Drosophila as a model system Flashcards
How can p-elements be used in mutagenesis?^
When they excise from the genome, they sometimes remove pieces of the DNA, making a mutant gene. In about 90% perfect excision, in 10% they take out more. The big advantage of that: control and mutant are genetically identic; good to reduce background noise
Mechanism of RNAi
dsRNA is detected by DICER –> cleaved in siRNA duplex –> RISC unwinds it, keeps one strand and binds to the corresponding mRNA –> cleaves the target –> mRNA degradation
Drosophila life cycle
egg/embryo - 1st instar larva - 2nd instar larva - 3rd instar larva - prepupa - pupa - adult
types of mutageneses in D.
- EMS (ethyl methanesulfonate; and other chemicals)
- P-elements (and other transposons)
- Deficiency kit (and other aberrations)
Why is Drosophila a good model for the study of human disease?
1) Rapid construction of transgenic models of human disease
2) Well established easy systems to drive kd/ko our over expression of gene expression in tissue or temporal specific patterns
3) able to rapidly identify modifier/bypass gene pathways via genetic screens for enhancers or suppressors of phenotypes
4) easy to culture cell lines - very-easy to dsRNA treat genes of interest
5) rapid determination of the molecular basis of disease mechanisms
6) rapid forward genetics - isolate mutants through transposons or chemical mutagenesis
D. as cancer model
great model -
- epithelial tumours -
- glial tumours, neuroblast tumours -
- myoblast tumours -
- tumours from haematopoietic precursors -
- and other
advantages of EMS
- random
- saturation
- different types of mutations
forward genetics
1) start with unkown system
2) mutations to identify genes needed
3) mutant phenotypes reveal function
4) map the genes
5) identify the genes in the region
6) find which of these is the “culprit”
mutagenic effects of deficiency kit
chromosome rearrangements (gene deletions)
selection
individuals not meeting the criteria don’t survive (or are otherwise eliminated from the population)
E.g. Looking for wingles fly mutants: open the vial and let the flies fly away
Lenght of Drosophile life cycle
10-12 days at RT
Ways to test drug candidates in flies
- inject -
- put in medium (solid or liquid)
- aerosol
- feed
- decapitation and application
reverse genetics
begins with gene/product, work backwards to figure out the process that is involved –> what we did for the project
advantages of deficiency kit
- small scale
- fast screening
- defined set
Food intake control in D. and humans
very conserved; e.g. CCK and NPS/Vasopressin/Oxytocin
High blood protein in Drosophila leads to
adipokinetic hormone (glucagon-like) release –> corpora cardiaca (pancreatic alpha cells) –> adipokinetic hormone –> stimulates glycogen breakdown
mutagenic effect of p-elements
DNA insertions (mostly hypomorphic)
P-element
a DNA-dependent transposon (no RNA intermediate; genomic DNA at original insertion site) flanked by inverted repeats (IRs)
transposase will cut out the transposon and insert it to another place in the genome
Using GAL4/UAS to get rid of a gene/ knock down a gene
Insert an inverted repeat of your targeted gene after UAS –> when it is transcribed, it will form dsRNA –> this will lead to RNAi –> inhibition of protein expression, because mRNA of the targeted gene is destroyed
Screen
Each member of the population is examined … does it fit the phenotype criteria that have been set up?
E.g. Looking for wingles fly mutants: look at each fly… wings present?
Why is drosophila a valuable model system?
- It’s an animal; can be used to study development, physiology, and behaviour -
- over 100 years of genetics -
- 70% of human “disease” genes have an homologue in Drosophila
The fat body is analogous to? ^
- adipose tissue -
- liver -
- immune and blood cells (part of the haematopoietic systems)
GAL4/UAS binary transgenic expression system
most used nowadays
- one fly with tissue specific promoter followed by GAL4 in p-element (i.e. flanked by IR) -
- one fly with UAS (upstream activating sequence that is the GAL4 target) followed by transgene in p-element -
- mate them -
- the progeny will express the transgene in cells also expressing GAL4 –> GAL4 expression activates UAS and transgene is turned on
Optogenetics in flies
no need for surgery, just shine strong enough light on them
How to make a transgenic fly using transposons
- -Add a plasmid carrying the transposon/detective p-element and a donor plasmid with your desired transgene and a marker flanked by IR into D. embryo before the germline forms -
- Hope that your transgene wil jump into the fly genome, mediated by p-element -
- screen/select for marker -
- at the excision site, either repair using a sister choromatid/homologous chromosome containing a P-element –> transposon remains in original position OR repair of gap using a homologous chromosome lacking a P element –> transposon no longer at original position
D. as a model for developmental defects
Char syndrome TFAP2B
advantage of P-elements
- fast gene identification
- flexible scale
mutagenic effect of EMS
base pair changes/ point mutations
Features shared by Drosophila and other animals
- obligate diploid -
- sexually dimorphic gametes -
- some genetic redundancy
What are transposons?
- Small pieces of DNA that can move from one site in the genome to another -
- All organisms have them (about 45% of our genome: transposon remnants) -
- jumping genes, selfish DNA -
- mechanism for evolutionary change
Oenocytes
Analogues to mammalian hepatocytes
How many chromosomes does D. melanogaster have?
4
disadvantages of P-elements
- no saturation
- non-random (hotspots)
Drosophila oenocytes are involved in
- lipid homeostastis -
- sugar homeostasis -
- feeding behaviour
Brainbow
“a recombinase-based fluorescence labeling technique to subdivide neural expression patterns” -
- UAS followed by multiple fluorescent sites with lox sites –> random KOs will be induced -
- Cre is activated using heatshock.
- Different colours are expressed –> individual colouring
Three types of transposable genetic elements
- DNA-dependent (prokaryotes & eukaryotes; DNA intermediates) -
- retroviruses (eukaryotes only, RNA intermediates) -
- retrotransposons (eukaryotes only, RNA intermediates)
disadvantages of deficiency kit
- slow gene identification
- no real saturation
disadvantages of EMS
- slow gene identification
- large-scale
High blood sugar in D. leads to
promotes insulin release –> insulin-producing cells (pancreatic beta cells) –> insulin-like peptides –> stimulates glucose uptake from blood
