MICE, a program to track and monitor animals in animal facilities

BACKGROUND: A growing number of laboratories are using the mouse as a model system in developmental biology as well as in molecular biology. Surprisingly, most of these laboratories do not have reliable computerized systems to track these animals, and the few commercial solutions available are expensive. We thus developed MICE (Mouse Information and Classification Entity), a program aimed at facilitating the monitoring of animals in animal facilities. RESULTS: This program consists of a virtual facility in which scientists can perform all the tasks done in the real world (i.e., receiving animals, breeding them, preparing cage labels, etc.). Recording of each animal (birth date, cage number, ID number, tail analysis number, parents, genetic status, genetic background, etc.) enables reliable tracking. According to any parameter of interest, animals can then be identified, grouped, sorted, moved, and so forth. Crossings are automatically processed by the program. For example, new genetic backgrounds, generation number, and anticipated due dates are determined. The program also reminds the user when new births are expected and entering newborn animals only requires a few clicks. The genealogy of each animal can be determined in two different ways, one being the visualization of a genealogical tree from which information of ancestors can be retrieved. CONCLUSION: This standalone program, that will be distributed free of charge to academic laboratories requesting a license, represents a new and valuable tool for all animal facility users, and permits simple and reliable tracking and retrieving of animals

REtools: A laboratory program for restriction enzyme work: enzyme selection and reaction condition assistance

BACKGROUND: Restriction enzymes are one of the everyday tools used in molecular biology. The continuously expanding panel of known restriction enzymes (several thousands) renders their optimal use virtually impossible without computerized assistance. Several manufacturers propose on-line sites that assist scientists in their restriction enzyme work, however, none of these sites meet all the actual needs of laboratory workers, and they do not take into account the enzymes actually present in one's own laboratory. RESULTS: Using FileMaker Pro, we developed a stand-alone application which can run on both PCs and Macintoshes. We called it REtools, for Restriction Enzyme tools. This program, which references all currently known enzymes (>3500), permits the creation and update of a personalized list of restriction enzymes actually available in one's own laboratory. Upon opening the program, scientists will be presented with a user friendly interface that will direct them to different menus, each one corresponding to different situations that restriction enzyme users commonly encounter. We particularly emphasized the ease of use to make REtools a solution that laboratory members would actually want to use. CONCLUSION: REtools, a user friendly and easily customized program to organize any laboratory enzyme stock, brings a software solution that will make restriction enzyme use and reaction condition determination straightforward and efficient. The usually unexplored potential of isoschizomers also becomes accessible to all, since REtools proposes all possible enzymes similar to the one(s) chosen by the user. Finally, many of the commonly overlooked subtleties of restriction enzyme work, such as methylation requirement, unusual reaction conditions, or the number of flanking bases required for cleavage, are automatically provided by REtools

Is the Vascular Network Discriminant Enough to Classify Renal Cell Carcinoma?

International audienceThe renal cell carcinoma (RCC) is the most frequent type of kidney cancer (between 90% and 95%). Twelve subtypes of RCC can be distinguished, among which the clear cell carcinoma (ccRCC) and the papillary carcinoma (pRCC) are the two most common ones (75% and 10% of the cases, respectively). After resection (i.e., surgical removal), the tumor is prepared for histological examination (fixation, slicing, staining, observation with a microscope). Along with protein expression and genetic tests, the histological study allows to classify the tumor and define its grade in order to make a prognosis and to take decisions for a potential additional chemotherapy treatment. Digital histology is a recent domain, since routinely, histological slices are studied directly under the microscope. The pioneer works deal with the automatic analysis of cells. However, a crucial factor for RCC classification is the tumoral architecture relying on the structure of the vascular network. For example, coarsely speaking, ccRCC is characterized by a ``fishnet'' structure while the pRCC has a tree-like structure. To our knowledge, no computerized analysis of the vascular network has been proposed yet. In this context, we developed a complete pipeline to extract the vascular network of a given histological slice and compute features of the underlying graph structure. Then, we studied the potential of such a feature-based approach in classifying a tumor into ccRCC or pRCC. Preliminary results on patient data are encouraging

Development of a new bicistronic retroviral vector with strong IRES activity

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, tagging, sequencing, and expression of any cDNA in the first cistron, while keeping a high level of expression from its IRES-dependent second cistron (here encoding eGFP)

