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.
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.
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
