Gene Expression in Zebrafish

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Questions and Answers

Name three methods used to alter gene expression in zebrafish.

Knock-down: introduce dsbreaks in genome by using ZFNs, TALENs, and Crispr/Cas9 (NHEJ).

Knock-in: introduce a new gene by inserting a template (conservative repair).

Knock-out: using the morpholino (25-mer antisense oligonucleotide with modified bases containing a morpholine ring). Morpholino knockouts are different from other knock outs, which should be taken into account. There is no compensation when doing the morpholino knock out.

How does a morpholino function to alter gene expression?

Morpholinos knock down genes by interfering with translation and splicing. The translation blocking is designed to 5’ UTR of mRNA or in coding region close to ATG and monitors loss of protein with an antibody or epitope tag. The splice blocking is designed to splice the site junction and monitor the loss of splicing by RT-qPCR, and sequence to determine if there is non-functional mRNA.

What is a morpholino, structurally?

It is a 25-mer antisense oligonucleotide with modified bases containing a morpholine ring.

Why did anti-vivisection movements lose momentum in the 20th century?

<p>The development of anesthesia, social conflicts/wars, and successful disease treatments using animal models.</p> Signup and view all the answers

How did the development of anesthesia impact the anti-vivisection movement?

<p>It reduced the perception of suffering in animal experiments, thus weakening the arguments of anti-vivisectionists.</p> Signup and view all the answers

What developments led to an increase in animal use during the 20th century?

<p>The development of new scientific disciplines, safety testing and quality control for pharmaceuticals.</p> Signup and view all the answers

What is the primary effect of the induction stage of anesthesia on an animal?

<p>The animal experiences analgesia and sedation.</p> Signup and view all the answers

What are two key indicators that an animal is in the excitation stage of anesthesia?

<p>Enhanced reflexes, increased muscle tension, pupils are dilated, and tear and saliva production increased.</p> Signup and view all the answers

Describe two physiological changes that occur in the surgical stage of anesthesia.

<p>Pupils narrow , respiration increases in frequency and depth, the eyelid and cornea reflexes are slower and disappear, muscle tension and toe reflexes decrease, and eventually reflexes are absent and anesthesia is sufficiently deep.</p> Signup and view all the answers

What happens to an animal in the hypoxic stage of anesthesia?

<p>Vital brain centers are repressed, breathing stops, and the heart stops contracting.</p> Signup and view all the answers

What is the 'Lee-Boot effect' observed in female mice?

<p>The 'Lee-Boot effect' is the tendency of female mice in a group to experience anoestrus, a suppression of their normal estrous cycle.</p> Signup and view all the answers

How does a decrease in productivity relate to the wellbeing of an animal?

<p>A decrease in productivity can indicate that something is wrong with the animal's health or wellbeing.</p> Signup and view all the answers

Describe the 'Whitten effect' in mice.

<p>The 'Whitten effect' is the synchronization of estrous cycles in female mice when a male is introduced, triggered by male pheromones.</p> Signup and view all the answers

What is the difference between the ultrasonic vocalizations of a calm and a stressed rodent?

<p>Calm rodents produce 50 kHz sounds, while stressed rodents produce 20 kHz sounds.</p> Signup and view all the answers

What is the 'Bruce effect' in mice, and what does it demonstrate about pheromonal influence?

<p>The 'Bruce effect' is the failure of egg implantation in a recently mated female mouse when a different male is introduced within 24 hours, which is caused by the pheromones of a new male. It demonstrates that pheromones can disrupt pregnancy.</p> Signup and view all the answers

What is an ethogram and how is it used in animal studies?

<p>An ethogram is a complete list of species-specific behaviors and is used as a tool to observe animal behavior.</p> Signup and view all the answers

When should animals be euthanized during an experiment?

<p>Animals should be euthanized when they have reached the humane endpoints.</p> Signup and view all the answers

Name two 'replacement' alternatives to using animals in research.

<p>No animals: exchange of info, physical and chemical techniques, mathematical and computer models, human studies, non-invasive techniques, slaughterhouse material, immunological methods, human simulators No protected animals: lower animals, early developmental stages, in vitro methods (tissue cultures)</p> Signup and view all the answers

How do pilot studies help in 'reducing' the number of animals used in an experiment?

<p>Pilot studies help by testing experimental conditions and seeing whether results are likely before commiting to larger experiments.</p> Signup and view all the answers

How does using inbred and transgenic strains of animals contribute to the reduction of animals used in research?

<p>Inbred animals have less variability between individuals, reducing the number of animals needed to observe a significant effect.</p> Signup and view all the answers

What is the general principle for choosing the 'types of animals' when refining research methods?

<p>The principle is to choose animals with the lowest degree of neurophysiological sensitivity, as 'if it hurts you, it probably hurts the animal'.</p> Signup and view all the answers

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Flashcards

Stages of anesthesia

The series of phases anesthesia progresses through: premedication, induction, maintenance, and recovery.

Induction stage

Stage 1 of anesthesia where analgesia and sedation occur, and the animal appears stunned with reduced pain perception.

Excitation stage

Stage 2 of anesthesia characterized by increased muscle tension, reflexes, and dilated pupils, occurring as the anesthetic takes effect.

Surgical stage

Stage 3 of anesthesia where the patient is sufficiently deepened in anesthesia, with decreased reflexes and more controlled respiration.

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Hypoxic stage

Stage 4 of anesthesia, also called the overdose stage, where vital functions are suppressed, leading to potential death.

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Measuring well-being

Assessing an animal's well-being based on biological parameters like reproduction, health, and stress levels.

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Stress hormones

Biological indicators that increase with stress, affecting health and well-being, such as elevated blood pressure.