A new generation of pPRIG-based retroviral vectors

<p>Abstract</p> <p>Background</p> <p>Retroviral vectors are valuable tools for gene transfer. Particularly convenient are IRES-containing retroviral vectors expressing both the protein of interest and a marker protein from a single bicistronic mRNA. This coupled expression increases the relevance of tracking and/or selection of transduced cells based on the detection of a marker protein. pAP2 is a retroviral vector containing eGFP downstream of a modified IRES element of EMCV origin, and a CMV enhancer-promoter instead of the U3 region of the 5'LTR, which increases its efficiency in transient transfection. However, pAP2 contains a limited multicloning site (MCS) and shows weak eGFP expression, which previously led us to engineer an improved version, termed pPRIG, harboring: i) the wild-type ECMV IRES sequence, thereby restoring its full activity; ii) an optimized MCS flanked by T7 and SP6 sequences; and iii) a HA tag encoding sequence 5' of the MCS (pPRIG HAa/b/c).</p> <p>Results</p> <p>The convenience of pPRIG makes it a good basic vector to generate additional derivatives for an extended range of use. Here we present several novel pPRIG-based vectors (collectively referred to as PRIGs) in which : i) the HA tag sequence was inserted in the three reading frames 3' of the MCS (3'HA PRIGs); ii) a functional domain (ER, VP16 or KRAB) was inserted either 5' or 3' of the MCS (« modular » PRIGs); iii) eGFP was replaced by either eCFP, eYFP, mCherry or puro-R (« single color/resistance » PRIGs); and iv) mCherry, eYFP or eGFP was inserted 5' of the MCS of the IRES-eGFP, IRES-eCFP or IRES-Puro-R containing PRIGs, respectively (« dual color/selection » PRIGs). Additionally, some of these PRIGs were also constructed in a pMigR MSCV background which has been widely used in pluripotent cells.</p> <p>Conclusion</p> <p>These novel vectors allow for straightforward detection of any expressed protein (3'HA PRIGs), for functional studies of chimeric proteins (« modular » PRIGs), for multiple transductions and fluorescence analyses of transduced cells (« single color/resistance » PRIGs), or for quantitative detection of studied proteins in independently identified/selected transduced cells (« dual color/selection » PRIGs). They maintain the original advantages of pPRIG and provide suitable tools for either transient or stable expression and functional studies in a large range of experimental settings.</p

A Novel Function for Fragile X Mental Retardation Protein in Translational Activation

Fragile X syndrome, the most frequent form of inherited mental retardation, is due to the absence of Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein involved in several steps of RNA metabolism. To date, two RNA motifs have been found to mediate FMRP/RNA interaction, the G-quartet and the “kissing complex,” which both induce translational repression in the presence of FMRP. We show here a new role for FMRP as a positive modulator of translation. FMRP specifically binds Superoxide Dismutase 1 (Sod1) mRNA with high affinity through a novel RNA motif, SoSLIP (Sod1 mRNA Stem Loops Interacting with FMRP), which is folded as three independent stem-loop structures. FMRP induces a structural modification of the SoSLIP motif upon its interaction with it. SoSLIP also behaves as a translational activator whose action is potentiated by the interaction with FMRP. The absence of FMRP results in decreased expression of Sod1. Because it has been observed that brain metabolism of FMR1 null mice is more sensitive to oxidative stress, we propose that the deregulation of Sod1 expression may be at the basis of several traits of the physiopathology of the Fragile X syndrome, such as anxiety, sleep troubles, and autism

Etude de l'expression et des modifications du produit de traduction du proto-oncogene c-ets-1

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Thérapie génique (stratégie suicide humanisée et évaluation du transfert de genes par vecteurs lentiviraux)

