Models Of Disease Flashcards
What does in vivo mean?
Cells growing in their normal biological context
Inside the body
What does ex vivo mean?
From the body but now outside the body
What does explantation mean?
Tissue growing outside the body in culture
Outside normal biological context
What does in vitro mean?
Latin for in the glass
Cells growing in culture outside the normal biological context
What is a tissue culture?
The growth of cells from a tissue from a multicellular organism in vitro
What are primary cells?
Cells isolated from a donor organism
How are cells maintained and grown in culture?
Given essential nutrients
Correct physio-chemical environment
Grown in substrate
Given growth factors
What are the necessary nutrients for cell growth in a culture?
Amino acids
Carbohydrates
Vitamins
Minerals
Hormones
Gases
What does the physio-chemical environment refer to?
pH
Osmotic pressure
Temperature
What is the difference between primary cells and immortalised cells?
Primary have a limited life span and will not proliferate indefinitely
Immortalised cells can proliferate indefinitely through random mutation or deliberate modification
What is senescence?
Undergo according to their Hayflick limit
The mean number of doubling times before division ceases
What is cell confluency?
The proportion of the culture dish or flask covered by adherent cells
Different cell confluencies
10% - ideal for planting/seeding
30% - good for transfection
50% - will need to split soon, not much more room left to grow
90% - cells touching, good barrier function tests and western blot to maximise protein harvest
What is an apoptotic cell?
A type of cell death in which a series of molecular steps in a cell lead to it death
What are the advantages to in vitro studies?
Relatively cheap and easy
High level of control
Test drugs easily and quickly
Potential to investigate disease models and cell interactions
What is a simple in vitro cell culture model?
2-D monolayers
What is a complex in vitro cell culture method?
3-D gel cultures and co-cultures
Transwell systems
What are the limitations of tissue culture?
Potential for cross species contamination
Introduction of viruses/prions
Animals have homeostasis - cultures don’t
Animals vary in genetics
What are the limitation of in vitro culture?
Loss of specialised extracellular matrix
Loss of paracrine stimulation and endocrine factors
Loss of contact mediated stimulation
What are the outcomes of induced pluripotent stem cells?
Reduce risk of immune rejection
Sidestep ethical concerns of using embryos
Opportunity to put right problem at the genetic level
Limitations of induced pluripotent stem cells
Are they mutation free?
Are they potentially cancerous?
Can they really be grafted back into a patient?
How long will they survive in vivo?
Will they actually solve the problem?
What is personalised medicine?
Drug testing on the individuals genome
Investigating the genetic origins of the disease
What are in vivo studies?
Any surgical procedure, imaging or behavioural experiment done on live animals
How do you induce a disease in in vivo experiments?
By surgery - partial pancreatectomy to induce diabetes
Chemicals and toxins - carcinogens
Dietary manipulation
Implantation of cells
How to begin an in vivo experiment
Breeding of and experimentation of naturally occurring genetic diseases
Genetic manipulation to change expression of the gene
Breeding of transgenic animals
In vivo experimentation can include the injection of what substances?
Saline
Drugs
Cells
What are some arguments for the use of animal experimentation?
Can replicate complex diseases
Closer to human physiology than cell cultures
Maintains cell to cell interactions
Can prove a concept better than in vitro
It is really healing to cure and treat cancer
Give some arguments against the use of animal experimentation
No animal experiment can absolutely predict what will happen in humans
We are not more important than animals what gives us the right to use them
What is the animal experimentation legislation?
Cruelty to animals act 1876
- enforced a licensing and inspection system for vivisection
Protection of animals act 1911
- largely repealed
- regulation regarding animal cruelty
Animals (scientific procedures) act 1986
- act of parliament which regulates the use of animals used for research in the UK
- all living vertebrates except humans
What does ASPA do?
Define legitimate purposes for animal use in experiments
Provides inspection of facilities and procedures
Ensures humane standards of husbandry are care
Ensures public accountability
At what point does ASPA take effect
From two thirds of the way through the gestation/incubation
When is an animal experimentation no longer regulated by ASPA?
