Development of a new bicistronic retroviral vector with strong IRES activity.
type: Dataset
description: BACKGROUND: Internal Ribosome Entry Site (IRES)-based bicistronic vectors are important tools in today's cell biology. Among applications, the expression of two proteins under the control of a unique promoter permits the monitoring of expression of a protein whose biological function is being investigated through the observation of an easily detectable tracer, such as Green Fluorescent Protein (GFP). However, analysis of published results making use of bicistronic vectors indicates that the efficiency of the IRES-controlled expression can vary widely from one vector to another, despite their apparent identical IRES sequences. We investigated the molecular basis for these discrepancies. RESULTS: We observed up to a 10 fold difference in IRES-controlled expression from distinct bicistronic expression vectors harboring the same apparent IRES sequences. We show that the insertion of a HindIII site, in place of the initiating AUG codon of the wild type EMCV IRES, is responsible for the dramatic loss of expression from the second cistron, whereas expression from the first cistron remains unaffected. Thus, while the replacement of the authentic viral initiating AUG by a HindIII site results in the theoretical usage of the initiation codon of the HindIII-subcloned cDNA, the subsequent drop of expression dramatically diminishes the interest of the bicistronic structure. Indeed, insertion of the HindIII site has such a negative effect on IRES function that detection of the IRES-controlled product can be difficult, and sometimes even below the levels of detection. It is striking to observe that this deleterious modification is widely found in available IRES-containing vectors, including commercial ones, despite early reports in the literature stating the importance of the integrity of the initiation codon for optimal IRES function. CONCLUSION: From these observations, we engineered a new vector family, pPRIG, which respects the EMCV IRES structure, and permits easy cloning, ta…
landing page: 1472-6750
keyword: infoeu-reposemanticsarticle
keyword: journal-articles
keyword: mesh-5-untranslated-regions
keyword: mesh-cell-line
keyword: mesh-genes
keyword: mesh-genetic-engineering
keyword: mesh-genetic-vectors
keyword: mesh-green-fluorescent-proteins
keyword: mesh-humans
keyword: mesh-kidney
keyword: mesh-promoter-regions-genetics
keyword: mesh-recombinant-fusion-proteins
keyword: mesh-research-support-non-us-govt
keyword: mesh-retroviridae
keyword: mesh-ribosomes
keyword: mesh-transfection
keyword: sdvbbmlife-sciences-q-biobiochemistry-molecular-biology
keyword: sdvbiolife-sciences-q-biobiotechnology
issued: 2026-05-08T16:37:43.038989
modified: 2026-05-08T16:37:43.038995
language: en
contact point: Organization
  fn: CCSD

test_moissonnage_selune
type: Agent

Development of a new bicistronic retroviral vector with strong IRES activity.
type: Distribution
format: HTML
access URL: https://inserm.hal.science/inserm-00089235
issued: 2026-05-08T16:37:43.079096
modified: 2026-05-08T16:37:43.014023