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Humane endpoints

Criteria used to determine when an animal should be euthanized to prevent unnecessary suffering during experimentation.

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Zinc finger nucleases

A method to create double strand breaks in zebrafish genome.

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TALENs

A technology for precise gene editing via double strand breaks.

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CRISPR Cas9

A modern method for editing genes by creating precise double strand breaks.

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Morpholino

A 25-mer oligonucleotide that modifies gene expression by blocking translation.

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Translation blocking

Interfering with mRNA to prevent protein synthesis.

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Splice blocking

Preventing splicing in mRNA to disrupt normal processing.

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Anti-vivisection movements

Movements aimed at opposing animal testing and experiments.

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Reasons for decline of anti-vivisection

Factors like anesthesia, wars, and success in disease control reduced movement strength.

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Lee-Boot effect

The tendency of female mice to enter anoestrus when grouped together.

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Whitten effect

The synchronization of oestrus cycles in female mice due to male pheromones.

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Bruce effect

Failure of egg implantation when a new male is introduced within 24 hours after mating.

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Reduction methods in studies

Strategies to minimize the number of animals used in research.

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Pilot studies

Small-scale preliminary studies to gauge the feasibility of larger experiments.

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Inbred strains

Genetically uniform animal strains that reduce variability in research.

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Statistical design

Proper planning of experiments to ensure valid and efficient use of animals.

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Refinement in research

Strategies to enhance animal welfare while achieving research objectives.

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Study Notes

Gene Expression in Zebrafish

  • Three methods exist to modify gene expression in zebrafish:
    • Introduce double-strand breaks using zinc finger nucleases, TALENs, or CRISPR-Cas9. Non-homologous end joining (NHEJ) causes a deletion, and a complete knock-down of the target gene is the result.
    • Introduce a new gene by inserting a template; conservative repair is required following the insertion.
    • Use morpholinos: 25-mer antisense oligonucleotides with a modified morpholine ring to block translation or splicing. This is fast but requires thorough control due to background effects, and is similar to RNAi.

Reasons for Decreased Power of 20th Century Anti-vivisection Movements

  • Increased use of animal models for research: the development of improved animal models and new scientific concepts.
  • Pharmaceutical safety testing and quality control: led to a decline in the urgency of opposing animal testing, as many advancements required such models.
  • Advancements in anesthesia: this reduced suffering during tests and made them more acceptable.
  • Social conflicts (wars): diverted attention away from animal rights issues, as public concern shifts to human conflicts.

Experimental Animals for MS Research

  • MS models are scored P3 due to the severity of discomfort, suffering, and distress.
  • Appropriate animal models depend on what is being investigated, such as inflammation, or clinical symptoms/neurodegeneration.

Preventing Animal Infections

  • Prophylactic measures:
    • Decreasing the concentration of microorganisms (MOs) per surface/volume.
    • Preventing spread of diseases throughout the building.
  • Medical prophylaxis:
    • Vaccinations (staff and animals - especially larger animals).
    • Preventive antibiotics (more for larger animals).
  • Sanitary prophylaxis:
    • Animal health certificates to ensure pathogen-free animals.
    • Use of germ-free or specific pathogen-free (SPF) animals.
  • Strict protocols and hygiene in the facility are also crucial

Stages of Anesthesia

  • Premedication/sedation: aims to reduce pain and anxiety.
  • Induction: introduces the anesthetic, the animal is stunned, and experiences less pain.
  • Excitation: many reflexes are enhanced and body responses increase.
  • Surgical: concentration of anesthetic increases, pupils narrow, respiration frequency & depth increases, reflexes slow and eventually disappear.
  • Hypoxic (overdose): vital brain centers are repressed, breathing stops and heart contractions decrease, eventually terminating the process.
  • Maintenance: recovery

Evaluating Animal Wellbeing

  • Monitoring physiological parameters: physical health, stress hormones, and immunological status.

Euthanasia Reasons

  • Reached humane endpoints.
  • Tumors exceeding 10% body weight.
  • Scientific importance outweighs the inconvenience (scoring system).
  • End of experiment, no further use.

Different Methods for Disinfecting Surgical Materials

  • Thermal: autoclave, boiling, alcohol & burn, dry heat sterilization.
  • Chemical: H2O2, liquid immersion, ethylene oxide.
  • Radiation: gamma radiation.

Spinal Cord Injury Differences in Animals and Humans

  • Contusion: impacting the spinal cord, relevant for rodents but cyst formation happens only in rats, unlike humans.
  • Compression: crushing the spinal cord, analogous to closed injuries in humans.
  • Laceration: cutting the spinal cord, less representative in humans.

Refinement of Animal Models

  • Researchers can perform similar procedures on transparent, small zebrafish larvae as mammals, but less invasively.
  • Pharmacologists can use fish to test new compounds before more invasive mammal trials.

Using Zebrafish in Genetic Research

  • Zebrafish are vertebrate and development is largely external, thus useful for studying embryonic development.

Lethal Phenotypes in Zebrafish

  • Embryos are transparent, allowing for visualization and study of development.
  • Easy to manipulate and screen for phenotypes (e.g., using fluorescent markers).
  • High throughput screening possible, allowing examination of large numbers.

Reasons for Euthanization

  • Severe suffering as there is not enough comfort possible from medication.

Validity of Animal Models for Disease

  • Construct validity: whether the model reflects the theoretical assumptions of the disease or condition.
  • Face validity: measures of similarity between the model and the actual condition.
  • Predictive validity: the ability of effects in the model to predict effects in humans.

Short & Long-term Memory Differences

  • Human short-term memory is 15 seconds or less.
  • Mice do not exhibit the same long and short-term memory phases that humans do.

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