Monitoring transfection efficiency by
green fluorescent protein (GFP) detection
with the Agilent 2100 bioanalyzer
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This Application Note describes how the Agilent 2100 bioanalyzer and
the Cell Fluorescence LabChip® kit can be used to determine the efficiency
of transfection of mammalian cells using green fluorescent protein
(GFP) as a reporter molecule. Transfection of CHO-K1 cells with
expression vectors encoding GFP from two different sources, and optimization
of transfection conditions for both plasmids were performed.
Histogram quality and the percent transfected cells determined, based
on the low number of fluorescent cells counted with the microfluidic
system, is in good agreement with data obtained with a conventional
flow cytometer. Detailed protocols and reagent recommendations for
analyzing transfection reactions are given. High reproducibility of chip
results, low cell consumption and ease-of-use are advantages the compact
Agilent 2100 bioanalyzer offers for monitoring transfection efficiency
using GFP.
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The Agilent 2100 bioanalyzer was
introduced by Agilent Technologies
as the first commercially
available lab-on-a-chip analysis
system for the life science laboratory
using LabChip®products,
developed by Caliper Technologies
Corp. Chip-based approaches
for a variety of separation-based
techniques have been introduced,
addressing DNA, RNA, and protein
separations. The Agilent 2100 bioanalyzer
is capable of two-color
fluorescence detection and runs
disposable microfluidic glass
chips. The application presented
here is based on the controlled
movement of cells through these
channels by pressure-driven flow.
Cells are hydrodynamically
focused in the channels before
passing the fluorescence detector
in single file. Each chip accommodates
several samples and data
acquisition of all samples is fully
automated while analysis allows
for user-specific settings. Specific
advantages of the instrument are
the low number of cells required
for analysis and the ease-of-use.
Among the first applications investigated
is the monitoring of cell
transfection efficiency.
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Transfection
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Transfection, the introduction of
foreign DNA into a eukaryotic cell,
is an important tool for studying
the regulation of gene expression
as well as protein expression and
function. In stable transfection,
the foreign DNA becomes integrated
into the genomic DNA of the
cell so that it is passed on in the
cell lineage and continues to express the encoded gene of interest.
More commonly used is transient
transfection, in which higher
copy numbers of the foreign DNA
and hence higher levels of gene
expression are present in the cell
for a brief period of time. There
are several methods available for
cell transfection such as formation
of complexes of the DNA with
either DEAE dextran or calcium
phosphate, to facilitate entry into
the cell by endocytosis, or electroporation,
which uses high voltage
pulses to form transient pores in
the cell membrane through which
the DNA can enter. Currently, the
most widely used method for
transfecting cells is with cationic
lipids that result in very high
transfection efficiencies with low
cytotoxicity.
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In order to determine the percentage
of cells in a transfection
experiment that have received and
are expressing the foreign DNA
sequence, a reporter gene can be
used. The reporter gene can be
present on the same vector as the
gene of interest or can be on a
separate plasmid. The reporter
gene can also be used to create a
fusion protein with the gene of
interest for protein localization
studies. A convenient reporter for
monitoring transfection efficiency
is the green fluorescent protein
(GFP). When excited by blue or
UV light, the protein emits bright
green fluorescence light through
cyclization of a tripeptide chromophore
embedded within the
complete amino acid sequence.
Genes encoding green fluorescent
proteins have been cloned from
various coelenterates such as the
jellyfish Aequorea victoria and the sea pansy Renilla reniformis. To
facilitate their use as reporters,
several GFP variants have been
developed by introducing amino
acid substitutions into the chromophore,
which result in a shift in
the emission wavelength as well
as an increase in fluorescence
intensity. Additional mutations
have been introduced to create
preferred human codons in order
to increase expression efficiency
in mammalian cells. Many expression
vectors containing these
humanized GFP variants are commercially
available. Expression of
GFP is typically detected by fluorescence
microscopy or flow
cytometry.
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In this Application Note, we
describe the use of the Agilent
2100 bioanalyzer to monitor transfection
efficiency using GFP as a
reporter. CHO-K1 cells were transfected
with GFP expression vectors
using a cationic lipid reagent,
and the percentage of GFP
expressing cells within the live
cell population was determined.
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Experimental
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Cell Culture
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CHO-K1 cells were obtained from
ATCC and cultured in F12 medium
containing 10 % FBS, 10 mM
HEPES, Pen/Strep, 1 mM sodium
pyruvate and 2 mM L-glutamine.
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Transfection
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pEGFP-C2 (Clontech) and phrGFP
(Stratagene) plasmid DNA was
purified using the Perfectprep
Plasmid XL kit (Eppendorf). Twenty
hours before transfection, CHOK1
cells were seeded in a 6-well
tissue culture plate at a density of
5 x 105 in 2 ml of growth medium
and incubated overnight. On the
day of transfection, 1 µg of plasmid
DNA was diluted into OPTIMEM
(Life Technologies) and
mixed with 6 µl of Lipofectamine
2000 (Life Technologies) according
to supplier’s protocol. Prior to
transfection, the growth medium
was replaced with 2 ml of OPTIMEM.
DNA-Lipofectamine complexes
were added to the cells and
incubated for 6 h. The transfection
medium was then replaced by
growth medium and cells were
incubated for an additional 18 h.
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Measuring transfection efficiency
with the 2100 bioanalyzer
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| 1. Cells were harvested and
resuspended at 1x106 cells/ml in
HBSS with 0.05 % pluronic acid
(Molecular Probes).
2. Cells were stained with 0.5µM
of the live cell dye carboxynaphthofluorescein
diacetate
(CBNF, Molecular Probes) in
the same buffer for 15 min at
room temperature.
