Animal models in research Flashcards
what do we need to consider when doing in vivo research (i.e. using animals)?
In vivo: experimental procedure done in a living organism
• Expensive; have to maintain animals etc.
• Ethical considerations
• Involves the use of laboratory animals – or humans
what do we need to consider when we do in vitro experiments?
in vitro: experimental procedure done within the confines of a test tube/flask/plate
- Reduced cost
- Allow the tight control of the physico-chemical environment than in vivo
- Animal use is very reduced (e.g. only initially collect cells)
- Often difficult to replicate the conditions cells encounter in a living organism
- Systemic effects cannot be tested
Both approaches are complementary. Often a test is first performed in vitro and then reproduced in vivo.
what are the desirable characteristics of a model organism?
- Relevance/representative–> if it is useful to understand human physiology
- Accessibility/availability
- Experimental manipulation
- Genetics–> should be well known
- Cost/space
describe the zebrafish
- Small tropical freshwater fish
- Originally from Asia – Eastern India, Ganges river
- Established as a model organism by George Streisinger at the University of Oregon, USA
what are the advantages of using the zebrafish as a model system?
- External fertilization; easy to collect and grow in petri dish
- Fast development
- Transparent embryos; can see under microscope, no need to open it up
- Genome completely sequenced
- Extensive genomic/transgenesis tools that allow us to manipulate its genome
- Allows high-throughput approaches (especially as 2 fishes can produce lots of embryo a day)
- Amenability to embryological and imaging approaches
describe the zebrafish’s life cycle
• embryo’s cell divides to make a mass of cells at the top of the yolk–> cells start spreading around the yolk sac–> anterior portion will be the head, eye and trunk at the end. Zebra fish shape will be the somites.
- > 4 days= swimming larvae able to feed and live by itself
- > 4 weeks of development= eyes
- 2 months= keep growing
- 3 months= adults ready to mate
- Embryonic period happens in just a few days
Describe the 24 hours post-fertilisation embryo
- Organogenesis is well underway
- Corresponding to a 45-50 days old human embryo
- Head, trunk, tail, eye and parts of brain, ear, spinal cord (NS) has developed with complexity
- Somites have formed- will differentiate to give rise to muscle and bone
- Underneath eye= heart will form–> can see how heart beats and blood moves around under microscope.
why is making mutants in zebrafish genes important for understanding organogenesis?
- fish are vertebrates!
- > 70% human genes have an ortholog in zebrafish
- We can learn about gene function by seeing what goes wrong when a gene is disrupted
- Mutagens such as the chemical ENU can be used to provoke mutations in the germ line: Sperm cells will be affected= mate the male with female= generated offspring that will carry mutations= mate 2 of these offspring carrying same mutation= subset of these offspring will have a mutation in homozygosis that will cause a morphology defect–>Only ¼ are carrying a mutation (mendelian genetics) as it is a recessive mutation- thus those that carry mutation in gene important in embryonic development= will see offspring that also have these defects.
what does ENU do?
- ENU causes RANDOM mutations i.e. anywhere in genome- have to try and find the gene effect by finding the defect in the genome
- Directed mutagenesis can also be performed (choose which is the gene we want to disrupt), to abrogate/get rid the function of a specific gene. E.g. CRYSPR can generate mutants by targeting any region of the genome= disrupt that part of the genome.
why are the fish embryos treated with small molecular compounds?
Why to treat embryos with drugs: Phenotypic drug screens –>
- morphological: to identify compounds that lead to a particular morphological defect during embryogenesis
- therapeutic: to identify compounds that ameliorate a disease phenotype
- pathway directed: to identify compounds that interfere/modify the activity of specific molecules or signaling pathways–> look for compounds that are specifically affecting a step-in signaling pathway
- behavioral: to identify compounds that have a specific behavioral consequence
define transgene
exogenous gene (derived from another organism) introduced in the genome of an organism
what is a green fluorescent protein?
used when making a transgenic zebrafish…
- Green fluorescent protein (GFP): a protein that glows green, derived from the jellyfish, and can incorporate into the region of genome you want it to be expressed to label specific regions of embryo to follow development. To direct expression to specific part of emnbryo, use–>
- Promoter: regulatory sequences in the genome controlling the expression of a given gene
Using the promoter of a gene specifically expressed in the eyes we made a transgenic fish line with glowing green eyes
i.e. GFP under the control of promoter sequences of gene that is expressed only in the eyes.
Going to go from fertilised embryo–> gastrula–> neurala–> mature fish embryo
1) 0-5 hpf: FROM FERTILISATION TO GASTRULATION
• The first 5 five hours: zygote undergoes the cleavage period (Cells synchronously divide= make a clump of 12,000 cells on top of the yolk sac) to become a blastula
2) 6-10hpf: GASTRULATION
• cells reorganise extensively, spreading around the embryo via a process known as epiboly.
When the cells cover halfway through the yolk, they start to rearrange more extensively, moving more towards one side of the embryo and keep extending–> generating:
1) the three germ layers (Ectoderm, endoderm and mesoderm)
2) Establishment of the anterior-posterior, dorsal-ventral and medio-lateral axes
describe cell movements during gastrulation
- Epiboly: pooling of cells surrounding the yolk sac via reorganisation of cells in the radial axis (radial intercalation: responsible for the thinning of multi-layered tissues during large-scale morphogenesis)
- Involution: occurs in one specific part of the embryo, consisting in some of the cells. the first step to generate the 3 germ layers- some of the cell stay outside, some are internalised.
- Convergence and extension: intercalation movement in the planar direction–> cells of the embryo start to intercalate towards one side. As all the cells are intercalated towards one side, all the cells are pushed to one side of the embryo, generating the dorsal-ventral axis of embryo.
what happens by the end of gastrulation?
- Generation of the three germ layers
- In fish: mesoderm and endoderm are still mixed up at the end of the gastrulation (will spread alone eventually)
- Establishment of the anterior-posterior, dorsal-ventral and medio-lateral axes
