A LUCIFERASE REPORTER MINIGENOME SYSTEM FOR
QUANTIFYING RESPIRATORY SYNCYTIAL VIRUS REPLICATION
Melanie J. Aston1, Michael H.Chi1, Monica K. Deterding1, Matthew M. Huckabee1,
Martin L. Moore2, and R. Stokes Peebles, Jr.2
1Department
of Biomedical Engineering, Vanderbilt University and 2Department of Medicine, Vanderbilt University
School of Medicine, Nashville, TN
Respiratory Syncytial Virus is the leading cause of respiratory tract infection in infants in
the United States and worldwide. There is currently no vaccine available to treat RSV.
The current method to determine RSV titer in the laboratory is the viral plaque assay, a
labor, materials, and time intensive procedure. There is a need for a high throughput,
inexpensive, and highly sensitive method to quantify infectious RSV. We engineered an
RSV minigenome containing a luciferase reporter for high-throughput quantification of
RSV replication. The minigenome is under control of the CMV promoter for constitutive
expression. HEp-2 cells were transfected, and stably-transfected cell lines were
selected. Luciferase bioluminescence was measured 48 hours after RSV infection in a
96-well plate. Luciferase activity was RSV-specific and dose-dependent.
Introduction
Respiratory Syncytial Virus is a paramyxovirus that consists of ten genes in a 15kbp
negative sense genome. Figure 1 shows a schematic of the RSV genome and its life
cycle within a cell. The genes in pink (N, P,
M2, and L) are necessary for the transcription
and replication of the virus. The viral life cycle
within the cell proceeds in several steps. First
the virus binds to the target cell via an
unknown receptor. The genome unfolds and
host cell machinery transcribes the viral
Figure 1: RSV genome and intracellular life cycle
genes. These genes are transcribed individually along a gradient. The mRNA’s are then
translated by the host cell machinery into protein. At some point, when the proteins build
up in the cell the negative sense genome is replicated into a positive sense antigenome,
which serves as a template for replication of many copies of the viral genome. These
new viral genomes are then packaged into new virus particles.
RSV is the leading cause of bronchiolitis and pneumonia in infants under one year of
age (1). Current estimates place the annual infection and mortality figures for RSV at 64
million and 800,000 respectfully. It is the leading cause of hospitalization, respiratory
failure, mechanical ventilation, and viral death in infants in the United States and
1
worldwide (1). There are currently no vaccines or drugs available to treat RSV. The
biggest challenge with creating a viable RSV vaccine is the bottleneck in laboratory
research. This bottleneck arises from the inefficiencies in determining viral titer.
The current method to determine RSV titer in the laboratory is the viral plaque
assay. The plaque assay is a labor, materials, and time intensive procedure. Plaque
assays can take up to 7 days to complete. Figure 2 shows a flow chart for the plaque
assay. In addition, the plaque assay is not highly sensitive. To count a plaque the
researcher must “eyeball” the
plate and manually count the
plaques or “holes” formed by
dead cells. This procedure
introduces subjectivity to the
measurement and as a result is
more prone to human error.
Clearly there is a need for a
high throughput, inexpensive, and
highly sensitive method to
Figure 2: Flow Chart for the Viral Plaque Assay (major delays
are in red)
quantify infectious RSV. We
propose a novel luciferase based minigenome reporter system.
A team at the National Institute of Health led by Dr. Peter Collins successfully
created a minigenome system that expresses a reporter gene in response to RSV
infection (6). This system involved an RSV minigenome under the control of the T7
promoter. The Collins group found that the N, P, M2-1 and L proteins were sufficient
and necessary for expression of the minigenome and reporter gene. We chose to adopt
this system because reporter gene activity was RSV specific and dependent.
2
Methods
This plasmid reporter was constructed from four separate sequences. They are:
a pcDNA3.1 vector, a leader sequence, a copy of the luciferase gene and a trailer
sequence. They were fabricated or isolated individually and finally ligated together. In
silco design was conducted using VectorNTI cloning software. We named our plasmid
pRSVlucM5.
The vector backbone selected was pcDNA3.1(+), a 5428 base pair plasmid
purchased from Invitrogen. This plasmid was chosen for the presence of unique
restriction sites, the CMV promoter,
and the geneticin and ampicillin
resistance genes to allow for selection.
Second, pGEM-luc was selected as
the luciferase gene source.
