Figure 3

Visualization and quantification of eGFP knockdown. (A) Schematic depicting the cross performed leading to ~ 50% of eGFP⁺ larvae in the offspring due to Mendelian inheritance. This cross was carried out for all experiments involving the eGFP gene. (B) Representative fluorescence images of 1 dpf larvae for each of the nine experimental conditions shown. Note the qualitative reduction in eGFP fluorescence in the eGFP shRNA-electroporated conditions when compared to control larvae, with the eGFP shRNA mixture (mix) condition and the individual shRNAs at 900 ng/µl yielding the highest reductions. eGFP⁻ larvae are outlined with dashed lines in the NE and scrambled shRNA controls for better clarity. NE non-electroporated. Scale bar 200 μm. (C) RT-qPCR results showing equivalent expression levels of eGFP mRNA in control samples. Scrambled shRNA eGFP expression levels were quantified relative to the NE control. (D) RT-qPCR results showing a clear reduction in the eGFP mRNA expression levels in all eGFP shRNA-electroporated conditions when compared to the scrambled shRNA control. When comparing the eGFP shRNA mixture condition with any of the three eGFP shRNAs electroporated separately at a concentration of 300 ng/μl each, the mixture yielded the lowest eGFP transcript level of all, significantly lower than eGFP shRNA 2 and 3 (p value ≤ 0.01), although not significantly lower than eGFP shRNA 1. When comparing the eGFP shRNA mixture condition with any of the three eGFP shRNAs electroporated separately at a concentration of 900 ng/μl each, all conditions yielded similarly low levels of eGFP transcripts, indicating comparable knockdown efficiencies between these four conditions. For all eGFP shRNA experimental conditions, eGFP expression levels were quantified relative to the scrambled shRNA control. All RNA samples used for RT-qPCR analyses were extracted from 1 dpf larvae. Bar heights represent mean values of at least three independent experiments and error bars show standard errors of the mean. Ns non-significant; ***p value ≤ 0.01.