Lecture 3: Screening using Zebrafish models Flashcards
Why do we use animals in research? Give an example?
- Cells behave differently in vitro to in vivo
- Vital in understanding animal ill-health + humans
- better quality of life and longevity to humans + animals.
Example:
Polio (poliomyelitis) acute, viral, infectious disease causes inflammation of spinal cord and brain stem leading to paralysis and death in some cases. After 40 years of research using mice, rats and monkeys a vaccine was finally developed in the 50s.
A vaccine programmed was introduced in the 80’s by the WHO which reduced the number of cases reported from 350,000 a year (1988) to just 480 a year (2002).
How is the use of zebrafish in line with 3R principle
Replace higher vertebrates, most research restricted to embryonic stages which isn’t covered under the act so reduction in use of animals. refinement to methods by adding drugs to medium rather than invasive interventions
What are Zebrafish and what are the advantages of using them for research?
Zebrafish are tropical freshwater fish originally found in slow streams and rice paddies in India and Burma.
What are the advantages of using Zebrafish for research?
Advantages
- The eggs are optically transparent and develop (Ex-utero) outside of the mother’s body:
- Researchers can follow development of embryo under microscope.
- With mice mother sacrificed to study embryonic development
- ZF embryos smaller than many vertebrate embryos, containing fewer cells so easier to trace development of individual cells.
- ZF Embryos are easily manipulated.
- Cells can be taken out and moved to a different area of the embryo to determine if they are committed to certain cell fate.
- Cells can be destroyed by lasers to determine their contribution to development.
- Gene expression and function can be knocked down using Morpholinos, or knocked out using mutagenesis screens and genome editing techniques or overexpressed by injecting RNA
- Gene products can be pharmacologically regulated by just administering drugs to fish medium, allows researchers to study temporal requirement for gene function during development.
- Zebrafish husbandry (care, cultivation, and breeding) is relatively cheap compared to other popular vertebrate models, e.g the mouse.
How does ZF compare up against other model organisms?
See table
Describe fish husbandry and egg collection?
Advanced with automated commercial systems. For breeding, parameters need to be optimal.
- Tecniplast system maintains fish at 28.5, pH 7, and conductivity of 500us using automatic pumps containing sodium bicarbonate, (to raise the pH, since fish wee they only ever make it acidic) or special sea salt.
- Approx 50:50 male to female ratio. Female fish: round whitish bellies, males thinner orangey/brown.
- Fish kept on a 14h day cycle. Night before collection of eggs, marbles placed inside each tank containing 20-30 male and females.
- lights on in morning à induces fish to start spawning.
- Females have an evolutionary disposition to lay eggs in gravelly area (to prevent other fish eating eggs). This is recreated using marbles à eggs roll down between marbles into tray à inaccessible to other fish in tank
- Then the eggs are collected using a small sieve, they’re washed and placed in a petri dish containing methylene blue, an anti-fungicide.
To look at gene function in fish a process of Gene Expression Analysis: In situ Hybridisation is used, describe it.
This helps us know where a gene is expressed
- Synthesis RNA riboprobe complementary to GOI
- Digoxigenin (steroid in plants) is used to label uridine
- This will stick to GOI mRNA
- Anti-DIG antibody conjugated to alkaline phosphatase is added
- NBT/BCIP substrate is added à from yellow to purple/blue
- This accumulates in cytoplasm of cells expressing GOI or that has RNA the probe has hybridised to.
mRNA expression doesn’t necessarily mean this is where the protein has its function.
So Protein localization is also an important part in allocating function to proteins:
- Describe a method for Protein localisation with purpose of Antibody Detection (Immunofluorescence)?
- Why are ZF optimal for use in fluorescent staining?
- What is a drawback of this method?
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Describe method?
- Permeabilisation step (puncturing of the cell membrane) allows primary Ab to enter embryo and cells à Ab binds to antigen
- Describe method? A secondary antibody conjugated with fluorophores added
* If the primary Ab made against zebrafish peptide was made in a rabbit then the secondary will be an anti rabbit IgG* - Optimal?
Zebrafish are particularly nice for performing fluorescent antibody staining as they are transparent and small enough to allow the whole embryo to be imaged on confocal microscopes, allowing high resolution clarity.
- Drawback:
Using antibody that are conjugated to a fluorescent dye against the protein of interest to localise it is one option however there are not that many zebrafish specific antibodies!
If there are not enough antibodies against protein of interest (POI) we can use transgenic fish.
Describe the generation of transgenic Fish using the tol2kit system?
Tol2kit system:
- Place the GFP gene after the promoter of GOI à so that wherever the GOI is expressed GFP is too.
- This construct (containing Tol2 recognition sites (cassette) at either end) is then injected with mRNA for transposase (protein that recognizes the tol2 sites) into embryo at the one cell stage.
- Transposase inserts the transposon (the cassete) randomly into genome.
- Embryos that show GFP expression are then grown up, crossed with wildtype fish and stable line is developed from the F1 progeny.
In biochemistry, the term oligonucleotide – or, informally, “oligo” – is used for….
…short, single-stranded nucleic acid fragments, such as DNA or RNA, or similar fragments of analogs of nucleic acids such as Morpholinos
Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining…
reverse transcription of RNA into cDNA and amplification of specific DNA targets using polymerase chain reaction (PCR).
What are MOs?
MOs: Synthetic oligonucleotides composed of chains of about 25 subunits
Similar to DNA and RNA oligonucleotides, except they have a morpholine ring rather than a ribose ring à can undergo base pairing but it offers other advantages:
- MOs are resistant to nucleases so remarkably stable
- They don’t carry a -vely charged back bone à less likely to interact non-specifically w/other components of cell and may be less toxic as a result
- MO target mRNA and block gene specific translation,*
- MO’s work by inhibiting translation. There are several different targeting types of MO, which are used in zebrafish to silence gene function.*
Describe the process of Gene knockdown using Antisense Morpholino Oligonucleotides
- MO designed to be complementary to AUG translational start site.
- MO bind to start site à block ribosomal machinery from starting translation à no protein, which can be tested using immunohistochemistry.
- Difficulties: proving knockdown of protein levels and thus have intended effect, this is due to lack of ZF specific Abs.
- Splice inhibiting MOs
- Advantage: can quantify efficacy of MO by RT-PCR (reverse transcriptase).
- Splice MOs prevent intron from being spliced à premature stop site à nonsense-mediated mRNA decay (a surveillance pathway that reduces errors in gene expression by eliminating transcripts that contain premature stop codons), or a truncated presumed non-functional protein is made.
What are the main issues of using Morpholinos?
Difficult to measure the efficacy of MOs without a good Ab
Difficult to rule out possibility that MO inhibits the function of an irrelevant gene.
It can be difficult to inject precise and reproducible volumes of MOs.
They are known to cause some off-target effects – therefore a few controls are needed to prove that this MO is specific to your GOI.
MO’s only last to 5days since they’re metabolized – so looking at later stages of development not possible unless use a mutant (see after next ques).
What are some appropriate controls?
- Comparison with existing mutants to reveal off-target MO effects. Not always possible!
- Loss of protein should be verified using antibody staining or other assays.
- Incorrectly spliced pre-mRNA should be verified by RT-PCR and altered splice product sequenced.
- At least 2 MOs per target gene should be used to ensure they give similar phenotypes, testing for synergism also good here.
- RNA rescue, co-inject MO and target gene RNA that is not recognized by Mos.
- Control MOs, use of a standard control that affects a gene not expressed in the cells of interest e.g. human beta globin/ 5 base mismatch/p53MO.