General Flashcards

1
Q

Why do scientists work on particular organisms?

A

Sometimes just by chance - e.g. they were abundant at the time of research.
There were already inbred populations available (collecting of mice with strange phenotypes was a hobby) - makes some specieis useful for genetics and genome sequencing (mendels peas)
Costs, life-cycles time
Easy to score disease traits/phenotypes
Simplification of complicated systems e.g. nervous system/heart
Easy to manipulate genetically or physically (big embryos)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What are the main organisms used in disease modelling and discovery and why?

A

Drosophila, zebrafish, chick, and mice - they share a remarkable degree of conservation with us - including the basic mechanism of gene action, the way our bodies are built and maintained, and basic metabolic pathways.
Zebrafish and chick embryos are transparent- so useful for cell lineage whilst the animal is alive
Choose animals with short gen times so we can look at the development of disease throughout the animal’s life and development and aging. Models also allow us to study gene x environmental interactions - these mean that diseases have an enormous number of variables - use animals to isolate and study how different variables affect the disease. - can also study these variables in large numbers due to high number of offspring.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

Explain how mice are being used to model Type 2 diabetes.

A

Animal model studies are trying to work out what can be a trigger even and how we can prevent T2D. Obesity is linked to the accumulation of excess fat in ectopic sites such as the liver and skeletal muscle, this is associated with the formation of T2D. Genetically modified mice with a lack of adipose tissue are characterized by hyperphagia, hyperglycemia and insulin resistance (type 2). However, note that due to a lack of functional adipose tissue, these mice are leptin-deficient - so the primary cause of T2D may be linked to brain dysfunction (lack of leptin secretion). In zebrafish and mice, people are making reporters that allow specific metabolic tissues to be visualized in real-time and space. This allows us to analyze the healthy animals, those with genetic mutations and those exposed to different environmental conditions (e.g. diet)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

How may crossing reporter lines be useful for the study of T2D?

A

Crossing of 3 reporter lines to make a fish where you can see the pancreas, liver and adipose tissue in a single fish. Feed high-fat diets and see which of the 3 tissues shows a fault first. Another use of reporter animals is to look at specific populations within a tissue, e.g.just the beta cells in the pancreas.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Explain how scientists discovered the cause of breast cancer in mice and what problems they faced.

A

A whole population of inbred mice were found to be particularly susceptible to breast cancer - was found to be because they were all infected with a retorvirus that inserted its cDNA before the proto-oncogene (intergration 1- Int1). It was predicted the proteins staeted with a signal sequence e.g. a growth factor or secreted factor (due to increasing growth). Initial attempts to purify this protien failed as the tech wasn’t available yet. and means they couldn’t do biochemical assays. Meanwhile, people using drosophila (Nusslein-wolhard and wischaus were doing a big mutagenesis screen to find function of different genes by mutating random ones and looking for phenotypes. They identified the segment polarity gene Wingless, which was found to be homologous to Int1 (named together as Wnt) - found to be an evolutionary conserved secreted factor. Its pathway was elucidated through analysis in model organisms and mammalian cultures. Using WNt transgenic reporter lines we can now see which cells in the body respond to Wnt pathway activation (many of which are in tissue-specific stem cell niches)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

Explain how the drug Erivdge was discovered and developed.

A

in 1940/50s people realised sheep being born with one eye was bc of cyclopamine in the corn lilies they were eating. In 1980s Wischaus etc found Shh was important for neural tube patterning and was expressed along the ventral midline. Mice born with cyclopia and holoprosencephaly due to Shh defect. - was then also found that cyclopamine was an antagonist of Shh pathway (hence why it was causing cyclopia). In 1990s, animal models systems demonstrated the importance of Shh signalling to the development of many tissues/organs. People also showed that Shh is usually down-regulated at end stages of development. Ideas around Shh being involved in cancer as its down regulated during differntiation, Was found that Smo was activated in BCCs. Therefore people thought cyclopamine may be able to reverse this (it inhibtis smo_. People developed drugs e.g. erivedge which was developed from cyclopamine to act as an antagonist of Smo. Its the first and only drug to show efficacy for BCC. Also now trials for it being used in other types of cancers linked to Shh (lung, pancreatic) However it is VERY expensive(so withdrawn from NHS- but will be ,ade cheaper one day)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

What models should be used for research into melanomas?

A

If you wanted to look at basic cell cycle machinery and how the mutations in these effect growth you could use yeast. If you wanted to look at the upstream controls of cell division/differentiation/death e.g. how MAPK/RAS effects these you could used drosophila or C. Elegans as it easy to track the lineage of cells. To look at tumour growth and metastasis, its best to use a model that has the same tissue context as it involves angiogenesis/lymph vessels/immune system- therefore best to use vertebrates e.g. mice (can then also use them for xenografts and KOs), or You could sue fish for large scale experiments, optics claristy and fast genetics, high throughput assays.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Explain how mice were used to model melanomas

A

Xenotransplants of human cells or tumours into nude mice (they have a mutation in foxn1 genes so have a very weak immune system- therefore easy to grow tumours. . They also used genetically engineered mice- make a mouse that has a mouse melanoma rather than a human one - make melanocytes that had specific expression of hRAS- was found that for melanomas to form you needed UV or CDKN2A o be inactive as well. People have also made mutant mice that has melanocyte-specific expression of BRAF - found that this caused extra moles but for them to become melanomas you had to inactivate PTEN too (a tumour suppressor) which induces melanomas quickly and causes the mice to die quickly.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

