Manipulation of Gene Expression

Question 1

Why would we want to manipulate gene expression?

Answer 1

To investigate the biological function of a gene
To create cell/animal models of disease
For gene therapy

Question 2

How can we study gene down-regulation?

Answer 2

‘Knock down’ gene expression
Look for a change in phenotype
Loss of function (LOF)

Question 3

What is the ‘gold standard’ for investigation gene function?

Answer 3

Gene down-regulation

Question 4

How can we study gene up-regulation?

Answer 4

‘Overexpression’ of a gene

Look for a change in phenotype

Question 5

How would you achieve stable overexpression of a gene?

Answer 5

By the insertion of an extra copy of the gene of interest into the genome

Question 6

What is a common way of delivery of extra copies of genes for stable overexpression?

Answer 6

Viral delivery, e.g. retroviral and lentiviral

Question 7

How can we get constitutive expression of a inserted gene?

Answer 7

Put it under the control of a constitutive promoter

Question 8

What are the disadvantages of gene insertion for stable overexpression?

Answer 8

The insertion of the gene may disrupt another gene
Is the effect caused by overexpression or by integration
Higher biosafety level than transient methods

Question 9

What is a very widely used approach for gene knockdown?

Answer 9

RNAi

Question 10

What does RNAi achieve?

Answer 10

Post-transcriptional gene silencing

Degradation of mRNA

Question 11

What does RNAi utilise?

Answer 11

A natural anti-viral mechanisms found within most cells

Question 12

What are the pros of using RNAi for gene knockdown?

Answer 12

Relatively cheap
Can be used both for transient and stable knockdown
Commercial pre-designed siRNA readily available
Targeting of specific splice variants possible

Question 13

What are the cons of using RNAi for gene knockdown?

Answer 13

Off target effects are increasingly being reported

Question 14

What are transcription like-activator effectors (TALEs)?

Answer 14

Synthetic transcription factor binding domains

Question 15

What is the role of TALEs?

Answer 15

They are programmed to recognise specific DNA motifs

Question 16

What are the pros of using TALEs?

Answer 16

Two TALEs required for FokI dimerisation

Very specific… fewer off-target effects than TALEs

Question 17

What are the cons of using TALEs?

Answer 17

TALEs need to be designed which is expensive and time consuming
They don’t always work

Question 18

What does CRISPR stand for?

Answer 18

Clustered regularly interspaced short palindromic repeats

Question 19

What is the CRISPR system?

Answer 19

A system found in bacteria to confer resistance to viral infection

Question 20

What is the protein associated with the CRISPR system and what does it do?

Answer 20

CRISPR-associated protein 9 (Cas9)

It is a nuclease that acts as a monomer to cause double strand breaks

Question 21

Does the CRISPR system temporarily or permanently modify DNA?

Answer 21

It is a permanent modification

Question 22

What is CRISPRi?

Answer 22

CRISPR interference
Also, commonly used to refer to CRISPR inhibition
It is reversible

Question 23

What is CRISPRa?

Answer 23

Activation of transcription

It is reversible

Question 24

Is RNAi an old or new technique?

Answer 24

Older technique but still widely used

Question 25

What are the pros of using RNAi?

Answer 25

Time efficient (shortest time to phenotype)
Highest level of off-target effects
Can target specific splice variants

Question 26

What are the pros and cons of TALEN?

Answer 26

More specific but is more difficult

Question 27

The CRISPR/Cas9 system has become very popular, why?

Answer 27

Because it is cheaper and easier than TALEN

Question 28

What are some of the considerations when trying to manipulate gene expression?

Answer 28

Short or long term knockdown to generate effect
Cell type: where is the gene expressed, use a primary or immortalised (cancer) cell line
Cell or animal model