Lecture 3: Screening using Zebrafish models Flashcards

1
Q

Why do we use animals in research? Give an example?

A
  • 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).

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2
Q

How is the use of zebrafish in line with 3R principle

A

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

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3
Q

What are Zebrafish and what are the advantages of using them for research?

A

Zebrafish are tropical freshwater fish originally found in slow streams and rice paddies in India and Burma.

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4
Q

What are the advantages of using Zebrafish for research?

A

Advantages

  1. 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
  1. ZF embryos smaller than many vertebrate embryos, containing fewer cells so easier to trace development of individual cells.
  2. 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.
  1. 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
  2. Gene products can be pharmacologically regulated by just administering drugs to fish medium, allows researchers to study temporal requirement for gene function during development.
  3. Zebrafish husbandry (care, cultivation, and breeding) is relatively cheap compared to other popular vertebrate models, e.g the mouse.
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5
Q

How does ZF compare up against other model organisms?

A

See table

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6
Q

Describe fish husbandry and egg collection?

A

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.
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7
Q

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

A
  1. Synthesis RNA riboprobe complementary to GOI
  2. Digoxigenin (steroid in plants) is used to label uridine
  3. This will stick to GOI mRNA
  4. Anti-DIG antibody conjugated to alkaline phosphatase is added
  5. NBT/BCIP substrate is added à from yellow to purple/blue
  6. This accumulates in cytoplasm of cells expressing GOI or that has RNA the probe has hybridised to.
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8
Q

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:

  1. Describe a method for Protein localisation with purpose of Antibody Detection (Immunofluorescence)?
  2. Why are ZF optimal for use in fluorescent staining?
  3. What is a drawback of this method?
A
  1. 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*
  2. 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.

  1. 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!

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9
Q

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?

A

Tol2kit system:

  1. Place the GFP gene after the promoter of GOI à so that wherever the GOI is expressed GFP is too.
  2. 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.
  3. Transposase inserts the transposon (the cassete) randomly into genome.
  4. Embryos that show GFP expression are then grown up, crossed with wildtype fish and stable line is developed from the F1 progeny.
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10
Q

In biochemistry, the term oligonucleotide – or, informally, “oligo” – is used for….

A

…short, single-stranded nucleic acid fragments, such as DNA or RNA, or similar fragments of analogs of nucleic acids such as Morpholinos

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11
Q

Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining…

A

reverse transcription of RNA into cDNA and amplification of specific DNA targets using polymerase chain reaction (PCR).

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12
Q

What are MOs?

A

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.*
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13
Q

Describe the process of Gene knockdown using Antisense Morpholino Oligonucleotides

A
  1. 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.
  1. 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.
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14
Q

What are the main issues of using Morpholinos?

A

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).

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15
Q

What are some appropriate controls?

A
  • 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.
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16
Q

What are the approaches to making mutant Zebrafish?

A

ENU:

  • Potent mutagen targeting spermatogonial stem cells.
  • Causes point mutations at a rate of 1 per 700 gametes.

Forward Genetics approach - ENU screening (phenotype based)

  • Forward genetics seeks to find the genetic basis of a phenotype/trait.
  • The first major forward genetic screens were performed on ZF by two groups which were published back to back in a 1996 zebrafish specific issue of Development.
  • Performed screens using ENU as a mutagen à induces point mutations, à by mapping for desired phenotype, researcher can identify a single candidate gene responsible for phenotype.
  • The point mutations occur at an approximate rate of 1 per 700 gametes.

Reverse Genetics: ENU screening (Genotype approach)

  • Reverse Genetics seeks to find out what phenotypes arise as a result of a particular genetic disturbance.

Male treated and mutagenized with ENU and then outcrossed to produce F1 males. Their sperm is then archived, fish fin clipped for genomic DNA and sequenced by exome capture.

17
Q

What are the 3 methods of Targeted mutagenesis (Gene-knockout/ genome editing)?

Are theses methods used in forward or reverse genetics?

A

Zinc Finger Nucleases (ZFNs): fusion of a Zinc finger DNA (recognize and bind to codons)-binding domain with the DNA cleavage domain from Fok1 restriction endonuclease. Induce targeted DNA double stranded breaks (DSBs).

Transcription Activator-Like Effector Nucleases (TALENs): fusion of DNA binding domains derived from TALE proteins with Fok1 cleavage domain. TALEs contain 33-35 aa repeats that each recognize a single base pair. Induce targeted DNA DSBs: https://www.youtube.com/watch?v=qbE-6x_Fgn0

CRISPR: uses a guide RNA which is non coding but required to guide cleavage by Cas9 endonuclease. The guide RNA contains a sequence the recognizes target. Generates DSBs.

All used in reverse genetics

18
Q

Forward Genetics vs Reverse Genetics

A

Forward genetics (or a forward genetic screen) used to identify genes (or set of genes) responsible for a particular phenotype of an organism.

Reverse genetics (or a reverse genetic screen) analyzes phenotype of an organism following disruption of a known gene.

Forward genetics starts with a phenotype and moves towards identifying the gene(s) responsible, whereas reverse genetics starts with a known gene and assays the effect of its disruption by analyzing the resultant phenotypes.

Both forward and reverse genetic screens aim to determine gene function

19
Q

How are the 3 methods of targeted mutagenesis similar and how do they differ?

A

Similar:

  • All 3 take advantage of imperfect endogenous repair mechanism called non-homologous end joining.
  • All 3 are designed to cause double stranded DNA breaks that due to imperfect nature of NHEJ cause a percentage that have been mis-repaired by adding or deleting nucleotides resulting in nonsense mutations |

Differ in the way they target the DNA

20
Q

Describe the CRISPR/Cas9 System (gene-editing)?

A

Clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) - It is discovered in bacterial adaptive immune system.

  1. Guide RNA which has a protospacer (Target sequence of guide RNA) is designed
  2. Protospacer binds to target DNA
  3. Cas9 endonuclease binds to non protospacer portion of gRNA + Protospacer Adjacent Motif (PAM) of DNA, causing targeted DSB 3bp upstream of PAM
  4. The cell then naturally repairs itself through – non homologous end joining (quick but error prone) or homology directed repair (slower, requires a template)
21
Q

IMPORTANT

Describe How ZF are being used to perform semi-high throughput screens (in drug discovery)?

A

Low-cost production of lots of eggs, ZF becoming a popular in vivo high through put model to test libraries of small molecules and compound drugs.

The evolutionary conserved genetic and biochemical pathways means that findings can be directly relevant to human development.

  1. Place eggs into a 96-well plate à 3/5 embryos in each well
  2. Allow them to grow and add drugs a certain time points into their medium
  3. Embryos left to incubate for a duration - Drug will diffuse passively into the embryo
  4. Phenotype analysed - observe the consequence of the drugs – for ex: toxicity

The screens can be performed on:

  1. Wildtype fish to assess affect on an organ or tissue type, perhaps labelled by a transgenic marker.
  2. Morpholino or mutant background to rescue a disorder or disease.

We can also use an in situ based drugs screens: (gene we know is upregulated in a disease and we want to find a drug to inhibit this gene expression)

22
Q

Describe Case studies where ZF have been used to screen for drugs for therapeutic medicine, behavioral profiles and drug targets

A
  1. Behavioural profiling reveals relationships between drugs and their targets, therefore this can be used to identify the effects of an unknown drug by comparing it to a known one.
  2. A Chemical Screen for Anti-Cancer drugs