1
Q
why are flies a good model?
A
- genetically amenable
- short life cycle so generations can be studied in a relat9ivley short time
- large numbers of offspring from each mating
- ease of growing and handling the organism
- lots of history
2
Q
what are the stages of the fl life cycle?
A
embryo, 1st, 2nd, 3rd instar larva, prepuppa, pupa, fly
3
Q
how many chromosomes does the fly have?
A
4 - small 4 and an x or a y
4
Q
what is in the larva stage?
A
third instar larvae
- contains imaginal discs which give rise to all the main appendages of the fly- have a very uniform morphology throughout the fly - they have been specified by this point
5
Q
what is good about the disc stage?
A
it is easy to manipulate so you can alter the disc stage when you want to target the morphology of a certain organ
6
Q
what is cellularisation in the fly embryo?
A
the early embryo consists of just a ball of nuclei and then these nuclei move to the outside of the embryo and then cell membrane forms and becomes cellularised
7
Q
has the fly genome been sequenced?
A
yes- around 14000 genes
8
Q
why is the fly good for looking at processes in the human? (2)
A
- more than half of the fly protein have homologs in mammals
- 60% of human disease genes have homologues in flies
9
Q
why is fly genetics straight forward in flies?
A
- there is normally one gene for each protein so when you get a loss of function you can be fairly certain that this is giving the phenotype
10
Q
how can p-element transposons be used in flies and for what?
A
- p elements are pieces of DNA surrounded by P -eleemnts and these tend to just around the genome and land in different places
- you can inject them into flies and use this jumping system- make plasmids with the p-element clones and then you clone a gene of interest and then inject this with a plasmid containing the transposase gene and inject into the embryo at the time when it is a big syncytium and this will hopefully insert into the fly embryo
11
Q
how do you make a transgenic fly
A
- p elements are pieces of DNA surrounded by P -eleemnts and these tend to just around the genome and land in different places
- you can inject them into flies and use this jumping system- make plasmids with the p-element clones and then you clone a gene of interest and then inject this with a plasmid containing the transposase gene and inject into the embryo at the time when it is a big syncytium and this will hopefully insert into the fly embryo
- you can inject into the posterior of the embryo into cells that will give rise to the germ line
- once it is in the germ line if will be inherited by the offspring
- the plasmid that you use with the gene of interest must have a marker gene in it such as red red (w+) and inject into the white eye mutant strain- so you know that the thing get inserted and you can score the offspring which you know has your p-element in
12
Q
how frequent is the insertion of the p elements into the genome?
A
5%
13
Q
how can you target where a p-element insert?
A
you can use certain p elements that will go into certain landing platforms
14
Q
why is is bad that the P-element inserts randomly?
A
can be inserted into a highly condensed region of DNA- low expression- cause mutations
15
Q
what cells in the fly embryo give rise to the germ cells?
A
pole cells
16
Q
how can you use a gene trap and p-elements? what are the downsides?
A
- can take a construct which contains a weak promoter upstream of GFP and you can jump the element in- and look for one that gives you just expression in certain neurons.
- because you know the sequence of the enhancer trap- you can easily clone the area of gene where it has inserted- you digest the DNA up with restriction enzymes- this linear DNA will then anneal and circularise- you then use a restriction enzyme known o cut in the marker gene- then use primer to amply up either side of the mare gene to get the region round the gene trap
- the jumping of the P-element to another gene by adding transpose each generation will normally only be 5%
17
Q
how can enhancer traps be useful for looking at development ?
A
can find specific expression regions of genes in homogenous looking cells-you can then look at what happens when you perturb the expression to find due development mechanisms etc
- can find enhancers for specific regions of a gene’s expression
- find genes that are expressed in the same regions other genes at the same time- unbiased
18
Q
where does the GAL4/UAS system come from?
A
yeast
19
Q
how does the GAL4/UAS system generally work?
A
- can have a tissue specific promoter in one fly upstream of Gal4 then a UAS binding site downstream of chosen gene- reporter or been for expression
- then you cross these two strains and then again to make them both homozygous
20
Q
how has the GAL4 system been made easier to use?
