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1

what are the advantages of using zebrafish as a model organism?

- they are easy to maintain in a lab - they only live 3-4 months so low generation time - there are large progeny clutches- foward genetic screens can be carried on on F1 (using techniques) - can survive for 3-4 days as a haploid - can be used to make haploid or gynogenetic offspring- they develop in clear eggs- they are transparent so can see the heart and blood system easily too

2

explain the protocol for a normal Tubingen screen.

1. expose a male fish to a mutagen in the hope the germ line will be effected 2. cross with a female 3. each F1 offspring will represent a different mutation and so will form a single family each 4. each F1 is crossed with a wilf type to produce families whichare 50% het. 5. random F2 crosses result in 25% of the F3 being homozygous mutant

3

give an example of a zebrafish mutant being used to model a human disease

anaemia- low blood cell count in fish

4

what two processes can be used to prevent having to screen for mutants in the F3, and in an earlier stage instead?

creating haploid offspring or parthenogenetic diploid offspring. They allow screening in the F2.

5

what is the protocol for making a diploid offspring for forward genetic screen?

You expose a male to mutagens and cross with a female to produce heterozygous F1s. You then squeeze a female F1 and make her lay her eggs. They you take UV treated sperm which contains no genetic material that can contribute to the offspring but can still activate the egg. Then this egg will continue as a haploid organism for a few days- allowing a mutant from the het chromosome to be seen in 50% of the offspring.

6

how can you use gynogenesis to shorten the length of genetic screening process?

-you can apply early pressure to inhibit meiosis ii or you can used heat shock to prveent mitosis i.

7

describe why you would use early pressure in a forward genetic screen.

This can be used to allow fish to be screened in the F2 rather than F3. During meiosis I, the sister chromosomes replicate and then the celll divides, creating a cell which only contains the two replicates of one chromosome of the het female. You then squeeze the female and extract the eggs and fertilise with UV treated sperm to activate meiosis II, however you inhibit meiosis II with early pressure. This means that the diplod oocyte doesnt divde and so enters mitosis I as a diploid and becomes a diploid organism which is homozygous for the areas of the chromosome other than those that can recombined. This results in a 50% chance of getting homozygous mutant offspring. However, as the position of the mutation moves further away from the centrosome, recombination is more likely so 50% decreases.

8

describe how you could use heat shock in a genetic screen.

This can be used to create a diploid homozygous mutation which is homozygous in 50% of the offspring without the trouble of recombination that early pressure provides. This is carried out by applying heat shock at mitosis I stage after UV treated sperm activate haploid oocytes. This means that the replicated chromosomes do not divide into 2 different cells so they are frozen as a diploid oocyte and therefore embryo.

9

explain why a mosaic screen would be used

if you have a mosaic animal then it may not die even though the mutation is lethal because the other non-mutant cells compensate but you may still see a phenotype

10

describe the process of a insertional mutagenesis with a retrovirus forward screen.

virsus are injected into 1000-2000 cells at the embryo stage. Hopefully these will affect the germ cells of the P fish which grow from these embryos. - these P fish are then crossed - these F1 fish and then screened via real time PCR or southern blot for multiple insertions and those containing multiple insertions are then intercrossed to produce fish that are heterozygous and wild type for a locus. - you then cross the F2s randomly and you will get the heterozygotes crossing to produce 25% homo mutant. But because at other loci there are mutations, some of the fish which are het or homo dom for the first mutation, may be homo recessive for another mutation and so increase the overall number of mutation phenotypes within the F3- multiple genes homo recessive if not linked

11

give three examples of fluorescence reporters that have been used in genetic screens

- to identify mutants that ar eunable to process phospholipids and cholestrol. a reporter for lipid processing, an engineered quesnched fluorescent moiety was placed in PLA2 cleavge site so that cleaving results in fluos. So mutants whom cant digest lipids properly will not emit flo when fed these phosphplipods . - fluorescent dies injected into the eyes of dat 5 larvae label the entire lengt of the retinal axons, which can be visualised in the skin. you can then screen F3;s for retinal path finding mutants - by linking green fluorescent protein to genes or promoters of interest, it is possible to visualise changes in gene expression in mutants

12

what kind of screens of zebrafish particularly good for and why?

- behaviour screens as they show social preference and hunting behaviour. This can be used to study things such as autism etc - because of their tranparency, optogeneics can also be used to allow the firing of different parts of the brain to be seen in response to visual or other stiimlu- allowing neural curcuitry to be studied.

13

how can pathways been analysed in screens?

you can use the concept of enhanced or suppressed phenotupes. This involves using a mutant line and then carrying out a sceond mutant screen for mutation sthat suppress (reverse) the mutation or enhance (amake it more extreme) to find genes that are implicated in the same pathway.

14

describe how allele specificity can assist dissceting a genetic pathway?

???

15

list some other techniques that can be used to look at mutant gene function.

1. temperature sensitive alleles2. using morpholinos for knock downs 3. ectopic gene expression by mRNA injection of viral transduction 4. can use chemicals which mimic genetic mutations in order to determine timing that a gene or protein is needed.

16

what disease can be replicated in zebrafish?

anaemia

17

what are the disadvantages of performing screens in zebrafish?

you have to screen in the F3, although you screen in the F3 for flies (F2 for c.elegans), fish are much harder to maintain in the lab that flies and therefore breeding requires more time and expense.

18

how long can zebrafish live for as a diploid?

3 days

19

what are the advantages of producing a haploid F2 fish?

they will expose the mutant phenotype and do not have to be crossed to make a homozygous

20

what defects do diploid zebrafish have that could complicate screening?

They are shorter, so if you were screening for growth mutants this would be confusing, they contain more and smaller cells, and can have other morphological defects,

21

what genes have experiments using diploid zebrafish uncovered?

genes involved in brain development

22

what stage in meiosis does early pressure inhibit and what is created as a result?

meiosis II, it prevents the diploid oocytes from doing their last division (they have previously been activated by UV-treated sperm) and so these diploid oocytes develop into diploid embryos

23

what point in meiosis does heat shock inhibit and what is created as a result?

after the haploid oocytes have developed normally, they then attempt to undergo mitosis, but heat shock prevents the cells from dividng after the chromosomes have divided so the haploid oocyte then becomes a diploid embryo

24

why are diploid embryos generated from heat shock preferable to early pressure?

they are homozygous for all loci so they are more likely to express a mutant phenotype for genes that are positioned away from the centromere.

25

what are the methods that can be used in zebrafish to screen in the F2?

gynogenesis (heat shock and early pressure) and haploid embryos

26

what are the drawbacks of gynogenesis

there is a low viability of the embryos, only 10-20% form from heat shock.

27

what is the difference between treating fish with ENU and then breeding fairly soon after mutagenesis and treating fish with ENU and then breeding a lot and then starting the screen procedure?

If you treat with ENU and then breed straight away, You are using post meiotic sperm cells and therefore, because ENU induces mutations on a single strand of DNA, the mutation only becomes fixed following several divisions after fertilisation. This means the resulting F1 are mosaic for a given mutation .This is good because mosaic fish can withstand a great mutational burden. if you mutagenise and then breed for a while then the mutation becomes fixed in the sperm of the pre meiotic germ cell and so the offspring are not mosaic

28

what are the advantages of breeding the mutagenised fish for a while following mutagenesis and then starting the screen procedure?

the fish aren't mosic so you dont have to worry about the mutant cells not being in the gonads of the F1s.

29

can the efficiency of loci mutation rate vary between genes with ENU?

yes

30

what are the downsides of using EMS in zebrafish?

it is less potent than in flies