Luciferase Based Plasmid Reporter System for the Detection and Quantification of Human Respiratory Syncytial Virus Progress Report 1, 12/6/2007 Melanie.

Slides:

Advertisements

Similar presentations

Respiratory Syncytial Virus is the leading cause of respiratory tract infection in infants in the United States and worldwide. There is currently no vaccine.

Advertisements

Luciferase Based Plasmid Reporter System for the Detection and Quantification of Human Respiratory Syncytial Virus Group 14: Oral Report 2, 1/24/2008 Melanie.

Luciferase Based Plasmid Reporter System for the Detection and Quantification of Human Respiratory Syncytial Virus Group 14: Oral Report 4, 3/13/08 Melanie.

Methods for DNA Transfer. Transferring Genes Vectors are used to move genes around Plasmids, Bacteriophage, Cosmids, YACs, BACs, Viruses are used E. coli.

Non-Influenza Respiratory Virus Vaccines Larry J. Anderson DVD, NCIRD, CDC Atlanta, GA, USA 41 st National Immunization Conference Kansas City, Missouri.

Principal Investigators: P. Murcia and J. F. Marshall

Invest. Ophthalmol. Vis. Sci ;44(3): doi: /iovs Figure Legend:

The use of reporter genes to define DNA regulatory elements

Volume 145, Issue 4, Pages e2 (October 2013)

Protocol for Generation of Stable Cell LinesStable Cell Lines.

Homocysteine inhibits endothelial cell growth via DNA hypomethylation of the cyclin Agene by Md S. Jamaluddin, Irene Chen, Fan Yang, Xiaohua Jiang, Michael.

Example of a DNA Array (note green, yellow red colors; also note that only part of the total array is depicted)

Volume 17, Issue 9, Pages (September 2009)

A B C D Supplementary Figure 1 CMV p300

Volume 129, Issue 3, Pages (September 2005)

Luciferase Based Plasmid Reporter System for the Detection and Quantification of Human Respiratory Syncytial Virus Group 14: Oral Report 3, 2/12/2008 Melanie.

Cdc42 Inhibits ERK-Mediated Collagenase-1 (MMP-1) Expression in Collagen-Activated Human Keratinocytes Maryam G. Rohani, Brian K. Pilcher, Peter Chen,

A Combinatorial CRISPR-Cas9 Attack on HIV-1 DNA Extinguishes All Infectious Provirus in Infected T Cell Cultures Gang Wang, Na Zhao, Ben Berkhout, Atze.

Volume 44, Issue 5, Pages (May 2006)

Volume 15, Issue 5, Pages (November 2001)

DEVELOPMENT OF A NOVEL VNT ASSAY USING QRT-PCR-BASED ENDPOINT ASSESSMENT FOR RAPID DETECTION AND TITRATION OF NEUTRALIZING ANTIBODIES AGAINST FMDV.

Volume 145, Issue 4, Pages e2 (October 2013)

Role of CRD-BP in the Growth of Human Basal Cell Carcinoma Cells

Respiratory syncytial virus nephropathy in rats

Molecular Therapy - Methods & Clinical Development

J. F Schaefer, M. S. , M. L Millham, M. A. , B de Crombrugghe, M. D

Volume 22, Issue 10, Pages (October 2014)

Volume 137, Issue 1, Pages (July 2009)

The Nogo Receptor NgR1 Mediates Infection by Mammalian Reovirus

Detection of human metapneumovirus and respiratory syncytial virus by duplex real-time RT-PCR assay in comparison with direct fluorescent assay P. Jokela,

Cell-Density-Dependent Regulation of Expression and Glycosylation of Dopachrome Tautomerase/Tyrosinase-Related Protein-2 Thomas J. Hornyak, Daniel J.

Volume 23, Issue 10, Pages (October 2015)

Volume 22, Issue 1, Pages (January 2014)

The mRNA bearing the IRES element is arranged in cytoplasmic clusters.

Prospective evaluation of a dot-blot enzyme immunoassay (Directigen RSV) for the antigenic detection of respiratory syncytial virus from nasopharyngeal.

Volume 1, Issue 3, Pages (September 2013)

Qian Wang, Heini Ilves, Pauline Chu, Christopher H

Volume 15, Issue 5, Pages (May 2007)

Takashi Fukaya, Hiro-oki Iwakawa, Yukihide Tomari Molecular Cell

Transcriptional Regulation of ATP2C1 Gene by Sp1 and YY1 and Reduced Function of its Promoter in Hailey–Hailey Disease Keratinocytes Hiroshi Kawada,

Keratinocyte Responsive Element 3: Analysis of a Keratinocyte-specific Regulatory Sequence in the 230-kDa Bullous Pemphigoid Antigen Gene Promoter Yasushi.

