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cours 2010 - TRANSGENESE

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    cours 2010 - TRANSGENESE cours 2010 - TRANSGENESE Presentation Transcript

    • TRANSGENESEI
      Drosophile
    • La transgenèse ET SES APPLICATIONS
      La transgenèse consiste à introduire dans un organisme vivant un gène qui lui est étranger – un transgène- de façon à lui conférer une nouvelle propriété qu’il transmettra à sa descendance. Chez les animaux, pour que le transgènepuisse être transmis, il doit être intégré dans les cellules produisant les gamètes (lignée germinale). La nouvelle propriété est celle donnée par la protéine que code le transgène.
      La transgenèse permet d’ajouter, de remplacer ou d’inactiver un gène particulier.
    • Le transfert des gènes est rendu possible grâce à l’universalité du support de l’information génétique (ADN) et du code génétique.
      Homologous proteins functioning interchangeably in the development of mice and flies.
      a fly protein used in a mouse
      Universal Mechanisms of Animal Development
    • Transgenic Fruit Flies
      Why is Drosophila such a good experimental organism?
      • Easy and inexpensive to culture
      • Very common and not harmful to humans
      • Generation time of 9-10 days at 25°C
      • Embryogenesis takes 22 hours
      • Short lived (~60 days is life-span under ideal conditions)
      • ~ 100 years of genetic studies & many unique techniques
      • Thousands of mutants and transgenic lines
      • Molecular genetics & cell biology are well known
      • The genomic sequence has been reported (Mars, 2000)
    • L’élément transposable P et la transgenèse permet d’utiliser la drosophile comme organisme-modèle pour étudier des gènes, des mutations et des maladies présentes chez l’humain.
      Spradling and Rubin (1982) used cloned transposable elements to generate transgenic flies and first rescue of a mutant phenotype by gene transfer.
      Foreign DNA can be incorporated into the Drosophila germ-line genome by the technique of P-element transformation.
      Incorporation of a single copy of the transgene into the Drosophila genome.
    • Chez Drosophilamelanogaster, les éléments P sont responsables d'un syndrome appelé dysgénésie hybride caractérisé par des taux de mutation élevés, des aberrations chromosomiques fréquentes ainsi que des individus stériles. Ceci correspond à la transposition des éléments P dans le génome.
         
      Cette instabilité intervient quand des mâles de type P sont croisés avec des femelles de type M. 
      P Cytotypeindividuals contain 30 to 50 copies of a DNA sequence known as the P transposable element.
      In dysgenic individuals, copies of the element are found in new locations.
    • HYBRID DYSGENESIS
      A complex genetic phenomenon, hybrid dysgenesis, led to the discovery of
      P transposable elements in Drosophila.
    • In a cross between a P element carrying female and a laboratory male [left], repressors in the maternally derived cytoplasm repress expression of the maternally inherited P elements. The resulting offspring show the wild-type phenotype.
      In a cross between a P-element carrying male and a laboratory female [right], repressors are absent in the maternally derived cytoplasm.
      The two zygotes are chromosomally identical but cytoplasmically different.
      In the right-hand cross, P elements are activated and undergo transposition in the genome, causing release of mutator activity and a variety of dysgenic phenotypes in the offspring.
    • -P elements are small transposons with terminal 31-bp inverted repeats
      -There are autonomous and non-autonomous P elements.
      -The autonomous P element is 2907 bp and is autonomous because it encodes a functional transposase.
      -The element generates 8-bp direct repeats of target DNA sequences upon insertion.
    • La transposase ne s'exprime que dans
      la lignée germinale.
    • Mécanisme d'excision-insertion.
      Le modèle de fonctionnement "couper-coller" fut proposé par le groupe d'Engels en 1990: le transposon est exprimé, et sa transposase vient l'extraire de son gène hôte. Le transposon, alors libre, peut s'insérer n'importe où ailleurs dans le génome. Cependant, il a laissé derrière lui une lacune (gap), qui sera comblée grâce à la copie du gène qui lui fait face, et dont les extrémités correspondent à celles de la lacune. Souvent ce gène qui sert de modèle pour le remplissage du trou est une copie du transposon porté par le chromatide soeur.
