The Functional Genome Flashcards
What is bioinformatics?
Bioinformatics: Candidate gene filtering using whole-exome sequencing (WES)
WES data is subjected to a prioritisation filtering protocol to find the causal variant
15-20,000 coding SNPs reduced to one or several candidate genes.
Checked for co-segregation (family members) and validated by Sanger sequencing.
There are a lot of assumptions we have to make for this process to find the causal gene including ignoring structural variants, assuming causal variant is coding, assuming causal variant has complete penetrance and that causal variant has complete detectance
What are the ways you can provide functional evidence in order to prove causal gene?
Detection of protein in patient samples
Tissue/cell expression
How does change affect protein behaviour
How does the change affect cell or development of tissues
Identification of molecular mechanism of action
Development of in vitro and in vivo models for dysfunction of GOI
Knockdown or overexpression effects
How do we analyse a genes role through protein involvement? (Example is included)
The first thing to find the reason for this gene association is to see whether its involved in a protein synthesising process
Blood or tissue biopsies
Example: Identification of MYL1 in patients with congenital muscular dystrophy by WES
P1; homozygous splice acceptor variant in intron 4, predicted to cause in-frame skip of exon 5 (this gene was present in each parent)
P2; homozygous missense mutation in exon 5, substitution of a highly conserved amino acid
Gene expressed in fast twitch muscle, patients have reduced fast muscle fibres
GOI (gene of interest) will not always be expressed in the blood and might not be in an accessible affected tissue!
Describe the in vitro cell culture technique to find the causal gene.
Removal of cells from an animal and subsequent growth in favourable conditions
Primary cells have finite division but can immortalised, using certain chemical treatments, to provide continuous source
Provides a cheap rapid and reproducible model for studying the normal physiology and biochemistry of cells
A good alternative to using animal models, reducing the numbers of animals being used in research, less restrictions
Many tissue specific cell lines commercially available
Describe the gene knockdown technique for finding a causal gene.
RNAi mediated gene silencing
Short hairpin RNA (ShRNA):
ShRNA are sequences that are encoded in a DNA vector and these can be introduced into the cell via transfection or firewall transduction
And because the ShRNA expression can be incorporated into this viral vector system, they can be integrated into a host genome for the creation of a stable cell line
And so ShRNAs are modelled on precursors of microRNAs which are involved in gene silencing and we can modify this in the lab to include a complementary sequence to the gene of interest
The viral vector that includes the gene of interest sequences is then transcribed under the control of a promoter, ubiquitously expressing promoters such as RNA Pol III
As a result within the cell, the ShRNAs are produced as a single strand and transcribed in the nucleus
Then they form a stem-loop structure consisting of 19 to 29 base pairs of double stranded region and then they have a region of single stranded RNA
Once transcribed it exits the nucleus via a protein called Exportin 5 then cleaved at the loop by a nuclease called Dicer (in the cytoplasm)
The cleaved segments bind to RNA induced silencing complex (RISC) and this directs cleavage and degradation of complementary mRNA
Short interfering RNA (siRNA); similar to shRNA, chemically synthesised, not vector based
Describe the protein localisation technique for finding a causal gene.
We want to know where the protein is as a change in the sequence might not change the protein but change where the protein is localised or how it behaves?
We can use antibody staining for this where a the protein of interest is made to produce an immune response with the body making antibodies, those can be detected using another fluorescent labelled antibody against that specific antibody
You can transfect cells with sequences with a plasmid, a vector, which has your gene of interest in as well as a small tag on it (which you can then detect using an antibody
Or you can tag your protein with GFP by putting you GFP tagged protein into some cells and you can monitor where the protein goes by shining UV light on it
You can do this with the wild type protein and the variant protein and compare
What are IPSCs and how can you culture them?
Cell culture: Induced Pluripotent Stem Cells (IPSCs)
Here you can take a skin biopsy from a patient and the fibroblasts from this can be grown in a dish and then, using certain chemicals and false gene expression, they can be caused to dedifferentiate and become pluripotent stem cells
They can then be reprogrammed to differentiate into a cell type of your choice based on a cocktail of genes known to be required for the differentiation of a cell
Now you can study whatever cell type you like from a specific patient
Why are cell cultures not enough for finding causal genes?
Cells behave differently in a petri dish/flask to how they behave in a whole organism. 2D environment.
Does not simulate the actual conditions inside an organism. Signals from other tissues.
