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 2, 1/24/2008
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 due to RSV infection,
which is about 91 per hour
There is no current vaccine available for RSV
Current method for quantification of infectious RSV:
Plaque Assay
VUSE Senior Design
Oral Report 2
Thursday, January 24, 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
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 2
Thursday, January 24, 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 2
90
Thursday, January 24, 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
Efficiency
30 samples/experiment
240 samples/experiment
VUSE Senior Design
Oral Report 2
Thursday, January 24, 2008
Methods
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Remove luciferase gene from pGEM-luc
VUSE Senior Design
Oral Report 2
Thursday, January 24, 2008
Methods
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Ligate luciferase and additional sequence together
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Blue: leader, NS1 gene start, and non-coding regions
Red: non-coding, L gene end, and trailer regions
VUSE Senior Design
Oral Report 2
Thursday, January 24, 2008
Methods
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Cut pcDNA3.1. Ligate luciferase, additional
sequences, and pcDNA3.1 together
VUSE Senior Design
Oral Report 2
Thursday, January 24, 2008
pRSVlucM5
VUSE Senior Design
Oral Report 2
Thursday, January 24, 2008
Methods
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Transfect cells with plasmid
Plasmid
VUSE Senior Design
Oral Report 2
Thursday, January 24, 2008
Methods
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Infect cells with various RSV concentrations
mRNA
mRNA
Luciferase
Luciferin
VUSE Senior Design
Oral Report 2
Thursday, January 24, 2008
Methods
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Measure luminescence
Plate Reader
VUSE Senior Design
Oral Report 2
Thursday, January 24, 2008
Development Costs
Item
Cost
pcDNA3.1 vector
$361.00
pGEM-luc
$83.00
Trailer minigenome plasmid
$274
Leader oligonucleotides
2x at $78.00 and 2x at $97.50
Cloning discs
2x at $29.30
Misc. chemicals and disposable lab equip.
$750*
TOTAL
$1877.60*
* Indicates an approximate value, many supplies are for general lab use
VUSE Senior Design
Oral Report 2
Thursday, January 24, 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
Possible E. coli rejection of RSV sequences
Sensitivity relative to plaque assay
VUSE Senior Design
Oral Report 2
Thursday, January 24, 2008
Alternate Solutions
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Try other E. coli strains
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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 2
Thursday, January 24, 2008
Current Progress
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Completed:
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Design of leader and trailer sequences
Design of final plasmid construct in silco
Purified pcDNA3.1 vector and luciferase insert
In Progress:
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Preparation of leader and trailer inserts
Gel purification of leader and trailer inserts
VUSE Senior Design
Oral Report 2
Thursday, January 24, 2008
Setbacks
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1/18/08
 Failure of oligonucleotide
ligation due to unknown
factors
 Failure of trailer double
digest due to unknown
factors
1/18
1/21/08
 Confirmation of ligation
failure due to lack of 5’
phosphorylation
 Success of trailer double
digest
1/21
VUSE Senior Design
Oral Report 2
Thursday, January 24, 2008
Future Work
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Phosphorylate and ligate leader insert parts
Cut out trailer insert from minigene plasmid
Quantify all four sequences
Ligate three sequences into pcDNA3.1vector
Transform e. coli with plasmids
Screen colonies with minipreps
Maxiprep correct colony to obtain high yield of final plasmid
Stably transfect cells with final plasmid
Test luminescence of cells using varying amounts of RSV
Optimize the system
VUSE Senior Design
Oral Report 2
Thursday, January 24, 2008