The leader and trailer regions of
Figure 3. (A) Diagram of the Trailer sequence, (B) Diagram of the
Leader sequence
the plasmid were designed based on the construct created by the Collins group (6).
Within the leader is a 44 nucleotide (nt) RSV leader region, a 9 nt gene start signal of
RSV nonstructural protein 1 (NS1), and a 32 nt nontranslated region of the NS1 gene
(Figure 3B). The trailer sequence includes 12 nt of the nontranslated region of the RSV
large polymerase (L) gene, the 12 nt L gene end signal, and the 155 nt RSV trailer
region (Figure 3A). The sequences were designed with the proper restriction sites
necessary for future ligation.
After individual design in silco the
sequences were compiled to create
the final plasmid construct (Figure 4).
The leader and trailer sequences
were fabricated by Integrated DNA
Technologies. The trailer sequence
was ordered as a 191 base pair
element in a minigene plasmid.
Fabrication of the leader sequence
Figure 4: Diagram of pRSVlucM5
proved to be more difficult. The
3
length of the leader sequence and the presence of a hairpin in the ribozyme made it
impossible to fabricate as a minigene plasmid. To get around this problem we
constructed the leader from four separate oligonucleotide fragments. This required
additional work before the leader
could be ligated.
The four oligonucleotides had
to be annealed together
(matching one end of the double
helix to the other) creating two
leader portions referred to as
Leader A (made up of oligos 1 &
2) and Leader B (made up of
oligos 3 & 4) (See Figure 5).
Initial ligations of Leader A
Figure 5: Diagram of the fabrication process
and Leader B proved
unsuccessful. This was due to the lack of 5' phosphorylation of the sequences. T4
polynucleotide kinase purchased from NEB was used to phosphorylate Leader A and
Leader B and the ligation was attempted again. The results of the ligation of Leader A
and Leader B following phosphorylation proved inconclusive.
The trailer sequence had to be isolated from the purchased minigene plasmid. It
was designed with the HindIII and XhoI restriction sites. Using the respective restriction
enzymes, the trailer was cut out of the minigene plasmid. The trailer digest was run
through a 2% agarose gel using an electrophoresis machine set at 100V for 2 hours and
45 minutes to separate the trailer from the remainder of the plasmid. The trailer
sequence was then extracted from the gel and saved for future use.
The luciferase gene was isolated from the pGEMluc vector in a similar fashion.
pGEM-luc was first cut with restriction enzyme XhoI followed by BamHI, resulting in two
separate pieces of the vector, the luciferase gene (1716 base pairs), and the remainder
of the plasmid (3215 base pairs). Gel purification was conducted in order to isolate the
luciferase gene. A 0.7% agarose gel was prepared and the luciferase digest was run at
4
80V for 1 hour, and then 100V for an additional hour. The luciferase gene was then
extracted from the gel and stored for later use.
pcDNA3.1 was also cut in order to allow for future ligation with the other
sequences. The vector was cut with HindIII, and then later with NotI. The enzyme cuts
created two fragments of the vector, one 5360 base pairs long (functional), the other 68
base pairs long (non functional). Another 0.7% agarose gel was prepared, loaded with
the digest, and run at 100V for an hour and a half. The functional 5360bp sequence
was extracted from the gel and stored for later ligation. Due to inconclusive results from
the Leader A and Leader B ligation we decided to conduct one large ligation of Leader
A, Leader B, luciferase, Trailer, and pcDNA3.1.
The four basic parts of RSVlucM5 (pcDNA3.1, luciferase, leader, and trailer)
were designed with ends such that when they were cut with the appropriate restriction
enzymes, they could only assemble in a particular way. However, it was possible that a
series of self-dimerizations could occur that would result in other viable alternatives to
pRSVlucM5. VectorNTI provided four viable alternate configurations. They were 10720
bp, 11090 bp, 11188 bp, and 21440 bp in length. From the design we know that our
plasmid is only 7495 base pairs in length. In order to screen for the correct plasmid an
additional digest with restriction enzyme SphI was conducted.
Bacteria were first transformed with the plasmid. This bacteria was grown on
ampicillin plates – this was done to weed out any bacteria that did not incorporate our
plasmid. We were then able to
screen for the correct plasmid from
all colonies that grew. An initial 24
colonies were selected for the first
round of screening.
