Examenvragen PDF

Examenvragen 1. **What are 3 methods to change gene expression in zebrafish?** There are 3 ways to delete a gene in zebrafish. We can introduce double strand breaks in the genome by using Zinc finger nucleases, TALENs, and Crispr Cas 9. If the double strand break undergoes Non homologous end join...

Examenvragen 1. **What are 3 methods to change gene expression in zebrafish?** There are 3 ways to delete a gene in zebrafish. We can introduce double strand breaks in the genome by using Zinc finger nucleases, TALENs, and Crispr Cas 9. If the double strand break undergoes Non homologous end joining, the site will become a deletion, and there is a full knock down of the gene. We can introduce a new gene by inserting a template ( the gene we want to introduce). For this the double strand breaks need to undergo conservative repair. Another way of changing the gene expression is by using the morpholino, which has a similar effect to RNAi. A morpholino is a 25-mer antisense oligonucleotide with modified bases containing a morpholine ring and is very stable. This can 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. However, morpholino knockouts are different from other knock outs, which should be taken into account. There is no compensation when doing the morpholino knock out. A tool for zebrafish is use of morpholino's. they are the zebrafish equivalent of RNAi. Create a 25-mer oligonucleotide for ur region of interest containing a morpholine ring. The then block translation or splice site junction. This is very quick. Morpholinos have to be controlled very thorough as they have background effects. **2. What are 3 reason why 20^th^ century anti-vivisection movements lost power?** In the beginning of the 20^th^ century there was an increase again in the use of animals. This is because of the development and improvement of animal models, new scientific disciplines and safety testing and quality control for pharmaceuticals. During this time, the anti-vivisection was gradually losing power because : - Anesthesia - Animals did not need to suffer as much anymore - Social conflicts and wars - As a consequence, people were not as interested in opposing against animal sciences anymore - Successful fight against disease - These successes showed that the animal experiments were valid and could lead to treatments **3. Which experimental animal would you use for an experiment with MS? What is the P-score in MS models and how can we improve this?** MS models are scored a P3 score as they undergo severe discomfort, suffering and distress. Animals that have a high P score can be administered to analgesia to minimize the pain, if it does not affect the test results + supplements to reduce weight loss + optimize scientific procedures by training staff Humane endpoints: \- score = 4.5 (paralysis to diaphragm, forefoot/forelegs paralyzed, weak breathing, apathetic, unable to eat/drink) for more than 2 days \- more than 25% weight loss compared to the highest value measured. Improvement: \- Administration of analgesia to minimize pain (does not affect test results) Mice/rodents as experimental animal for a study with MS: \- 75% of mouse genes have equivalent in humans \- 90% of the mouse genome could be lined up with a region on the human genome \- 99% of mouse genes turn out to have analogues in humans. Model depends on what you want to investigate - Inflammation/clinical symptoms/neurodegeneration: EAE/ viral models - De-/ remyelination : toxin induced models - Screening of compounds for remyelination: zebrafish, in vitro models Animal models for MS - Toxin induced (de-/ remyelination) - Cuprizone: Cuprizone: oral administration which then affects oligodendrocytes which die leading to a loss of myelin. Advantage is reproducible model of de- remyelination and its easy to induce. Disadvantage is the time it takes, theres no clinical readout so u cant use clinical parameters and little inflammation so u cant monitor this. - Lysolecithin: induced by focal injection In the white matter of spinal cord, optic nerve or brain. Advantage: reproducible de- remylination. Focal, rapid demyelination. Disadvantage: no clinical readout, more difficult to induce and large lesions or poor techniques could induce massive axonal loss which is not pleasant. - Virus induced encephalomyelitis: induction by injection of virus into the CNS  oligodendrocytes and neuronal loss by viral infection, killing by CTLs. Advantage is that in can be used for inflammation and neurodegeneration aswell as re-demyelination. Disadv: use of virus (not safe), labor intensive and mice are only animals who are susceptible. - EAE(most frequent used) (generally rat not used because transgenic lines only in mice available): induced either by immunization with myelin proteins in combination with adjuvants (need to develop EAE). Adoptive transfer in which T cells are isolated from EAE