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1
Reporter Half-Life as a Dominant Factor in
Determining Assay Sensitivity
(November 2005)
Geneviève Gavigan

Abstract—Reporter gene technology provides a method to
measure the intracellular activity of transcription factors.
HOXB4 is a transcription factor under investigation for its
potential to expand human hematopoietic stem cells (HSCs) in
vitro. In this study, a TAT-HOXB4 fusion protein was added to
human embryonic kidney (HEK) 293 cells in vitro, and a
luciferase reporter gene was used to quantify its intracellular
activity. This system was used in a model to assess the reporter’s
ability to monitor a transient system. The half-life of the reporter
was found to be a dominant factor in determining the assay
sensitivity.
Index Terms— hematopoietic stem cell, reporter gene
sensitivity, reporter half-life, TAT-HOXB4
I. INTRODUCTION
H
OXB4 is an important transcription factor in
hematopoietic stem cell (HSC) expansion, however, little
is known about its molecular mechanism.[1] It is
suggested that there is a “therapeutic window” and dosedependent effects for HOXB4 expression on HSC
proliferation.[2] Additional studies are needed identifying
threshold levels, and an optimum delivery protocol for
HOXB4.
This necessitates a reporter to measure the
intracellular activity of this transcription factor at different
doses and delivery modes.
Reporter assays have been used to monitor promoter activity,
gene transfer, gene expression, and signaling pathways.[3]
These assays exploit reporter genes, which are genes coding
for proteins that can be easily measured and monitored
because of a detectable signal they produce, such as the
emission of light or a colour change.[4] Promoter regions on
these reporter genes can be modified with response elements
that are activated by the protein of interest, initiating the
transcription pathway of the reporter gene.[3] Quantification
of the resulting signal is therefore indicative of the intracellular
activity of the protein.
Manuscript received November 7, 2005.
G. Gavigan is with the Institute of Biomaterials and Biomedical Engineering,
University of Toronto, Toronto, ON M5S 3G9 Canada
(genevieve.gavigan@ utoronto.ca).
A green fluorescent protein (GFP) reporter has been used to
determine transfection efficiency for a retrovirus encoding
both GFP and HOXB4.[1] Although this system enabled the
proliferation of transfected cells to be monitored, it provided
no information on the dose of HOXB4 the cells were
receiving. Also, GFP exhibits low sensitivity, as no signal
amplification is observed.[3]
Another study investigated the binding interactions between
the transcription factor HOXB1 and PBX1, a cofactor.
Luciferase reporters have high specific activities [3], and this
assay used this reporter with a HOX-PBX binding site [5]. It
was found that HOX-PBX may act as both an activator and an
inhibitor.[5] A reporter system with a higher sensitivity may
help uncover these molecular mechanisms. It has been
suggested that a more responsive reporter system may be
achieved by destabilizing the reporter mRNA and reporter
protein, because the long half-lives of these molecules may
contribute to a signal delay and dilution.[6]
A deterministic model was completed to test the hypothesis
that the half-life of the reporter is a dominant factor in
determining the sensitivity of the assay. The model predicts
the transcription of a luciferase reporter with different delivery
schemes of TAT-HOXB4. The model’s components are
introduced, and background is provided for its design.
Simulations were conducted to test the hypothesis and results
are presented. Finally several simplifying assumptions and
their significance are discussed, and conclusions are offered.
II. MATERIALS AND METHODS
A. TAT-HOXB4
Exogenous HOXB4 is not able to translocate into the cell’s
nucleus, and hence affect DNA transcription. This has been
solved with TAT-HOXB4, a recombinant human fusion
protein, with the ability to translocate into a cell’s nucleus.[7]
The TAT-HOXB4 delivery scheme is the model’s input.
B. Luciferase Reporter
Firefly luciferase is a reporter protein that catalyzes a
reaction between D-luciferin and oxygen, in the presence of
ATP and Mg2+, to produce a luminescence signal:
2
, Mg
Luciferin  O2  ATP Luciferase


