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International audience; Lentiviruses have been extensively used as gene delivery vectors since the mid-1990s. Usually derived from the human immunodeficiency virus genome, they mediate efficient gene transfer to non-dividing cells, including neurons and glia in the adult mammalian brain. In addition, integration of the recombinant lentiviral construct into the host genome provides permanent expression, including the progeny of dividing neural precursors. In this review, we describe targeted vectors with modified envelope glycoproteins and expression of transgenes under the regulation of cell-selective and inducible promoters. This technology has broad utility to address fundamental questions in neuroscience and we outline how this has been used in rodents and primates. Combining viral tract tracing with immunohistochemistry and confocal or electron microscopy, lentiviral vectors provide a tool to selectively label and trace specific neuronal populations at gross or ultrastructural levels. Additionally, new generation optogenetic technologies can be readily utilized to analyze neuronal circuit and gene functions in the mature mammalian brain. Examples of these applications, limitations of current systems and prospects for future developments to enhance neuroscience knowledge will be reviewed. Finally, we will discuss how these vectors may be translated from gene therapy trials into the clinical setting.

Les auteurs ont produit et caractérisé la première banque de BAC (chromosome bactériens artificiels) chez une espèce de bananier (#Musa balbisiana# 'Pisang Klutuk Wulung', d'où le nom de la banque PKW). Un protocole nouveau et amélioré d'isolement des noyaux a été employé pour surmonter les problèmes causés par l'abondance de polyphénols et de polysaccharides dans les tissus foliaires. L'emploi de la cytométrie en flux pour purifier les noyaux a éliminé la contamination aux métabolites secondaires ainsi que l'ADN plastidique. De plus, l'utilité d'un vecteur inductible, pCC1BAC, en vue de l'obtention d'une plus grande quantité d'ADN de BAC a été démontrée. La banque PKW compte l'équivalent de neuf génomes du #M. balbisiana# et la taille moyenne des inserts est de 135 kb. Elle est composée de deux sous-banques; la première (la sous-banque SN avec 24 960 clones) ayant été préparée en suivant un protocole standard amélioré pour l'isolement des noyaux tandis que la seconde (sous-banque FN avec 11 904 clones) a été obtenue à partir des noyaux purifiés par cytométrie en flux. Le criblage de la banque avec 12 sondes RFLP, connues comme étant situées sur huit groupes de liaison chez le #Musa acuminata#, a permis d'identifier une moyenne de 11 clones par sonde. Cette observation permet de valider l'estimé quant à la couverture du génome et indique une forte conservation entre les génomes des deux espèces du genre #Musa#. La localisation sur les chromosomes mitotiques de clones BAC choisis au-moyen d'hybridations FISH a permis de montrer que cette banque de clones BAC constitue une ressource utile pour la cartographie cytogénétique. Dans le cadre d'une démarche de clonage positionne] d'un facteur génétique qui est impliqué dans l'activation de séquence pararétrovirales intégrées appartenant au virus de la striure du bananier genre badnavirus (BSV), le locus BSV exprimé (BEL; "BSV expressed locus") a été délimité physiquement. La banque BAC PKW constitue un outil disponible publiquement et est présentement employé pour révéler les mécanismes d'intégration et d'activation de séquences du BSV et pour étudier la structure et l'évolution du génome du bananier.

The influence of CaCl2 was investigated on Agrobacterium tumefaciens-mediated gene transfer in Hevea brasiliensis friable calli which are usually proliferated on maintenance medium (MM) containing 9 mM CaCl2. Five A. tumefaciens strains (C58pMP90, C58pGV2260, AGL1, LBA4404 and EHA 105) and two binary vectors (pGIN and pCAMBIA2301) were tested and the strain EHA105pC2301 was selected to conduct further experiments. The calli were precultured on MM containing a range of CaCl2 concentrations, then inoculated with Agrobacterium suspension. Transfer of friable calli from MM containing 9 mM CaCl2 to calcium-free medium significantly enhanced the transient bêta-glucuronidase activity. Interestingly, the use of calcium-free Agrobacterium resuspension medium to inoculate friable calli again dramatically increased the transformation efficiency. Induction of Agrobacterium's virulence with acetosyringone remained an important factor to stimulate transformation.

