Drosophila as a model system Flashcards Preview

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Flashcards in Drosophila as a model system Deck (39)
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1
Q

How can p-elements be used in mutagenesis?^

A

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

1
Q

Mechanism of RNAi

A

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

2
Q

Drosophila life cycle

A

egg/embryo - 1st instar larva - 2nd instar larva - 3rd instar larva - prepupa - pupa - adult

2
Q

types of mutageneses in D.

A
  • EMS (ethyl methanesulfonate; and other chemicals)
  • P-elements (and other transposons)
  • Deficiency kit (and other aberrations)
3
Q

Why is Drosophila a good model for the study of human disease?

A

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

3
Q

D. as cancer model

A

great model -

  • epithelial tumours -
  • glial tumours, neuroblast tumours -
  • myoblast tumours -
  • tumours from haematopoietic precursors -
  • and other
3
Q

advantages of EMS

A
  • random
  • saturation
  • different types of mutations
4
Q

forward genetics

A

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”

5
Q

mutagenic effects of deficiency kit

A

chromosome rearrangements (gene deletions)

6
Q

selection

A

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

7
Q

Lenght of Drosophile life cycle

A

10-12 days at RT

7
Q

Ways to test drug candidates in flies

A
  • inject -
  • put in medium (solid or liquid)
  • aerosol
  • feed
  • decapitation and application
8
Q

reverse genetics

A

begins with gene/product, work backwards to figure out the process that is involved –> what we did for the project

9
Q

advantages of deficiency kit

A
  • small scale
  • fast screening
  • defined set
10
Q

Food intake control in D. and humans

A

very conserved; e.g. CCK and NPS/Vasopressin/Oxytocin

12
Q

High blood protein in Drosophila leads to

A

adipokinetic hormone (glucagon-like) release –> corpora cardiaca (pancreatic alpha cells) –> adipokinetic hormone –> stimulates glycogen breakdown

13
Q

mutagenic effect of p-elements

A

DNA insertions (mostly hypomorphic)

15
Q

P-element

A

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

16
Q

Using GAL4/UAS to get rid of a gene/ knock down a gene

A

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

17
Q

Screen

A

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?

19
Q

Why is drosophila a valuable model system?

A
    • 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
20
Q

The fat body is analogous to? ^

A
    • adipose tissue -
  • liver -
  • immune and blood cells (part of the haematopoietic systems)
21
Q

GAL4/UAS binary transgenic expression system

A

most used nowadays

  1. one fly with tissue specific promoter followed by GAL4 in p-element (i.e. flanked by IR) -
  2. one fly with UAS (upstream activating sequence that is the GAL4 target) followed by transgene in p-element -
  3. mate them -
  4. the progeny will express the transgene in cells also expressing GAL4 –> GAL4 expression activates UAS and transgene is turned on
22
Q

Optogenetics in flies

A

no need for surgery, just shine strong enough light on them

23
Q

How to make a transgenic fly using transposons

A
  1. -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 -
  2. Hope that your transgene wil jump into the fly genome, mediated by p-element -
  3. screen/select for marker -
  4. 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
24
Q

D. as a model for developmental defects

A

Char syndrome TFAP2B

24
Q

advantage of P-elements

A
  • fast gene identification
  • flexible scale
25
Q

mutagenic effect of EMS

A

base pair changes/ point mutations

26
Q

Features shared by Drosophila and other animals

A
    • obligate diploid -
  • sexually dimorphic gametes -
  • some genetic redundancy
27
Q

What are transposons?

A
  • 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
29
Q

Oenocytes

A

Analogues to mammalian hepatocytes

31
Q

How many chromosomes does D. melanogaster have?

A

4

33
Q

disadvantages of P-elements

A
  • no saturation
  • non-random (hotspots)
34
Q

Drosophila oenocytes are involved in

A
    • lipid homeostastis -
  • sugar homeostasis -
  • feeding behaviour
35
Q

Brainbow

A

“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
36
Q

Three types of transposable genetic elements

A
    • DNA-dependent (prokaryotes & eukaryotes; DNA intermediates) -
  • retroviruses (eukaryotes only, RNA intermediates) -
  • retrotransposons (eukaryotes only, RNA intermediates)
37
Q

disadvantages of deficiency kit

A
  • slow gene identification
  • no real saturation
38
Q

disadvantages of EMS

A
  • slow gene identification
  • large-scale
39
Q

High blood sugar in D. leads to

A

promotes insulin release –> insulin-producing cells (pancreatic beta cells) –> insulin-like peptides –> stimulates glucose uptake from blood