Oxyluciferin  hv  AMP  PPi  CO2 .
(1)
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This light emission has a maximum at 562 nm and can be
measured with a luminometer or a microplate reader.[8] In
addition to its advantage of having a high specific activity,
luciferase has a broad dynamic range and no endogenous
activity in mammalian cells.[3] Evaluating the intracellular
activity of TAT-HOXB4 is accomplished with a luciferase
reporter protein that has a promoter region with a TATHOXB4 binding site.
C. Hematopoietic Stem Cells
HSCs are responsible for the constant renewal of blood and
immune cells. Although it is desired to understand the
intracellular activity of TAT-HOXB4 in HSCs, they will not
be utilized as the reporter cell line. This is due to their low
abundance and the difficulty maintaining them in culture for
the purpose of producing and selecting a stably transfected cell
line.
D. HEK 293 Cells
Human Embryonic Kidney (HEK) 293 cells are used as the
reporter cell line as they are robust, abundant, and more likely
to survive the reporter gene vector transfection than are the
HSCs. Determined intracellular activity of TAT-HOXB4 with
a specific dosing scheme in the HEK 293 cell line can then be
correlated to the proliferation affect the same dosing scheme
had on HSCs in a parallel experiment. In this way the HEK
293 cells act as a vehicle to the luciferase reporter. A
fundamental assumption in this system is that the intracellular
activity of TAT-HOXB4 in HEK 293 cells can be correlated to
that in HSCs. This model only examines the intracellular
activity of TAT-HOXB4 in the HEK 293 reporter cell line.
Correlations to the affect of TAT-HOXB4 on HSCs are
beyond the scope of the model.
E. Model Design
To accurately interpret the intracellular activity of TATHOXB4, a sensitive reporter able to monitor a transient system
is required. Certain factors add complexity to the correlation
between the measured signal and the actual TAT-HOXB4
activity. TAT-HOXB4 has a half-life, t(HOXB4), of
approximately 1.1 h in cell culture.[7] While it is expressed,
each TAT-HOXB4 may bind sequentially to several promoter
regions, each time initiating the transcription of a signal
protein. DNA transcription generates mRNA for the signal
protein with a half-life of t(mRNA). Next this mRNA may be
translated several times to construct luciferase enzymes, which
each has a half-life t(Luc). Figure 1 represents the cascade of
events that must occur, in the in vitro system with the HEK
293 cells, for a luminescent signal event.
2
TAT-HOXB4
Expression in
System
TAT-HOXB4
Translocation
into Nucleus
TAT-HOXB4
Degradation
t(HOXB4)
TAT-HOXB4
Binds to
DNA
DNA
Transcription
mRNA
Translation
mRNA
Degradation
t(mRNA)
Luciferase
Catalyzes
Reaction
Luciferase
Degradation
t(Luc)
Luminescent
Signal
Fig. 1. Schematic sequence of events to occur for a resulting signal, where
t(HOXB4), t(mRNA), and t(Luc), represent the half-lives of TAT-HOXB4, the
mRNA, and luciferase, respectively.
The deterministic model was designed in Matlab and
followed the number of molecules of TAT-HOXB4, mRNA,
and luciferase enzyme in the system at time i, with the
following equations:
 ln 0.5

TATHOXB 4(i)  TATHOXB 4(i  1) exp 
 i 
 t ( HOXB 4)

(2)
 ln 0.5

mRNA(i)  mRNA(i  1)  k1  TATHOXB 4(i)  i exp 
 i 
 t (mRNA)

(3)
 ln 0.5
 (4)
Luciferase(i)  Luciferase(i  1)  k 2  mRNA(i)  i exp 
 i 
t
(
Luc
)


