Germline transmission of R2Ol in zebrafish. a A Founder F0 fish was crossed with WT to obtain F1 progenies. F1 embryos or adults were used for the germline mosaicism analysis. b PCR screening of 3′ junction of R2Ol-positive fish (Red asterisks) using genomic DNA from F1 caudal fins. c 5′ junction sequence of inserted R2Ol. Black box, 28S rDNA sequence; Gray box, R2Ol sequence; Vector, vector sequence; Non-templated, non-template sequence. Truncated or deleted sites are indicated by nucleotide numbers. d Left panel: Southern blot analysis of PCR-positive F1 fish. Right panel: Schematic diagram of three detected R2Ol insertions. P, PvuII site; thick line, probe; thin lines and numbers, signal lengths. Red boxes indicate vector or non-templated sequence

The transgenic EGFP is expressed in zebrafish. a EGFP expression pattern of the F2 progeny of two R2Ol–EGFP(R) transgenic lines. GRm-1-32 (i), GFm-14-1(ii) and − 14-7 (iii and iv) F1 females were mated with WT. hpf, hours post-fertilization. Dpf, days post-fertilization. Low, middle and high indicate the EGFP expression level. b Heat-shocked F2 progeny of a cross between the R2Ol–GAL4-R (heterozygous) and UAS-GFP lines (homozygous). dpf, days post-fertilization. Each image was taken from an independent mating

A scheme for transgenesis in zebrafish using R2Ol.

Experimental approach for analysis of hsp-GAL4:UAS-EGFP expression induced by heat shock. A Schematic diagram of hsp-GAL4 construct. thick line, probe. B Transgenic progeny analysis from R2Ol-hsp-GAL4 founder. C Two lines R2Ol-hsp-GAL4 and UAS-GFP were crossed and yielded embryos were heat shocked at 37 ? for 1, 3, 6, hours.