A
there are strains that are ready which you can just order for whatever tissue you ant to use
21
Q
how can you prove the role of a gene in a process ? give an example?
A
to express in another tissue and see if it has the same affect- expressing the eyeless gene in the fly of a fly forms an eye- good for homeobox genes - master regulatory genes
22
Q
how can you test master regulatory genes in other species? use an example
A
- expressed the human pax6 (vert) you make fly eyes in the fly- this is profound because it tells you there must be a common precursor for eyes during evolution- the original eye could be a very simple eye - this would be pax 6- this would example why if you take human and fly pax 6 you get an eye- conserved eugenic cascades
23
Q
what is the principle of a loss of function approach to fly genetics?
A
1) Induce mutation using a chemical, Xrays or P-element
2) Score phenotype (i.e. consequence of losing the gene function)
3) Identify the mutated gene (reverse genetics)
24
Q
how did people go about finding out how you build the body plan of the fly embryo?
A
- people knew that the fly was highly segmented
- they did screens for segmentation difference in the embryo
- if you look at the larvae you can see denticle belts which are reflections of the segmentation- this is easy to look at
- they looked for mutation which affect these things
- they cloned many genes and found many maternal gene - int he F3
25
what are sequence of events which give rise to the segmentation?
- gene called bicoid becomes localised to one side of the egg and marks the anterior pole (maternal genes)- then these stimulate the gap genes which then stimulate the stripping of the pair rule genes and then these give the segments their polarity
26
why would you us the FLP/FRT system?
to study an embryonic lethal mutation - you create homozygous in adult that were heterozygous in the early development
27
what does FLP do?
FLP catalyses recombination at FRT sites
28
how do you get the FRT site into the chromosome
- can do targeted P-element targeting with a construct
29
how does the FLP/FFRT work?
you put FRT sites onto an arm in a certain position.in a paternal and a maternal chromosome- you start with a het with a mutant and cross with a homo that expresses a reporter downstream of the FRT site so that half will be het for the mutant with the FRT site on both - you then cross and you get an offspring which have a het for the alleles, each with FRT at the same site. then when mitosis happens then normally each daughter cell will inherit the same two het alleles - but if you express FLP then recombination will occur between the FRT sites between the two paternal an maternal chromosomes then this means that two daughter cells actually receive homozygous alleles for the site downstream of the FRT site because this has been swapped over
- you will know which cells ate the home mutant because they won't express the GFP reporter (the cells that do are called the twin spot) - so these cells will continue to dividing to produce a mosaic of mutant homo and non homo
- so you express FLP with a heat shock promoter
30
how can you kill the wild type cells in FLP/FRT
you can put a homo lethal allele on the on one of the wild type that you cross with to kill the wild type cells
31
give an example of the FLP FRT being used?
can look in the eye for cancer genes- or gene that give a growth advantage or disadvantage - hippo pathway- if you keep the mutant het flies apart then you can easily go back t them and use them as a strain
32
how would you go about doing a screen using the FRT-FLP system?
you carry out a screen using chromosomes that already contain FRT sites - each chromosome arm is screened one at a time by crossing to a strain with the relevant FR site for each arm being screened - each arm contain the FRT will contain a reporter aswell. so then each arm is screened and can be crossed
33
how can you get hold of FRT chromosomes for each arm?
you can order ready made ones which or formed using P-element mediated transformation
34
where is the FLP gene and heat shock promoter expressed during the FLP/FRT method?
- it is expressed in the cell lines hat hasn't been mutaginsed but is crossed to
35
if you have a gene of interest and you want to carry out reverse genetic approaches to see if is involved in a certain process using FLP/FRT, how would you do this?
- you would isolate a
36
what is the problem with following mutations in flies?
they are diploid and most mutations of recessive so you want to be able to follow the mutations by linking them to genetic markers such as white eye or ebony body- but recombination can occur is the mutation is not very close to the marker
37
how can you get around the issues with following mutants by linking them to markers?
