level: Animal Models
Questions and Answers List
Flashcards on the case and lecture on Animal Models
level questions: Animal Models
| Question | Answer |
|---|---|
| what are the 3Rs? | replacement: the use of animals should be completely (or partially) avoided. reduction: the number animals should be minimised. refinement: the pain, suffering, distress and lasting harm that animals might experience should be minimised. |
| what is needed/should be done before an animal experiment can be performed? | - a course certificate of an animal course. - an establishment and project licence is needed from the CCD (centrale commissie dierproeven)/article 9 WoD licence. - a master in a relevant discipline. |
| in which categories can animal models be divided? | 1. animals in which disease is induced, through pharmacological injection, stress induced, biological injection or lesion induction. 2. spontaneous animal models. 3. genetically modified animals. 4. negative animal models (animals that will not develop the disease, but can be used to study disease susceptibility, e.g. pigs and SARS-CoV-2). 5. healthy animals. |
| what are the advantages of the use of animal models? | - contribute to the understanding of pathological and biological processes. - enable development of drugs, vaccines and surgical techniques. - have a short lifespan. - can be used for animal production, to create animals that are bigger (generate more consumption meat) or produce more milk. - can be used to decrease environmental pollution (e.g. pigs that generate less phosphate). |
| what are the disadvantages of the use of animal models? | - high costs and time consuming. - ethics. - low translatability of findings in animals to humans. - difficult to assess behavioral and cognitive tests in animals. |
| what is the difference between active immunization and passive transfer in the generation of animal models? | in active immunization, an animal model is induced through exposure to the antigen, after which the animal develops its own antibodies to this antigen. in passive transfer, an animal model is induced through the administration of serum, purified immunoglobulins, monoclonal antibodies or antibody-producing cells, which are isolated from a diseased organism. antibodies are present directly, and this model can be used to study the acute effects of a disease. |
| how can the efficacy of a MG animal model be assessed? | - clinical score: score of animal weakness prior and after induction, scores from 0 (no weakness) through 4 (death). - measures of weakness and fatigability, through grip meter or mesh test. - electromyography: measurement of muscle action potential, and thus muscle weakness/strength. - immunofluorescence or electron microscopy of the neuromuscular junction to assess absence of AChR. - radioimmunoassay to measure AChR concentration. |
| what are recommended methods to sacrifice rodents? | - overdose of injectable agents (sodium pentobarbital) - overdose of inhalational agents (halothane) - inhalation of carbon dioxide gas (con: creates stress, increased HPA axis) - inhalation of carbon monoxide gas (con: dangerous to humans) - decapitation (con: brain activity still present after 14s) - cervical dislocation - blunt force trauma (con: skilled lab personnel needed) - focus beam microwave irradiation, heating of the rodent's brain |
| in which fields of study are different animals commonly used? | - rhesus monkeys and other nonhuman primates: emerging infectious diseases, serious (brain) diseases, life-threatening situations. - nematodes (C. elegans): metabolic diseases (esp. obesity). - zebrafish: endocrinology and metabolic diseases, neurodevelopmental conditions. - drosophila: neurobiological processes. - pigs: studying and performing animal-to-human transplants. - rodents: infection and immunity, endocrinology, metabolism, cancer, pharmacology and therapeutics, neuroscience, surgical techniques. |
| what is the definition of a transgenic animal? and what is the process of transgenesis? | transgenic animal: an animal whose genome has been changed to carry genes from another species or to use techniques for animal genome editing for specific traits. transgenesis: introduction of foreign DNA sequences into the genome of transfected cells ensuring that the DNA sequences are integrated and transmitted to the offspring. |
| how does the DNA microinjection technique of transgenesis work? | 1. collect fertilized oocytes or embryonic stem cells (from fertilized blastocysts). 2. inject the genes of interest in the oocytes or inner cell mass of the stem cells. 3. culture the oocyte into a blastocyst or insert the cells into a blastocyst. 4. perform genetic tests, freeze the cells or transplant them into a recipient. |
| how does sperm-mediated gene transfer work? | a spermatozoon is incubated with foreign DNA, which is then used in fertilization to produce a transgenic animal. or a spermatozoon is treated with a detergent in a way that the membrane is altered so that foreign DNA can enter. this spermatozoon is then used for fertilization. |
| how does somatic cell nuclear transfer work? | somatic cell nuclear transfer is essentially cloning: 1. one oocyte is enucleated. 2. a second enucleated oocyte is injected with isolated fibroblasts containing the gene of interest. 3. the two oocytes are merged. 4. a complete embryo is formed. |
| what is a knock out model and what has been found using knock out models? what is a knock in model? | knock out: an animal model in which researchers have inactivated an existing gene by replacing or disrupting it with an artificial piece of DNA. it has been found that most genes are pleiotropic; they are expressed in different tissues, in different ways and at different times and that knock outs of many genes in the mouse genome can be compensated for. knock in: an animal model in which a gene sequence of interest is altered by one-for-one substitution with a transgene, or by adding gene sequences that are not found within the locus. |
| how can DNA be edited? | - via the generation of a DNA vector through homologous recombination to make sure that the vector is incorporated into the genome and random integration is prevented. - via the use of recombinases, such as the CRE recombinase - via the use of nucleases, such as CRISPR-Cas9, to induce deletions or insertions. |
| how does CRE recombinase work? | CRE recombinase is a tool to edit DNA. it recognises loxP sequences. when these sequences are in opposite directions, CRE reverses the gene sequence. when these loxP sequences are in the same direction, CRE cuts out one of the loxP sequences, and can thereby remove the gene in between the loxP sequences. |
| how to produce an animal model for McArdle disease? | 1. produce a recombination vector including the mutation. 2. make the vector into a linear DNA sequence. 3. introduce the vector into stem cells by electroporation to allow for cell permeabilization. 4. grow the cells (under neomycin selection). 5. identify the recombinant clones using PCR. 6. confirm that homologous recombination has occurred using southern blot; the lengths of the wild-type and mutant DNA should be different, as measured by a probe. 7. perform a karyotype analysis. |