Zebrafish General Flashcards
Why are zebrafish embryos transparent unlike xenopus and chick?
The embryo develops on top of the yolk. This is beneficial because the yolk is seperate from the embryo, in contrast to xenopus and chick which have yolk inside the embryonic cells that prevents them from being transparent.
What is SPIM/new imaging techniques? And how is it useful?
- Selective plane illumination microscopy. Is a type of light sheet fluroescence microscopy.
- It offers a high speed imaging capabilities alongside high resolution that makes it extremely useful for embryonal development of whole living animals.
- Combined with computer techniques that can focus on predetermined dots (e.g. the nucleus), allow for study of morphogenesis.
- Parameters such as direction and speed of movement can be colour coded
- Cell division can also be monitored by labelling proteins in certain stages of cell devision, e.g. G2
What were the first mutant phenotypes identified and studied in a forward genetic scren in the zebrafish and why? How can less obvious phenotypes be studied?
- Cyclopia/holoprosenephaly phenotypes. They were the first studied because they had such obvious phenotypes.
- Less visually obvious phenotypes can be studies by staining methods. E.g. Optic commissure stain in Pax2 gene mutations
Why can lethal mutation be studied in the zebrafish and not in mammals?
Embryonic lethal phenotypes can be studied in the fish because the embryos are laid in eggs. In mammals an embryonic lethal mutation will cause the embryo to be absorbed in utero or spontaneously aborted.
How can single mutations that do not cause obvious phenotypes (obvious and by using staining methods)?
- Animals tend to have robust systems that require a few genetic peturbations to cause a phenotype.
- Genetic enhancer screens can be employed
- Animals with a know mutational background are mutagenesied.
- They chance of achieving a double mutant is 1/16. This makes the zebrafish advantageous because it has a large clutch
- E.g. tcfa-/- mutants were mutagenised to uncover hidden genes involved in the Wnt signalling pathway
After carrying out next generation sequencing to map mutations (homozygousity mapping) what questions are asked to identify the exact mutated gene causing the phenotype? What techniques can answer these questions?
- NGS is likely to narrow down the region where the mutant gene lies. (if you are lucky there may be one candidate but it is likely there will be many)
- Is the gene expressed in the region where the phenotype is observed?
- in situ hybridisation to detect time and place of gene expression
- Is there tight linkage to the mutation?
- Is there gene tightly linked to the mutation in a large number of mutants (1000s)?
- Does anti-sense abrogtion of gene activity phenocopy the mutant phenotype?
- morpholinos and RNAi
- Is there a mutation in the gene?
- Can WT RNA rescue the phenotype?
What are the concerns of antisense approaches?
- Off-target effects
- Not 100% realiable
How can animals with mutations in specific genes be identified/generated?
- Mutagenised fish to generate libraries of fish/sperm with mutation in every gene (5000-10000 fish)
- Cryoperserve sperm
- Identify which of the fish/sperm samples carry a mutation in the GOI
- Array DNA in 96 well plate
- PCR
- Sequencing of DNA
- Recover mutant line (with mutation in GOI) from frozen sperm.
- Alternatively new reverse genetic techniques such as ZFNs, TALENs and CRISPRs can be used.
- These approaches hijack endogenous DNA repair mechanisms.
- NHEJ
- Homologous repair
- These approaches is now taking over the ES approach in mice
- Can be used to make deletions (mutations), insertions (transgenics)
- or Cas9 can be modified so it doesn’t cut but brings a transcription factor to the region of DNA. This means it can active transcription. Have also been used in similar manner to alter chromatin
In what animals can embrylogical manipulation occur?
- All model systems??
Are zebrafish good models for chemical drug screening? And what areas of research can be studied using this method?
- Excellent models for drug screening
- Embryos are small can be placed in 96 well plate
- Chemicals can be delivered via water
- development
- timing of action
- dose control
- epistasis-like experiments with mutants
- disrupt all copies of a given gene of family
- Large scale probing of drug effects on behaviour
What does single cell electroporation allow?
enables expression of DNA in individual cells of the intact brain
How can the expression of transgenic lines be orientated within the animal?
Transgene expression can be anchored by standard markers of axons, neuropil, and/or nuclei
What is GCaMP? And how can it be advantageously used in the zebrafish compared to mice?
- GFP is fused with calmodium. Calmodium binds Ca2+. Therefore, this constructs indicated cell activity.
- Can be used to track neuronal activity in the brain
- In zebrafish the whole circuitary can be monitored. However, in mice it is only possible to analyse a more localised area.
Are zebrafish superior for high-throughput behavioural assays?
Yes. They can be monitored in a fully automated manner which cannot be achieved in the other model systems.