The SphI digest cut the
DNA from the 24 selected
colonies. A 0.7% agarose gel was
again prepared and all 24 cut
samples were loaded. Based on
the in silco design a digest of our
Figure 6: Gel image of the total plasmid digest with bands indicated
5
plasmid with SphI, would result in five segments of the following lengths 4908bp,
1146bp, 795bp, 574bp, and 72bp. We then analyzed the digest gel for this band pattern.
Figure 6 shows this digest gel with the correct pattern highlighted. Over half of the
colonies showed the correct digest pattern. Colonies 2 and 3 were chosen for future
use.
To determine if the plasmid would in fact produce luciferase in response to RSV
infection, initial tests on the system were conducted. Human epithelial cells (HEp-2
cells) were plated into 30 wells of a clear 96 well plate. The plasmid was then applied
via transient transfection in a gradient concentration. Four different trials were
conducted using Green Fluorescent Protein (GFP) as a control for positive transfection.
After a growth period of 24 hours, the cells were infected with RSV at a
multiplicity of infection (MOI) of 1, following a 24 hour infection period a luciferase assay
was conducted. A luciferase assay kit, from Promega Corporation, was used. The cells
were transferred to a luminometer tube, reagent was added, and then each sample was
read using a luminometer, courtesy of the Blackwell Laboratory.
Our main goal
was to generate a
stably transfected cell
line for future use
during vaccine
research. This work
was done concurrently
with the transient
transfection. In a 6-well
plate, additional HEp-2
A
B
C
D
E
F
G
H
pfu/mL
mock
10
101
102
103
104
105
106
1
2
RSV INFECTION
3 4 5 6 7 8
9
REOVIRUS
10 11 12
mock
5*10-1
5*10
5*101
5*102
5*103
5*104
5*105
24 hour analysis
48 hour analysis
72 hour analysis
Figure 7: Plate diagram for the stable transfection assay
cells were grown and then transfected with 4μg or 8μg of plasmid. Twenty four hours
following transfection, neomycin was added to the wells to kill any cells that did not take
up the plasmid. Cloning discs were used to select 17 colonies to screen for specific
bioluminescence activity. These colonies were grown up and plated on a 96 well plate
and then a luciferase assay was conducted using a plate reader.
6
Further study of the stable transfection was conducted by studying the system
over 72 hours post infection. The specificity of the system was studied by concurrently
using a different virus for infection in addition to RSV. A strain of Reovirus, obtained
from Terry Dermody was used in 24 of the wells (Figure 7). Cells were plated in 96
wells, and infected with RSV or Reovirus. The first luciferase assay was conducted at
24, 48 and 72 hours post infection. Both the cells and the supernatant were measured
for luminescence. After transferring each sample to an opaque plate, luciferase assay
reagent was added and the samples were read using the plate reader.
Results and Discussion
Confirmation of pRSVlucM5 Function by Transient Transfection
A transient transfection of pRSVlucM5 into human epithelial (HEp-2) cells was
performed to confirm that the engineered plasmid worked as designed in HEp-2 cells.
Different amounts of pRSVlucM5 were transiently transfected into HEp-2 cells. The
cells were subsequently infected with 106 PFU/mL of RSV and a luciferase assay was
performed 24 hours later.
The luciferase assay works
bioluminescence, which
correlates to the amount of
luciferase produced by the
cells. As shown in Figure 8,
the amount of
bioluminescence measured
increased as the amount of
Bioluminescence (RLU)
by measuring
3000
2500
2000
1500
1000
500
0
0
0.2
0.4
0.6
0.8
1
Amount of DNA Transfected (ug)
pRSVlucM5 transfected into
the cells increased. These
data show that the
Figure 8. Three replicate transient transfections (dashed lines) show an
increase in luciferase as the amount of DNA transfected increases, as
measured by bioluminescence. The average of the three replicate
experiments is shown as the solid line.
pRSVlucM5 plasmid does
work correctly in HEp-2 cells. That is, it was confirmed that HEp-2 cells transfected with
the plasmid pRSVlucM5 express luciferase when infected with RSV. Furthermore, it
can be gathered from the data that an increase in the amount of DNA transfected
7
results in an increase in the number of plasmids taken up by the HEp-2 cells. The
increase in copy number per cell results in the increasing bioluminescence seen in the
data. This relationship between copy number and bioluminescence could play a key
role in the optimization of the system when screening stably transfected cell lines.