- there is no recombination in male flies
| - you can use a balancer chromosome which contain many inversions which suppress recombination
38
how can you track balancers?
have a dominant marker on the balancer - such as cy+ (curled wing)
- you can also have a double homo on the balancer so that all the double homos die
39
how can you use a balancer screen to determine is a mutant is recessive lethal?
1- mutagnise a fly that is homo white eye mutant
2- cross with a het fly that has a balancer on one and a DTS (temp sensitive dominant lethal)
3- you will get some flies that are mutant with either curlywing or DTS (some will be WT over witness too)
4- you cross your F1s with a DTS/balancer het
5- grow at 29 degrees so that you are only left with mutants that are red eyed
6 cross siblings
7 will get white eyes (double mut) or red eyes and curly wings
- if there are no homo white eye- know they are lethal
- can just keep the red eye/balancer het and maintain as stock
40
can you freeze flies? why is this bad
no- you have to keep maintaining the stock by transferring food
41
why would you sue mosaic analysis
- can allow analysis of cell-type specific gene function - people just tend to guess rather htan checking(especially for lethals)
- cell autonomous vs non autonomous (being neighbouring cells being affected instead - receiving signals)
- correlation vs causation gene expression
42
what is the problem with the standard way of having the non mutants expressing GFP?
- this is good if you have one clone and its big but if you have a small clones or in a complicated tissye with lotsof clones- it is not easy to see a non fluouresing clone in a fluourescing background
43
what is the FLP out system? and how is a FLP out experiment carried out?
FRT sites are on the same chromosome but flank a stop codon. when you activate recombination- instead of the gene being on- its off
- you can have a FRT surrounding a stop upstream of an RNAi
- You have one strain that expresses the FLP downstream of a chosen promoter region, In this same strain there is a region holding an actin promoter upstream of a FRT'd stop codon, which is upstream of a Gal4. You then cross this with a strain that expresses a UAS site upstream of your desired cDNA. This will result in the cDNA being expressed in a certain tissue
- alternativley you can simply have instered a construct into one strain of a tissue specific promoter driving the expression of cDNA but it is blocked by a FRT'd stop. When you cross with a FLP being expressed in a temp sensitive manner you can trigger the tissue specific expression
44
how can you use the GAL80 and Gal4 system in conjunction with the FLP FRT system in order to label the mutant cells rather than the non mutant cells?
you have a line which express GAL80 upstream of a FRT site. you then have a mutant on the same chromosome arm as that with the FRT site+ Gal80. of the other member of the chromosomal pair.
- on the same chromosome as the mutant but not on thr FRT arm, there is a UAS driven GFP site. on the same chromosome as the gal80 driver but not within the FRt site there is a Gal4 gene diriven by actin promoter (this doesnt have to be in the same chromosome but just not within the FRt site- cant be expressed in Mutant strain ebcause then UAS will get turned on) - so you cross a homo for gal80 FT - act-Gal4 with a strain expressing the mutant wiht the FRT and then witht he UAS-GFP region. SO you would have this goind on with the temp or tissue sensitive FLP too
45
in what tissues is it important to have the mutant expressing the GFP not the non mutants?
the brain- complicated tissue
46
what are flies particulalry good at?
maternal mutations
47
what is the process by which the oocyte is formed in the mother?
a stem cell in the gonad becomes a cytoplasm which then divides and 4 rounds of cell division occur to create 16 cells- 1 of these becomes an oocyte and 15 a nurse cell
48
how can you use somatic recombination in the germline?
you can induce the formation of homozygous mutants in the germline by having a het mother which has a mutant downstream of the FRT and a FS donwstream of the FRT on the other chromosome- can induce only mut eggs to be produced in the mother
49
what is the role of gurken in fly development?
it controls the formation of the- AP and DV axis- Grk is transported along microtubules, and localises via dynein motor to MT minus ends
50
what does GRK encode?
a TGF-alpha-like ligand that signals to overlying follicle cells .
51
how does Grk act to define the two poles in the fly?
AP: Defines posterior follicle cellsA PFCs signal back to oocyte &
repolarise MT cytoskeleton
DV: Grk drives overlying FCs to be dorsal.
