FLPe functions in zebrafish embryos

Transgenic Res. 2011 Apr;20(2):409-15. doi: 10.1007/s11248-010-9410-9. Epub 2010 Jun 15.

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Animals, Genetically Modified*
  • DNA Nucleotidyltransferases / genetics
  • DNA Nucleotidyltransferases / metabolism*
  • Embryo, Nonmammalian / metabolism*
  • Female
  • Fertilization / genetics
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Male
  • Microinjections
  • Mosaicism
  • Recombination, Genetic
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / metabolism
  • Transgenes / physiology*
  • Zebrafish / genetics
  • Zebrafish / metabolism*

Substances

  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • DNA Nucleotidyltransferases
  • FLP recombinase