Generation of HEp-2RSVM5 Cell Line by Stable Transfection
HEp-2 cells were transfected with pRSVlucM5, and neomycin was applied in
order to select stably transfected cells. Cells were cultured as separate cell lines and
tested for luciferase activity when infected with different concentrations of RSV. A
preliminary screening
6000
was performed with
the resulting data
from the luciferase
assay shown in
Figure 9. The HEp2RSVM5.4 cell line
showed significant
amounts of
luciferase, while the
remaining cell lines
Bioluminescence (RLU)
test of 17 cell lines
5000
4000
3000
2000
1000
0
1000
10000
100000
1000000
RSV Concentration (PFU/mL)
Figure 9. HEp-2RSVM5.4 (black) shows increased bioluminescent activity,
while the remaining cell lines (blue, pink, light blue, maroon) show
insignificant amounts of luciferase.
did not show
bioluminescent activity significantly greater than the blank used from the luciferase
assay. The bioluminescence of HEp-2RSVM5.4 was expected to increase with
increasing RSV concentration increased, but the data does not match with the expected
results. As the only cell line tested that exhibited luciferase after RSV infection, HEp2RSVM5.4 was chosen for further testing in order to determine if the relationship
between bioluminescence and RSV concentration is optimal.
HEp-2RSVM5.4 cells were again cultured and infected with different
concentrations of RSV. A luciferase assay was performed and the resulting data is
shown in Figure 10. An increase in bioluminescence was shown as the RSV
concentration increased. These data suggest that HEp-2RSVM5.4 has the potential to
8
be the optimized final product. Further testing, however, was needed to determine the
bioluminescence time dependence as well as to verify RSV specificity.
HEp-2RSVM5.4
infected with different
concentrations of RSV
and Reovirus.
Luciferase assays were
performed 24, 48, and
72 hours after infection.
230
Bioluminescence (RLU)
cells were cultured and
225
220
215
210
205
200
195
190
Figure 10 shows the
1
100
measured 24 hours
1000000
RSV Concentration (PFU/mL)
results. When the
bioluminescent activity is
10000
Figure 10. HEp-2RSVM5.4 shows an increase in bioluminescent activity as
the RSV concentration increases.
post-infection, no trend can be found. A spike in bioluminescence is found 48 hours
post-infection for an RSV concentration of 1x105 PFU/mL, with bioluminescence dying
out at higher RSV
800
hours post-infection, no
increase in
bioluminescence is
detected with increasing
RSV concentration.
Similar to the 48 hours
post-infection data,
Bioluminescence (RLU)
concentrations. At 72
700
600
500
400
300
200
100
0
1
100
10000
1000000
bioluminescence dies out
RSV Concentration (PFU/mL)
at high RSV
concentrations. A
luciferase assay was also
Figure 11. HEp-2RSVM5.4 exhibits different bioluminescence-RSV
concentration relationships depending on the amount of time between
infection and luciferase assay. The times shown are 24 (black), 48 (red), and
72 (blue) hours.
performed on HEp2RSVM5.4 cells infected with Reovirus. The results are shown in Figure 11, and
9
compared to the data from the RSV infected cells. It was found that the HEp2RSVM5.4 cell line is specific to RSV infection, as there is no increase in
bioluminescence with Reovirus infection.
Further testing is required to determine the reasons why no trend is found at
lower RSV concentrations. The significant decrease in bioluminescence at high RSV
concentrations is most likely due to the large amount of cell death due to infection.
luciferase produced within
them, resulting in decreased
bioluminescence measured
by the luciferase assay.
It can be seen in
Figure 12 that there is
background luciferase in the
Bioluminescence (RLU)
Cells dying release the
800
700
600
500
400
300
200
100
0
1
100
10000
1000000
HEp-2RSVM5.4 cells, as
there is significant
bioluminescence measured
at extremely low RSV
Amount of Virus (PFU/mL)
Figure 12. Bioluminescence is shown to be specific to RSV infection (blue),
as shown by no increase in bioluminescence with Reovirus infection (pink).
concentrations. This is most likely due to the incorporation of the pRSVlucM5 plasmid
into the HEp-2 DNA downstream of a native promoter. This native promoter would then
drive the transcription of a positive sense luciferase mRNA, which would in turn be
translated into luciferase without the presence of RSV.
