How are marmosets used as a disease model?
- injected with MPTP chemical –> kills cells in substantia nigra –> Parkinson’s disease
What model was used to investigate CML?
Myeloid cells –> injected with BCR-Abl transgene
What are the limitations of using petri dish cells as disease models instead of humans? (2D VS 3D)
- no cell to contacts
- diff cell signalling/ interactions
- petri dish = rigid matrix but body is flexible
- petri dish cell environment = atmospheric 02 –> stressful
- where as body cells normoxic conditions
Benefits/Limitations of using marmosets as disease models
Benefits - allows therapy testing - develop similar symptoms to humans Limitations - can't study how Parksinson's develops - Lesion is not progressive
Benefits/limitations of myeloid cells as disease model
Benefits - cheap and quick to grow - easy to manipulate - allows drug testing -understand pathophysiology Limitations - 2D model - can't learn about CML onset - more opportunity for cells to develop mutations - cell lines vary over time
What are the benefits of using Zebrafish as a disease model?
- good vasculature –> can investigate variables involving bloodflow
- transparent –> can track therapies/chemicals
- genetically tractable –> can easily introduce mutations
- cheap
What have Zebrafish been used as a model to investigate?
Wound healing/ tissue damage and how it may be correlated with tumor growth SEE NOTES
How can use as mice as T1DM disease models?
inject them with a chemical that causes B-cell destruction –> induce hyperglycaemia
Why might inbred mouse strains be used? Give an example
- can mimic aspects of human disease
- NOD mouse (non obese diabetic mouse)
What is a limitation of using inbred mouse strains?
- complex genetic background
- so may not reflect cause of human disease
How may mutant mice be used as a disease model?
- use CRISPR to knockout specific genes
- investigate genetic diseases such as cystic fibrosis –> introduce CTFR gene
- investigate genetic variants that predispose to Alzheimer’s
Why may mice with an introduced mutant CTFR gene still not develop cystic fibrosis the same procedure in pigs works?
- ATPase pump not upregulated in mice
- no mucosal acidification
- resident immune cells can still function
- so no thick mucous/bacteria build up
What are the limitations of using mutant mice? (4)
- specific strains are used
- time of experimentation can have different effects
- depending on gender of mice feeder/carer –> diff responses
- mice have clean cages –> no influence of diff microbiomes like in humans
What is a benefit of using mutant mice? Use a specific example
- SCID-NOD mice
- have no immune system
- so can introduce patient human tumour cells (no rejection)
- allows investigation of diff therapies
- personalised medicine
- enhance understanding of tumour biology
What are the limitations to using SCID-NOD mice for PDT X? (4)
- can’t predict affect of immune system on therapies
- can’t investigate immunotherapies
- expensive
- time consuming
How could you repopulate the immune system of a SCID-NOD mouse?
Take a graft of human stem cells from bone marrow –> introduce into mice
*see other ways in notes
Give and explain an example of a personalised model.
Avatars
- patient tumour cells DNA sequenced
- creates a mutation profile
- create cancer specific mutation in mice/flies
- test specific therapies
What are organoids derived from?
- primary tissue
- OR embryonic/ induced pluripotent stem cells
Outline the steps in creating an organoid (4)
- harvest tissue sample
- take epithelial/ embryonic stem/ induced pluripotent stem cells
- give 3D collagen support
- treat with hormones/selection of factors that promote differentiation into cell type of interest
List the uses of organoids (5)
- omics profiling
- study host-microbe interaction
- high throughput drug screening
- disease model
- gene editing –> targeted correction of mutations
What are the benefits of using organoids as disease models?
- can study in vivo biological processes e.g. tissue renewal/drug response
- physiologically more similar to humans
How does the sgRNA flag the 20 bp target sequence?
- has RNA complementary to the target seq
- so will base pair with target seq and displace other strand
How is the CRISPR machinery created and introduced into a target cell?
- DNA coding for sgRNA and Cas9 binding seq introduced into plasmid downstream of a promoter
- plasmid also has cDNA for Cas9 with promoter
- introduced into target cell using transfection/viral transduction
What are the 2 types of mammalian DNA repair?
- homologous end joining - v quick can cause INDELS
2. homologous recomb.
