Transcript TRANSGENESE - univ

TRANSGENESE
I
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 transgène puisse 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 Drosophila melanogaster, 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 Cytotype individuals 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 Drosophila
melanogaster
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
overexpress it 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.
(A) 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 modular overexpression
screen.
(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.
Plasmid Rescue
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.
Plasmid Rescue
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 digested gDNA but do not
add a plasmid.
4) Transfrom the ligation into E. coli (+ Amp) and look for colonies. Any cells that survive
antibiotic selection must have some flanking chromosomal DNA in the plasmid.
wwwhite
Gal4
White
+ amp
Amp ori
UAS(Gal4 binding sites)
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
Neurodegenerative Diseases
(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-Dependent Neurodegeneration
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.
(A) 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 genespecific silencing.
Examples