Functional Genome Flashcards
What is the effect of knockdown on PDZRN3?
Inhibition of myotube formation and MHC expression
What is whole genome sequencing for?
Used to capture the sequence of the coding region of the genome
How do you prove that a gene variant causes dysfunction?
→ Knock out gene
→ over expression of gene
How can primary cells be kept?
They can be immortalised
How do you filter out mutations in WES?
→ Remove the synonymous mutations
→ Filter out variants in databases that are common
→ Look at family members genotypes
What are the assumptions in WES?
The variant is within the coding region
Why is only isolating a gene not sufficient for a diagnosis?
You need to prove that the gene causes disease
Why are biopsies of affected tissues not always available?
→ Gene isn’t expressed in the blood
→ tissue is not accessible
What does in vitro mean?
Removal of cells from an animal and subsequent growth in favorable conditions
What is an advantage of in vitro?
→ Cheap
→ Rapid
→ Reproducible model
Describe how RNAi gene knockdown works?
1) a sequence is introduced that is complementary to the gene of interest
2) it is packaged within a plasmid and contains a promoter (RNA polymerase III)
3) they are transfected into the nucleus
4) the RNA is transcribed and exits the nucleus through the pores with exportin 5 protein
5) It then is cleaved by Dicer
6) it leaves the complementary DNA
7) the DNA binds to the RISC complex
8) and this seeks the gene of interest RNA and silences it by cutting it
What is the advantage of using IPSCs for cell culture?
Uses patient’s own cells for study after differentiation
What is involved in Duchenne?
One or more exons (parts of the gene) are missing, causing errors in the instructions for making dystrophin
Why is cell culture not enough to study the functionality of the genome?
→ Cells behave differently in a petri dish/flask to how they behave in a whole organism.
→ 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
Which animals are most used for in vivio studies?
- mouse
- rats
- zebra fish
- chicks
Why is the mouse most used for in vivo studies?
→ Accelerated lifespan -1yr/30 human years
→ Small, reproduce quickly, relatively easy to handle and transport
→ More ethical than using larger animals/non-human primate/humans
→ Mammals: genetically similar to humans
→ Lots of mouse strains and models already exist
What type of cells are used in mutant mice?
Embryonic stem cells four days after fertilisation
Why are ES cells used for mutant mice making?
→ They are able to differentiate into nearly any type of adult cell, which means that if a gene is knocked out in an ES cell, the effects can be observed in any tissue in an adult mouse
→ ES cells grown in the lab can be used to make knockout mice as long as 10 years after they were harvested
What are the two ways mutant mice are made?
→ Gene targeting/ homologous recombination
→ Gene trapping
→ both are carried out in vitro
How does homologous recombination for mutant mice work?
→ Introducing an artificial piece of DNA that shares identical, or sequence to the gene
→ Homologous sequence flanks the existing gene’s DNA sequence both upstream and downstream of the gene’s location on the chromosome
→ Cell’s own nuclear machinery automatically recognizes the identical stretches of sequence and swaps out the existing gene or portion of a gene with the artificial piece of DNA
→ The artificial DNA is inactive, bearing only a genetic tag, or “reporter gene,” designed for use in tracking, the swap eliminates, or “knocks out,” the function of the existing gene.
How is gene trapping used for mutant mice?
→ Random process
→ A piece of artificial DNA containing a reporter gene is designed to insert randomly into any gene
→ The inserted piece of artificial DNA prevents the cell’s RNA “splicing” machinery from working properly,
→ Prevents the existing gene from producing its designated protein
→Track the activity of the artificial reporter gene to figure out the existing gene’s normal pattern of activity
What do both gene trapping and homologous recombination involve?
→ Consists of a modified viral vector or a linear fragment of bacterial DNA
→ After the artificial DNA is inserted, the genetically altered ES cells are grown in a lab dish for several days
→ Injected into early-stage mouse embryos.
→The embryos are implanted into the uterus of a female mouse and allowed to develop into mouse pups.
How does the FLEx system work?
→ If LoxP sites flank a gene in the same direction, recombination will result in the gene being excised- irreversible.
→ If LoxP sites flank a gene in opposing orientations, recombination results in gene inversion.
What is MO?
→ PMOs have the same nucleic acid bases found in RNA, they are bound to six-sided morpholine rings instead of five-sided ribose rings.
→Very stable as they do not carry negative charge
