luciferase based plasmid reporter system for the detection and quantification of human respiratory...

Luciferase Based Plasmid Reporter System for the Detection and Quantification of

Human Respiratory Syncytial Virus

Group 14: Oral Report 2, 1/24/2008Melanie 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 RSV Current method for quantification of infectious RSV:

Plaque Assay

Background

Thursday, January 24, 2008VUSE Senior Design Oral Report 2

The Problem Viral plaque assay is

Labor intensive Costly Time consuming Partially subjective

Need high throughput, inexpensive system to quantify infectious RSV

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

Thursday, January 24, 2008VUSE Senior Design Oral Report 2

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

Thursday, January 24, 2008VUSE Senior Design Oral Report 2

ComparisonPlaque 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

Thursday, January 24, 2008Oral Report 2VUSE Senior Design

Methods Remove luciferase gene from pGEM-luc

Thursday, January 24, 2008VUSE Senior Design Oral Report 2

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

Thursday, January 24, 2008VUSE Senior Design Oral Report 2

Cut pcDNA3.1. Ligate luciferase, additional sequences, and pcDNA3.1 together

Methods

Thursday, January 24, 2008VUSE Senior Design Oral Report 2

pRSVlucM5

Thursday, January 24, 2008Oral Report 2VUSE Senior Design

Transfect cells with plasmid

Methods

Plasmid

Thursday, January 24, 2008VUSE Senior Design Oral Report 2

Infect cells with various RSV concentrations

Methods

mRNA

Luciferase

mRNA

Luciferin

Thursday, January 24, 2008VUSE Senior Design Oral Report 2

Measure luminescence

Methods

Plate Reader

Thursday, January 24, 2008VUSE Senior Design Oral Report 2

Development CostsItem 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*

Thursday, January 24, 2008Oral Report 2VUSE Senior Design

* Indicates an approximate value, many supplies are for general lab use

Factors Affecting Success 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

Thursday, January 24, 2008Oral Report 2VUSE Senior Design

Alternate Solutions Try other E. coli strains PCR - polymerase chain reaction

Proven to work for the detection and quantification of viruses

Limitations: Measures amount of nucleic acid (cannot differentiate

between live virus and dead virus) Low throughput Costly

Thursday, January 24, 2008VUSE Senior Design Oral Report 2

Current Progress Completed:

Design of leader and trailer sequences Design of final plasmid construct in silco Purified pcDNA3.1 vector and luciferase insert

In Progress: Preparation of leader and trailer inserts Gel purification of leader and trailer inserts

Thursday, January 24, 2008VUSE Senior Design Oral Report 2

Setbacks 1/18/08

Failure of oligonucleotide ligation due to unknown factors

Failure of trailer double digest due to unknown factors

1/21/08 Confirmation of ligation

failure due to lack of 5’ phosphorylation

Success of trailer double digest

1/18

1/21

Thursday, January 24, 2008VUSE Senior Design Oral Report 2

Future Work 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

Thursday, January 24, 2008VUSE Senior Design Oral Report 2