animal lymph nodes and restimulated with BMP or MOG in-vitro. Or u can use transgenic models, these develop EAE spontaneously - Myelin proteins + adjuvants: inflammation,demylenitation and axonal damage depending on model. Acute rat EAE (MBP + adjuvant), model for inflammation but no demyelination/ axonal damage and only acute. Cronic (rMOG in IFA In DA rats). Model for inflammation, axon damage and demyelin. However is expensive and high variation. Chronic MOUSE eae (MOG). Chronic with CNS inflammation, limited neurodegeneration/demyelin. Disadvantage also variable - Adoptive transfer: develops EAE without adjuvant but labor intensive and several animals needed to reinject one animal. - Genetical models: not physiological - Marmoset EAE model: close to humans so good for preclinical tests but is a higher animal so ethically not very pleasant. - Novel models: zebrafish: most used for drug screening for remyelination. Can test compounds. Expression of myelin can be visualized. This is good to do first test to see if a certain drug can effect myelin. Not good for immune and all the other things. **4. How can we prevent infections in the animals facility?** - By prophylaxis and health screenings - Prophylaxis - Decreasing infection pressure = decreasing concentration microorganisms (MO) per surface/volume - Protection from pathological MO - Prevent spreading of (possible) diseases throughout the building - Medical prophylaxis - Vaccination: staff (tetanus, Hepatitis B) and/or animals (especially in larger animals, less in rodents) - Preventive antibiotics - Usually used with larger animals - Not common used for rodents - Sanitary prophylaxis - Sources of infections - Lab animals - Make sure they are clean - They are usually accompanied by a health certificate (proving they\'re pathogen free) - 1\. Only use animals that are free from pathogens - Germfree = completely free from MO - Gnobionts = harbour a fully known microflora - Specific Pathogen Free (SPF) = free from a number of specified (potentially) pathogenic MO - This one is usually used in animal facilities - Free from a number of specified potential pathogenic microorganisms - FELASA has stated recommendations of which pathogens should be excluded from the animal facility - Only the ones that would have an effect on the results of the experiments - Conventional animals = microbiological status is unknown and uncontrolled - These are also specific pathogen free animals - Difference between clean and conventional animals in our case - Only certain types of pathogens are tolerated in the conventional area - Conventional animals could have any disease - In our conventional area, we only let certain types of pathogens be tolerated - Zoonoses are always excluded - Exclude highly contagious pathogens - Exclude pathogens causing severe symptoms for the animals - 2\. Quarantine - 'Quarantine' = French for 40 days of isolation - Nowadays: at least 12 weeks of isolation (which takes into account the incubation period of diseases) - Quarantine room is physically isolated from other animal rooms - Strict hygiene measures (zoonoses) - Screening (sentinel mice) - 3\. Clinical investigation upon arrival - 4\. Forbidden for staff and researchers to keep pets of the same species (responsibility!) - The pets at home probably carry a lot of pathogens. Staff members could carry them over to the animals in the lab - Staff - Can carry bacteria - E.g.: S. aureus: carried around on skin - When person is healthy: it poses no problem - When the person has wounds or its immune system is not effective: s. aureus can cause problems - This is the same with rodents - So wear gloves when touching animals - 1\. Strict protocols and rules (e.g. staff members can't enter other rooms within a period of 48 hours) - 2\. Personal hygiene - Lab coat, hair and/or beard cap, clogs, gloves - Wash and disinfect hands - Biological materials - 1\. Only use sera, cells, tissues, tumours, etc. that are free from pathogens - Cells you take in should not be infected with pathogens - 2\. Screening (MAP-test/RAP-test) - MAP = Mouse Antibody Production - RAP = Rat Antibody Production - Principle - Inoculation of virus antibody free animal - After 4 weeks: serum sampling and screening - Price: €600-1200/cell line - Vermin - Keep out wild rodents, but also mosquitos, spiders and other small insects - 1\. Design of building - Corridors to keep vermin out - 2\. Pest control - Boxes filled with poison to kill any wild rats/mice in the neighborhood - Other materials - E.g.: if you want to take a microscope into the animal facility, make sure it's clean (via H~2~O~2~) - Health screening - Clinical investigation (e.g. appetite, fur, behavior, defecation, respiration, \...) - See what the animal looks like - Additional investigation (e.g. biopsies, fur and skin swabs, oral swabs, feces, blood sample, \...) - Take oral swaps, blood samples