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ZFIN ID: ZDB-PUB-100621-27
FLPe functions in zebrafish embryos
Wong, A.C., Draper, B.W., and Van Eenennaam, A.L.
Date: 2011
Source: Transgenic Research   20(2): 409-415 (Journal)
Registered Authors: Draper, Bruce, Van Eenennaam, Alison L., Wong, Andrew C.
Keywords: FLP, FLPe, Recombinase, Zebrafish
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*
PubMed: 20552273 Full text @ Transgenic. Res.
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 degrees C.
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