The functional genome Flashcards
Describe the process of candidate gene filtering for whole exome sequencing?
- From all of the single nucleotide variants within the exome that are identified you first remove the synonymous variants (ones that don’t change amino acid sequence)
- You then remove previously identified variants
- Finally you remove the variants that don’t fit into dominant/recessive style of inheritance (mendelian inheritance)
What assumptions have to be made during candidate gene filtering for whole exome sequencing?
- The first assumption made is that genetic variant is within the coding region of the genome
- Another assumption that is made is that the genetic variant is non-synonymous - it alters the amino acid encoded within the protein sequence
- A third assumption made is that the genetic variant being identified, as well as the ones being removed, have complete penetrance - By assuming this you rule out the variant being a previously identified variant because if it was any other disease-causing variant they would exhibit the symptoms of that disease instead
- Final assumption that is made is that the genetic variant has complete detectance
Once you have filtered it down to one candidate gene is it enough to say that the variant within that gene is disease-causing?
- No it isn’t because there may have been variants in the non-coding region that are associated/cause the disease you’re looking at
- Also, you haven’t definitvely proved that this variant within the candidate gene causes the diease by identifying the candidate gene you need to carry out experiments to validate this
What are some techniques that can be used to calidtae whether or not a variant within a candidiate gene is disease-causing?
- Development of in vitro and in vivo models
- Seeing whether the genetic variant affects development of protein by taking blood/tissue biopsies
- Gene knockdown
- Seeing whether the genetic variant affects protein behaviour - looking at protein localisation
What is the problem with taking blood/tissue biopsies to see whether a variant within a candidate gene causes the protein produced by that candidate gene to not be expressed?
- Problem is that the candidate gene may not always be expressed in the blood or may not be accessible within the affected tissue
What is cell culture?
The removal of cells from an animal and then subsequently growing those cells in an artificial environment with favourable conditions
During cell culture why do the cells initially taken from an animal, primary cells, usually immortalised?
- They can only divide a finite no. of times so you immortalise them so you can make them last longer by delaying the amount of times that they divide
What is gene knockdown?
An experimental technique by which the expression of one or more of an organism’s genes is silenced (reduced).
What is one of the methods that can be used for gene knockdown?
RNAi (RNA interference) mediated gene knockdown.
Briefly explain the process of RNAi mediated gene kncokdown?
- First you deliver a shRNA (short hairpin RNA) sequence, which is complementary to the gene of interest sequence, into the nucleus which is then transcribed by RNA polymerase III
- Once the shRNA has been produced it is then delivered from the nucleus into the cytoplasm by Exportin-5.
- Once the shRNA is in the cytoplasm it’s cleaved into small segments by a nuclease called Dicer
- The shRNA segments are then bound to by the RNA-induced silencing complex (RISC)
- As the shRNA segments are complementary to the target mRNA to be silenced, the RISC uses the shRNA as a template for locating the target mRNA.
- After the RISC locates to the target mRNA it cleaves the mRNA preventing it from being used in translation - this means that the gene of interest cannot be expressed as a protein product can’t be produced.
During RNAi mediated gene knockdown what are the two ways in which shRNA can be integrated into the host cell?
- A lentivirus containing the shRNA sequence can deliver it to the nucleus where it will then be reverse transcribed to form shDNA. The shDNA will then be integrated into the DNA of the mammalian cell which then expresses the shDNA sequence via RNA polymerase III to produce shRNA
- sHDNA sequence can be present within a plasmid which can then be integrated into the nucleus and expressed by RNA polymerase III to produce shRNA
What other type of RNA can be used instead of shRNA and how are these two types of RNA different?
- Short interfering RNA (siRNA) can also be used instead of shRNA.
- siRNA is similar to shRNA but it is chemically synthesised rather than being vector based.
Explain how you can check whether a genetic variant within a candidate gene affects protein localisation?
- To do this you transfect some cells with a DNA plasmid containing the normal gene of interest tagged with GFP (green fluorescent protein)
- You also transfect other cells of the same cell type with a DNA plasmid containing the gene of interest that contains the genetic variant and tag that with GFP also.
- Once the cells have produced the protein products the cells are viewed under luminance to see where the protein has localised within the cell compared to the dysfunctional protein, protein coded for by gene of interest with genetic variant.
How can cell culture be used to produce pluripotent stem cells?
- You take a skin biopsy from a patient with a particular genetic variant
- You then isolate fibroblasts from the skin biopsy and then culture and amplify them.
- You then use specific proteins to reprogramme and dedifferentiate the fibroblasts.
- This produces pluripotent stem cells
- The pluripotent stem cells can then be redifferentiated to produce a particular cell type
When trying to validate whether a particular varinat found with a candidate gene causes disease why aren’t cell culture techniques enough to validate this?