etc. - Post-mortal investigation - Sentinel programme - Principle - 1\. Sentinel animal = same species, susceptible strain, immunocompetent, female - In the past: we would take samples or send the complete animal to a lab that would take samples - If animal is positive: look in the entire room for which strains are infected and which are not - A negative results was not a big reassurance - We only selected a small sample from the population (e.g. 3 out of 200 mice) - We later switched to a sentinel programme instead - Sentinel = animal of same species than the rest in the room - The sentinel strain you use has to be more susceptible and immunocompetent - Immunodeficient animal would not be able create antibodies → don\'t use it because you check on antibodies against the antigen present - Use female groups - You can easily house them in groups without having the risk of fighting animals - Use at least 2 sentinel mice in each cage - One can die - Rodents usually don\'t live alone (group housing) - Individual housing is not good for the animal welfare - 2\. Bring in contact with other animals: either direct or indirect (via dirty bedding material) - Direct: place the sentinel mouse/rat into the cage of other animals - Disadvantages: - If the sentinel would be positive due to previous contact in another cage, it can spread the infection to other cages where you introduce this sentinel - If you have a cage with male mice/rats and you would introduce another male → they would start fighting - If you have a cage with male mice/rats and you would introduce a female → the males would start fighting over the female & the female mice could get pregnant - Indirect: via collecting bedding material from a (potentially) infected cage and place it into cage of sentinel - Sentinel gets in contact with feces and urine of other (potentially) infected animals - Sentinel can take up infections very easily (because it's susceptible) - Infections can get spread easily via feces - 3\. 12 weeks later: take samples from sentinel animal (e.g. fur swab, oral swab, faeces, blood sample) - Take a sample and send it to a lab to get results - 4\. Screen for pathogens (FELASA recommendations) **5. What are the stages of anesthesia?** - [Premedication/ sedation] - [Induction (introduction)] - Stage 1: **induction stage** - Analgetic and sedation stage: the animal is stunned and experiences less pain - Stage 2: **excitation stage** - many reflexes are enhanced - muscle tension is increased - pupils are dilated - tear and saliva production increase - Stage3: **surgical stage** - the concentration of anesthetic in the brain increases - the pupil is narrowed - respiration frequency and depth increase - Eyelid reflex and cornea reflex are slower and disappear - muscle tension and toe reflex decrease - eventually reflexes are absent and anaesthesia is sufficiently deep - Stage 4: **hypoxic stage / overdose stage** - Vital brain centres are repressed - Breathing stops - Contraction of the heart muscle stops - overdose anaesthetic administered = the animal dies soon = euthanasia - [Maintenance] - [Recovery] **7. How can we see the wellbeing according to biological parameters?** [Measuring well-being based on biological parameters] - Productivity decreases: if something is going wrong, this is noticed in the reproduction very quickly - Physical health - Physiological criteria (stress hormones, immunological status, etc) - Breathing is affected easily by an overload of stress - Stress hormones and blood pressure will be increased and the immunological status is impaired → will have an effect on health → renders the animal more susceptible to disease - Mice and rats produce ultrasonic sounds - These sounds differ depending on the situation the animal is in - Animals that are calm and relaxed produce 50 kHz sounds - Animals that are stressed produce 20 kHz sounds Ethogram ? (complete list of species-specific behavior: active/maintenance/inactive/alert ) **8. What are 4 reasons to euthanize the animals?** We cannot euthanize too early in the experiment, because then the animals will be wasted, but we should not do it too late as it will cause unnecessary suffering. When need to euthanize the animals when: - When it has reached the **humane endpoints**. Death cannot be used as an endpoint! - When a **tumor has reached 10% body weight** - Is the **scientific importance is outweighed by the inconvenience**. We can also set up a scoring system (degrees of inconvenience), and when the animal has reached a certain score, we have to euthanize. - When we have reached the **end of the experiment** and the animals are no longer of use **9. Define experimental animal and animal experiment.**  **10. What are different methods to disinfecting your surgical material?