The Functional Genome Flashcards
What is the prioritisation filtering protocol?
The protocol that WES data is subjected to. It reduces the candidate genes to find the region that might cause a disease. It filters out those that are already in the database or common variants. It also does this by looking at family members etc.
Why is the prioritisation filtering protocol important?
It is important for developing drugs for personalised medicine
Why is more functional evidence required?
WES does not prove causality
How is WES used in patients?
It can be used with blood or tissue samples to find out how proteins are affected. Some proteins will not be expressed in blood and this will show which genes are affected.
Why are cell culture techniques used?
- In vitro (artifical environment)
- Removal of cells from an animal and subsequent growth in favourable conditions
- Primary cells have finite divisions but can immortalised to provide continuous source.
- Provides a cheap, rapid and reproducible model for studying the normal physiology and biochemistry of cells
- It is a good alternative to animal models so less restriction.
- Many tissue specific cell lines commercially available - different tissue types such as neuronal and myogenic
What is gene knockdown?
microRNA based gene silencing technique
What is the microRNA based gene silencing technique?
microRNA adapted shRNA joins with a simple hairpin shRNA. This makes a protein which is exported out of the nucleus. It forms a mature microRNA duplex, then the mature microRNA which cleaves and does gene silencing.
What is a short hairpin RNA (shRNA)?
- Based on endogenous microRNA gene silencing
- Modified to include GOI complementary sequence
Explain how a shRNA is used
Packaged in a DNA plasmid, expression is controlled by an RNA polymerase III promoter ubiquitously.
- 50-70nt. Transcribed. Exits nucleus, cleaves it through the nuclear pore called Expoitin-5; cleaved by a nuclease called Dicer in the cytoplasm
- Cleaved segments bind to RNA induced silencing complex (RISC) and direct cleavage and degradation
of complementary mRNA -> gene of interest.
What is short interfering RNA?
SiRNA similar to ShRNA, chemically synthesised, not vector based
Give an example of a shRNA
PDZRN3
Where is PDZRN3 found?
It is found to be developmentally regulated in skeletal muscle
What are C2C12 cells and what do they do?
They are immortalised mouse myoblasts that differentiate into muscle
What causes upregulation of PDZRN3 and MCH?
PDZRN3 blocks SiRNA upregulation of PDZRN3 and MHC.
What happens when PDZRN3 is knocked down?
Inhibition of myotube formation and MHC expression if it is knocked down, the myotubes don’t form.
How is the gene of interest encoded protein localised?
Through Antibody staining find the:
- Protein of interest
- Downstream target
Transfect DNA plasmid inserted into cell cells with GFP tagged GOI (CMV promoter)
Transfect cells with GFP tagged mutated GOI (CMV promoter)
What is dysfunctional protein behaviour?
With the mutation, the protein is localised to specific areas
Why is cell culture not enough?
- Cells behave differently in a petri dish/flask to how they behave in a whole organism.
- Doesn’t stimulate the actual conditions inside an organism.
- No information about gene expression and function, with regards to developmental phenotypes
What percentage of research is non-animal based?
90%
Why are animals used in research and what are the benefits?
- Cells behave differently in vitro to in vivo.
- Most medicines we have today come from animal research
- Contribute to 70% of the Nobel prizes
- Polio
- Anything found is also used in veterinary science for animal ill-health
- Research scientist seek to alleviate pain and suffering
Which animals are used in research?
- 2.11M procedures on mice
- 0.28m procedures on chicks
- 4,481 procedures on dogs
- 0.6m procedures on zebrafish
- 0.32m procedures on rats
- 10,424 procedures on horses
- 159 procedures on cats
- 3,207 procedures on monkeys
What is the animals scientific procedures act (ASPA)?
Formed in 1986.
- The UK parliament permitted the use of animals in scientific procedures.
- it regulates the use of protected animals in any experimental or other scientific procedure that may cause pain, suffering, distress or lasting harm to the animal.
- Animals cared for with the best standards of modern animal husbandry
What are the animals protected under the ASPA 1986 act?
Any living vertebrae animal (other than man) and any living cephalopod (squid and octopus).
Why are mice used as a mammalian model for human genetic disease?
- Accelerated lifespan (1year = 30 human years)
- Small
- Reproduce quickly
- Easy to handle and transport
- More ethical than larger animals/non-human primates/humans
- Modern genetic engineering have allowed for precise mutation to recreate a disease
- Lots of mouse strains and models already exist
- Mammals: genetically similar to humans
- Mice have been used in BM research for 100+ years
