FLPe functions in zebrafish embryos

Andrew C. Wong, Bruce W. Draper, Alison L. van Eenennaam

Research output: Contribution to journalArticle

18 Scopus citations

Abstract

To assay the efficiency of the FLP/FRT site-specific recombination system in Danio rerio, a construct consisting of a muscle-specific promoter driving EGFP flanked by FRT sites was developed. FLPe capped RNA was microinjected into transgenic single cell stage zebrafish embryos obtained by crossing hemizygous transgenic males with wild-type females. By 48 h post fertilization (hpf), the proportion of embryos displaying green fluorescence following FLPe RNA microinjection was significantly lower (7.7%; P < 0.001) than would be expected from a cross in the absence of the recombinase (50%). Embryos that retained fluorescence displayed marked mosaicism. Inheritance of the excised transgene in non-fluorescent, transgenic embryos was verified by PCR analysis and FLPe-mediated recombination was confirmed by DNA sequencing. Sperm derived from confirmed transgenic males in these experiments was used to fertilize wild-type eggs to determine whether germline excision of the transgene had occurred. Clutches sired by FLPe-microinjected males contained 0-4% fluorescent embryos. Transgenic males that were phenotypically wild-type produced no fluorescent progeny, demonstrating complete excision of the transgene from their germline. FLPe microinjected males that retained some fluorescent muscle expression produced a small proportion of fluorescent offspring, suggesting that in mosaic males not all germline cells had undergone FLPe-mediated transgene excision. Our results show that FLPe, which is derived from Saccharomyces cerevisiae, is an efficient recombinase in zebrafish maintained at 28.5°C.

Original languageEnglish (US)
Pages (from-to)409-415
Number of pages7
JournalTransgenic Research
Volume20
Issue number2
DOIs
StatePublished - Apr 2011

Keywords

  • FLP
  • FLPe
  • Recombinase
  • Zebrafish

ASJC Scopus subject areas

  • Biotechnology
  • Genetics
  • Agronomy and Crop Science
  • Animal Science and Zoology

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