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Fig. 6 An IRES sequence is required for independent expression of the reporter gene.
(A) The ECMV/IRES element in the pSPL3-Trap(intron) vector was deleted to generate the pSPL3-Trap(intron)ΔIRES. SD1, splice donor for exon1; IR/DR(L) and IR/DR(R), left and right inverted repeat/directed repeat of the SB transposon; SA, splice acceptor; IRES, internal ribosome entry site; EGFP, enhanced green fluorescence protein gene; poly(A), poly(A) signal; SA2, splice acceptor for exon2. (B) pSPL3-Trap(intron) and pSPL3-Trap(intron)ΔIRES constructs were transfected into HeLa cells at 80% confluence, respectively. Images were taken under a Nikon TE2000 fluorescent microscope at 48 h after transfection and cell numbers in three independent transfections were counted. (C) Zebrafish embryos at one-cell stage were microinjected with the pSPL3-E3/Trap(exon). Injected embryos at 24 hpf were imaged under a SteReo Lumar V12 microscope form Zeiss and total embryos in three dishes were counted. (D) Statistical analysis of EGFP-expressing cells in (B) or embryos in (C). Each construct was tested three times and each experiment was done in triplicate. Data are given as means ± standard Deviation. ** indicate P<0.01 versus the corresponding control.