The data collected thus far for the HEp-2RSVM5.4 cell line shows that stably
transfected cells can exhibit some relationship between bioluminescence and RSV
concentration. The cell line, however, does not show a significant relationship between
the two. It also, exhibits background luciferase that decreases the sensitivity of the
system. In order to optimize the system, more stably transfected cell lines must be
cultured and screened in order to find a line with better sensitivity and no background
luciferase. More testing will need to be done with the candidate cell lines in order to
characterize them for use.
10
Economics
The current market for our luciferase reporter system would mainly consist
of research and development companies and academic researchers. Companies
specifically interested in RSV research would be able to use our plasmid instead of the
more costly and less time efficient plaque assay. Companies currently working on
vaccines and antiviral drugs for RSV include Wyeth, MedImmune, Virion Systems,
Novartis, Alnylam, Sanofi Pasteur, and Tibotec and could all be seen as a potential
market for our luciferase reporter system.
The development costs of our reporter system were based on the prices of the
segments, reagents, and lab equipment used. The cost of the pcDNA3.1 vector was
$361. The cost of the pGEM-luc necessary for luminescence of the plasmid was $83.
The minigenome plasmid containing our trailer cost $274. The four leader
oligonucleotides cost $78 (2x) and $98 (2x). The cloning discs were purchased for $29.
Finally, miscellaneous chemicals and disposable lab equipment cost approximately
$750, yielding a development cost of $1878.
The future maintenance cost of this system is extremely low. The cells needed
for transfection can be frozen in liquid nitrogen and used whenever needed. The
plasmid would be stored in the -80oC freezer. This storage would allow for the
maintenance of our system with little additional cost. We are responsible for most of the
marketing distribution costs. We estimate that the cost of marketing will again be
relatively low. Marketing of our system will most likely occur by attending scientific
meetings and conferences. The RSV research community is relatively small so
marketing could be done on an individual basis. By expanding our network of contacts
through scientific conferences, marketing of our system can be cheap and feasible.
The benefits of this system are shown in the comparison between our system
and the plaque assay method. Our system increases the objectivity of experiments. It
reduces the time from 10 hours over the course of a week to 2.5 hours over the course
of 2 days. We estimate that for every eight dollars spent on the plaque assay, one dollar
will be spent on the luciferase system. Our system also increases throughput from 30
samples per experiment to 240 samples per experiment. The benefit for using the cell
line to screen for anti-RSV drugs would be enormous. However, further research and
11
development of the cell line is necessary before the full benefits can be understood.
Based on assumptions over the course of a year the benefit to cost ratio was found to
be 4.24 (See Appendix B). This demonstrates how greatly the benefits of our system
outweigh its cost. This analysis was carried out assuming that our system would have a
lifetime of 1 yr, however, because our system has the possibility to last many years the
benefit cost ratio could increase.
The life cycle for this system is dependent on the resources available to those
individuals or those labs that use the system. If the labs are capable of passing the cells
purchased and then freezing them, the cells are capable of lasting as long as
necessary. The purchaser needs only to buy our system once in order to carry out tasks
for a lifetime.
As with all new technology, safety concerns must also be taken into account. The
minigenome that we have created is not replicative on its own and is not infectious. So
safety concerns for the actual system are nonexistent. However, it is a RSV reporter
and so any lab using it must implement the proper biohazard containment precautions.
This reporter cell line is more of a tool for vaccine and drug delivery research and will
not be used in humans at any point, thus there are no FDA regulations for our system.
Future Directions
We recommend that this project be continued. As the results indicate there is
significant room for the improvement of this assay system. Possible future
improvements might be to optimize the transient transfection while controlling for
transfection efficiency. Other avenues to consider might be to determine the sensitivity
and specificity of the transient and stably transfected reporter. We also feel that
significant work should be invested in selecting other stably transfected cell lines in the
hopes of lowering background luciferase activity.
Despite these drawbacks the authors feel that this reporter system will eventually
serve as a surrogate for the plaque assay. It is also possible that this system might be
employed to screen small molecule inhibitors of RSV replication.
Acknowledgements
We would like to thank the following people from Vanderbilt for their contributions to the
project: Kasia Goleniewska (laboratory support), Kirk Lane (project advice), Jim Crowe
12
(plate reader), and Terry Dermody (reovirus). The authors further wish to thank Peter
Collins from the NIAID for his guidance on this project.
References
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Appendix A (Innovation Workbench)
Ideation Process
I.
II.