Ventral FCs synthesise a specialised vitelline membrane that selectively sequesters signalling component. After fertilisation, ventral proteolytic cascade activates signalling that generates DV transcription factor gradient.
52
how does Gurken control the DV patterning of the fly?
- it drives overlying cells to be dorsal- ventral FCs synthesis a specialised vitelline membrane that selectivley sequested signalling components. After fertlisation, ventral proteotylic cascade activates signalling that generates DV transcription
53
explain how the dorsal ventral axis and segments are patterned in the fly?
...
54
how can you do an RNAi screen in flies?
- you use the gal4 system and UAS, to drive an inverted repeat with a linker for a gene- this will trigger the degradation of the mRNA transcripts that are related to the hairpin
55
how can you get RNAi into the fly?
you can inject the DNA construct into the embryo syncitium using the p-element system
56
what makes using a tissue specific gal4 driver easy in flies
there are libraries that you can use
57
are there RNAi fly libraries available?
yes - makes it easy
58
advanatges of RNAI, disadvantages
- multiple fly lines for each gene- you can have multiple lines targeting different parts of the gene- specific
- can do tissue specific
- you know your gene (but you can just use sequencign now)
dis adv:
- WG screen would mean 30,000 fly screens that you have to use which then means you have to also maintain all of these strains
- knock down not
59
what is good about screening for dominant mutations?
- saves 2-3 fly generations
- they are rare but you can screen many many more flies
-
60
how can you carry out a dominant screen to make dominant mutations more likley?
use a modifier screen- on a mutant bakcground that has an intermeidate phenotype or involved in the process of interest- suppressor or enhancer screen
61
how can you carry out a modifier screen?
- you drive over expression of a gene in the eye and if it disrupts eye development then you get a rough eye (for example hairy)
- you do this by using a eye specific promoter to drives its expression
- you can then screen for mutations that enhance or suppress the eye expression
- or you can do this with genes that you think may act with the gene in the modifier
62
how can you use modifier screen to find certain interaction domains in a protien?
you can express the gene homo mutant in the fly eye - get rough eye- then you do lots of screens which alter the phenotype- if you find they are all the same gene but different alleles- you can look at these allele and where the mutation is and identify the important regions/domains of the protein
63
how can you use modifier screens in the eye to look at disease pathways?
- you express a known mutant (involved in a disease)gene in the eye and look to see if it expresses a mutant eye phenotype (but not the normal gene) and then you can screen for genes that rescue or enhance the eye phenotype - then you look for where these two interactions could happen physiologically in what tissues- by staining or something- then you can do KO in this tissue and binding investigations etc
64
how can you make sure that p-elements insert at a specific site?
this is important because the differences you see in your mutants may be due to the insertion of your reporter gene being inserted somewhere or something else
- you can put a recipient sequence in you fly and the recipient sequence in your plasmid- it will always go to the same place - and if you know that this site doesnt cause an effect- then you know the insertion f your gene isnt causing the phenotype
65
can you sue CRISPR cas9 in flies?
yes
66
what can you use the FRT/FLP/ Gal80- system for if you aren't looking at mutants?
you can use this system to label the clonal fates- you can stimulate FRT so that one daughter expresses GFP and the other doesnt- this means you can follow the lineages and how they specify in cells over time if you stimulate the the FLP at different times
67
how can you just use the FLP/FRT system to follow and compare the expression of two daughter cells?
you have a het cell which on chromosome 1 has pink label-FRT-green label. You have a second chromocome which has green maker-FRT- pink marker. You activate FLP recombinase via heat shock- you get a green daughter a pink, daughter or a yellow daughter in those that avoided recombination
68
why would you want to do the DFS FLP/FRT system?
provides a very powerful way to find mutants in maternally supplied components that are essential for embryonic development. You induce a homozygous germ cell in a het mother-
69
if people want to look at the effect of development on a certain fly organ, how would they normally do this?
they would look at the effect on the imaginal discs- or use a gene KO etc in this organ
Mechanisms o f Development
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