Schematic representation of Gal4/Vp16-UAS expression vectors (A), comparison of induction levels between transient and transgenic approaches (B-E′) and transient Gal4/Vp16 mediated reporter expression in medaka embryos induced by heat-shock (F-K). A; Gal4/Vp16 activator units driven by a 1.5 kb (pCG5.0WCS) or a 600 bp (pCG6.0WCS) heat-shock promoter fragment followed by a SV40 polyadenylation signal are shown on the left. Reporter units are separated from the activator units by the pBSII backbone. YFP (pCG3.0Y) or CFP (pCG5.0WCS/6.0WCS) open reading frames are placed downstream of 4 UAS elements and followed by a SV40 polyadenylation signal. Entire expression cassettes are flanked by the IR/DRs of the SB transposon system. Additionally, I-SceI meganuclease sites flank the expression cassettes of activator vectors. Abbreviations and actual sizes of each vector are given. B-E′; Wild type medaka embryos were injected with pCG5.0WCS and MN and subjected to heat-shock treatment (B, B′) or with MN and Gal4/Vp16 mRNA without treatment (C, C′). Similarly, the transgenic activator line pCG6.0WCS/T was injected with Gal4/Vp16 mRNA (D, D′) or subjected to heat-shock treatment (E, E′). Anterior is to the top (B-E′). DNA and RNA concentrations are indicated together with the developmental stage and the duration of heat-shock treatment. Microinjection of the activator/reporter plasmid pCG5.0WCS results in activation of CFP according to the distribution of plasmid DNA (B-C′). In contrast, induction of activator and reporter in the transgenic line by mRNA injection or heat-shock treatment results in ubiquitous and entirely uniform expression of CFP (D-E′). F-K: Activator and reporter vectors were injected into one-cell stage medaka embryos. Anterior is to the left (F-K). Developmental stage at heat-shock induction and duration of treatment is indicated. HS treatment of up to 2 hours did not interfere with embryonic development but yielded detectable transgene expression (F-K). CFP (internal reporter) shows the expression pattern of the activator Gal4/Vp16. YFP shows activation of the independent reporter. Co-injection of activator pCG5.0WCS and independent reporter pCG3.0Y (100 ng/μl each) resulted in mosaic activation of reporter gene expression (F-H) only. Co-injection of activator pCG5.0WCS (5 ng/μl) and independent reporter pCG3.0Y (100 ng/μl) with I-SceI resulted in a broad range of different levels of mosaicism. Notably, 16% of co-injected embryos showed highly uniform expression (I-K). Abbreviations: CFP, cyan fluorescent protein; HS, heat-shock; hpf, hours post fertilization; IR/DR, inverted/direct repeats; MN, meganuclease; pA, SV40 polyadenylation signal; pBS, pBluescriptII; st, developmental stage; UAS, upstream activating sequence; zf, zebrafish.

Kinetics of activator and reporter induction (A) and activation of an independent reporter in pCG6.0WCS/T (B-J). A; Transcriptional induction of the activator Gal4/Vp16 and the internal reporter (CFP) in pCG6.0WCS/T was analyzed by RT-PCR. Embryos were heat-shock for 90 seconds at st20/31.5hpf and were allowed to recover for the indicated periods of time. Activator transcripts were detectable already 10 minutes after induction and the levels increased up to 3 hours. Degradation to undetectable levels was complete after 20 hours. Activator dependent transcription of the internal reporter CFP was observed only after 2 hours and levels were still increasing after 25 hours. C-actin was used as an internal control. (B-J); The independent reporter plasmid pCG3.0Y was injected into the transgenic Gal4/Vp16 activator line. Anterior is to the left (B-J). Expressions of both internal and independent reporters were observed in a weak to strong ubiquitous manner (B-J). Occasional higher levels of internal reporter expression were observed in some parts of the embryonic body. These higher levels were paralleled by independent reporter expression (B-D, H-J). Mosaic clones exerting stronger YFP expression reflect locally higher plasmid concentrations (B-J). Abbreviations: h, hours; hpf, hours post fertilization; HS, heat-shock; M, size marker; st, developmental stage.