In all simulations, the time step Δi was one minute. Rate
constants for transcription (k1) and translation (k2) were
estimated from literature as 0.45 min-1 and 18 min-1,
respectfully. Since both processes involve multiple steps, such
as initiation and elongation, these values correspond to rates
for the limiting initiation step.[9] The rate of transcription
depended on the number of TAT-HOXB4 molecules, as each
initiated transcription. Also, the rate of translation was
dependent on the number of mRNAs available.
Three injections of TAT-HOXB4 at a regular time interval
Δh, were input to the system. Each injection was an input of
1000 molecules and was one minute in duration. To asses the
reporter sensitivity for different mRNA and luciferase halflives, the smallest Δh that resulted in a luciferase signal with
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three peaks was determined. Also, the maximum height of the
luciferase peak was recorded. Figures 2, 3, and 4 are sample
plots of the total TAT-HOXB4, mRNA, and luciferase
molecules, respectively, in the system. These plots were
output from the simulation where t(mRNA)=t(HOXB4)=t(Luc),
corresponding to trial 1 in Table 1. In this case, the time
interval Δh was determined as 160 minutes and the plots
illustrated follow this dosing scheme.
Fig. 4. Determination of the smallest Δh (160 min. for this simulation) that
results in three distinguishable regions in the luciferase signal. Shown here is
the total luciferase enzymes in the system for the TAT-HOXB4 injection
scheme shown in Figure 1, and t(Luc)=t(HOXB4).
III. RESULTS
Fig. 2. Total TAT-HOXB4 molecules in the system for an injection interval
of 160 min. The three peaks represent the three injections of 1000 molecules
each of one minute duration.
Simulations were conducted for different mRNA and
luciferase half-life ratios of t(HOXB4). Table 1 lists the
resulting Δh and maximum signal for each case.
TABLE 1
SENSITIVITY VALUES
Trial
t(mRNA)
t(Luc)
Δh
Max Signal Height
(#)
*
*
(min.)
(# luciferase enzymes)
1
t(HOXB4)
t(HOXB4)
160
4.1 x 107
2
0.5 t(HOXB4)
0.5 t(HOXB4)
100
1.5 x 107
3
2 t(HOXB4)
2 t(HOXB4)
225
1.1 x 108
4
0.5 t(HOXB4)
t(HOXB4)
125
2.5 x 107
5
t(HOXB4)
t(HOXB4)
185
6.5 x 107
6
t(HOXB4)
2 t(HOXB4)
185
6.5 x 107
7
t(HOXB4)
0.5 t(HOXB4) 125
* where t(HOXB4)=1.1 h
2.5 x 107
IV. DISCUSSION
Fig. 3. Corresponding total mRNA molecules in the system for the TATHOXB4 injection scheme in Figure 2. In this simulation, t(mRNA)=
t(HOXB4).
A. Reporter Sensitivity
From Table 1 it is evident that increasing the reporter halflife, increases Δh, consequently decreasing the sensitivity of
the reporter to the transient system. Conversely, as the
reporter half-life decreases, the maximum signal output is also
reduced, diminishing the reporter’s sensitivity. The latter is an
important factor when determining if the assay measurement
device, such as a luminometer, will correctly distinguish
between the assay signal and the background, when the assay
signal is low.
Also, an increase in the mRNA half-life caused the same
output as an increase in the luciferase protein half-life. This
may be due to the rates of transcription and translation being
of similar orders of magnitude.
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B. Model Assumptions
In the development of the model, many simplifying
assumptions were made. A fundamental assumption of the
system was that the intracellular activity of TAT-HOXB4 in
HEK 293 cells could correlate to that in HSCs.
Also, as the actual intercellular activity of TAT-HOXB4
was unknown, the reporter’s sensitivity to the number of TATHOXB4 molecules input to the system was determined. This
sensitivity was assumed to be proportional to that of the
biological intracellular activity.
The system was assumed to be well mixed and gradients
were not considered. In systems at equilibrium with simple
binding kinetics, it has been found that local ligand
fluctuations are often negligible.[10] Since a transient system
was modeled for this analysis, a more accurate and complex
model should include the probabilistic factor of concentration
gradients and extrinsic noise.
Dissociation events were not considered. The consequence
of this simplification was that the signal produced for each
simulation was greater than it should have been. This is
because with each coupling event in the cascade, there is the
possibility of an uncoupling event.
Furthermore, production of the luminescent signal was
dependent on the availability of luciferin, ATP, oxygen, and
Mg2+, however, these were assumed to be present in the system
in excess. The signal was also modeled as the rate of luciferase
production for simplicity, when in fact the catalyzed reaction
between D-luciferin and oxygen must take place to produce
the luminescent signal. This reaction has its own rate