Gene therapy is a new approach of Medicine creating the notion of "gene-medicine". During my thesis, I mainly participated to development and evaluation of four essential components of gene therapy strategies : transfer, expression, and control of the therapeutic gene, as well as the future of the therapeutic targets, the transduced cells. In a first part of this work, we try to develop a system of induced and targeted apoptosis. This system relies on the expression of an inducible caspase 8 controlled by a "specific" promoter, and transfered in target cells by a viral vector. The main point of this strategy is the evaluation of an inducible caspase 8 as a "therapeutic" gene. We compared two transcriptional regulation systems, and a post-translational one. We eventually proposed a modified form of human caspase 8 inducible by an homodimerization regulated system as an alternative system to suicide gene HSV-tk. We also considered controlling expression of this proapoptotic gene with a promoter "specifically" active in cells transformed by a mutated form of Ras, our first cellular model. In a second part, we were interested in gene transfer systems, and more particularly in entiviral vectors. Our first study model was the 3T3-L1 adipocyte system, one of the main cellular model used in obesity and diabetes fields of research, but hardly transfectable by usual transfection methods. We show that HIV-1 derived lentiviral vectors efficiently transduce quiescent and fully differentiated 3T3-L1 adipocytes, as well as 3T3-L1 dividing pre-adipocytes. Furthermore, contrary to adenoviruses, there's no overt signs of cytotoxicity, as confirmed by the glucose uptake physiological assay. Moreover, we checked that capacity to differentiate is not affected by the transduction of 3T3-L1 pre-adipocytes. We thus show that 3rd generation of lentiviral vectors are the system of choice for genetic modification of 3T3-L1 pre-adipocytes and mature adipocytes. Finally, we established several collaborations offering attracting applications for such a system, not only as gene vectorisation system, but also as a stable ectopic expression system that can be combined with other technologies like siRNA. To conclude, it's important to note that if clinical applications in this kind of approaches are long-term prospects, even very long-term, for security and efficiency reasons, such technologies can find multiple applications in various fields of research as a commun tool of molecular biology.La thérapie génique est une nouvelle approche de la médecine créant la notion de gènes-médicaments. Au cours de ma thèse, j'ai participé principalement au développement et à l'évaluation de quatre composants essentiels d'une approche de thérapie génique : le transfert, l'expression et le contrôle du gène thérapeutique, ainsi que le devenir des cibles thérapeutiques, les cellules transduites. Dans une première partie de notre travail, nous avons tenté de développer un système d'apoptose inductible et ciblée. Ce système est basé sur une caspase inductible dont l'expression est contrôlée par un promoteur " spécifique ", et transférée dans la cellule cible par un vecteur viral. Le point central de cette stratégie a été l'évaluation d'une caspase 8 inductible comme gène " thérapeutique ". Nous avons comparé pour cela des systèmes de régulation transcriptionnelle ainsi qu'un système de régulation post-traductionnelle, et nous proposons finalement une caspase 8 humaine inductible par un système d'homodimérisation régulée comme une alternative au gène suicide HSV-tk. Nous avons également considéré le contrôle de l'expression de ce gène grâce à un promoteur " spécifique " de cellules transformées par une forme activée de Ras, notre premier modèle d'étude. Dans une deuxième partie, nous nous sommes intéressés au système de transfert de matériel génétique et plus particulièrement aux vecteurs lentiviraux. Notre premier modèle d'étude a été le système adipocytaire 3T3-L1, un des principaux modèles cellulaires pour les études concernant l'obésité, le diabète, mais très difficilement transfectable par les méthodes usuelles. Nous montrons que des adipocytes 3T3-L1 quiescents et pleinement différenciés, aussi bien que des pré-adipocytes 3T3-L1 en division, sont efficacement transduits par les vecteurs lentiviraux dérivés du HIV-1. De plus, contrairement aux adénovirus, les lentivirus n'engendrent aucun signe de cytotoxicité apparent, comme le confirme le test physiologique d'absorption du glucose en réponse à l'insuline. Nous avons également vérifié que la transduction des pré-adipocytes 3T3-L1 n'affecte en rien leur capacité à se différencier. Nous montrons donc que les vecteurs lentiviraux de 3ème génération constituent le système de transfert de choix pour la modification génétique des pré-adipocytes et adipocytes matures 3T3-L1. Enfin, nous avons établi plusieurs collaborations offrant des applications attrayantes pour un tel système, non seulement en tant que stratégie de vectorisation de gènes, mais aussi comme outil d'expression ectopique stable pouvant notamment être combiné avec d'autres technologies comme les siRNA. En conclusion, il est important de noter que si les applications cliniques de ce type d'approche restent des perspectives à long terme, voire très long terme pour cause d'aspects sécuritaires et d'efficacité encore insuffisants, une telle technologie peut trouver de multiples applications dans des domaines très divers comme outil commun de biologie moléculaire.NICE-BU Sciences (060882101) / SudocSudocFranceF