Project Initiation
a. Project name: Luciferase Luciferase Based Plasmid Reporter System for
the Detection and Quantification of hRSV
b. Project timeline:
i. October 29th - November 2nd: Select plasmid and place order
ii. October 29th - November 16th: Design and Test insertion
sequence
iii. November 19th-23rd: Order insertion sequence
iv. November 26th - December 7th: Insert insertion sequence into
plasmids and replicate
v. December 10th - January 18th: Select for cells that take up plasmid
and test cells for efficacy (make new insertion sequence and
plasmids if necessary)
vi. January 21st - February 8th: Test cells and establish best cell line
vii. February 11th -29th: Establish luminescence-titer correlation
c. Project team: Melanie Aston, Michael Chi, Monica Deterding, Matt
Huckabee
Innovation Situation Questionnaire
a. Brief description of the situation
The current method for determining the amount of RSV in a sample is
complex and time consuming. This serves as a major bottleneck to RSV
vaccine development.
b. Detailed description of the situation
The current method for determining the amount of RSV in a sample is
complex and time consuming. It involves a lengthy plaque assay that
requires 10 man hours over seven days. It is only partially objective and
is thus prone to error.
i. Supersystem - System - Subsystems
1. System name:
Luciferase Based Plasmid Reporter System for the
Detection and Quantification of hRSV
2. System structure:
The system involves:
Cells stably tranfected with plasmid
Plate Reader
Luciferin
3. Supersystems and environment
N/A
4. Systems with similar problems
The problem has been solved another way but it is
inefficient because it measures the number of virus
particles not infectivity.
15
ii. Input - Process - Output
1. Functioning of the system
Our plasmid reporter system will be composed of a
mammalian promoter, a luciferase gene, an RSV
promoter, and a gene for neomycin resistance. The
mammalian promoter will induce the production of
mRNA when the plasmid is introduced to the host
nucleus (mRNA will be produced even when the cell is
not infected with RSV). This mRNA will travel to the
cytoplasm where it will wait. Once the cell is infected, the
virus will activate the viral promoter on the mRNA and
cause it to make another mRNA strand that codes for
the luciferase protein. The standard cellular machinery
(i.e. ribosomes) will translate the mRNA into protein.
This mechanism is novel because luciferase will only be
expressed when the cell is infected with RSV. We will
then detect the bioluminescence from this protein with a
luminescence detector and establish a correlation with
viral titer.
2. System inputs
RSV
3. System outputs
Luminescence data
iii. Cause - Problem - Effect
1. Problem to be resolved
Inefficiency of plaque assay
2. Mechanism causing the problem
The nature of the plaque assay
3. Undesirable consequences if the problem is not resolved
Wasted time and money
4. Other problems to be solved
N/A
iv. Past - Present - Future
1. History of the problem
The use of plaque assays in the quantification of RSV
has been well reported. It's problems and issues have
always been apparent.
2. Pre-process time
Work has already begun on our system so there is no
pre-process time.
3. Post-process time
The post-process time will begin once the plasmid
reporter system has been developed and tested. We
estimate this to be around the end of February.
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III.
c. Resources, constraints and limitations
i. Available resources
All laboratory equipment and materials necessary in the design
and creation of the luciferase reporter system will be provided
by Dr. Peebles' Lab
ii. Allowable changes to the system
None
iii. Constraints and limitations
Biological functions of cells
iv. Criteria for selecting solution concepts
None
Problem Formulation and Brainstorming
a. Directions for Innovation
Find an alternative way to obtain a Plaque Assay that provides or enhances RSV
Quantification.
We chose to synthesize a new system
Find a way to reduce Time.
Find a way to reduce Money.
Find a way to eliminate, reduce, or prevent Subjectivity.
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Appendix B (Calculations)
Benefit Cost Calculation (B/C)
Given
Our system saves $7 per experiment and 5 days per experiment.
Initial investment = $1878
Assumptions
 each lab carries out 2 plaque assays per month and about 15 experiments with
our system per month
 maintenance costs per year are $500
 average salary for lab technician per hour is $15
Calculations
B/C per year = [(benefitsmaterials + benefitstime) – maintenance costs]/(initial investment)
Benefitsmaterials = $7/experiment x 15 experiments/month x 12months/yr = $1260/yr
Benefitstime = $15/hr x 40hrs/month x 12months/yr = $7200/yr
Therefore, B/C = ($1260+$7200-$500)/($1878) = 4.24
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