Transcript Luciferase Based Plasmid Reporter System for the

Luciferase Based Plasmid Reporter System for the Detection and
Quantification of
Human Respiratory Syncytial Virus
Group 14: Oral Report 3, 2/12/2008
Melanie Aston, Michael Chi, Monica Deterding, Matt Huckabee
Background
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Human Respiratory Syncytial Virus is the most
common cause of bronchiolitis and pneumonia in
children under 1 year of age (CDC)
~800000 children die per year (~91 per hour) due to
RSV infection
There is no current vaccine available for RSV
Current method for quantification of infectious RSV:
Plaque Assay
VUSE Senior Design
Oral Report 3
Tuesday, February 12, 2008
The Problem
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Viral plaque assay is
 Labor intensive
 Costly
 Time consuming
 Partially subjective
Need high throughput, inexpensive system to
quantify infectious RSV
VUSE Senior Design
Oral Report 3
Tuesday, February 12, 2008
Our Solution
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Novel plasmid based reporter system
A luciferase plasmid and cell line that will luminesce
when infected with RSV
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Stable transfection of plasmid into cell
Optimization of system protocol
VUSE Senior Design
Oral Report 3
Tuesday, February 12, 2008
Comparison
Plaque Assay
Luciferase System
Detection Method
Staining/Counting
Luminescence
Objectivity
Partial
Yes
Time (work/total)
10 hours/7 days
2.5hrs/2 days
Materials Cost
$8
$1
Throughput
30 samples/experiment
240 samples/experiment
VUSE Senior Design
Oral Report 3
Tuesday, February 12, 2008
Comparison: Evaluation Chart
Plaque Assay
Criteria
Weight (1-5)
Value
Luciferase System
Product
Value
Product
Quick
5
2
10
4
20
Low Cost
3
2
10
4
20
Objective
3
4
20
5
25
Efficient
4
3
15
5
25
Total
VUSE Senior Design
55
Oral Report 3
90
Tuesday, February 12, 2008
Methods
RSV Genome
NS1NS2
N
3’
SH
P
M
M2
G
F
L
5’
RSV Genome (truncated)
NS1
3’
L
NS1 Start
5’
L Stop
pcDNA
(Synthesized)
Methods
Luciferase Gene (luc)
L Stop
NS1 Start
luc
pRSVlucM5
selection
pRSVlucM5
VUSE Senior Design
Oral Report 3
Tuesday, February 12, 2008
Development Costs
Item
Cost
pcDNA3.1 vector
$361
pGEM-luc
$83
Trailer minigenome plasmid
$274
Leader oligonucleotides
2x at $78 and 2x at $98
Cloning discs
2x at $29
Misc. chemicals and disposable lab equip.
$750*
TOTAL
$1878*
* Indicates an approximate value, many supplies are for general lab use
VUSE Senior Design
Oral Report 3
Tuesday, February 12, 2008
Factors Affecting Success
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There are 5 possible plasmids resulting from the
combination of our four DNA molecules; we must
screen for the correct one: pRSVlucM5
Unforeseen problems with designed sequences
Sensitivity relative to plaque assay
VUSE Senior Design
Oral Report 3
Tuesday, February 12, 2008
Alternate Solutions
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PCR - polymerase chain reaction
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Proven to work for the detection and quantification of
viruses
Limitations:
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Measures amount of nucleic acid (cannot differentiate
between live virus and dead virus)
Low throughput
Costly
VUSE Senior Design
Oral Report 3
Tuesday, February 12, 2008
Current Progress
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Completed:
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Design of all plasmid constituents in silco
Purified all plasmid constituents by gel electrophoresis
Quantify all four sequences
Ligate three sequences into pcDNA3.1vector
Transform e. coli with plasmids
Screen colonies with minipreps
In Progress:
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Maxiprep correct colony to obtain high yield of final plasmid
Submit Information Disclosure forms to Office of Tech Transfer
VUSE Senior Design
Oral Report 3
Tuesday, February 12, 2008
Screening
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Cut with SphI
1146bp
795bp
574bp
4908bp
72bp
VUSE Senior Design
Oral Report 3
Tuesday, February 12, 2008
Future Work
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Stably transfect cells with final plasmid
Test luminescence of cells using varying amounts of RSV
Optimize the system
VUSE Senior Design
Oral Report 3
Tuesday, February 12, 2008