Avoidance of reporter assay distortions from fused dual reporters

Abstract

Positioning test sequences between fused reporters permits monitoring of both translation levels and framing, before and after the test sequence. Many studies, including those on recoding such as productive ribosomal frameshifting and stop codon readthrough, use distinguishable luciferases or fluorescent proteins, as reporters. Occasional distortions, due to test sequence product interference with the individual reporter activities or stabilities, are here shown to be avoidable by the introduction of tandem StopGo sequences (2A) flanking the test sequence. Using this new vector system (pSGDluc), we provide evidence for the use of a 3’ stem loop stimulator for $\textit{ACP2}$ readthrough, but failed to detect the reported $\textit{VEGFA}$ readthrough.This work was supported by grants from Science Foundation Ireland (12/IP/1492 and 13/1A/1853 to J.F.A.), National Institutes of Health (R01GM114291 and R21ES022716 to M.T.H.), Wellcome Trust (106207 to A.E.F.) and the European Research Council (646891 to A.E.F.)