Volume 22, Issue 4, Pages (April 2014)

Aortic wall cell proliferation via basic fibroblast growth factor gene transfer limits progression of experimental abdominal aortic aneurysm Katsuyuki.

Keratinocyte growth factor promotes goblet cell differentiation through regulation of goblet cell silencer inhibitor Dai Iwakiri, Daniel K. Podolsky

Molecular Therapy - Nucleic Acids

Volume 12, Issue 5, Pages (November 2005)

Juxtaposition of GFP-Rab1b with IRES-containing RNA.

Volume 16, Issue 12, Pages (June 2006)

Shrimp miR-34 from Shrimp Stress Response to Virus Infection Suppresses Tumorigenesis of Breast Cancer Yalei Cui, Xiaoyuan Yang, Xiaobo Zhang Molecular.

Light-Dependent Interactions between the Drosophila Circadian Clock Factors Cryptochrome, Jetlag, and Timeless Nicolai Peschel, Ko Fan Chen, Gisela Szabo,

Volume 15, Issue 11, Pages (November 2007)

Hairpin & reporter systems.

Molecular Therapy - Nucleic Acids

Regulation of human renin gene promoter activity: A new negative regulatory region determines the responsiveness to TNFα Ling-Sing K. Chen, Michael P.

Changyu Zheng, Bruce J. Baum Molecular Therapy

The lncRNA PDIA3P Interacts with miR-185-5p to Modulate Oral Squamous Cell Carcinoma Progression by Targeting Cyclin D2 Cheng-Cao Sun, Ling Zhang, Guang.

Volume 10, Issue 6, Pages (December 2004)

Position-Dependent Function for a Tandem MicroRNA miR-122-Binding Site Located in the Hepatitis C Virus RNA Genome Catherine L. Jopling, Sylvia Schütz,

RNAi screening formats.

Volume 9, Issue 2, Pages (February 2004)

Volume 17, Issue 5, Pages (May 2013)

Figure S1. Effect of lipid-based transfection reagents on genome editing Figure S1. Various amount of Cas9 protein and gRNA of HPRT were transfected.

Etiology of acute respiratory infections in children.

Fig. 1 Prophylactic, neutralization, and therapeutic efficacy of HPAC.

Volume 18, Issue 2, Pages (February 2010)

Suppression of Keratinocyte Growth and Differentiation by Transforming Growth Factor β1 Involves Multiple Signaling Pathways Alison L. Dahler, Lois L.

Comparison of Akt1 and Akt3 for their abilities to activate the Fra-1 promoter. Comparison of Akt1 and Akt3 for their abilities to activate the Fra-1 promoter.

αvβ6 functional assays in the β cell line using blocking antibodies.

Optimization of incubation time for visibly stained spot formation in Vero-E6 infected by four serotype DENVs and the efficiency of DENV-4 plaque formation.

Presentation transcript:

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 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 Wednesday, November 14, 2018

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 1:10 1:102 1:103 Talk about the “system” as a whole, meaning the plasmid and how to use it Oral Report 1 Wednesday, November 14, 2018 3

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 Talk about the “system” as a whole, meaning the plasmid and how to use it Oral Report 1 Wednesday, November 14, 2018

Methods Remove luciferase gene from pGEM-luc Oral Report 1 Wednesday, November 14, 2018

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 Wednesday, November 14, 2018

Methods Cut pcDNA3.1. Ligate luciferase, additional sequence, and pcDNA3.1 together Oral Report 1 Wednesday, November 14, 2018

Methods Transfect cells with plasmid Plasmid Oral Report 1 Wednesday, November 14, 2018

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 Wednesday, November 14, 2018

Methods Infect cells with various RSV concentrations mRNA mRNA Luciferase Luciferin Oral Report 1 Wednesday, November 14, 2018

Plate Reader Methods Measure luminescence Oral Report 1 Wednesday, November 14, 2018

Comparison: Evaluation Chart Plaque Assay Luciferase System Criteria Weight (1-5) Value Product Quick 5 2 10 4 20 Low Cost 3 Objective 25 Efficient 15 Total 55 90 Oral Report 1 Wednesday, November 14, 2018

Comparison Plaque 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 Oral Report 1 Wednesday, November 14, 2018

Current Progress Completed: In Progress: 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 Wednesday, November 14, 2018

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 Wednesday, November 14, 2018