Notes de cours
Medical Biotechnology Lecture Notes
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This is my lecture notes on following the course, also a summary I made when I prepared myself for the exam.
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Medical biotechnologyLE1 – Vaccination
Cancer cells produce neoepitopes there are vaccines for cancer
Types of vaccines:
1. Killed whole organism
Killed but still carry the epitope of the virus to trigger the immune system
Presents the whole bug so the immune system can choose what to do with it
The way of killing could destroy epitopes that are there in the alive one
Incomplete inactivation
2. Life attenuated (weakened/inactivated)
Alive vaccine but cannot harm the patient
It is maximally there to trigger the immune system
Denaturing or killing not required
Takes ages to modify test to see if it is still effective expensive process
Most effective vaccine type, relatively safe
3. Vector vaccines
Taking a safe bug environment and modify with surface coat protein ex. Put a spike
protein on the membrane
Example: AstraZeneca vaccine
Can also be used for bacteria
4. Subunit vaccine
One component from the bug (subunit) and hope that it is immunogenic
Recombinant protein, so it doesn’t look like the bug anymore
Inject the purified protein
5. Peptide vaccines
A part of the whole protein that is known to be immunogenic
Make a big particle that shows many of these peptides
6. Nucleic acid vaccine
Genetic material and code the peptide DNA/RNA vaccines
Inject expression plasmids into a cell creates protein expresses on the membrane
RNA very unstable and vulnerable (short-lived) so it must be modified in such it is more
stable in the cells (making it less prone to RNases)
People question the side effects of the modified nucleotides that are injected into the
body, but cancer patients show no complications
Inject the RNA via nanoparticles (vesicles) side effect of nanoparticles still unknown
Efficacy vs safety slide 15
Safety, production (have to be in large quantity), storage, administration
RNA vaccines need to be stored at -20˚C so it is hard to be distributed
,LE2 – Genetic modification: transfection & transduction
Genetic modification:
1. Episomal transfer/addition
If the addition is not being integrated into the genome of the virus
2. Insertion
3. Deletion
4. Replacement
Modifications can be temporal, permanent, random, or targeted
Random insertion can cause mutation, can damage the genome, can cause cancer
Conditional change (in between transient and stable) modify genetic content only if a
condition is present to maintain that modification ex. Antibiotic testing
Transient: DNA inserted into the cell but then in a few hours degraded by the organism.
Follow up in 24-72h period.
Targeting can be done in 2 ways: homologous recombination (repair breaks just by the sister
chromatid, makes double strand break), site-specific recombination
Plasmids:
Can be manipulated
Easily isolated large quantities
Transfection methods:
1. Physical
- Microinjection (transgenic mice)
with a fine needle and pinch into the cell where you want to modify the DNA or RNA.
Laborious one cell at a time
You never know where it ends up, how it will be expressed, how it affects stability
- Electroporation (knock-out mice)
Using electrical resistance to make pores which allows ion to flow in and out.
Reorient lipids in the membrane so pores appear
Using high electrical energy pulse, DNA flow in the cell
Can do millions of cells at once get thousands of colonies
Half of the cells fails to repair membrane and die
- Particle bombardment (plants, genetic vaccination)
Shoots gold particles coated with DNA using a particle gun (He pressure) and can be
used in live animals
No inflammation, mild, no disease
It is so mild that the DNA must code a very strange protein to induce immune
reaction
Only hit a few cells, have to open up the target tissue
2. Chemical
- Calcium phosphate precipitation
Package DNA into calcium phosphate crystal that can be endocytosed by the cell
DNA need to be pure
, pH has to be controlled
Crystal too big rock, crystal too small it will float in extracellular matrix
- DEAE dextran uptake
Short term
Lipid-sugar will complex with DNA and digested into the cell
Long-term it is toxic
- Lipofection
Preferred way in terms of efficiency, can get up to 100%
We take lipid-like components
Very expensive
3. Biological
- Cell fusion (hybridoma)
Combining the genome of 2 cells
Monoclonal antibody (spleen cell and cancer cell)
- Virus infection (gene therapy)
It has its dangers
Retrovirus randomly insert into the gene banned
Lentiviruses tend to integrate at spots in between genes allowed for trials
Transgenesis: can pass down the alteration into next generation
1. Viral infection
Taking embryo and infect it with viruses
In 60’s-70’s used to study which gene areas are important for development
2. Microinjection
Insert DNA directly into oocyte
3. ES cells
Embryonic stem cells culture to proliferate (not develop)
Use electroporation and look for the one that takes up
Inject it back into the animal
4. SCNT: somatic cell nuclear transfer
Laborious and not work very well
Take nucleus from one cell and inject to a cell where the nucleus is taken out
Injecting it back to the mouse
If you take somatic nucleus, the DNA have gone through epigenetically reprogramming
which causes aging problem with the offspring
Can be used for mitochondrial disease prevention:
Healthy donor egg cell take out nucleus insert nucleus from the diseased mother
Already done in the UK
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