La présente invention concerne un vecteur recombinant pour l'expression et la sécrétion, par une bactérie propionique, d'une ou plusieurs séquences d'acides aminés d'intérêt, ledit vecteur comprenant au moins : - sous le contrôle d'au moins un promoteur approprié, - au moins une séquence nucléotidique codant un peptide signal de bactérie propionique et, en fusion traductionnelle avec celle-ci, - une ou plusieurs séquences nucléotidiques codant la ou lesdites séquences d'acides aminés d'intérêt. L'invention s'intéresse également aux utilisations d'un tel vecteur dans le domaine pharmaceutique ou pour la production à grande échelle de peptides ou protéines d'intérêt.

International audience; The recent advances in molecular genetics, pathogenesis and immunology have provided an optimal framework for developing novel approaches in the rational design of vaccines effective against viral epizootic diseases. This paper reviews most of the viral-vector based antigen delivery systems(ADSs) recently developed for vaccine testing in veterinary species, including attenuated virus and DNA and RNA viral vectors. Besides their usefulness in vaccinology, these ADSs constitute invaluable tools to researchers for understanding the nature Of protective responses in different species, opening the possibility of modulating or potentiating relevant immune mechanisms involved in protection.

For correspondence fax +33 467 615 605 ; email dhont@cirad.fr; International audience; Modern sugarcane (Saccharum spp.) is an important grass that contributes 60% of the raw sugar produced worldwide and has a high biofuel production potential. It was created about a century ago through hybridization of two highly polyploid species, namely S. officinarum and S. spontaneum. We investigated genome dynamics in this highly polyploid context by analyzing two homoeologous sequences (97 and 126 kb) in a region that has already been studied in several cereals. Our findings indicate that the two Saccharum species diverged 1.5–2 million years ago from one another and 8–9 million years ago from sorghum. The two sugarcane homoeologous haplotypes show perfect colinearity as well as high gene structure conservation. Apart from the insertion of a few retrotransposable elements, high homology was also observed for the ontranscribed regions. Relative to sorghum, the sugarcane sequences displayed colinearity, with the exception of two genes present only in sorghum, and striking homology in most non-coding parts of the genome. The gene distribution highlighted high synteny and colinearity with rice, and partial colinearity with each homoeologous maize region, which became perfect when the sequences were combined. The haplotypes observed in sugarcane may thus closely represent the ancestral Andropogoneae haplotype. This analysis of sugarcane haplotype organization at the sequence level suggests that the high ploidy in sugarcane did not induce generalized reshaping of its genome, thus challenging the idea that polyploidy quickly induces generalized rearrangement of genomes. These results also confirm the view that sorghum is the model of choice for sugarcane

One of the communication tools of the EC-funded Biosafenet project is a series of six Biosafenet Seminars that will be held 2007–2009. Their purpose is to convene small groups of scientists to consider emerging issues related to the potential impact of genetically modified organisms (GMOs). The first Seminar was organized by the ICGEB at the MasterCampus of the Fondazione Cassamarca in Ca’ Tron di Roncade (Italy) on 6th–8th June 2007, and focused on the potential impact of both naturally occurring and transgenic viral sequences in plant genomes.