constants, and the luminescent signal also has a half-life.
C. Stochastic Events
A deterministic model was created although stochastic events
may also be important. The stochasticity in the intrinsic
biochemical process of gene expression has been demonstrated
to impact the overall variation in a system. This causes cellcell variability in the amount of protein produced in isogenic
populations. For gene expression, this intrinsic noise should
decrease as the amount of transcript increases. [11] This
agrees with the chemical Langevin equation in that
randomness may go unnoticed if the population is large. [12]
From Figures 3 and 4, as well as the maximum signal in Table
1, the mRNA and luciferase populations are on the orders of
magnitude of approximately 104 and 107 molecules,
respectively. This may be large enough to assume that
stochastic events may go unnoticed.
No randomness was attributed to the cells, even though cell
populations are highly heterogeneous. [11] All cells behaved
identically, contained the same number of reporter genes, and
had the same rates of DNA transcription and translation.
Certain factors such as cycling status, may contribute to
variation in these parameters. Furthermore, any stochastic
factors at play may have different outcomes and resulting
variance in the HEK 293 cells versus the HSCs.
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V. CONCLUSION
The half-life of the reporter affects the sensitivity of the
assay. When designing or selecting a reporter for use in an
assay, certain factor must be balanced. A reporter with a short
half-life has a higher sensitivity for changes in transient
systems. However, a reporter with a long half-life has a higher
maximum signal, and may be easier for the measurement
device to distinguish from background noise.
When using reporter assays to measure promoter activity,
gene transfer, gene expression, and signaling pathways, an
input is deduced from the reporter’s output signal. However,
from Figure 4, it may not be trivial to work backwards and
predict the dosing scheme observed in Figure 2. This may
have implications on the accuracy of the reporter assay.
For these reasons, studies optimizing and understanding the
relationship between the transcription factor input and the
luciferase signal output must be accomplished before the
reporter may be exploited for delicate measurements such as
the therapeutic window of TAT-HOXB4 and its dosedependent effects. Additional investigations on the promoter
and its interaction with the transcription factor may also
provide insight. Slight alternations in the promoter region may
affect the transcription rate and may prove helpful for the
creation of a sensitive reporter.
VI. REFERENCES
[1]
Beslu, N., Krosl, J., Laurin M., Mayotte, N., Humphries, K.R., and
Sauvageau, G. (2004) Molecular interactions involved in HOXB4induced activation of HSC self-renewal. Hematopoiesis, 104, 8, 23072314.
[2] Klump, H., Schiedlmeiyer, B., and Baum, C. (2005) Control of SelfRenewal and Differentiation of Hematopoietic Stem Cells: HOXB4 on
the Threshold. Annals New York Academy of Sciences. 1044, 6-15.
[3] Naylor, L. H. (1999) Reporter Gene Technology: The Future Looks
Bright. Biochemical Pharmacology, 58, 749-757.
[4] Ignowski, J.M., and Schaffer D.V. (2004) Kinetic Analysis and
Modeling of Firefly Luciferase as a Quantitative Reporter Gene in Live
Mammalian Cells. Biotechnology and Bioengineering, 86, 7: 827-834.
[5] Saleh, M., Rambaldi, I., Yang, X.-J., and Featherstone, M. (2000) Cell
Signaling Switches HOX-PBX Complexes from Repressors to
Activators of Transcription Mediated by Histone Deacetylases and
Histone Acetyltransferases. Molecular and Cellular Biology, 20, 22,
8623-8633.
[6] Voon, D.C., Subrata, L.S., Baltic, S., Leu, M.P., Whiteway, J.M.,
Wong, A., Knight, S.M., Christiansen, F.T., and Daly, J.M. (2005) Use
of mRNA- and protein-destabilizing elements to develop a highly
responsive reporter system. Nucleic Acids Research. 33, 3.
[7] Krosl, J., Austin, P., Beslu, N., Kroon, E., Humphries, R.K., and
Sauvageau, G. (2003) In vitro expansion of hematopoietic stem cells
by recombinant TAT-HOXB4 protein. Nature Medicine, 9, 11, 14281432.
[8] MGT (2005) Product Information Sheet (0626-005): Live Cell Luciferase
Assay Kit-Product M0626.
[9] Bower, J.M. and Bolouri, H. (2001) Computational Modeling of
Genetic and Biochemical Networks. The MIT Press.
[10] Lauffenburger, D.A. and Linderman, J.J. (1996) Receptors: Models for
binding, trafficking, and signaling. Oxford University Press.
[11] Elowitz, M.B., Levine, A.J., Siggia, E.D., and Swain, P.S. (2002)
Stochastic Gene Expression in a Single Cell. Science, 297, 5584, 11831186.
[12] Gillespie, D.T. (2002) The Chemical Langevin and Fokker-Planck
Equations for the Reversible Isomerization Reaction. J. Phys. Chem.
106, 5063-5071.