The circulation has stopped permanently or the brain is completely destroyed
What are the 3Rs of ASPA?
Replace
Reduce
Refine
What are the three points of the first R - replace
Experimenting on cell cultures instead of whole animals
Use computer models, humans, population studies instead
Try non-traditional methodologies
What are the three point for the second R - reduce?
Improving experimental technique
Improving techniques of data analysis
Sharing information and tissues with other researchers
What are the three points for the third R - refine?
Better medical care and living conditions
Minimise distress
Minimise pain
What is constitutive or conventional KO?
Where every cell in the body lacks functional expression of a gene of interest at the protein level
What is CrispR-Cas9?
It was designed to find and cut a unique gene sequence
When is CrispR-Cas9 used?
Carried out on an egg or embryonic stem cell to delete or insert DNA one one allele
What does CrispR-Cas9 result in?
Resultant embryo has 1 dysfunctional gene
Implant embryo and grow until birth
Identify successful KOs
What are the steps to breeding KO animals?
Breed (-,+) with (-,+) to get (+,+) and (-,-) for experimentation
Keep some (-,+)
Genotype each pup
Breed (-,-) with (-,-) so all offspring are (-,-)
Don’t need to genotype every pup
Wildtypes needed for control
How do you identify protein loss?
Immunohistochemistry (IHC)
Western blot
In situ hybridisation
How does immunohistochemistry and western blot work?
Depend on antibodies recognising a site on the protein after the point of KO but not all proteins have selective antibodies targeting these regions
How does in situ hybridisation work?
Recognises mRNA
Designed to find the mutation
Expensive and more difficult to do but also more accurate
Which are the easiest KOs to genetically identify?
Transgenders with large insertions/deletions
Easily ID using PCR of genomic DNA
Transgenic animals as models of disease
Technological advances have increased our ability to create models of human disease
Whole body - conventional knockout
Inducible whole body knockout
Tissue specific knockout
Gene reporter
Knock-ins
What are the advantages of whole body knockouts?
Relatively easy
Fairly cheap
Easy to genotype using PCR
Every cell is affected
Easy to determine main functions of gene
Good for in vivo work
Simple controls
What are the disadvantages to whole body knockouts?
Can’t say the effect in solely due to loss of one action
Loss of expression can be compensated for by other genes
CrispR site may not be specific to the gene of interest
15% of KOs are embryonically lethal
How to avoid embryonic lethality in KO therapy
Gene of interest is floxed by inserting LoxP restriction sites on either side of gene of interest in the embryo
Grow into the animal but gene is KOed when you tell it to be
How to produce an inducible knockout?
Breed CRE animal with floxed animal to get Cre-LoxP
What are the advantages of inducible KO?
Avoids embryonic lethality
Comparative study of before and after KO in the same animal
Very flexible
Mimic pathology of late onset
Less problems with compensatory expression
What are the disadvantages of inducible KO?
Differential penetration of trigger compound into tissues - need to prove changes at protein level
Adding the LoxP site risks modification or disruption of splicing regulation
Tetracycline inducible systems may be leaky
Proteins may be promiscuous
What are the advantages of tissue specific KO?
Study gene function in one specific organ/tissue/cell
Mimic pathologies caused by gene activation
Create tissue specific phenotype for multifunction protein
Investigate signally pathways in certain cell types
What are the disadvantages to tissue specific KO?
No gene is really specific to one cell type
Added expense
Proving cell specific KO is difficult in tissues of mixed cell types
Embryonic lethality
What are reporter genes?
Identify where proteins are expressed
Identify cells/embryos which possess the trans gene
Give an example of what a reporter gene could be
A gene which provides resistance to antibiotics so KOed embryos could be positively selected with high does antibiotic
What are the advantages of knock-in models of disease?
Can prove role of mutation in diseases
Can investigate human diseases in animals
Can add back in a deleted gene and prove no compensation
What are the disadvantages of knock-in models of disease?
Difficult to make accurately
May need sequencing to prove successful mutation
What are the four types of transgenic models?
Conventional
Inducible
Tissue specific
Knock-in