    • L'élément P, vecteur de transformation germinale.
      Structure du plasmide portant le transgène
    • Generation of transgenic fruit flies by
      P-element transformation.
      To make transgenic fruit flies, the appropriately modified DNA fragment is injected into a very young fruit fly embryo (less than 1 hour old) along with a separate plasmid containing the gene encoding the transposase. When this is done, the injected gene often enters the germ line in a single copy as the result of a transposition event
    • Transgenesis upgrades for Drosophilamelanogaster
      Koen J. T. Venken and Hugo J. Bellen
      Development 134, 3571-3584 (2007)
      + transposase
      white eyes
    • Flies that develop from injected embryos (G0) will carry
      some germ cells that have incorporated the transgene;
      some of their progeny will carry the transgene in all somatic and germ-line cells, giving rise to pure transgenic lines.
      Individuals carrying the transgene are recognized by expression of a marker gene (white+) that is present on the donor DNA.
    • GAL4/UAS Expression System
      • To study the function of gene it is desirable to 1) misexpress and/or overexpressit in different tissues at different times or 2) express altered forms of a gene
      • The yeast GAL4 transcription factor and its target sequence (UAS) have been introduced into Drosophila.
      • GAL4 does not activate native Drosophila genes but when a gene is placed next to a UAS sequence (cloning or transposition), it can be controlled by GAL4.
      • Wide-range of GAL4 lines that vary in amount, timing and tissue of expression have been produced.
    • Exemple
    • Le détecteur d'enhancer GAL4
      Rechercher un enhancer revient à détecter un gène, non pas sur le phénotype de mutations, visibles ou létales, mais directement sur le profil de leur expression.
    • ENHANCER TRAP
    • The advantage of this system is that such enhancer traps can drive expression of any transgene under control of the UAS promoter recognized by GAL4. This thus provides a technique for cell-specific expression of transgenes in an intact organism.
    • The GAL4/UAS technique for controlled gene misexpression in Drosophila.
      Although one can achieve the same result by linking a copy of the H regulatory sequence directly to the G coding sequence, the GAL4/UAS approach allows a strategy that is more efficient in the long run.
    • (B) The enhancer trap technique
      Two separate “libraries” of transgenic flies are constructed, one containing GAL4 inserts driven by a variety of regulatory sequences of different genes A, B, C, etc., the other containing UAS inserts driving a variety of different coding sequences X, Y, Z, etc. By mating a fly from one library with a fly from the other, any desired coding sequence can be functionally coupled to any desired regulatory sequence.
      To generate the library of flies with GAL4 insertions at useful sites, flies are first produced with GAL4 insertions at random locations in their genome. These are then mated with flies containing a UAS element linked to a reporter gene with an easily detectable product. Expression of the reporter reveals whether GAL4 has been inserted at a site that brings its expression under the control of an interesting enhancer; flies showing interesting reporter patterns are kept and studied.
      This is called the enhancer trap technique, because it provides a way to hunt out and characterize interesting regulatory sequences in the genome.
    • The modularoverexpressionscreen.
      (A) A specific pattern line is crossed to a large number of independent target lines. In progeny carrying both a pattern and a target element, Gal4 encoded by the pattern element should bind to Gal4 binding sites within the target element enhancer and activate an adjacent endogenous gene. Each independent target element insertion thus targets one endogenous gene for expression.
      (B) Structure of the EP target P-element. The plasmid rescue sequences and the unique EcoRI site allow rapid cloning of flanking genomic DNA.
    • An EP element is a transposable P element containing a basal promoter and 14 copies of the yeast UAS sequence, which responds to the transcription factor GAL4.
      As P-elements have a tendency to insert in the 5’ end of genes, misexpression of an endogenous gene occurs in a large proportion of EP lines.
      This system has been adapted to carry out genetic screens for genes that give phenotypes when misexpressed in a particular tissue (modular misexpression screens).
      Example:
      Each EP line was crossed to a fly line that expresses GAL4 and the green fluorescent protein (GFP) in all post-mitotic neurons. This generated F1 flies in which GAL4 drove high expression of the genes adjacent to the EP element, with GFP enabling visualisation of the neurons.