- Because cells behave differently in a petri dish/flask to how they behave in a whole organism - there are different signals coming from the 3D environment inside an organism compared to the artificial 2D environment that is the petri dish.
- Looking at cells in a dish gives no information about gene expression and function.
Wwhat scientific reason is there for a scientist seeking to alleviate the pain of animal during animal testing?
Animals that suffer during testing may release stress hormones which could affect the experiment
What are some of the main animals used during animal testing?
- Mice
- Rats
- Fish
- Chickens
What is the Animals Scientific Procedures Act (ASPA) 1986 and what does it state?
- An act of the UK Parliament that permits the use of animals in scientific procedures
- It regulates the use of protected animals in any experimental or other scientific procedure which may cause pain, suffering, distress or lasting harm to the animal.
What are some of the conditions that need to be met in order for an institution to be granted permission to perfrom animal testing?
- Benefit of experiment has to outweigh the cost.
- There are no non-animal alternatives that can be used
- Research premises have necessary facilities to care for animals.
- Use animals with lowest sensitivity to pain possible
Why are mice a good animal to use to model human disease?
- They have an accelerated lifespan (1 year equivalent to 30 human years)
- They are mammals - genetically similar to humans
- They are small, reproduce quickly and are easy to handle/transport
- Lots of mouse strains/models already exist
- More ethical to use compared to larger animals/primates
How can Cre-lox recombination be used to produce a mutatnt mouse?
- First you extract embryonic stem cells from a mouse and select the cells that are expressing the desired gene
- Then you flank the desired gene with identical LoxP sites that are in the same orientation.
- You then inject the modified embryonic stem cells into the inner cell mass of the blastocyst
- Implant blastocyst into psuedopregnant mouse
- Mother gives birth to mouse with LoxP sites flanking desired gene in every cell
- You then breed LoxP mouse with a mouse expressing Cre recombinase in a particular cell type.
- In the mouse produced from this breeding, in cells that express Cre recombinase the cre recombinase enzyme will recognise the LoxP sites flanking the desired gene and recombination wil occur
- This recombination event will result in the gene being excised, removed, thus froming the mutant mouse.
Why are zebrafish used in animal testing?
- They develop really quickly
- They develop ex-utero - This means that you can visualise the development of their offspring by looking down a microscope
- They are transparent - This means you can see the cells moving throughout the whole organism
- They are relatively easy to genetically manipulate
What is a morpholino?
A morpholino is a nucleic acid analog, has structure similar to nucleic acids, that is used to modify gene expression.
What is the effect of having a morpholine backbone instead of a ribose one for the zebrafish?
Makes the DNA of the zebrafish incredibly stable
How can morpholinos affect gene expression?
- They can block the start site of a gene which prevents transcription
- They can prevent snRNPs from binding to splice sites on introns so introns get included into mature mRNA
- They can prevent the formation of the lariat-like structure formed during splicing
How can you make a mutant zebrafish?
- Bathe male zebrafish in a potent mutagen called N-ethyl-N-nitrosourea (ENU).
- ENU targets spermatogonial stem cells, precursors to sperm cells, and causes point mutations at a rate of 1 per 700 gametes.
- You then cross the mutant male zebrafish with the females and screen their offspring to see wha mutations have been inherited
What are the 2 ways in which the offspring of mutant zebrafish can be screened to see whether they’ve inherted a particular mutation?
- Phenotype based screen (forward genetics) - looks for visible changes in the phenotype
- Genotype based screen (reverse genetics) - Involves whole exome sequencing Zebrafish DNA to find mutations and then looking for changes in the phenotype based on those mutations
What is the CRISPR/Cas9 system?
A system adapted from the bacterial immune system that is able to edit the genome at specific points by producing double stranded breaks in the DNA
What are the different components of the CRISPR-Cas9 system?
- Cas9 nuclease
- A single guide RNA (sgRNA), which is made up of a CRISPR-RNA (crRNA) - trans-activating crRNA (tracrRNA) duplex
How is the CRISPR/Cas9 system able to edit the genome?
- sgRNA forms a complex with Cas9 nuclease.
- The CRISPR-RNA (crRNA) portion of the sgRNA guides the Cas9 nuclease to the target sequence (protospacer) via recognition of the protospacer adjacent motif (PAM) which is a small section of DNA that is next to the target DNA sequence.
- The tracrRNA then ensures that the Cas9 nuclease binds to the target sequence.
- Once bound to the target sequence the Cas9 nuclease produces a double strand break in the DNA
- The double strand break is then repaired either by non-homologous end joining (NHEJ) or homology-directed repair (HDR)