Un programme de transformation génétique d'Hevea brasiliensis a été initié suite, i) à la mise au point d'un protocole de régénération de plantes génétiquement modifiées à partir de cals embryogènes, ii) à la caractérisation d'un promoteur spécifique d'un gène codant une protéine majeure du latex, l'hévéine, contenue dans les cellules laticifères de l'hévéa. La transformation génétique vise désormais à surexprimer deux gènes de résistance au stress oxydatif, facteur limitant la production de latex en exploitation, tout comme le rendement d'obtention de plants in vitro. Deux enzymes ayant un rôle primordial dans les voies de détoxication des espèces réactives de l'oxygène (ROS), responsable du stress oxydatif chez Hevea brasiliensis et chez les végétaux en règle générale ont été choisies: la super oxyde dismutase (SOD) et la [gamma]-glutamyl cystéine synthase ([gamma]-ECS). Mon stage consiste à réaliser l'étape initiale nécessaire à la transformation génétique, c'est-à-dire, i) la préparation d'un vecteur contenant une cassette d'expression génétique comprenant les deux gènes cibles - CuZnSOD et [gamma]-ECS - sous la dépendance du promoteur du gène codant l'hévéine Hev2.1 et du terminateur de la Nopaline synthase et, ii) l'intégration de cette construction dans un vecteur binaire d'Agrobacterium tumefaciens.

Rice is the most important food crop of the developing world. Whilst rice demand is increasing continuously, rainfed rice farmers in most African countries still harvest around one ton grain per hectare. Their crops, mainly Oryza sativa, are affected by multiple stresses and pests. Oryza glaberrima appeared to be more adapted to these constraints. Although not highly productive, and threatened by extinction, O.glaberrima cultivars continue to be cultivated by many resource-poor farmers because of its grain quality and yield stability. In the early nineties, using O.glaberrima (accession CG14) as donor, breeders at the West African Rice Development Association (WARDA), created many backcrossed introgressed lines O.sativa/glaberrima exhibiting several adaptive traits in the field, assumed to come from O.glaberrima. We are currently constructing a CG14 genomic library at the Centre de Coopération Internationale en Recherche Agronomique pour le développement (CIRAD) using a transformation-competent artificial chromosome (TAC) vector system (pYLTAC17). We aim at generating 36,864 clones representing at least 10 genome equivalents coverage. This library should benefit all marker-assisted selection breeding programs using O. glaberrima as a donor of useful genes for biotic and abiotic traits. Once constructed and as a first application, this library will be exploited for the cloning of the first nematode resistance gene mapped in rice. Screening and clone distribution will be ensured by the John Innes Centre (JIC) Genome laboratory as a worldwide service on a cost-recovery basis for public use. This project, funded by the Rockefeller Foundation and CIRAD, is a collaboration between JIC, CIRAD and WARDA. (Texte intégral)

Rice is a major crop and a plant model for monocotyledons genomic, and especially for cereals. A new field has to be developed with the functional genomic, to take advantage of the growing number of genomic sequences. The potential of plant virus-based transient expression vectors is reported in fundamental virology but also for plant biology. At present plant virus vectors offer number of benefits for the expression of foreign genes, by overexpress (gain of function mutant) or suppress (loss-of-function mutant) gene expression. The potential of such vectors for plant molecular biology is substantial particularly to identify previous unknown genes. Rice yellow mottle virus (RYMV) is a single-stranded-positivesense RNA virus that specifically infects rice leaves and causes serious disease in irrigated rice systems in East and West Africa. The purpose of this work is to develop for rice a transitory expression vector based on an infectious full-length cDNA clone of RYMV to assess gene function. Three main RYMV-based vector constructs is being generated. The first approach consists in producing a fusion between a reporter gene (encoding GUS or GFP) and the viral coat protein. With such construct, gene expression can be monitored in planta. In a second approach, to over-express a foreign gene, we consider duplicating sub-genomic mRNA promoter of the coat protein in order to improve gene expression thanks to an additional stem-loop structure. The third approach is the construction of an interesting virus-based vector for gene complementation, a construct devoid of its infectious elements like genes encoding viral coat protein and/or movement protein. Accumulation of recombinant virus is evaluated by RT-PCR in electroporated rice protoplasts and mechanically inoculated plant. Then, recombinant vectors will be challenged for the ability to express levels of viral proteins, GUS and GFP expression in protoplasts and in planta. To improve gene expression all these constructs will be cloned under the control of constitutive promoters. In parallel, as we also want to better understand molecular mechanisms involved in gene silencing induced by RYMV, we are studying inhibition of gene silencing by P1 protein of different RYMV-isolates. The purpose of the study is to estimate how the inhibition of gene silencing by RYMV occurs, in order to improve vectors expression. With this aim in view, we will inoculate these transgenic rice lines with different RYMV-isolates, to show variability on the suppressor of silencing expression and thus, highlight isolates that generates the main effect on silencing inhibition. (Texte intégral)

