The Functional Genome Flashcards
(22 cards)
What are two examples of next generation sequencing?
We have:
- Whole Exome Sequencing (WES), which is used to capture the sequence of the coding region of the genome.
- Whole Genome Sequencing (WGS), which captures the whole thing (it is not always necessary)
They are rapid modern methods for high throughput DNA sequencing
They aim to identify potential disease-causing genetic variants: personalised medicine
WES and WGS identify candidate causative genes…functional follow up experiments required for proof of pathogenesis and a genetic diagnosis.
Describe candidate gene filtering using WES.
WES data is subjected to a prioritisation filtering protocol.
During this process, we make multiple assumptions:
- we remove any variants that are common in the database, so we assume that the mutation is rare
- we also assume that the people who have the variant also have the disease
- we also assume that the disease is being caused by a change in the protein rather than a change elsewhere (e.g. promoter sequence)
15-20,000 coding SNPs are reduced to one or several candidate genes. They are checked for co-segregation (if family members have it as well) and validated by Sanger sequencing.
The filtered WES variants do not proven causality. We still need further functional evidence.
List some of that evidence.
- tissue/cell expression: see if the gene is actually expressed in that cell type
- detection of protein in patient samples?
- how does change affect protein behaviour?
- how does the change affect cell or development of tissues
- identification of the molecular mechanism of action: common pathways
- development of in vitro and in vivo models for dysfunction of GOI
- knockdown or overexpression affects phenotype?
Describe cell culture techniques.
It is the removal of cells from an animal and their subsequent growth in favourable conditions.
It provides a cheap, rapid and reproducible model for
studying the normal physiology and biochemistry of cells.
Primary cells have finite divisions but can immortalised to provide a continuous source.
It’s a good alternative to using animal models, reducing the numbers of animals being used in research, as there are less restrictions on this technique.
Many tissue specific cell lines are commercially available.
How do we study gene knockdown?
We study it using RNAi (interference) mediated gene silencing. This can be achieved using two RNAs:
- ShRNA (short hairpin RNA)
- SiRNA (short interfering RNA)
Describe short hairpin RNA (ShRNA).
It’s based on endogenous microRNA gene silencing. It’s modified to include GOI complementary sequence
It’s packaged in a DNA plasmid, and its expression is
controlled by an RNA Polymerase III promoter
It exits the nucleus, gets cleaved by a
nuclease called Dicer (found in the cytoplasm). The cleaved segments bind to the RNA induced
silencing complex (RISC) and direct
the cleavage and degradation of the complementary mRNA.
How can you find out where your GOI’s encoded protein is localised?
You can use antibody staining:
- for the protein of interest
- or for a downstream target
You can also transfect cells with GFP-tagged GOI (in a CMV promoter).
Expand on animals in research.
Ideally, we wouldn’t use animals in research. Up to 90% of research uses non-animal methods.
However, the cells behave differently in vitro to in vivo.
Most of the medicines we have today come from animal research. They contributed to 70% of the Nobel prizes.
This is research scientists seek to alleviate pain and suffering for these animals in the experiments, so it has rigorous HO monitoring.
Expand on mice as models for human genetic diseases.
- they have an accelerated lifespan (1 yr = 30 human years)
- they’re mammals, so they’re genetically similar
to humans - lots of mouse strains and models already exist
- modern genomic engineering has allowed for precise
mutation to recreate a disease - it’s deemed more ethical than using
larger animals/non-human
primate/humans - they’re small, reproduce quickly, and are relatively easy to handle and transport
List some reasons as to why zebrafish are a good model for human genetic disease.
- you can add drugs to their water
- they’re an inch in length (so don’t take up space)
- you can take their cells out and implant them into another organisms to see if their fate is determined
- they develop ex-utero, so you can monitor their develop microscopically
- they’re easy to genetically manipulate
- lay eggs on a weekly basis
How would you make a mutant mouse?
1) Put lox sites on either sides of the exons, or within the introns of your GOI
2) You have another mouse with Cre-recombinase under the control of a ubiquitously expressing promoter or a tissue-specific promoter
3) You reintroduce ES cells into the mouse (into the cell mass), which will then proliferate and be put into a donor mother, then grow up.
4) You hope some of the mutations enter the germ line, and you select for those that have and develop a stable, mutant transgenic mouse.
When analysing mutations, what are the two ways in which you can do it?
- FORWARD GENETICS (phenotype-based): when you try to find the genetic cause of a phenotype
- REVERSE GENETICS (genotype-based): when you try to find the phenotypic consequence of a genotype change
What is the difference between the siRNA and shRNA techniques?
The difference between shRNA and siRNA is how they’re introduced into the cell.
SiRNA is similar to shRNA; it is chemically synthesised, but it is not vector-based. We can simply transfect it into the cell, skipping out on the transcription phase.
What are some in vitro techniques for studying gene function/dysfunction?
cell culture siRNA shRNA IPSCs (induced pluripotent stem cells) CRISPR
What are some in vivo methods for studying gene function/dysfunction?
mouse zebrafish ENU screens morpholinos CRISPR
Give examples of functional assays
Immunohistochemistry
cell biology and behaviour
protein interactions
transcriptomics
What are and how do we get induced pluripotent stem cells (IPSCs)?
Induced pluripotent stem cells (iPSCs) are adult cells that have been genetically reprogrammed to an embryonic stem cell–like state by being forced to express genes and factors important for maintaining the defining properties of embryonic stem cells.
You first perform a skin biopsy where you isolate adult fibroblasts.
You then culture them and amplify them, where they are then reprogrammed
They are dedifferentiated to form IPS cells and then differentiated to form differentiated cells
Why is cell culture not enough?
Cells behave differently in a petri dish/flask to how they behave in a whole organism
Do not stimulate the actual conditions inside an organisms. You can get signals from other tissues
No information about gene expression and function with regards to developmental phenotypes
Who approves licences for animal work and off what basis?
Home office
Only approved if:
- Benefit outweighs cost.
- If there is no non-animal alternative.
- Minimum number of possible animals used.
- Using animals with lowest sensitivity to pain possible. -Pain is minimised.
- Research premises have necessary facilities to care for animals.
What are the downsides to using mice?
Mice experiments can get expensive
they develop inutero (mother has to be culled - selectively slaughtered)
only produce average 8 pups per litter (low)
What do we study with zebrafish?
Gene knockdown:
MOs block gene specific translation or splicing
However they can have off-target genetic effects, so a mutant is gold standard
Mutants: using forward ad reverse genetics
How can targeted mutations be achieved?
Using CRISPR system where you have:
CRISPR - Clustered regularly interspaced short palindromic repeats
CRISPR associated protein 9 (Cas9)
Bacterial adaptive immune system
Protospacer (Target sequence of guide RNA)
Protospacer Adjacent Motif (PAM)
Guide RNA binds to strand of genomic DNA, Cas9 endonuclease binds to non protospacer portion of gRNA + PAM of DNA, DSB 3bp upstream of PAM
- Mutations can be introduced through NHEJ and HDR