    • PlasmidRescue
      The purpose of plasmid rescue is to isolate the chromosomal DNA adjacent to an inserted piece of DNA. There are 4 steps to this process.
    • PlasmidRescue
      1) At the beginning, a piece of DNA has inserted into the chromosome:
      2) Isolate the genomic DNA (gDNA) and digest it with a restriction enzyme that will cut at the edge of the plasmid portion of the insert. In this example, you would use Sac1, Sal1, Hind3, etc…
      3) Clean and concentrate the DNA, and do a ligation on the digestedgDNA but do not add a plasmid.
      4) Transfrom the ligationinto E. coli (+ Amp) and look for colonies. Anycellsthat survive antibioticselection must have someflankingchromosomal DNA in the plasmid.
    • wwwhite
      Gal4
      White Ampori UAS(Gal4 binding sites)
      + amp
    • Drosophila have recently emerged as model system for studying mechanisms of neurodegeneration in several major neurodegenerative diseases. These models are also excellent in vivo systems for the testing of therapeutic compounds. Genetic studies using these animal models have provided novel insights into the disease process.
      Identifying new genes in the fly with a role in maintaining adult CNS integrity is perhaps the fastest way to identify them in humans, given the ease of genetic screens for brain integrity mutants and range of genetic tools available for assessing gene function.
      Numerous Drosophila mutants have been isolated that profoundly affect neural viability and integrity of the nervous system with age.
      Additionally, many transgenic strains have been developed as models of human disease conditions.
      D. melanogaster will remain a key tool for the analysis
      of genes required for CNS integrity.
    • Schema for Generation of Fly Models of
      NeurodegenerativeDiseases
    • (A). GMR-GAL4 is a driver line which uses regulatory DNA that responds to the glass transcription factor to induce expression of GAL4 in all cells of the eye
      (B). The cDNAs encoding human disease-associated genes, such as that for Atx3, which causes SCA3, are subcloned into the UAS expression construct and used to generate transgenic flies.
      When these flies (B) are crossed with those of a driver line (A), GAL4 is expressed in the progeny and induces expression of the disease gene, producing an abnormal eye phenotype that can readily be scored under the dissection microscope.
      A stable tester stock can be established that expresses both of these transgenes and used to carry out genetic screens.
      (C). In the case of the EP element, in the presence of GMR-GAL4 (A), genomic sequences near the EP element can be identified that modulate severity of the tester stock.
    • Atx2 Enhances Atx3-DependentNeurodegeneration
      Genotype: A: Gmr-Gal4/, B: Gmr-Gal4/+; UAS-SCA3trQ78(w)/+, C: Gmr-Gal4/UAS-Atx2
      D: Gmr-Gal4/UAS-Atx2; UAS-SCA3trQ78(w)/+.
    • Transgenic RNAi in Drosophila. The generic GAL4/UAS system is used to drive the expression of a hairpin RNA (hpRNAs). These double-stranded RNAs are processed by Dicer into siRNAs which direct sequence-specific degradation of the target mRNA.
    • RNAi phenotypes.
      Control with GAL4 driver only.
      (B) GAL4 driver + UAS-eyRNAi, targeting the eyeless gene. The eye is missing, as in the eyeless mutant.
    • A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila
      Georg Dietzl et al 2007
      Nature 448, 151-156
    • Strategy used to generate UAS-IR constructs. Restriction sites in the original PCR primers were used to digest and ligate PCR products, followed by ligation of the inverted repeat into the pMF3 vector. pMF3 contains 10 GAL4-responsive UAS elements, the basal hsp70 promoter, the 150 bp second intron of ftz and the SV40 polyadenylation signal. Most, but not all, inverted repeats were cloned in the antisense-sense orientation using EcoRI and XbaI as indicated. IR-L and IR-R indicate the primer pairs used to amplify the left or right halves, respectively, of the inverted repeat. P3 and P5 indicate P-element ends.
      The library comprises 22,247 transgenic Drosophila strains, each containing an inducible UAS-RNAi construct against a single protein coding gene. 12,251 genes, or 88.2% of the Drosophila genome, are represented in this collection. All insertions have been molecularly validated, and a sample also functionally validated. We estimate that >80% of the lines provide potent and gene-specific silencing.
    • Examples