Name advantages of the use of Drosophila as an animal model
- short generation time
- easy to maintain
- 4 large chromosomes
- cheap to maintain
- easily identifiable markers (mutant phenotypes)
What are reasons to be aggressive?
- access to mates
- access to food
- access to territory to gain mates and foot
- maintain social hierarchy
- defence against predation
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
Drosophila life cycle
egg/embryo - 1st instar larva - 2nd instar larva - 3rd instar larva - prepupa - pupa - adult
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
Lenght of Drosophile life cycle
10-12 days at RT
mutagenic effect of p-elements
DNA insertions (mostly hypomorphic)
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
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
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
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
advantage of P-elements
- fast gene identification
- flexible scale
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
How many chromosomes does D. melanogaster have?
disadvantages of P-elements
- no saturation
- non-random (hotspots)
Three types of transposable genetic elements
- - DNA-dependent (prokaryotes & eukaryotes; DNA intermediates) -
- retroviruses (eukaryotes only, RNA intermediates) -
- retrotransposons (eukaryotes only, RNA intermediates)
Name behaviours of male flies
How do males behave when the external cue is female?
- virgin --> mate
- mated --> ignore
How do males behave when the external cue is male?
- dominant --> flee
- submissive --> attack or ignore
- unknown --> attack
How do males behave when the internal cue is satiated?
- female --> mate
- male --> ignore or attack
How do males behave when the internal cue is hungry?
- female --> ignore
- male --> attack
Name some molecules that affect aggression in flies
- ocotopamine (noradrenaline)
- NPF (NPY)
- 11 cis-vaccenyl acetate (cVA)
What does CREB regulate in flies?
- inhibits mating
- induces aggression
- induces lipid storage
- induces feeding
it mediates the starvation genotype
Which GTPase signals in the cVA pathway?
Why do males put cVA on females?
- makes males more aggressive --> they are not interested in mating anymore
- when it builds up, the males will leave, so the females end up having all the food they need for the offspring
When is Rac2 active/inactive?
- active: bound GTP
- inactive: bound GDP
Name characteristics of Rac2 mutants
- mating behaviour defect (they want to mate NOW)
- not aggressive
- lean phenotype
- lower lipid content
- upstream of Rac2
- lipid storage defect
- more susceptible to infection as they age
How does diet influence the behaviour of flies?