DNA Elements Tetris: A Strategy for Gene Correction

Transposable elements (TEs) are mobile genetic sequences that are able to move in the genome from one location to another. TEs were first regarded as junk or selfish DNA, as they comprise the largest molecular class within most metazoan genomes having no genomic function. It was necessary to wait until whole genome sequencing to provide new insights about the origin, diversity, and impact of TEs on the genome function. Thus, due to advances in molecular technology, TEs have been shown to create new regulatory sequence networks. Although nowadays most TEs present in the human genome are silenced, particularly DNA transposons, it does not mean that these sequences are dead. In this review, we detail how DNA transposons could be emphasized to create a new tool for gene correction. DNA-based transposon vectors are derived from three models: Sleeping Beauty, piggyBac, and Tol2, which all work via a “cut-and-paste” mechanism where transposase enzyme is alone able to catalyze the transposition process, which means integrating the genes of interest in chromosomal DNA. Limitations and improvements of the systems are discussed, particularly the latest way to target a specific integration site, showing that the DNA transposon-derived system and its engineering, are powerful tools for gene correction

Factors acting on Mos1 transposition efficiency

<p>Abstract</p> <p>Background</p> <p><it>Mariner-</it>like elements (<it>MLEs</it>) are widespread DNA transposons in animal genomes. Although <it>in vitro </it>transposition reactions require only the transposase, various factors depending on the host, the physico-chemical environment and the transposon sequence can interfere with the <it>MLEs </it>transposition <it>in vivo</it>.</p> <p>Results</p> <p>The transposition of <it>Mos1</it>, first isolated from <it>drosophila mauritiana</it>, depends of both the nucleic acid sequence of the DNA stuffer (in terms of GC content), and its length. We provide the first <it>in vitro </it>experimental demonstration that MITEs of <it>MLE </it>origin, as small as 80 to 120-bp, are able to transpose. Excessive temperature down-regulates <it>Mos1 </it>transposition, yielding excision products unable to re-integrate. Finally, the super-helicity of the DNA transposon donor has a dramatic impact on the transposition efficiency.</p> <p>Conclusion</p> <p>The study highlights how experimental conditions can bias interpretation of <it>mariner </it>excision frequency and quality. <it>In vitro</it>, the auto-integration pathway markedly limits transposition efficiency to new target sites, and this phenomenon may also limit events in the natural host. We propose a model for small transposons transposition that bypasses DNA bending constraints.</p

Optimisation de la biosécurité du vecteur transposon piggyBac pour le transfert de gène (utilisation des ARN messagers et des insulateurs.)

Les progrès en biotechno]ogie ont permis le développement d outils pour le transfert de gène intégratif en transgénèse, bioproduction et thérapie génique. Cependant, trois challenges majeurs doivent être relevés pour garantir un système sécurisé : l innocuité et l efficacité du transfert, l intégration ciblée et contrôlée dans le génome, le niveau et la durée d expression du transgène au cours du temps. Dans ce but, mes travaux de thèse ont consisté à tester des solutions pour améliorer la biosécurité du transposon piggyBac qui nécessite un plasmide porteur du gène d intérêt à insérer dans le génome et une source de transposase catalysant la réaction d intégration du transgène. Une des stratégies de ma thèse repose sur l apport de la source de transposase sous forme d ARN messager au lieu d ADN afin d améliorer la stabilité de l intégration et de réduire les effets génotoxiques en limitant la transposase dans les cellules. Pour la première fois, la biodisponibilité de l ARNm de la transposase et les conditions optimales d utilisation en cellules humaines ont été déterminées pour augmenter la biosêcurité du système. Le second objectif de mes travaux consiste à améliorer l expression du transgène en ajoutant des insulateurs connus pour s opposer à l extinction de l expression des gênes. En termes de biosécurité, cette stratégie permet de réduire le nombre de copies du transgène nécessaires pour obtenir une expression suffisante. Deux candidats ont été identifiés pour améliorer l expression du transgène. La combinaison des approches ARNrn et insulateurs est prometteuse pour sécuriser le transfert de gène médié par piggyBac et pour maintenir l expression du gène d intérêt.Advances in biotechnology have enabled the development of tools for gene transfer applicable to transgenesis, bioproduction and gene therapy. But, 3 major challenges must be met to ensure a secure system: the safety and effectiveness of the transfer. the targeted and controlled integration into the genome. and the level of transgene expression over time. In this aim, my thesis project was to validate solutions to improve the biosafety of the piggyBac transposon, which requires a plasmid carrying the gene of interest to be inserted in the genome, and a source of transposase which catalyzes the transgene integration. One approach of my thesis work is to deliver the source of piggyBac transposase as an mRNA molecule instead of DNA. This strategy aims to improve the stability of the integration and reduce the genotoxic effects by limiting the transposase in the cells. For the 1st time, the bioavailability of the transposase rnRNA and the optimal conditions for its use in human cells were determined to increase the biosafety of the transposon system. The 2nd objective ofmy project is to improve the expression of the transgene by adding insulators known to counteract the transgene silencing. This strategy reduces the number of integrations required ta get a sufficient expression of the transgene and thus, improve biosecurity. Two candidates have been identified to improve transgene expression. The combination of the mRNA and insulator strategies is promising to secure the piggyBac-mediated gene transfer and to maintain the expression of the gene of interestTOURS-Bibl.électronique (372610011) / SudocSudocFranceF

Transposable elements as tools for reshaping the genome: it is a huge world after all!

chapitre 1absen

Mise au point d'un système dérivé du transposon Mos1 comme vecteur non viral de transfert de gène en cellules eucaryotes

TOURS-BU Sciences Pharmacie (372612104) / SudocSudocFranceF

Transcriptional expression of mannose receptor gene during differentiation of human macrophages.

International audienceMannose receptor is a differentiation marker of macrophages. Circulating monocytes isolated from plasma are devoid of this receptor; upon culture this receptor is rapidly expressed. Its expression is modulated by a variety of inflammatory and anti-inflammatory agents. In the present study, we investigated its transcription level during the differentiation process. Mannose receptor mRNA was monitored by quantitative RT-PCR on freshly harvested monocytes and on monocytes cultivated up to four days. No transcription was detected in freshly harvested cells, the transcription increased during the first 24 h upon adhesion and then decreased

Cytoplasmic transposase RNA transfer and insulated transgenes for safe and efficient trangenesis

National audienc

Properties of the various Botmar1 transcripts in imagoes of the bumble bee, Bombus terrestris (Hymenoptera: Apidae).

Botmar1 elements are mariner-like elements (MLEs), class II transposable elements that occur in the genome of the bumble bee, Bombus terrestris. Each haploid B. terrestris genome contains about 230 Botmar1, consisting entirely of 1.3-kb and 0.85-kb elements. During their evolution in the B. terrestris genome, two Botmar1 lineages have been differentiated in terms of their nucleic acid sequences and the differences found in their 5' untranslated regions suggest that they could be transcribed differently in B. terrestris. Here, we show that small amounts of Botmar1 mRNA occur in RNA extracts purified from B. terrestris imagoes. This indicates that the Botmar1 transcription is either weak in imagoes, or is restricted to very few cells. The cloning of several mRNAs reveals that only lineage-2 Botmar1 elements are transcribed. This transcription is specific, and uses cardinal initiators and terminators of eukaryotic elements in the Botmar1 elements. The intrastrand stem-loop folds in the mRNA theoretically synthesized by elements of the first lineage suggest that mRNA maintenance in cells might be self-regulated by RNA interference