Current Practice in Bicistronic IRES Reporter Use: A Systematic Review

Figure 1. PRISMA flow diagram of eligible studies for this systematic review. Two hundred and fifty-seven studies were identified and screened. Twenty-eight studies were excluded after screening the title and abstract. Fifty-three additional studies were excluded on the basis of violation of inclusion criteria. A total of 176 full text articles were included in this study. The identity of all 257 articles and the 176 included studies can be found in Table S1.

Figure 2. Bicistronic reporter design considerations and reporter gene use over time. (A) Relevant reporter design parameters are depicted. The choice of promoter and use of a chimeric intron and/or enhancer alters the mRNA expression levels, stability, and translation efficiency. The reporter genes themselves, as well as their positioning, can influence the observed IRES activity. The exact IRES sequence, for example EMCV sequence variants, can also have large effects. Potential read-through can be inhibited with (multiple) stop codons behind the first cistron (in all three reading frames) or inclusion of an inhibitory hairpin (second arrow). The first cistron always contains a Kozak sequence, but in some specific plasmid backbones, we identified a second Kozak sequence at the second cistron. A common criticism on the bicistronic reporter assay is the possibility of cryptic splicing due to specific reporter element (*) or cryptic promoter activity of the IRES sequence that can lead to false positive results. The former can be evaluated with Northern blot, RT-PCR, or 5′-RACE and ruled out with a monocistronic reporters or a cell-free translation system; the latter can be ruled out by using…

Figure 3. An overview of extracted information from included studies. (A) Plasmid, IRES, and the number of IRESs/publication were quantified and depicted in pie charts. (B) Cellular translation assay parameters. Transfection methods were combined in four categories for easier visualization. Time of transfection refers to the measurement time point of the reporter post-transfection. (C) Cell-free translation assay parameters. Note: total numbers do not always match the number of included studies due to the use of multiple parameters in one study.

Figure 4. Data representation possibilities and overview. (A) Schematic of a bicistronic IRES reporter mRNA. (B) Visualization options for bicistronic data. Left: raw data from separate cistrons or the calculated ratio. Right: normalized cistrons separately and a normalized ratio. The latter is easier to interpret, however, relevant information is also lost about the measurement and relative signal differences. (C) Data representation in analyzed publications. (D) Normalization strategies and control use. Note that multiple normalization strategies or controls were combined in a large number of studies. The prevalence of the top eight normalization or control methods is depicted in the pie chart.

Figure 5. Potential misinterpretation of activity ratios without individual cistron data. Reporting of ratios alone can be misleading due to different underlying reasons for regulation. For example: (A) IRES ratio induction, caused by an increase of second cistron activity. (B) IRES ratio induction, due to a decrease of first cistron activity. Note that IRES activity in this hypothetical example is very low and might be close to the detection limit or background levels. Naturally, both first and second cistrons can also be induced or reduced, wherein slight differences can lead to IRES induction or repression. Additional normalization strategies can be used to correct for changes in cap translation (monocistronic βGal co-transfection), cell number, or protein content. Graphs were generated for illustration purposes using hypothetical data, on the basis of our own experience with the bicistronic reporter assay.