Alternative-splicing-based bicistronic vectors for ratio-controlled protein expression and application to recombinant antibody production

In the last decade polycistronic vectors have become essential tools for both basic science and gene therapy applications. In order to co-express heterologous polypeptides, different systems have been developed from Internal Ribosome Entry Site (IRES) based vectors to the use of the 2A peptide. Unfortunately, these methods are not fully suitable for the efficient and reproducible modulation of the ratio between the proteins of interest. Here we describe a novel bicistronic vector type based on the use of alternative splicing. By modifying the consensus sequence that governs splicing, we demonstrate that the ratio between the synthesized proteins could easily vary from 1 : 10 to 10 : 1. We have established this system with luciferase genes and we extended its application to the production of recombinant monoclonal antibodies. We have shown that these vectors could be used in several typical cell lines with similar efficiencies. We also present an adaptation of these vectors to hybrid alternative splicing/IRES constructs that allow a ratio-controlled expression of proteins of interest in stably transfected cell lines

An upstream open reading frame within an IRES controls expression of a specific VEGF-A isoform

Vascular endothelial growth factor A (VEGF-A) is a potent secreted mitogen critical for physiological and pathological angiogenesis. Regulation of VEGF-A occurs at multiple levels, including transcription, mRNA stabilization, splicing, translation and differential cellular localization of various isoforms. Recent advances in our understanding of the posttranscriptional regulation of VEGF-A are comprised of the identification of stabilizing mRNA-binding proteins and the discovery of two internal ribosomal entry sites (IRES) as well as two alternative initiation codons in the 5′UTR of the VEGF-A mRNA. We have previously reported that VEGF-A translation initiation at both the AUG and CUG codons is dependent on the exon content of the coding region. In this report, we show that the expression of different VEGF-A isoforms is regulated by a small upstream open reading frame (uORF) located within an internal ribosome entry site, which is translated through a cap-independent mechanism. This uORF acts as a cis-regulatory element that regulates negatively the expression of the VEGF 121 isoform. Our data provide a framework for understanding how VEGF-A mRNAs are translated, and how the production of the VEGF 121 isoform is secured under non-hypoxic environmental conditions

Etude in vitro et in vivo de l'expression du VEGF-A (Vascular Endothelial Growth Factor-A) / Stéphanie Bornes ; Hervé Prats, Christian Touriol

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

To build a new Lactobacillus culture collection of human origin and farther screening on cellular and Caenorhabditis elegans models

International audienc

Etudes in vivo des propriétés probiotiques et anti-Candida albicans de Lactobacillus rhamnosus Lcr35®

National audienceLes probiotiques sont définis par la FAO et l’OMS comme des microorganismes vivants conférant des effets bénéfiques à l’hôte comme par exemple Lactobacillus rhamnosus Lcr35®. Ce dernier est notamment commercialisé pour ses propriétés anti-Candida albicans. De plus, ses propriétés sont potentialisées par des composés inclus dans les formulations. Cependant, comme pour la plupart des probiotiques, les mécanismes d’action de Lcr35® ne sont pas encore décrits. Les identifier devient aujourd’hui une priorité. Dans ce but, des études in vivo utilisant un organisme modèle, Caenorhabditis elegans, ont été réalisées : (i) étude de la survie du nématode en présence du probiotique potentialisé ou non, (ii) étude des effets préventifs et curatifs de Lcr35® contre une infection à Candida chez C. elegans et (iii) approches transcriptomiques pour identifier les voies de signalisation impliquées dans les effets anti-Candida de Lcr35®.La pathogénicité de C. albicans est généralement liée à la filamentation induisant la destruction des cellules de l’hôte. De Barros et al. (2018) ont montré que C. elegans, prétraité avec L. paracasei, était moins sensible à une infection à C. albicans. Cependant, une réduction significative de la filamentation n’a pas été observée. De ce fait, des expérimentations sur la réduction de la filamentation de C. albicans par Lcr35® seront réalisées

Caenorhabditis elegans, a Host to Investigate the Probiotic Properties of Beneficial Microorganisms

International audienceCaenorhabditis elegans, a non-parasitic nematode emerges as a relevant and powerful candidate as an in vivo model for microorganisms-microorganisms and microorganisms-host interactions studies. Experiments have demonstrated the probiotic potential of bacteria since they can provide to the worm a longer lifespan, an increased resistance to pathogens and to oxidative or heat stresses. Probiotics are used to prevent or treat microbiota dysbiosis and associated pathologies but the molecular mechanisms underlying their capacities are still unknown. Beyond safety and healthy aspects of probiotics, C. elegans represents a powerful way to design large-scale studies to explore transkingdom interactions and to solve questioning about the molecular aspect of these interactions. Future challenges and opportunities would be to validate C. elegans as an in vivo tool for high-throughput screening of microorganisms for their potential probiotic use on human health and to enlarge the panels of microorganisms studied as well as the human diseases investigated

