sequencing to confirm transgene excision and recombination.

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  • Assessment of germline transmission of the excised transgene through subsequent crosses.

    • Main Results:

    • FLPe microinjection resulted in significantly lower EGFP expression (7.7%) compared to controls (50%) at 48 hours post fertilization.
    • Retained fluorescence in embryos exhibited significant mosaicism.
    • PCR and DNA sequencing confirmed FLPe-mediated recombination and transgene excision.
    • Germline analysis showed 0-4% fluorescent embryos in clutches sired by FLPe-microinjected males, indicating efficient germline excision in most cases.

    Conclusions:

    • The FLPe recombinase is an efficient tool for site-specific recombination in zebrafish at 28.5°C.
    • The FLP/FRT system demonstrates high efficacy for transgene excision in both somatic and germline cells of zebrafish.
    • Mosaicism observed in some embryos suggests variability in FLPe activity within individual organisms.