Functional Genome Flashcards

1
Q

What is the effect of knockdown on PDZRN3?

A

Inhibition of myotube formation and MHC expression

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What is whole genome sequencing for?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

How do you prove that a gene variant causes dysfunction?

A

→ Knock out gene
→ over expression of gene

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

How can primary cells be kept?

A

They can be immortalised

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

How do you filter out mutations in WES?

A

→ Remove the synonymous mutations
→ Filter out variants in databases that are common
→ Look at family members genotypes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What are the assumptions in WES?

A

The variant is within the coding region

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

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

A

You need to prove that the gene causes disease

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Why are biopsies of affected tissues not always available?

A

→ Gene isn’t expressed in the blood
→ tissue is not accessible

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What does in vitro mean?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What is an advantage of in vitro?

A

→ Cheap
→ Rapid
→ Reproducible model

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Describe how RNAi gene knockdown works?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What is the advantage of using IPSCs for cell culture?

A

Uses patient’s own cells for study after differentiation

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

What is involved in Duchenne?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

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

A

→ 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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Which animals are most used for in vivio studies?

A
  1. mouse
  2. rats
  3. zebra fish
  4. chicks
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Why is the mouse most used for in vivo studies?

A

→ 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

17
Q

What type of cells are used in mutant mice?

A

Embryonic stem cells four days after fertilisation

18
Q

Why are ES cells used for mutant mice making?

A

→ 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

19
Q

What are the two ways mutant mice are made?

A

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

20
Q

How does homologous recombination for mutant mice work?

A

→ 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.

21
Q

How is gene trapping used for mutant mice?

A

→ 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

22
Q

What do both gene trapping and homologous recombination involve?

A

→ 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.

23
Q

How does the FLEx system work?

A

→ 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.

24
Q

What is MO?

A

→ 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

25
Q

What are two ways MOs are used for gene knockdown?

A

→Inhibit splicing
→block gene specific translation

26
Q

What are the two molecules involved in CRISPR?

A

→Enzyme called CAs9
→gRNA

27
Q

What is guide RNA?

A

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

28
Q

What is the role of guide RNA?

A

→ ‘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

29
Q

What happens after guide RNA finds target DNA sequence?

A

→ 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