progress report 1, 12/6/2007 melanie aston, michael chi, monica deterding, matt huckabee
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Luciferase Based Plasmid Reporter System for the Detection and Quantification of Human Respiratory Syncytial Virus. Progress Report 1, 12/6/2007 Melanie Aston, Michael Chi, Monica Deterding, Matt Huckabee. Background. - PowerPoint PPT PresentationTRANSCRIPT
Luciferase Based Plasmid Reporter System for the Detection and Quantification of
Human Respiratory Syncytial Virus
Progress Report 1, 12/6/2007Melanie Aston, Michael Chi, Monica Deterding, Matt Huckabee
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 hRSV Need quick cheap way to quantify RSV
Background
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Current Method: Viral Plaque Assay Procedure
Culture cells in 12 well plates (2 per sample) Infect cells Continue culturing cells in media with methyl cellulose
for 5 days Stain with hematoxylin and eosin
RSV Sample
1:1
1:1
1:1
1:10
1:10
1:10
1:102
1:102
1:102
1:103
1:103
1:103
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Our Solution Novel plasmid based reporter system A luciferase plasmid and cell line that will
luminesce when infected with RSV Stable transfection of plasmid into cell Optimization of system protocol
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Methods Remove luciferase gene from pGEM-luc
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Methods Ligate luciferase and additional sequence
together Blue: leader, NS1 gene start, and non-coding
regions Red: non-coding, L gene end, and trailer regions
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Cut pcDNA3.1. Ligate luciferase, additional sequence, and pcDNA3.1 together
Methods
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Transfect cells with plasmid
Methods
Plasmid
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Plate cells onto a 96-well plate
Methods
Pipette cells into rows/columns of
plate
Tests run intriplicate for each
concentration
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Infect cells with various RSV concentrations
Methods
mRNA
Luciferase
mRNA
Luciferin
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Measure luminescence
Methods
Plate Reader
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Comparison: Evaluation Chart
Plaque Assay Luciferase System
Criteria Weight (1-5) Value 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 55 90
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ComparisonPlaque Assay Luciferase System
Detection Method
Staining/Counting Luminescence
Objectivity Partial Yes
Time (work / total)
10 hours / 7 days 2.5 hours / 2 days
Materials Cost $8 $1
Efficiency 30 samples/experiment
240 samples/experiment
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Current Progress Completed:
Literature research Laboratory training Proof of Concept experiment Purchased stock plasmids
In Progress: Design of insertion sequences Maxiprep purchased plasmids and create glycerol
stocks
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Future Work Finalize insertion sequences and test on
computer Order insertion sequences
Remove luciferase gene from pGEM-luc Ligate insertion sequences and luciferase into
pcDNA3.1 Test luminescence of cells using varying
amounts of RSV Optimizing the system
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