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. 2021 May 14;22(10):5193.
doi: 10.3390/ijms22105193.

Current Practice in Bicistronic IRES Reporter Use: A Systematic Review

Guus Gijsbertus Hubert van den Akker  1 , Federico Zacchini  2   3 , Bas Adrianus Catharina Housmans  1 , Laura van der Vloet  1 , Marjolein Maria Johanna Caron  1 , Lorenzo Montanaro  2   3   4 , Tim Johannes Maria Welting  1
Affiliations

Current Practice in Bicistronic IRES Reporter Use: A Systematic Review

Guus Gijsbertus Hubert van den Akker et al. Int J Mol Sci. .

Abstract

Bicistronic reporter assays have been instrumental for transgene expression, understanding of internal ribosomal entry site (IRES) translation, and identification of novel cap-independent translational elements (CITE). We observed a large methodological variability in the use of bicistronic reporter assays and data presentation or normalization procedures. Therefore, we systematically searched the literature for bicistronic IRES reporter studies and analyzed methodological details, data visualization, and normalization procedures. Two hundred fifty-seven publications were identified using our search strategy (published 1994-2020). Experimental studies on eukaryotic adherent cell systems and the cell-free translation assay were included for further analysis. We evaluated the following methodological details for 176 full text articles: the bicistronic reporter design, the cell line or type, transfection methods, and time point of analyses post-transfection. For the cell-free translation assay, we focused on methods of in vitro transcription, type of translation lysate, and incubation times and assay temperature. Data can be presented in multiple ways: raw data from individual cistrons, a ratio of the two, or fold changes thereof. In addition, many different control experiments have been suggested when studying IRES-mediated translation. In addition, many different normalization and control experiments have been suggested when studying IRES-mediated translation. Therefore, we also categorized and summarized their use. Our unbiased analyses provide a representative overview of bicistronic IRES reporter use. We identified parameters that were reported inconsistently or incompletely, which could hamper data reproduction and interpretation. On the basis of our analyses, we encourage adhering to a number of practices that should improve transparency of bicistronic reporter data presentation and improve methodological descriptions to facilitate data replication.

Keywords: IRES; bicistronic reporter; dicistronic reporter; mRNA translation; ribosome; systematic review.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
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
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 promotorless control constructs or a cell-free translation system. Cryptic promoter activity can be reduced with SPA sites, although this can lead new cryptic splicing events. (B) Reporter gene configuration was summarized by allocation to six functional groups in five-year bins. The first publication was from 1994, which explained the low number of publications in the first bin. βGal = β-galactosidase, CAT = chloramphenicol acetyl transferase, Luc = any luminescent protein, GFP = green fluorescent protein, Fluc = Firefly luciferase, Rluc = Renilla luciferase.
Figure 3
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
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
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.
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