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Progress Report 1, 12/6/2007 Melanie Aston, Michael Chi, Monica Deterding, Matt Huckabee

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.

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Progress Report 1, 12/6/2007 Melanie Aston, Michael Chi, Monica Deterding, Matt Huckabee

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  1. Luciferase Based Plasmid Reporter System for the Detection and Quantification ofHuman Respiratory Syncytial Virus Progress Report 1, 12/6/2007 Melanie Aston, Michael Chi, Monica Deterding, Matt Huckabee

  2. Background • 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 Oral Report 1

  3. Current Method: Viral Plaque Assay 1:1 1:10 1:102 1:103 RSV Sample 1:1 1:10 1:102 1:103 1:102 1:1 1:10 1:103 • 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 Oral Report 1

  4. 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 Oral Report 1

  5. Methods • Remove luciferase gene from pGEM-luc Oral Report 1

  6. 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 Oral Report 1

  7. Methods • Cut pcDNA3.1. Ligate luciferase, additional sequence, and pcDNA3.1 together Oral Report 1

  8. Methods • Transfect cells with plasmid Plasmid Oral Report 1

  9. Methods • Plate cells onto a 96-well plate Tests run in triplicate for each concentration Pipette cells into rows/columns of plate Oral Report 1

  10. Methods • Infect cells with various RSV concentrations mRNA mRNA Luciferase Luciferin Oral Report 1

  11. Methods • Measure luminescence Plate Reader Oral Report 1

  12. Comparison: Evaluation Chart Oral Report 1

  13. Comparison Oral Report 1

  14. 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 Oral Report 1

  15. 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 Oral Report 1

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