FLPe functions in zebrafish embryos

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.

Fig. 1

a Schematic drawing of FRT mylz2-EGFP construct for assaying FLPe-mediated site-specific recombination. Black triangles represent FRT sites. The Tol2Kit (Kwan et al. 2007), which utilizes Gateway cloning (Invitrogen), was used to create this construct. Tol2 sites at either end of the construct facilitated zebrafish transgenesis. b Founder zebrafish embryos microinjected with the construct, FRT mylz2-EGFP, displayed mosaicism by 48 hpf. c Hemizygous F₁ embryos derived from mosaic founder fish that genotyped positive for the transgene in their sperm displayed even distribution of EGFP expression throughout

Fig. 2

Mosaic EGFP expression after FLPe recombinase RNA microinjection. Forty-eight hours post fertilization, mosaic expression of fluorescence can be observed in FLPe RNA-injected transgenic zebrafish embryos (a, b). Individual muscle cells that have not had the FRT-flanked transgene excised can be seen fluorescing green. In contrast, non-injected transgenic controls (c, d), show evenly distributed EGFP expression throughout the muscles of the zebrafish. e PCR confirmation of recombinase-mediated site-specific recombination. Genomic DNA from finclips of FLPe RNA-injected transgenic zebrafish was used as a DNA template for PCR to confirm excision of the FRT-flanked EGFP transgene. In FLPe-injected zebrafish (1–8), site-specific recombination produced an 803-bp fragment, indicative of excision. In the non-injected transgenic control, an unexcised 3.7-kb band is expected. Excision products were cloned and sequenced to verify precise FLPe-mediated site-specific recombination in zebrafish