FLPe functions in zebrafish embryos

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

Research output: Contribution to journalArticle

17 Citations (Scopus)

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

Fingerprint

Zebrafish
Danio rerio
embryo (animal)
Embryonic Structures
Transgenes
transgenes
genetically modified organisms
germ cells
Recombinases
Genetic Recombination
Fluorescence
fluorescence
RNA
site-specific recombination
Muscles
muscles
Mosaicism
Microinjections
DNA Sequence Analysis
Fertilization

Keywords

  • FLP
  • FLPe
  • Recombinase
  • Zebrafish

ASJC Scopus subject areas

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

Cite this

Wong, A. C., Draper, B. W., & van Eenennaam, A. L. (2011). FLPe functions in zebrafish embryos. Transgenic Research, 20(2), 409-415. https://doi.org/10.1007/s11248-010-9410-9

FLPe functions in zebrafish embryos. / Wong, Andrew C.; Draper, Bruce W.; van Eenennaam, Alison L.

In: Transgenic Research, Vol. 20, No. 2, 04.2011, p. 409-415.

Research output: Contribution to journalArticle

Wong, AC, Draper, BW & van Eenennaam, AL 2011, 'FLPe functions in zebrafish embryos', Transgenic Research, vol. 20, no. 2, pp. 409-415. https://doi.org/10.1007/s11248-010-9410-9
Wong AC, Draper BW, van Eenennaam AL. FLPe functions in zebrafish embryos. Transgenic Research. 2011 Apr;20(2):409-415. https://doi.org/10.1007/s11248-010-9410-9
Wong, Andrew C. ; Draper, Bruce W. ; van Eenennaam, Alison L. / FLPe functions in zebrafish embryos. In: Transgenic Research. 2011 ; Vol. 20, No. 2. pp. 409-415.
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