Exploitation of viruses

Question 1

What is the use of reverse transcriptase in molecular biology?

Answer 1

helps to understand transcriptome of cell

add to the cDNA library by creating cDNA

Question 2

What is RT-PCR?

Answer 2

reverse transcriptase
specific negative sense primers
amplify coding sequence
clonse dsDNA into plasmid

Question 3

What are the viral features of plasmid expression vectors?

Answer 3

T7 promoter
CMV immediate early promoter
SV40 origin of DNA replication
SV40 polyadenylation site

Question 4

What are the advantages of virus expression vectors?

Answer 4

‘natural’ delivery system
efficient
can infect majority of cells

Question 5

What are the disadvantages of virus expression vectors?

Answer 5

cytotoxicity
biological hazards
regulations - COSHH
time consuming
technically demanding

Question 6

What are the 2 types of virus vectors?

Answer 6

Retroviruses (lentiviruses)

Baculoviruses

Question 7

What part of the retrovirus life cycle is needed for a successful vector?

Answer 7

Just use early stages from virus binding to nuclear transport regulation to express YFG

Question 8

What kind of system is used for production of lentiviral vectors?

Answer 8

3-plasmid system

Question 9

How is the 3-plasmid system for retroviral (lentiviral) vectors created?

Answer 9

3 plasmids transfected into HEK293 cells
1 - Env
2 - genes
3 - Gag-Pol

Question 10

How can you generate a stable cell line?

Answer 10

select GFP positive cells
FACS

if trying to inhibit YFG expression - select with puromycin
siRNA inhibits expression of target gene

Question 11

What is gene therapy?

Answer 11

introduction of genes encoding toxins that are activated in target cells
augmentation of immune responses to tumours or virus-infected cells

Question 12

What is ex vivo gene therapy?

Answer 12

Remove cells from tumour/blood/bone marrow
culture cells in vivo
infect cells with retrovirus
re-inject into patient

EX VIVO - SELECTIVE ADVANTAGE

Question 13

What was the first gene therapy experiment?

Answer 13

1990
ADA
single gene defect in DNA metabolism = SCID
corrected cells = growth advantage
leukocytes from 4yr old bone marrow
infected ex vivo 
PEG-ADA

Question 14

What was the set-back for retroviral gene therapy?

Answer 14

X-SCID - absence of IL2Rgammac
mature T and B cells cannot develop
cutured in vivo - worked for T but not for B

HOWEVR - T-cell Acute lymphoblastic leukaemia developed = integration of retrovirus in LMO2 oncogene

LMO2 expressed all the time = tumour

Question 15

What are baculovirus vectors?

Answer 15

insect viruses
dsDNA genome appro. 134kb
express large amounts of late proteins - polyhedrin, p10

Question 16

How are baculoviruses constructed?

Answer 16

AcMNPV - restriction digest to make non-infectious - cut at Bsu36I sites

Plasmid containing part of baculovirus genome - replace polyhedrin with YFG - now under control of strong polyhedrin promoter

Homologous recombination - doesn’t need polyhedrin as in working tissue culture environment

Question 17

What are the advantages of baculovirus expression system compared to E.coli?

Answer 17

• large proteins expressed
• express multiple proteins
• low protease abundance = aided purification
• Eukaryotic expressions
• proteins expressed in soluble form

Question 18

What are the disadvantages of baculovirus expression system compared to E.coli?

Answer 18

• Time consuming - <1month
• Technically demanding
• Yields low