2. The Need for Model Organisms

Question 1

What is a model organism?

Answer 1

A non-human species studied to understand biological phenomena, with the expectation that findings in the model will provide insight into workings of humans

Question 2

When are model organisms used to study human disease?

Answer 2

When human experimentation is unfeasible/unethical

Question 3

Why are we able to use model organisms to study human disease?

Answer 3

Due to common descent of all living organisms and the conservation of developmental and metabolic pathways through evolution

Question 4

Why are particular organisms selected as models?

Answer 4

1. By chance (what was available at time)
2. Inbred populations within species to decrease genetic variation (allows rapid genome sequencing)
3. Low cost and short lifespan
4. Easy to induce mutations
5. Easy to score phenotypes
6. Easy to analyse disease traits
7. Easy to manipulate

Question 5

Why are Drosophila, Zebrafish and Mouse used as models to study human disease?

Answer 5

• All share a degree of gene conservation with humans
• Basic mechanisms of gene action (proteins and pathways) are conserved
• Development and maintenance of body and core metabolic pathways are conserved
• Easy to interfere with gene function in model organisms
• Can study gene expression in real time (e.g. optic clarity in zebrafish)

Question 6

What are the 6 opportunities afforded by model organisms?

Answer 6

1. Provide models where it is possible to examine individual over life course
2. Provide models where it is possible to examine genetic x environmental interactions
3. Can understand mechanisms from correlation to cause
4. Allow us to make transgenic reporter lines
5. Large numbers of animals and reporter lines aid the development of new therapeutics
6. Enable a whole organism or systems approach to studying disease states

Question 7

What is the significance that most diseases studied are chronic diseases that develop over time?
What does this mean for model organisms?

Answer 7

• Need to study disease in relation to ageing process

- Require model organisms with short lifespans to trace progression of disease through life

Question 8

What does an individual’s genotype confer?

Answer 8

Genotype confers the potential for health across life course and the high likelihood of developing a specific disease - e.g. BRCA2 mutation confers high susceptibility of developing breast cancer

Question 9

How do environmental factors interact with the genotype?

Answer 9

Interact with genotype to trigger/exacerbate disease

Question 10

What are diseases likely to arise from?

Answer 10

Gene x environment interactions

Question 11

Why are animal models useful for modelling complex diseases with many variables?

Answer 11

• Genetically tractable - CRISPR can make transgenic animal with specific mutation in specific gene to model human susceptibility genes
• Can be examined in large numbers - outcomes can be concluded as statistically significant

Question 12

What do genome-wide association studies (GWAS) identify?

Answer 12

Mutations that correlate with disease susceptibility

Question 13

Give an example of a GWAS

Answer 13

• 10 individuals, 5 healthy and 5 with T2D
• Sequence genomes and identify mutations that correlate with T2D individuals but not healthy individuals
• Identifies correlation between genome variation and T2D susceptibility

Question 14

What do GWAS studies not show?

What can be used for this?

Answer 14

• When and where gene is expressed or how gene product may trigger/exacerbate disease
• Use an animal model

Question 15

What techniques can be used to identify which cells/tissues express a specific gene?
What does this tell us in relation to disease?

Answer 15

• In situ hybridisation shows where and when mRNA is expressed
• Immunohistochemistry shows where and when protein is synthesised
• Can then infer from location of expression what may be happening in dysfunction, e.g. if expressed in hypothalamus, disease may be caused by hypothalamus dysfunction

Question 16

What techniques can be used to identify gene function?

What does this tell us in relation to disease?

Answer 16

• Loss/Gain of function genetic approaches

- Function of cells/tissues that is controlled by the gene product

Question 17

What is the process for generating a tissue-specific (conditional) knockout?

Answer 17

1. Gene to be KO’d is flanked by lox sites and 1st transgenic mouse is made in which WT gene is replaced with engineered gene
2. Identify tissue-specific promoter governing expression of gene that is specifically expressed in the cells in which you wish to KO gene
3. Clone the coding sequence of the enzyme Cre recombinase downstream of this promoter and 2nd transgenic mouse is made in which Cre recombinase is expressed only in the required cells
4. Cross 2 transgenic mice - Cre recombinase acts on lox sites like scissors and removes flowed allele so that gene is KO’d only in cells expressing Cre recombinase

Question 18

What are transgenic reporter lines?

Answer 18

Animals whose genomes have been engineered to give a colour report of expression of a specific gene

Question 19

What is the main advantage of reporter lines?

Answer 19

Animal is alive therefore can visualise gene expression in real time

Question 20

What do reporter lines enable?

Answer 20

• Visualisation of cells expressing specific gene in real time
• Isolation of labelled cells by FACS

Question 21

What do reporter lines depend on?

Answer 21

The fact that genes are differentially transcribed as a function of the interaction of their promoter/enhancers and cell-specific transcription factors

Question 22

What is the process for generating a transgenic reporter line?

Answer 22

• Identify a tissue-specific promoter in cells to be followed
• Clone coding sequences of fluorescent reporter proteins (e.g. GFP, Kaede, RFP) downstream of this tissue-specific promoter
• Make transgenic animal in which this transgene is stably incorporated into the genome and reporter will be expressed only in cells which have correct transcription factors for the promoter

Question 23

What can transgenic reporter lines be used for?

Answer 23

• Follow cells over time in health, disease, after insult or drug administration
• Analysis of cell behaviour (proliferation, migration)
• Analysis of cell function (hormone secretion, response to signalling)

Question 24

What does understanding of disease and deterioration require?

Answer 24

A comprehensive understanding of the whole organism and the complex and dynamic interactions between different systems, organs and cells

Question 25

What is a technique for studying cells in their normal environments?

Answer 25

Generate multiplex transgenic reporter lines, where different cells express a different reporter colour

Question 26

What are in vitro cell lines used for?

Answer 26

• Drug screening for new therapeutics
• Single cell type studies
• Generation of organoids

Question 27

What do the opportunities afforded through model organisms allow us to do?

Answer 27

• Identify molecular pathways in normal cells/tissues
• Identify normal and aberrant cellular outputs e.g. proliferation
• Identify disease progression e.g. primary cause and secondary consequences
• Perform high-throughput screens for drug development