Les projets de séquençage des génomes végétaux sont en plein essor, mais l'accès aux fonctions codées reste limitant pour le développement de la génomique. Ainsi, la mise en place d'outils pour faciliter la détermination de ces fonctions devient primordiale et les vecteurs d'expression constituent un élément de choix pour atteindre cet objectif. Cette technologie, très peu développée chez les monocotylédones, permet d'étudier finement l'expression des gènes en suivant les produits de traduction au niveau cellulaire, grâce à des gènes rapporteurs. De plus, la sur-expression ou l'extinction des gènes peut conduire à un phénotype caractéristique de la fonction codée. Nous avons développé un vecteur viral d'expression basé sur un virus fonctionnel chez le riz: le virus de la panachure jaune du riz, Rice yellow moule virus (RYMV), un Sobemovirus. La forte spécificité qu'il présente avec les cellules de riz, l'importante stabilité dans son hôte, ainsi que son génome de petite taille et de structure simple, font du RYMV un candidat intéressant pour la construction de vecteurs viraux d'expression. Deux types de vecteurs ont été construits à partir de clones infectieux sous dépendance du promoteur T7: (i) des vecteurs pour la sur-expression des gènes dont la fonction est à évaluer (ii) des vecteurs utilisant la propriété des virus à induire du VIGS (virus induced gene silencing) pour éteindre ces gènes. Ces mêmes vecteurs pourront être optimisés grâce à l'étude de la levée du silencing par le virus. Plusieurs constructions sont disponibles. Une fusion entre un gène rapporteur (GUS ou GFP) et la CP, permettra d'évaluer la fonctionnalité d'un tel vecteur (taille de l'insertion) et de suivre l'expression de la CP in planta. L'expression élevée de l'ARN sub-génomique sera exploitée pour sur-exprimer spécifiquement un gène rapporteur. L'analyse de ces vecteurs est en cours, au niveau cellulaire et in planta. Afin de mieux comprendre les mécanismes moléculaires impliqués dans le gene silencing et son effet sur la pathogénie, nous envisageons d'étudier la suppression du silencing par le RYMV, sur des plantes dont les transgènes (GUS ou GFP) ne s'expriment pas. Dans cet objectif, les plantes transgéniques seront inoculées avec différents isolats de RYMV variables sur le gène de la P1, laquelle est impliquée dans la suppression du silencing. (Texte intégral)

The prospect of the deliberate environmental release of genetically manipulated microorganisms has given rise to a great deal of polemic. Amongst the rational scientific concerns are those concerned with the fate of the released bacteria, the fate of the recombinant genes that they carry, the selective pressures acting upon them in different environmental situations and the long term effects on the environment and human health. All recombinant DNA is carried by vectors (plasmids, transposons or bacteriophage or remnants of these). Thus the way in which recombinant constructions are made may itself lead to potential biosafety concerns, irrespective of the host bacterium and the recombinant DNA fragment of primary interest. The purpose of the present review is to assess progress in improved vector design aimed at eliminating risks due to the way recombinant vectors are constructed. Improved vector constructions include the avoidance of the use, or removal, of antibiotic resistance genes, the use of defective transposons rather than plasmids in order to reduce horizontal transfer and the development of conditionally lethal suicide systems. More recently, new site-specific recombination systems have permitted transposon vectors to be manipulated following strain construction, but before environmental release, so that virtually all recombinant DNA not directly involved in the release experiment is eliminated. Such bacteria are thus pseudo-wild type in that they contain no heterologous DNA other than the genes of interest.

