FLPe functions in zebrafish embryos.
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.
Wong
Andrew C
AC
Department of Animal Science, University of California, Davis, CA 95616, USA.
Draper
Bruce W
BW
Van Eenennaam
Alison L
AL
D000818:Animals; D030801:Animals, Genetically Modified; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D004625:Embryo, Nonmammalian / Q000378:metabolism*; D005260:Female; D005306:Fertilization / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D008845:Microinjections; D009030:Mosaicism; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D019076:Transgenes / Q000502:physiology*; D015027:Zebrafish / Q000235:genetics / Q000378:metabolism*
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase