Download The Use Of Nitroreductase And Fluorescence Activated Cell Sorting

The Use Of Nitroreductase And Fluorescence Activated
Cell Sorting To Select Transiently Transfected Cells
Ian D. Goodyer*, Adrian Cushing, Nick Thomas, Paul Michael, Rhian Martin, Rahman Ismail, Helen Cox, Hayley A. Tinkler, Stephen Game
Amersham Biosciences UK Limited, The Maynard Centre, Forest Farm, Whitchurch, Cardiff, CF14 7YT, U.K.
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Fig. 1 Schematic representation of the nitroreductase reporter
assay. Activation of an intracellular signaling pathway will
ultimately lead to gene expression of the gene reporter.
E.coli nitroreductase B, a 48kDa flavoprotein may be
expressed in a wide variety of mammalian cells. Many
fluorescent molecules may be quenched by the addition of
nitro-groups. Reduction of these groups by nitroreductase
restores fluorescence. This mechanism has been used to
develop a novel, flexible, fluorescence reporter gene system
that can be used, in parallel with other fluorescence
techniques, to monitor molecular events in single living cells.
This system is based on the use of bacterial nitroreductase
(NTR) and cell permeable cyanine Fluor analogues.
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Relative Red fluorescence
Fig.3. FACS analysis of SKOV cells. Parental SKOV cells
and cells stably expressing NTR under the control of the
CMV promoter were incubated in 2 µM Cy5Q ethyl ester
(Cy5Qee) at 37ºC for 2 hours before sorting by flow
cytometry. NTR expressing cells exhibited increased red
fluorescence relative to the control cells allowing them to be
sorted based on their level of expression.
1. Mix NTR and GFP
expression vectors
10 4
10 3
101
10 2
In a dual transfection (Figure 4) it would be expected that the
relative expression levels of the two constructs would be
proportional. The highest expressing NTR cells would therefore
be expected to also express the gene of interest to the highest
level. Figure 5 shows HeLa cells that were transfected with
GFP and NTR. By selecting for NTR expressing cells it is
possible to collect the highest expressing GFP cells. This has
traditionally been difficult without a lengthy selection procedure.
This method has a number of applications including:
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10 4
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10 2
10 1
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R1
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1 01
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1 01
R3
§
R2
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1 03
No cells are
transfected with
NTR only
§
Dual transfected
cells
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Using NTR it is possible to select for cells transiently expressing
the construct and following the sort to return them to culture or
perform experiments on only transfected cells.
By selecting for cells that have been transfected noninformative cells are removed from the analysis. Statistically
this increases the power of the experiment as a higher
proportion of cells will respond and therefore the difference
between a positive response and controls (the signal to noise
ratio) will be increased.
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1 01
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Relative Green fluorescence (log scale)
NAD(P)H
R
N O2
Selection of cells expressing a gene of interest that is not
inherently fluorescent and where there is no nondestructive detection system available.
Selection of cells that have been transfected with a toxic
gene that needs to be silenced.
Selection of cells where the expression of the gene of
interest requires induction as part of the assay.
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10 4
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R2
Untransfected
cells
Number of events
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R3
10 2
SKOV NTR stable cell line
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GFP transfected
cells
NTR transfected
cells
Parental SKOV
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R1
Untransfected
cells
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Results
Figure 3 shows that on the FACS a population of cells
expressing NTR can be distinguished from non-expressing
cells. By collecting the brightest cells it is possible to select only
cells expressing NTR. With the current NTR substrate it is not
possible to collect lower expressing cells as the background
cells overlap with the low expressing NTR cells. Future
developments in the NTR technology will increase the signal to
background ratio and allow a finer discrimination of expressing
and non-expressing cells.
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reporter gene is inserted into a suitable plasmid vector. The
inducible transcriptional control element drives the
expression of the reporter gene. The vector DNA is
introduced into cells by transient or stable transfection and
the level of expression of the reporter is assayed by an
enzyme reaction.
Most reporter gene assays are
destructive (i.e. they require cell lysis) as the enzyme
substrate either does not work under physiological
conditions or it is cell impermeable.
In contrast the
nitroreductase gene reporter system uses a cell permeable,
non-toxic substrate.
Red fluorescence (log scale) Red fluorescence (log scale) Red fluorescence (log scale)
Introduction
The regulation of gene expression in mammalian cells is a
complex process involving contributions from one or more
signal transduction pathways. To study such complex control
processes it is advantageous to have the capability to study
multiple events occurring in the same cell. Reporter gene
technology is widely used to monitor the cellular events
associated with signal transduction and gene expression. A
reporter gene is a gene with a readily measurable phenotype
that can be distinguished easily over background of
endogenous proteins.
NAD(P)
R
NHOH
Fig.2. The enzyme action of nitroreductase. In the presence
of NADH or NAD(P)H, one or more –NO2 groups on an
organic molecule are reduced to hydroxylamine, which may
subsequently be converted to an amine.
2. Add cationic lipid to form
liposomes
All liposomes contain a
mixture of both
expression vectors
3. Transfect into cells
NTR enzymes have been shown to catalyze the general
reaction shown in figure 2.
Principle
In a reporter gene assay the reporter gene is placed under
the transcriptional control of a promoter. The promoter plus
Fig.4.The principle of dual transfection. As the DNA is
mixed prior to transfection any cells that are transfected will
express both constructs.
*The w ork above is described in patent WO 0157237. This product is developed under license from University of Iowa Research Foundation under patents US 5168062 and US 5385839.
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Fig.5. Transient expression of GFP and NTR in HeLa
cells. Both genes are expressed under the control of
the CMV promoter. In the top panel the cells were
transfected with GFP only – as expected the cells show
a range of green fluorescence corresponding to a range
of GFP expression levels and very little red
fluorescence is observed. In the central panel cells
were transfected with NTR only. As expected only low
green fluorescence is observed and a range of red
fluorescence levels are obtained. In the lower panel the
cells were co-transfected with GFP and NTR. No cells
are seen that express NTR and that do not express
GFP.
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CONCLUSION
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Cells expressing nitroreductase can be separated from nonexpressing cells by flow cytometry following addition of
Cy5Qee.
Following selection by flow cytometry NTR expressing and
Cy5Qee stained cells ar e viable
Co-transfection using two plasmids produces a population of
cells that express both constructs. Very few cells that
express NTR alone are produced.
This allows cells
expressing the gene of interest to be selected by flow
cytometry when it is not inherently fluorescent or when it is
silenced.
Selection of transiently expressing cells from non-expressing
cells in the population will increase the sensitivity of the
assay by increasing the % of responding cells and
increasing the signal/noise ratio.
This poster was presented at the 8 annual Conference of the Society of Biomolecular
Screening, 22-26 September 2002. The Hague, Netherlands. Poster #2436
* To whom all correspondence should be addressed.