FLPe functions in zebrafish embryos
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FLPe functions in zebrafish embryos Andrew C. Wong • Bruce W. Draper Alison L. Van Eenennaam
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Received: 31 July 2009 / Accepted: 19 May 2010 / Published online: 15 June 2010 Ó Springer Science+Business Media B.V. 2010
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 A. C. Wong A. L. Van Eenennaam (&) Department of Animal Science, University of California, Davis, CA 95616, USA e-mail: [email protected] B. W. Draper Department of Molecular and Cellular Biology, University of California, Davis, CA 95616, USA
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. Keywords Zebrafish
FLP FLPe Recombinase
Abbreviations EGFP Enhanced green fluorescent protein FLPe Enhanced FLP recombinase FRT FLP recognition target hpf Hours post fertilization
Introduction Already established as an important developmental model, zebrafish (Danio rerio) are emerging as a powerful genetic model as well. Zebrafish possess certain characteristics that make them ideally suited for developmental and genetic studies, namely short generation time, large number of offspring, and optical clarity of externally fertilized embryos. Embryogenesis is finished in about 24 h, organogenesis is completed in about 5 days, and sexual maturity is
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Transgenic Res (2011) 20:409–415
reached at 3 months. A single female can provide hundreds of embryos at a time, either by natural crosses or by in vitro fertilization. Eggs are externally fertilized, develop synchronously outside of the mother and remain optically transparent up to
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