** There are 3 approaches for disinfecting the surgical materials: - **Thermal** : autoclave, boiling for 20-30 min, alcohol and burn, dry heat sterilization - **Chemical** : H202, liquid immersion, ethylene oxide - **Radiation**: gamma radiation Instrument sterilization: - **Thermal sterilization (heat)** - Denaturation of cellular proteins and nucleic acids by heat - Lower heat is effective in presence of a humid environment - Always a time-temperature relationship 1. Dry heat sterilization (2h, 180°C) ([not]: gummi, latex, pvc) a. Bad for sharp instruments (scissors) 2. Alcohol and burn b. Almost never used anymore 3. 20-30 min boiling c. Almost never used anymore 4. Steam under pressure: **autoclave** d. Time-temperature-pressure relationship: different programs/cycli e. Most reliable procedure f. The steam must come in direct contact to every inner surface of the instrument/material (correct containers have to be used! Correct loading is important!) g. Air inside the autoclave prevents proper sterilization (air is heavier than steam, and is pulled out of the autoclave by a temperature dependent valve at the bottom of every autoclave) h. Not possible for all gummi, latex and pvc - **Chemical sterilization** - Liquid immersion (alcohol, dettol 5%, umonium, 2% glutaraldehyde) - Not perfect for spores, viruses, Tubercle bacillus - Ethylene oxide (EtO, gas)(very active): alkylating, toxic and irritating - Not really practical - Effective against all known microorganisms - Gas concentration/time/temperature/humidity - Used for delicate surgical instruments - Rubber and plastic tubes - Plastic syringes - Plasma sterilisation (H~2~O~2~) - **Radiation sterilization** - Gamma radiation (Cobalt 60) **11. What are the differences in spinal cord injuries in animal models and people?** Spinal cord injuries in animal models can be achieved in 3 ways. **Contusion** : this is when a **weight is dropped on the spinal cord causing the injury**. This can be done in mice and rats, but the formation of cysts only happens in rats. This formation of cysts is relevant because this also happens in humans **Compression**: this is when the **spinal cord is crushed**. This is analogue to a closed injury in humans. **Laceration**: this is when the **spinal cord is cut,** causing total axonal disruption and retraction of the spinal cord. This technique however is not very representative for humans, as the way of conceiving the injury doesn't happen like this. --------------------------- ------------------------------------ **Humans= heterogeneity** **Animal models= standardization** --------------------------- ------------------------------------ 12. **5 vragen van marshal:** - Is the experiment necessary? - Does it have the possibility of achieving the desired results? - Can the protocol be modified to reduce discomfort? - Has the experiment been done before? - Will the protocol produce valid results? 13. **Explain the different types of suture materials** Today all sutures are connected almost directly to the needle: - Eyed needle: - Not in use anymore - Swaged needle Suture material: Ideally: - Easy to handle - Easy to make a knot, flexible - Minimal tissue reaction - High tensile strength - High knot security which doesn't get loosed - Non-reactive absorption/tissue incorporation → no reaction - Noncapillary, nonallergetic, noncarcinogenic - Sterile - Cheap It is impossible to combine these ideal properties in 1 and the same suture material. There are different suture materials and divided in different properties:  - Advantage: The possibility of making a good knot that doesn't get loosed - If you have multiple uncoated strands → infection can move through the twisted strands! **Absorbable suture materials:** - Natural absorbable material - Give more tissue reaction - Unpredictable absorption time - Synthetic absorbable material - Less tissue reaction - Predictable absorption time Absorption of suture materials can happen in a different time - Fast absorbable - 1-2 weeks - Long-term absorbable - 3-8 weeks (almost 60 days) - Non absorbable - More than 8 weeks (more than 60 days) - Truly non absorbable - Rate of absorption ≠ loss of tensile strength - Some products can lose their strength already a long time before they are absorbed  14. **5 voordelen van het gebruik van vissen in genetisch onderzoek** Zebrafish model system: - **Fish are vertebrate, development ex vivo** - We can study bone development compared to drosophila - Their development is external: - Fertilization and development are external so you can follow the complete embryonic development from fertilization upon adulthood. - In a mouse you cannot do this because the first part of the development is in the uterus. - **You can study lethal phenotypes** - Because you can study them from conception on - You cannot do this in mice because lethal phenotypes don't get born - **Rapid development** - Embryogenesis = 24 hour - From the fertilization till the moment you have eyes and somites = 14-18 hours - In a few hours you have a