Ebola virus 3′ UTRs enhance luciferase reporter activity in transfected cells and injected zebrafish embryos. A, Transfection assay. 1.5 × 105 293T cells were seeded per well of a 12-well plate and transfected 1 day later with 100 ng of pMIR-REPORT firefly luciferase plasmids containing the indicated 3′ UTRs and 100 ng of a pMIR-β-gal expression plasmid as a transfection control. pMIR-REPORTempty containing no added gene-specific 3′ UTR served as the negative control (Empty). Cell lysates were harvested 2 days posttransfection and measured for luciferase and β-gal activity. Luciferase values were normalized to β-gal activity (converted to milliunits), and the normalized luciferase values for the negative control were set to one. The normalized luciferase values for each individual 3′ UTR-luciferase reporter construct were then compared to the negative control, which was set to one. Statistical analysis was performed with the GraphPad Prism software, using a 1-sample t test analysis; n = 5 for all conditions. B, Zebrafish embryo injection assay. A schematic of the assay is depicted on the right. One-cell stage embryos were each injected with 5 pg of mRNA encoding nanoluciferase and the indicated 3′ UTR together with 50 pg of firefly luciferase mRNA. Six hours after injection, embryos were pooled in groups of 7 and collected in lysis buffer. Multiple groups were assayed in 2 injection rounds performed on 2 different days. Nanoluciferase values were normalized to firefly luciferase values and compared to the negative control (empty), which was set to one. Circles and rhombi represent the individual groups of 7 embryos injected with the same mRNAs on 2 different days. Statistical analysis was performed with the GraphPad Prism software, using 1-way ANOVA. NS P value > .05, * P value ≤ .05, ** P value ≤ .01, *** P value ≤ .001, **** P value ≤ .0001. Error bars in A depict standard deviation. Error bars in B depict the standard error of the mean. Abbreviations: β-gal, β-galactosidase; hpi, hours post injection; IL-6, interleukin 6; NP, nucleoprotein; NS, not significant; UTR, untranslated region.
