Gene therapy Flashcards
Replication-incapable viruses are used as vectors. What does this mean?
They have been GMed so that essential viral components have been deleted so they cannot replicate.
Give 5 major components of a viral vector.
- MCS
- Selection gene
- Marker gene
- Promoters
- Enhancers to increase expression of selection gene
Viruses are commonly used as vectors in gene therapy. Define an adenovirus.
A non-integrating DNA virus: it does not integrate itself into the genome and instead uses the cellular machinery to make viral proteins.
Are adenoviruses replicated at cell division of the host?
No.
Adenoviruses do not elicit an immune response from the host. True or false?
False, they are highly immunogenic.
Viruses are commonly used as vectors in gene therapy. Define a retrovirus.
Viruses that use reverse transcriptase to incorporate their genetic material into the genome.
How does a retrovirus incorporate its DNA into the genome?
It uses reverse transcriptase to make cDNA from its RNA genome which is them incorporated into the host DNA.
The inserted DNA of retroviruses is replicated during cell division of the host. True or false?
True, retroviruses affect dividing cells.
What can insertional mutagenesis cause?
Cancer.
What is are lentiviruses?
A subset of retroviruses that can infect non-diving cells.
Give some examples of non-dividing cells.
Terminally differentiated cells like neurons etc.
What kind of illnesses do lentiviruses produce? Give an example.
Those with a delayed onset of symptoms after infection. HIV (human immunodeficiency virus) is an example of a lentivirus.
HIV-based vectors can be used in gene therapy. How is this possible without causing AIDS?
The packaging and replication elements of the virus are separately transfected into the same cell - no complete HIV viruses are produced thus it cannot be replicated.
What is pseudotyping?
A process that allows us to specify the envelope proteins expressed by a virus.
What is the point of pseudotyping?
The pseudotyped virus appears, based on its cell-surface proteins, as something it is not, allowing it to transfect most cell types.
Can a pseudotyped virus replicate itself?
Yes but it will not pass on the new cell-surface proteins to its progeny.
What is the general procedure of viral transfection?
- Insert the desired DNA into a plasmid, grow in bacterial hosts
- Insert plasmid into viral vector
- Use virus to transfect desired recipient of target DNA
Define Gateway Cloning.
Allows DNA transfer to different cloning vectors whilst maintaining the reading frame. It uses specific recombination sequences called ‘gateway sites’.
Gateway cloning is based on proteins from which organisms?
Lambda and E. coli.
Define an entry clone.
A plasmid containing your gene of interest flanked by entry sites.
Define a destination vector.
A vector that will be inserted with the desired gene. The desired gene will replace a section of DNA in the destination vector.
What is the advantage to gateway cloning?
Entry clone libraries have been created. This means that every time you want to insert a particular gene into a vector you don’t have to go through the whole process, you can just use the entry clone to transform the vector.
In gateway cloning, gateway sites are sometimes referred to as what?
‘att’
In entry clones the gateway sites are referred to as…?
attL
In destination clones the gateway sites are referred to as…?
attR