little animal (you can see eyes, tail, brain...) - **Transparent embryos** - Slide 21: Embryos are transparent during first days → nice for imaging - They are easy to manipulate so you can make great transgenes - You can mark almost every organ system of interest with a GFP or other fluorescent marker. - **Cost efficient** - Way cheaper than mice (1/1000) - Keeping a mouse is 1000 times more expensive than keeping a fish - 1 fish costs 1 euro per year. - You keep many more fish than mice but still it's way cheaper - **Large offspring, hardy and easy to culture** - Genetic study has to be fast. - 1 couple can give up to 1000 offsprings in 1 mating - They can handle fluctuations in their extracellular environment - E.g. temperature drop - They are resistant to the external environment and therefore easy to culture. - They are very small, therefore a lot of fish in 1 room. - They choose fish for genetic screening experiments because they get a lot of offspring and are easy to handle. - **3R's** - **Replacement**: The evolutionary strategy of zebrafish is to develop extremely rapidly to attain an adult-like stage within 72 hours, in order to be able to escape predation. Thus, by 4 dpf they are already able to see and to swim to escape predation. As a consequence, at unlicensed stages, non-neuronal organs such as the heart are well developed, allowing for functional assessments to be performed. - Zebrafish should be treated the same at day 4 and 6 but you don't need an ethical approval - You can replace a big amount of the real animal experiments because you can start using them very early (reducing the amount of animals you use) - Before day 5 it is not considered as animal experiment = replacement - **Refinement:** Researchers can take advantage of the size and transparency of zebrafish larvae to perform similar procedures as those performed in mammals at licensed stages but using less invasive methods. - Zebrafish are small and transparent and you can do way many more experiments with fish than with mammals - **Reduction:** - They (pharmacologists) are now being used to first test new compounds before they go on testing them on higher animals because a lot of compounds fail in transition to animals. - Use zebrafish as intermediate between cells and higher animals. - **Powerful genetics** - Genome totally sequenced, 2 big stock centers that provide mutants available, Crispr/Cas tools... - First it was forward genetics where we study phenotype - Now it is more the tool for doing reverse genetics in which you manipulate your gene of interest. - Reporter lines make them easy to use for imaging: look at entire animal in vivo 15. **Uitgebreid onderzoek naar Alzheimer, hoe zit het met validity?** **Validity of models/tests** 1. [Construct validity] = the measures in the model/test reflect theoretical assumptions a. E.g. AD mouse is based on amyloid precursor protein mutation → you can only test the effect of a compound on these amyloid precursor protein constructs. You could not be able to see something on Tau pathology 2. [Face validity] = degree of similarity between measures in the model/test and in humans b. A mouse is not a small human! There are differences in short term memory and different brain regions study the same brain functions 3. [Predictive validity] = the effects of drugs or lesions in the model/test predict the effect of those drugs or lesions in humans **[3.1 Construct validity]** When you test an Alzheimer mouse model for a treatment if it has effect on AD. - Use an APPswePS1dE9 mouse → you can only study amyloid deposition - This model will only heal amyloid plaques - AD is defined by plaques and tangles - You cannot study the effect on tangle formation in this mouse model - It is not always obvious - If the substrate is different in a model → bad example **[3.2 Face validity]** Mind the gap! A mouse is not a small human - We can test a lot on mice but does this mean everything is the same in mice compared to humans? No - Example: - Short term **human memory** - STM and WM is 15 seconds of our time and if we couple it to attention we will make it in long term memory. - First information and when the eyes have sensory input brings it to the hippocampus. If you bring this into a loop it will be rehearsed, this will form memory and will go to long term memory. If you don't rehearse it, you will forget about it.