Figure 3.

Validation of intron based RNAi approach on rescuing co-expression of fluorescent markers. (A–D), (E–H) and (I–L) Heterozygous Tg(ubi:mCherry:SPmiR137) embryos with ubiquitous mCherry expression were injected with Tol2 transposase and 50 pg of 4× anti-miR137 miRNAs construct without intron (plasmid 154) and with intron (plasmid 156), including a control (plasmid 157). A minimum of five larvae were imaged at 4 dpf using a confocal microscope to correlate the presence of 4× anti-miR137 expressing eGFP (with and without intron) to decrease/absence of mCherry fluorescence. Pixel intensities (grey values) of green versus red fluorescence (presented as a percentage) plotted in graphs H demonstrate no correlation of fluorescence in embryos injected with plasmid 154, similar to control 157. Comparatively, a linear correlation of green versus red fluorescence (L) is evidenced in embryos injected with plasmid with intron (plasmid 156).