Exploitation of Viruses Flashcards
How do we find out which mRNA molecules are active?
Oligo-dT column (agarose beads which can be purified and conjugated and bound to a string of Ts). mRNAs bind - shows all mRNAs present in cell
How do we find one specific mRNA?
RT-PCR
PCR amplification with specific + and - sense primers which can be cloned into a plasmid
Which part of the retrovirus life-cycle is needed for a successful vector?
Nuclear transport and integration
How do you make a retrovirus vector?
The vector plasmid, contains a cDNA copy of the whole genome, remove anything unnecessary and add YFG and marker for neomycin
What do we replace gag, pol and env with?
CMV promoter
What is the 3-plasmid system for production pf retroviral vector?
Transfect HEK293 cells with 3 plasmids, do not have to make the stable cell line so is easier.
What is the broader definition of gene therapy?
introduction of genes encoding toxins that are activated in target cells and augmentation of immune responses to tumours or virus-infected cells.
What was the first gene therapy experiment?
Adenosine deaminase deficiency, SCID manifested in bone marrow (easy to manipulate in vitro)
what causes SKID?
Absence of IL2 receptor y subunit. Mature and T and b cells cannot develop
What was the set back for treating SKID?
They all developed T-cell acute lymphoblastic leukaemia
What % of the baculovirus protein is polyhedron?
50%, remove and express gene of interest would produce a lot of protein
Give 5 advantages of using baculovirus in comparison to E.coli
- Eukaryotic expression (post translational modification)
- Proteins are expressed in a soluble form
- Can express large proteins
- Can express multiple proteins
- Purification is aided by low abundance of proteases in insect cells.
Give 3 disavantages of using Baculovirus in comparison to E. coli?
- time consuming
- Technically demanding
- Low yields