\  - In **mice**: - STM is not defined as first 15 seconds, but to the first 3 hours - STM \< 3 hours. In the first 3 hours we have no protein synthesis, you only have neurotransmitter release → nothing structurally changes in those mice. - Long term memory \>3 hours. You have protein synthesis (brain derived neurotrophic growth factor which introduces more branching, more synaptic connections). There is structural change in the brain → making us remember for a long time - WM: no phonological loop. If we like to remember a phone number, we try tof make a phonological loop in our head (repeating the number all over again). Mice cannot do this. - Difference is face validity → you cannot study differences in STM only in the first 15 seconds. You cannot study LTM in 1 hour (this is still considered STM in mice). - You should not make mistakes against construct validity or face validity - Other example : - If you want to study memory function in the [dorsolateral prefrontal cortex] (executive control of both STM and LTM) - In the mouse brain, the variant for the dorsolateral PFC of the human brain, is located in the medial PFC - Brain structures do not overlap - You are at a wrong side - You have to make the correct translation from mice to humans and also the other way around **[3.3 Predictive validity]** = If treatments were shown to be effective in animals, the next step is to test their effectivity in humans. - It doesn't mean that a new treatment which is effective in animals is also automatically effective in patients. [3.4 Validity example] A research group investigates tau hyperphosphorylation in an APPwePS1E9 mouse model for AD. - Mistake against construct validity - It is important to translate the information from human to mice and back 16. **Anatomische verschillen tussen vlieg en mens** Flies have second thoracal ganglion, making up a split neuronal system. Their spinal cord also runs on the ventral side compared to dorsal side in humans. Their breathing is different (WHY). Flies have a open circulatory system, ours is closed. Also the heart of flies is located in the abdomen. Flies don't have endoskeleton, lungs or a acquired immunity (so no antibody respons). They are also [different in several ways] a. [They have a second thoracic ganglion,] making up a split neuronal system b. [Our nerve cord goes dorsally, in flies this is eventually located in the middle of the body ] c. [Flies do not have lungs → they breathe with their trachea] d. [Flies do have a heart → but it is an open circulatory system] i. Ours will separate our blood from our body fluids in a filter mechanism. ii. In flies it is not that way → only the shuttle, the body fluid that pass around in the whole organism iii. The heart is also located in the abdomen (tube that you see is the heart → more like a long aorta) e. Flies don't have an endoskeleton, they have a exoskeleton. iv. When they are growing they need to undergo metamorphosis processes and molting. f. Flies don't have an acquired immunity, so no antibody response. v. They do have innate immunity 17. **Welke factoren van EAE modellen en MS zorgen voor een verschil in therapie?** Difference between mice and humans. Differences between EAE and MS. Difference in study design: dose, route of administration and timing. Also company/researcher interests Afbeelding met tafel Automatisch gegenereerde beschrijving Difference in timing: MS treatment starts only after clinical presentation which is a late stage in disease development, in mice treatment is started shortly after or even before disease induction. Pathological differences in MS and EAE: - Inflammation: - MS focal, EAE diffuse - MS CD8/ EAE CD4m MHS class II expression on T-cells and endothelial cells in MS - Demyelination - MS widespread, in EAE little demyalination - Cortical lesions in MS - Few foamy macrophages in EAE - Remyelination - MS occasionally, EAE largely absent - Axonal damage: - MS widespread, EAE some models 18. **Welk criterium wordt gebruikt om pijn/ distress te evalueren? (P-score of iets anders?)** - How to classify P0-P3 - **P0: non recovery ≠ no pain/discomfort\ **Procedures which are performed entirely under general anaesthesia from which the animal shall not recover consciousness shall be classified as non-recovery - **P1: Mild**\ Procedures on animals as a result of which the animals are likely to experience short-term mild pain, suffering or distress, as well as procedures with no significance impairment of the wellbeing or general condition of the animals shall be classified as mild. (not or mild suffering = P1) - **P2: Moderate**\ Procedures on animals as a result of which the animals are likely to experience short-term moderate pain, suffering or distress, or long-lasting mild pain, suffering or distress as well as procedures that are likely to cause moderate impairment of the wellbeing or general condition of the animals shall be classified as moderate - **P3: Severe**\ Procedures on animals as a result of which the animals are likely to experience severe pain, suffering or distress, or long-lasting moderate pain, suffering or distress as well as procedures that are likely to cause severe impairment of the wellbeing or general condition of the animals shall be classified as severe (Humane Endpoints are also set up, animals must be euthanized if it has more discomfort than is strictly necessary for the experiment and cannot be limited in