Manipulation of Gene Expression Flashcards
Why would we want to manipulate gene expression?
To investigate the biological function of a gene
To create cell/animal models of disease
For gene therapy
How can we study gene down-regulation?
‘Knock down’ gene expression
Look for a change in phenotype
Loss of function (LOF)
What is the ‘gold standard’ for investigation gene function?
Gene down-regulation
How can we study gene up-regulation?
‘Overexpression’ of a gene
Look for a change in phenotype
How would you achieve stable overexpression of a gene?
By the insertion of an extra copy of the gene of interest into the genome
What is a common way of delivery of extra copies of genes for stable overexpression?
Viral delivery, e.g. retroviral and lentiviral
How can we get constitutive expression of a inserted gene?
Put it under the control of a constitutive promoter
What are the disadvantages of gene insertion for stable overexpression?
The insertion of the gene may disrupt another gene
Is the effect caused by overexpression or by integration
Higher biosafety level than transient methods
What is a very widely used approach for gene knockdown?
RNAi
What does RNAi achieve?
Post-transcriptional gene silencing
Degradation of mRNA
What does RNAi utilise?
A natural anti-viral mechanisms found within most cells
What are the pros of using RNAi for gene knockdown?
Relatively cheap
Can be used both for transient and stable knockdown
Commercial pre-designed siRNA readily available
Targeting of specific splice variants possible
What are the cons of using RNAi for gene knockdown?
Off target effects are increasingly being reported
What are transcription like-activator effectors (TALEs)?
Synthetic transcription factor binding domains
What is the role of TALEs?
They are programmed to recognise specific DNA motifs
What are the pros of using TALEs?
Two TALEs required for FokI dimerisation
Very specific… fewer off-target effects than TALEs
What are the cons of using TALEs?
TALEs need to be designed which is expensive and time consuming
They don’t always work
What does CRISPR stand for?
Clustered regularly interspaced short palindromic repeats
What is the CRISPR system?
A system found in bacteria to confer resistance to viral infection
What is the protein associated with the CRISPR system and what does it do?
CRISPR-associated protein 9 (Cas9)
It is a nuclease that acts as a monomer to cause double strand breaks
Does the CRISPR system temporarily or permanently modify DNA?
It is a permanent modification
What is CRISPRi?
CRISPR interference
Also, commonly used to refer to CRISPR inhibition
It is reversible
What is CRISPRa?
Activation of transcription
It is reversible
Is RNAi an old or new technique?
Older technique but still widely used
What are the pros of using RNAi?
Time efficient (shortest time to phenotype) Highest level of off-target effects Can target specific splice variants
What are the pros and cons of TALEN?
More specific but is more difficult
The CRISPR/Cas9 system has become very popular, why?
Because it is cheaper and easier than TALEN
What are some of the considerations when trying to manipulate gene expression?
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