Escherichia coli$ DnaK (Hsp70) cooperates with DnaJ and GrpE in its essential role as a molecular chaperone. Function of mitochondrial Hsp70 (mHsp70) in protein folding and organellar import in eukaryotes is critically dependent on GrpE. We clone two genes from tobacco (#Nicotiana tabacum$) BY2 cells based on peptide sequences from a purified protein. The predicted amino acid sequences of both clones resembled that of GrpE from #E. coli$ and its homologues from eukaryotes, and a cDNA clone from #Arabidopsis thaliana$. One gene (Type 1) encoded a deduced protein that was identical to the purified protein while the other (Type 2) encoded a deduced protein that has 80% sequence identity to Type 1. Both tobacco and #Arabidopsis thaliana$ GrpE homologues bound to DnaK and ATP inhibited this binding. The tobacco GrpE homologue contained a typical N-terminal mitochondrial target presequence of 64 residues and the presequence directed the green fluorescent protein to tobacco mitochondria. The tobacco GrpE homologue also associated with mHsp70 when reintroduced into BY2 protoplasts, and this association was disrupted by ATP. A three-dimensional structure for the tobacco GrpE homologue was modeled based on the X-ray structure of #E. coli$ GrpE complexed with DnaK. The modeled structure has the same overall structure as #E. coli$ GrpE. We propose that the tobacco GrpE homologue interacts wih mHsp70 in a manner analogous to #E. coli$ with DnaK and designate it as tobacco mitochondrial GrpE (NtmGrpE). (Résumé d'auteur)

International audience; Defective avian leukosis-based vectors expressing the bacterial lacZ gene were used as helper-free preparations to infect early stage Brown-Leghorn embryos. Both in toto X-gal staining and DNA analysis using Southern blot technique were applied to detect virus integration and expression. Our results demonstrate a low efficiency of in vitro infection in early stages of embryonic development. Southern blot analysis reveals that only 1% of embryonic cells integrate the vector genome after infection using 2 to 12 virus particle per embryonic cell. In situ expression of the lacZ marker gene was detected in only 0.06% of embryonic cells. These results lead us to conclude that only 6% of infected cells express efficiently the lacZ marker gene. This low level of expression could result from avian leukosis virus LTRs inhibition in chicken embryonic cells at an early stage of development. In spite of the low efficiency of infection, no evidence for tissue restrictive expression was observed. However, vector containing LTRs from RAV-2 virus allows preferential expression of provirus vector in neural tube tissue, whereas cardiac localization of the preferential expression was observed using vector containing the RAV-1 LTRs. The chronological analysis of the marker gene expression in terms of location of expression foci and sizes of these foci, lead us to hypothesize the putative regulation of retrovirus expression linked to embryonic development.

Within the MusaGenomics Consortium three BAC libraries were constructed from the genomes of Musa acuminata (diploid Calcutta-4, triploid Cavendish) and Musa balbisiana (diploid PKW). To study the structure and evolution of Musa genomes in relation to those of rice and sorghum, 50 Sorghum bicolor cDNA clones (EGRAM genes), were used as probes to screen the M. acuminata BAC library. Nine BAC clones were selected using EGRAM probes of known function, analysed by BAC-end sequencing and RFLP-fingerprinting, and sequenced. In addition, BAC clones believed to arise from the homeologous region on the A and B Musa genomes were identified using M. acuminata probes encoding for genes of agronomic interest. We have completely sequenced thirteen Musa BACs (~1.4 Mb), having a gene density of approximately one per 5 kb. The base composition of the CDSs ranges from 35% GC to 75% GC and shows a bimodal distribution. A subset of the genes shows a gradient in GC content along the coding sequence similar to rice, while the other set has a uniform base composition like Arabidopsis. At least one region of microsynteny was observed between Musa and rice, in which 6 genes are found in the same order of rice chromosome 5. Comparison of presumed homeologous BACs from the A and B genomes showed almost complete conservation of gene content and order. Retrotransposon content of these gene rich BACs was low. Preliminary phylogenetic analysis of individual genes suggests that Musa is as close to members of the Asparagales as to the Poales. (Texte intégral)