one way or another. key signals to set up a score: score is assigned to different degrees of inconvenience, critical border is assigned to the final score, after reaching it the animal is euthanised) 19. **5 voordelen van vliegen** a. Have many progeny: up to 200 eggs per day per female b. Have a fast development: 10 day generation time c. Having lots of flies allows for high throughput experiments and detection of rare phenotypes d. Small, cheap, easy to culture (kept in large population sizes) e. Basic functional units and building blocks are the same in flies and mammals, for example the brain similar repertoire of cell types, neurotransmitters and guidance cues fly neurological response to drugs is similar to that of mammals. f. Very powerful model organism for forward and reverse genetic approaches complete genome sequence = 132 Mbp, \~18000 genes and 4 chromosomes\ 50% of fly protein sequences have mammalian homologs\ 75% of known human disease genes have a recognizable match in the fly genome. (well-annotated genome + extensive bio informatics resources) A lot of expertise available: technology, toolkit development , optimized protocols.. making it practical. g. Flies can be used for mutagenesis screening in forward genetics and Gal4-UAS system in reverse genetics. 20. **What influence do pheromones have?** Female mice in group tendency to anoestrus (Lee-Boot effect). When a male is introduced oestrus cycle will synchronise due to pheromones of the male ( Whitten effect). After a succesful mating, if another male is introduces into the cage withing 24 hours, there will not be implantation of the egg duet o the pheromones of the second male. This disrupts the cycle of the famle and the implantation will fail. (= Bruce effect) The Bruce effect is only found in mice, not in rats. 21. **what can you do to reduce the number of animals in your study?** There are 4 reduction methods: **3R's: [reduction] alternatives?** - If we can't completely avoid animal experimentation, how can we reduce the number of animals used - 1\. internal communication system (sharing) - Share results, exchange ideas - 2\. change of research strategy (pilot studies) - Performing pilot studies - Instead of defining the effect of drug A on cardiac function in 50 animals - Start by defining the effect in 6 animals, you can immediately see whether there is a trend or not - 3\. inbred and transgenic strains - Inbred animals - Variabilities between individuals will be reduced - You will need less animals to see a difference maybe - 4\. proper statistical design and analysis: systematic reviews? - How many animals would you need to see a potential statistical effect - 5\. using/writing systematic reviews **3R's: what are the [replacement] alternatives? - summary** - no animals - no protected animals - 'lower' animals - Have a lower sensitivity - early development stages - Embryos in mammals from the 3rd trimester are considered experimental elements - Before this period, they are not considered experimental animals - *in vitro* methods: 'tissue culture' - other - non-invasive techniques (MRI) - slaughterhouse material - immunological methods - Human simulators **3 R's: strategies for [refining] *in vivo* methods** - 1**. Choose the proper type of animal** - It is possible to use animals with the lowest degree of neurophysiological sensitivity "If it hurts you, it probably hurts the animal" - Purpose-bred animals - **2. Animal housing and husbandry** - Animals have to be housed in appropriate environments, with natural conditions - The animals have to be healthy and experience an as low as possible level of stress → leads to consistent results - **3. Scientific procedures** - Has to be performed by someone who has had appropriate training and experience - This reduces stress and pain for the animal - Anesthesia and analgesia - To decrease pain of animals - Define humane end-points (instead of death!!) - Humane sacrificing methods should be used - **4. Minimum number of animals** - = Strategy of reduction 22. **Meerkeuze ethiek: marginal case? (testen op disabled people juiste antwoord?)**  Human marginal cases have interests comparable to non-human animals consider them moral patients (small children and babies, severe mentally disabled people and people who cannot defend their own interests) Ethical consistency: treat animals like marginal human cases Critique: species as a functional category + functional and ontological difference between disabled human life and non-rational but self-sustaining life 23. **Merkeuze ethiek over dat geld beter aan armen dan aan onderzoek gegeven wordt. Verschillende approaches gegeven** Moral ? 24. **Meerkeuze HC 10: stel ziekte door transgeen, wat doe je dan?** ? 25. **cDNA kan niet gebruikt worden om transgeen... te maken. Waarom niet?** Geen splice acceptor/donor site. (doe zelftest van genetica HC12 want da kan op examen komen)