W4 - The Functional Genome Flashcards
What is next generation sequencing?
WES and WGS
Rapid modern methods for high throughput DNA sequencing.
Whole Exome Sequencing (WES) used to capture the sequence of the coding region of the genome.
Whole Genome Sequencing (WGS) captures the whole thing!…….not always necessary
Aim to identify potential disease causing genetic variants: personalised medicine
In Bioinformatics, how are gene candidates filtered using WES?
WES data is subjected to a prioritisation filtering protocol
15-20,000 coding SNPs reduced to one or several candidate genes.
Checked for co-segregation (family members) and validated by Sanger sequencing.
Is the protein affected?
Blood or tissue biopsies
Ravenscroft et al 2018 Human Molecular Genetics
GOI will not always be expressed in the blood and might not be in an accessible affected
tissue!
Example: Identification of MYL1 in patients with congenital muscular dystrophy by WES
Gene expressed in fast twitch muscle, patients have reduced fast muscle fibres
P1: homozygous splice a cceptor variant predicted to cause in-frame skip of exon 5
P2: missense mutation in Exon 5, substitution of a highly conserved a.a
GOI will not always be expressed in the blood and might not be in an accessible affected
tissue!
What are some in vitro culture techniques?
Removal of cells from an animal and subsequent
growth in favourable conditions.
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
continuous source
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
What is cell culture: gene knockdown?
Based on endogenous microRNA gene silencing
Modified to include GOI complementary sequence
Packaged in a DNA plasmid , expression
controlled by a RNA polymerase III promoter
50-70nt. Exits nucleus, cleaved by a
nuclease called Dicer (cytoplasm)
Cleaved segments bind to RNA induced
silencing complex (RISC) and direct
cleavage and degradation of
complementary mRNA
Short interfering RNA (SiRNA): similar to ShRNA,
chemically synthesised, Not vector based
Where is GOI’s encoded protein localised?
Antibody staining
-Protein of interest
-downstream target
Transfect cells with GFP
tagged GOI (CMV promoter)
Transfect cells with GFP
tagged mutated GOI
(CMV promoter)
How are Induced Pluripotent Stem Cells cultured?
Take a skin biopsy from a patient. This isolated adult fibroblast can be grown in a dish. Using certain chemicals and gene expression, the fibroblast can be used to dedifferentiate pluripotent stem cells. They can then be programmed to be redifferentiated into a cell type of your choice. You can now study the behaviour of those cells.
Why is cell culture not enough?
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.
Why is cell culture not enough?
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.
What is the position of animals in research?
Ideally we wouldn’t……..
- 90% research uses non animal methods
- cells behave differently in vitro to in vivo
- Most of the medicines we have today come from animal research.
- contributed to 70% of the Nobel prizes
- animal ill-health
- Research scientist seek to alleviate pain and suffering ……rigorous HO monitoring
Which animals are used in research?
1%: Cats,dogs, horses and primates (specially protected)
Procedures:
2.11million - Mice
0.6m - Fish
0.32m - Rats
0.28m - Birds
4,481 - Dogs
3, 207 - Monkeys
159 - Cats
10,424 - Horses
Why, as a mammalian model for human genetic diseases is a mouse important?
Classic go too is a mouse.
Accelerated lifespan (1yr/30 human years)
Mice have been used in BM research for 100+yrssmall, reproduce quickly, relatively easy to handle and transport
Mammals: genetically similar to humans
Lots of mouse strains and models already exist
More ethical than using larger animals/non-human primate/humans
Modern genomic engineering have allowed for precise mutation to recreate a disease.
What is making a mutant mouse?
Method 1)
DNA exists in Embryonic Stem Cells (ES). They are selected for cells expressing desired gene. They inject transformed ES cells into inner cell mass, which forms the Blastocyst. You take the inner cell mass and implant it in uterus of the mouse.
Method 2)
We take a pronuclei in a fertilised off with the desired gene (with vector) and implant in uterus of a mouse.
The offspring are tested for the presence of the gene. They mate heterozygous offspring to produce homozygous transgenic mice.
- Targeting vector constructed (containing a DNA cassette) and introduced into the nucleus of plutipotent ES cells
- HR integrates in the cassette. ES selected, based on ABr
- +ve ES cells grown to blastocystes and implanted into a pseudopregnant recipient mice
What are tissue specific KO mice?
- Mouse carrying a floxed allele ( meaning, inactivatable in specific cell types in a certain tissue) and a different mouse carrying Are recombinase under a heart specific promoter exists. These two genes go in the same direction. If they have an offspring together, they would have both.
Mice experiments can get expensive, they develop inutero (mother has to be culled), only produce on average 8 pups per litter (low n)
How are zebrafish used?
A vertebrate model for human genetic disease.
They exist in tropical freshwater- Ganges, India
They have lots of transparent eggs.
They also progress rapidly - from an embryo they nearly resemble an adult fish after 72 hours!
Embryos contain fewer cells than other vertebrates.
Their genome is easier to manipulate and cheaper.
When it comes to zebrafish gene knockdown can be done through Translation Blocking MOs (Morpholino oligonucleotides) . This is where the MOs block gene specific translation or splicing occurs. We can also splice inhibiting MOs. This means they can have off-target genetic affects - a mutant is the gold standard.
When you look at the stains for muscle fibre for the control vs dmd-MO, you would be able to see big holes where the fibres has degraded and apoptosis had occurred.
