Functional Genome

Question 1

What is the effect of knockdown on PDZRN3?

Answer 1

→ Inhibition of myotube formation and MHC expression

Question 2

What is whole genome sequencing for?

Answer 2

→ Used to capture the sequence of the coding region of the genome

Question 3

How do you prove that a gene variant causes dysfunction?

Answer 3

→ Knock out gene

→ over expression of gene

Question 4

How can primary cells be kept?

Answer 4

→ they can be immortalised

Question 5

How do you filter out mutations in WES?

Answer 5

→ remove the synonymous mutations
→filter out variants in databases that are common

→look at family members genotypes

Question 6

What are the assumptions in WES?

Answer 6

→ The variant is within the coding region

Question 7

Why is only isolating a gene not sufficient for a diagnosis?

Answer 7

→ you need to prove that the gene causes disease

Question 8

Why are biopsies of affected tissues not always available?

Answer 8

→ Gene isnt expressed in the blood

→ tissue is not accessible

Question 9

What does in vitro mean?

Answer 9

→ Removal of cells from an animal and subsequent growth in favorable conditions

Question 10

What is an advantage of in vitro?

Answer 10

→ Cheap
→ rapid

→ reproducible model

Question 11

Describe how RNAi gene knockdown works?

Answer 11

→ a sequence is introduced that is complementary to the gene of interest
→ it is packaged within a plasmid and contains a promoter (RNA polymerase III)

→ they are transfected into the nucleus
→ the RNA is transcribed and exits the nucleus through the pores with exportin 5 protein
→ It then is cleaved by Dicer
→ it leaves the complementary DNA
→ the DNA binds to the RISC complex
→ and this seeks the gene of interest RNA and silences it by cutting it

Question 12

What is the advantage of using IPSCs for cell culture?

Answer 12

uses patient’s own cells for study after differentiation

Question 13

What is involved in Duchenne?

Answer 13

one or more exons (parts of the gene) are missing, causing errors in the instructions for making dystrophin

Question 14

Why is cell culture not enough to study the functionality of the genome?

Answer 14

→ 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

Question 15

Which animals are most used for in vivio studies?

Answer 15

1. mouse
2. rats
3. zebra fish
4. chicks

Question 16

Why is the mouse most used for in vivo studies?

Answer 16

→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

Question 17

What type of cells are used in mutant mice?

Answer 17

Embryonic stem cells four days after fertilisation

Question 18

Why are ES cells used for mutant mice making?

Answer 18

→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

Question 19

What are the two ways mutant mice are made?

Answer 19

→Gene targeting/ homologous recombination
→gen trapping
→ both are carried out in vitro

Question 20

How does homologous recombination for mutant mice work?

Answer 20

→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.

Question 21

How is gene trapping used for mutant mice?

Answer 21

→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

Question 22

What do both gene trapping and homologous recombination involve?

Answer 22

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.

Question 23

How does the FLEx system work?

Answer 23

→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.

Question 24

What is MO?

Answer 24

→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

Question 25

What are two ways MOs are used for gene knockdown?

Answer 25

→Inhibit splicing

→block gene specific translation

Question 26

What are the two molecules involved in CRISPR?

Answer 26

→Enzyme called CAs9

→gRNA

Question 27

What is guide RNA?

Answer 27

consists of a small piece of pre-designed RNA sequence (about 20 bases long) located within a longer RNA scaffold

Question 28

What is the role of guide RNA?

Answer 28

‘guides’ Cas9 to the right part of the
genome
→makes sure that the Cas9 enzyme cuts at the right point in the genome
→it has RNA bases that are complementary to those of the target DNA sequence

Question 29

What happens after guide RNA finds target DNA sequence?

Answer 29

Cas9 follows the guide RNA to the same location in the DNA sequence and makes a cut across both strands of the DNA.
→DNA repair machinery to introduce changes to one or more genes