Give examples of how mice models were used to develop treatments to melanoma

A

Cancer genome project identified BRAF was discovered as a major drive behind melanoma - leading to a specific inhibitory mutation was developed. USed cell culture to look at the effects of this mutation of lots of different tumors. Found many of them are sensitive to this inhibitor (most melanoma sand one breast cancer). They then transplanted lots of melanoma cell lines into mice and found that a compound which blocked BRAF dramatically reduced tumour formation and survival. Tested in humans and was really promising until the tumors gained resistance to it in week 23 and came back.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

Explain how zebrafish were useful in melanoma research

A

Used CRISPR/Cas9 to make KOs in zebrafish, Chemical screen for compounds that interfere with melanocyte precursor formation, thought they might be able to find a compound that you could combine with the promising one to stop tumours becoming resistant to it. (looked for Crestin which a neural progenitor marker and melanoma cells have a neural precursor identify) - found one compound (which is already on the market) did. Zebrafish are useful for high-throughput screens like this because you don’t have to kill and dissect each one bc they’re see-through.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Why are zebrafish good models for heart disease and development?

A

Zebrafish heart development happens quickly, is quite simple and is easily watched/tracked. The eggs are also big enough that you can inject them with things e.g. vector and stains, however, they’re not big enough to operate on (can’t add stents etc). Able to oxygenate via diffusion (so can actually stop the heart working and they will stay alive), looking at moving blood without dyes. However, they do not have all of our genes and there is no spontaneous CVD in zebrafish.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What are the disadvantages of non-zebrafish models for heart disease/development?

A

Mice models are extremely expensive, only partial success in modelling disease in mice - good for atherosclerosis, moderate for hypertension, poor for valve defects. Big organisms like pigs are difficult to manipulate genetically and are very expensive, but can be good for valves. Mice etc difficult to see vasculature (have to kill and dissect or use angiogram, expensive

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

How were zebrafish models used to study innate immunity?

A

Zebrafish neutrophils are similar to humans (polymorphic). You can make transgenic zebrafish (GFP in neutrophils), then injure their tail and watch how they migrate. You can also infect the zebrafish and watch whole body migration. It was found that hypoxia enhances inflammation and is necessary for robust anti-bacterial response. If you turn on hypoxia signal with DAGO you suppress apoptosis, They did a screening assay with transgenics where they injured the zebrafish and tracked neutrophils and then added different drugs to each well to see how these drugs affect inflammation,. they found a drug called Tashinone which is known for treating heart disease but its exact mechanism is unknown. They first tested in human neutrophils which lead to reduced apoptosis even in the presence of cytokines. Migration assay showed that tashinone increased reverse migration (made neutrophils leave site)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Why was the discovery of how to differentiate stem cells into motor neurons important?

A

Meant that we can now grow purified cultures of motor neurones, which you can’t do just by taking a sample of cells. Means we can use them to test drugs and look at development. Long term goal would be to use these to rescue cells in neurodegenerative diseases. Motor neurones re sourrounded by glial cells that secrete neurotrophic factor to keep it healthy- so in MND we don’t know whether its the glial cells or the motor neurones themselves that are going wrong.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Explain how stem cells were used to model diseases like microcephaly,

A

Mouse mutants fail to recapitulate the condition, so used reprogrammed stem cells from microcephaly patients to make cerebral organoids. It was found that there were fewer neural progenitors in patient organoids. Models like this can also be used for ALS, heart disease, alzheimers and parkinsons. increases in microcephaly was linked to the Zika virus outbreak. People infected cerebral organoids with the zika virus and found that it increased the death of neural cells. It was found that the drug Emricasan blocked zika virsu infection and helped reduce the chance of microcephaly in unborn babies infected.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

What are some challenges in modelling disease with stem cells?

A

Some diseases have multiple genetic causes e.g. autism. Some diseases have complex phenotyeps e.g. cleft pallet. Some have late-onset e.g. parkinsons. There is a lack of efficient differentiation protocols e.g. blood cell lineages.

17
Q

What are some challenges faced in cell replacement therapy?

A

Progenitors Vs differentiated cells. Immune reaction/tumour formation. Mechanism: replacement or regeneration of already present stem cells. Positional identity (the cells don’t always settle in the right place)

18
Q

What are the strengths and weaknesses with mouse models in healing?

A

Strengths - complexity - cell types, 3D structure, angiogenesis, hair follicles gives more information than just growing in a dish. Short gen time.
Weaknesses- Losse-skinned (slightly different structure), contraction rather than re-epithelialization due to loose skin. Size is very different to humans- not a great model for diabetes (better blood flow so not good models for extremity neuropathy), age - mice only live 3 years. Pigs are a better model for wound healing - however, you can’t make KO pigs because they live too long.

19
Q

What are the limitations of using stem cells for regeneration?

A

Culturing cells induces genetic changes, transplantation, and organogenesis is very complex.

20
Q

Why are zebrafish a good model for regenerative biology?

A

Fins are an excellent model because they are easy to work with, and trimming the fin is not life-threatening They also contain multiple cell types so you can look at how they interact easily (fin is see-through)