Highlighting of the molecular mechanisms involved in probiotic properties of Lactobacillus rhamnosus Lcr35

National audienceProbiotics are defined by the World Health Organization as live microorganisms which, when administered in adequate amounts, confer a health benefit on the host (1). Although their beneficial effects on health are proven, knowledge on their mechanisms of action is becoming a priority today. This is the case with L. rhamnosus Lcr35, whose probiotic and anti- Candida albicans properties have been demonstrated (2, 3). Lcr35 is genetically and physiologically well-characterized and it has been proven that ingredients included in the formulations is able to potentialize Lcr35 probiotic effect. In order to investigate the molecular mechanisms involved in the Lcr35 strain probiotic properties, in vivo studies have been carried out in C. elegans : the analysis of the nematode survival in the presence of the probiotic strain potentialized or not for the anti- C. albicans properties, and a preliminary analysis of the modulation of the immune response by Lcr35.The survival analyzes showed that the strain Lcr35 allowed a significant increased lifespan of the nematode in comparison with E. coli OP50 whereas C. albicans impaired survival. The activation of insulin and p38 MAPK pathways has been shown using RT-qPCR. Then we will go further into the investigations to understand the molecular mechanisms related to these properties by studying in particular the preventive and curative effects of Lcr35 with respect to a C. albicans infection. In addition, a complete analysis of the transcriptome of C. elegans will be carried out by RNA sequencing in order to precisely identify the signaling pathways involved in the interaction between the host and the probiotic or the pathogen microorganisms. Moreover, because of C. elegans genetic homology with humans and in particular its intestinal physiology close to humans, our work lead us to evaluate the potential of C. elegans to be a relevant in vivo model to characterize the molecular mechanisms involved in the probiotic potential of a microorganisms and then to screen microorganisms collections for their potential probiotic capacities.1. FAO, WHO. 2001. Health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. Food Nutr Pap 85:71.2. Nivoliez A et al. 2012. Influence of manufacturing processes on in vitro properties of the probiotic strain Lactobacillus rhamnosus Lcr35. J Biotechnol 160:236–241.3. Nivoliez A et al. 2015. Influence of manufacturing processes on cell surface properties of probiotic strain Lactobacillus rhamnosus Lcr35. Appl Microbiol Biotechnol 99:399–41

Evaluation du modèle Caenorhabditis elegans pour estimer la qualité d’une eau eau. Application à l’eau de la Dordogne sur la Communauté de Communes de Sumène-Artense

International audienc

Etude des propriétés biologiques de fractions de fromage à l’aide d’un modèle in vivo innovant.

International audienc

Caractérisation in vivo des mécanismes probiotiques de Lactobacillus rhamnosus Lcr35®

National audienceLa levure Candida albicans est un pathogène opportuniste retrouvée dans la flore commensale de 80% de la population. Elle est l’agent d’un grand nombre d’infections digestives et gynécologiques appelées candidoses. La prise d’antifongique, bien qu’efficace, peut entrainer une dysbiose favorable à une récidive ultérieure. Lactobacillus rhamnosus Lcr35® est un probiotique dont les propriétés antimicrobiennes, notamment anti-C. albicans, ont été démontrées à la fois in vitro et lors d’essais cliniques. Cependant, les mécanismes moléculaires expliquant ces propriétés n’ont pas encore été décrits. L’objectif de ma thèse est d’identifier ces mécanismes en utilisant un modèle in vivo physiologiquement proche de l’Homme, le ver Caenorhabditis elegans.Ce nématode modèle sera présenté suivi des différentes stratégies expérimentales mises en œuvre : (i) l’étude du temps de vie en présence du probiotique ou du pathogène, (ii) l’étude des effets préventifs et curatifs du probiotique vis-à-vis d’une infection fongique puis (iii) des études omiques (transcriptomique et métabolomique).Les premiers résultats ont mis en évidence que Lcr35® induit un effet positif sur la durée de vie de C. elegans à l’inverse de C. albicans qui induit un effet négatif. De plus, un effet protecteur du probiotique vis-à-vis d’une infection à C. albicans est observé. En effet, en présence de Lcr35®, le nématode résiste à l’infection et voit son temps de vie augmenter. En perspective, une étude transcriptomique de la réponse globale du ver à l’administration du probiotique et/ou du pathogène est envisagée dans le but d’identifier les mécanismes moléculaires responsables des effets observés