No information about gene expression and function, with regards to developmental phenotypes
How do we make a mutant mouse?
Targeting vector is constructed which contains a DNA cassette and introduced into the nucleus of pluripotent ES cells
The DNA cassette is flanked both upstream and downstream with DNA sequences that match perfectly with the site that you are trying to integrate in within the genome
These homology arms recognise sequences in the endogenous gene or regional investigator wants to replace
In the case of a knockout allele, typically the engineered cassette will replace a coding exon with a selectable marker such as neomycin cassette resistant gene
This enables the researcher to select and enrich for positive embryonic stem cells containing the cassette you’ve put in
So the targeting vector is then introduced into the nucleus of a pluripotent stem cell where the homology arms match up with the sequence in the endogenous gene
In a process similar to what occurs during meiosis, the aligned regions cross over and incorporate the engineered cassette into the endogenous gene locus and remove the endogenous sequence
These are put in the germline in place of the reference sequence where the embryonic cells are integrated through screening using the selectable marker e.g. the neomycin resistance
The positive embryonic clones are then grown to blastocysts and then implanted into a pseudopregnant recipient mouse
How do we create tissue specific KO mice?
The mouse with the targeted floxed allele carrying the two loxP flanking a targeted region can be crossed with the transgenic mouse with a Cre recombinase under control of a tissue specific promoter (in this case a heart promoter)
The offspring produced will carry both the floxed targeted allele for the gene of interest as well as the recombinase
In tissues where the recombinase is expressed, the region flanked by the loxP will be excised, generating a tissue restricted, tissue specific, conditional knockout mouse
In tissues where the Cre promoter is inactive and the floxed gene is still intact and there is that it won’t get cut out and will remain functionally intact
Mice experiments can get expensive, they develop in-utero (mother has to be culled), only produce on average 8 pups per litter (low n)
(summary version)
Why do we use zebrafish in biological experimentation?
Zebrafish: a vertebrate model for human genetic disease
The eggs are developed ex-utero so we can follow their development
The embryos develop in 24 hours and in 5 days are independent feeders
Females can produce up to 200 eggs at a time
Cheap
Easy to manipulate
How do we perform gene knockdown in zebrafish?
We can use antisense morpholino technology
Morpholinos are synthetic oligonucleotides, composed of chains of about 25 nucleotides
Rather than having a ribose backbone they have a morpholine backbone which makes them incredibly stable and resistant to new cases but still allows them to undergo base pairing and do not carry a negative charge (so do not stick to random places in a cell)
Morpholinos can work via two ways:
MOs block gene specific translation or splicing.
What are the two types of morpholinos and how do they work?
a) Translation Blocking Mos
They can block gene-specific translation by targeting the initiation site on the mRNA so blocks the ribosomal machinery from translating the messenger RNA into protein
b) Splice Inhibiting Mos
Morpholinos can be designed against splicing sites and by doing that, generally they are designed against the splice donor site and that then blocks the spliceosome from doing its job and we get an intronic inclusion rather than the intron being spliced out
If you choose it carefully you pick an intron with a stop codon so you can get a truncated protein
Exon1 = 326bp
Intron1 = 9013bp
Exon2 = 134bp
Intron2 = 740bp
Exon3 = 363bp
What is the CRISPR/Cas9 system and how does it work?
-Clustered regularly interspaced short palindromic repeats (CRISPR)
It works by having two parts:
A guide RNA- the guide RNA has a sequence which is complementary to your target DNA
The target sequence, or protospacer, must have a pan sequence (any nucleotide with GG), and this dictates a target for the guide RNA
The guide RNA will have this sequence which will bind to your genomic DNA
It also has a sequence which forms a binding site and architecture for a Cas9 endonuclease to bind to
So what happens is that the guide RNA and the Cas9 endonuclease bind together to form this nuclear protein complex which can move along the genome to find complementary sequences
Once its found its complementary sequence, the Cas9 will then cause a double-stranded break within the genomic DNA, three base pairs upstream from the pan sequence
Once there is a double-stranded break, there is a process of repair which kicks in
The natural cells process of repair which is the quickest and most efficient way called non-homologous end joining which essentially sticks them back together
This is an imperfect repair mechanism so it often causes deletions and insertions of nucleotides and that’s how we get our mutations
We can use a DNA template to cause a specific mutation and push towards homology directed repair which uses that template to fix the damaged DNA