3. Afterwards, cells were washed
in HBSS with 0.05 % pluronic
acid.
4. Stained cells were centrifuged
(500 x g, 5 minutes) and resuspended
in cell buffer (supplied
with the Cell Fluorescence
LabChip kit) at 2 x 106 cells/ml
5. 10 µl of the cell suspension was
applied to the sample wells of
the cell assay chip and analyzed
on the Agilent 2100 bioanalyzer. |
Approximately 600-800 cell events
were counted per sample. Parallel
samples were prepared for conventional
flow cytometer measurement.
Cells were photographed on
a Nikon fluorescence microscope.
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Results and Discussion
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For successful transfection of
GFP plasmid DNA into CHO-K1
cells, the optimal DNA:lipid ratio
was initially determined. As a
result, 6 µl of Lipofectamine 2000
was chosen as the optimal amount
for transfection of 1 µg of DNA in
6-well culture plates.
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Assay of transfection efficiency on
the 2100 bioanalyzer
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In this experiment, pEGFP-C2,
which encodes a red-shifted variant
of wild-type Aeqourea GFP
was used for transfection of CHOK1
cells. Mock- or EGFP-transfected
cells were stained with the live
cell dye CBNF and analyzed using
the 2100 bioanalyzer. Approximately
600 cell events were collected
for each sample. Figure 1A
shows the dot plots of the fluorescence
data (CBNF versus EGFP)
of the control and EGFP-transfected
cells. The population within the
rectangular region represents live
(CBNF-positive) and EGFPexpressing
CHO-K1 cells. The data
can also be displayed as frequency
histograms, as depicted in figure
1B. In this case, in order to determine
the percentage of EGFPexpressing
cells, live cells in the
CBNF-positive population were
cross-gated onto the EGFP histogram.
A parallel measurement of
the same cell samples on a flow
cytometer yielded a comparable
result (figure 1C). On average, 600
to 800 cell events were collected
for each sample run on the cell
assay chip, whereas 10,000 events
were collected using the flow
cytometer. The histogram quality
on both instruments was very
similar (figure 1C). Figure 2 shows
a photograph of the cell sample
obtained with a fluorescence
microscope.
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Reproducibility of GFP transfection
efficiency assay
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To determine the reproducibility
of the assay method using the
2100 bioanalyzer, the same mockor
EGFP-transfected CHO-K1 cells
were run on multiple cell assay
chips. On each chip, mock-transfected
cells were loaded in cell
well 1 and EGFP-transfected cells
in cell wells 2 to 6. Figure 3A
shows the individual data points
from 15 chips run on three different
instruments (5 chips per
instrument). Chip to chip reproducibility
of the assay is shown in
figure 3B. Figure 3C is a summary
of the transfection data from 75
measurements of the same cell
preparation assayed on the 2100
bioanalyzer. The transfection efficiency
as determined on the 2100
bioanalyzer is identical to the
result obtained using a conventional
flow cytometer.
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Using the 2100 bioanalyzer to monitor
optimization of transfection conditions
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Measurement of the transfection
efficiency and expression of GFP
in cells are typically done by fluorescence
microscopy or flow
cytometry. Each of these methods
has its drawbacks. Here we
demonstrate that the 2100 bioanalyzer
can be used to monitor quantitatively
the expression of GFP in
an exercise to develop an optimized
transfection protocol for
CHO-K1 cells. In the experiment,
titration of Lipofectamine 2000
reagent was performed to determine
the optimal DNA:lipid ratios
that gave the best transfection efficiency in 6-well culture plates.
In order to see if there are differences
in transfection and expression
levels of GFP from different
species, reporter plasmids encoding
green fluorescent proteins
from Aequorea (EGFP) and Renilla
(hrGFP) were used to transfect CHO-K1 cells. While keeping the
amount of GFP (EGFP or hrGFP)
plasmid DNA at 1 µg, the
DNA:lipid ratio was varied from 1
to 8 (ratios of 1, 2, 4, 6, and 8).
Twenty hours after transfection,
control cells and the 5 transfected
cell samples were stained with CBNF and loaded into the wells of
a cell assay chip and analyzed on
the 2100 bioanalyzer. Figure 4
shows the results obtained when
EGFP was used for transfection.
At a DNA:lipid ratio of 1, expression
of the reporter gene was
barely detectable. There was a 20
to 25-fold enhancement in activity
when the ratio was increased to 8.
It indicated that optimal transfection
and expression of EGFP were
achieved when a DNA:lipid ratio of 6 or 8 was used. A comparable
result was obtained when parallel
cell samples were measured on a
flow cytometer. The 2100
bioanalzyer results also correlated
with the those obtained by fluorescence
microscopy.
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When CHO-K1 cells were transfected
with phrGFP, which
encodes a humanized Renilla
GFP, there was a similar enhancement
in transfection and expression of the reporter gene as the
DNA:lipid ratio was increased
from 1 to 8 (figure 5A).
However, when compared to
EGFP, the transfection efficiency
of hrGFP was reduced (figure 5B)
and the hrGFP-expressing population
as a whole also showed a
substantially diminished fluorescence
intensity (figure 5C).
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Conclusion
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The Agilent 2100 bioanalyzer
microfluidic lab-on-a-chip system
can be used to monitor the transfection
efficiency and expression
of the GFP reporter gene in mammalian
cells. It is particularly useful
as a tool for optimization of
transfection conditions for different
transfection methods or new
cell lines. In contrast to fluorescence
microscope techniques, the
2100 bioanalyzer system provides
accurate, quantitative data for
optimization experiments, without
tedious manual cell counting. The
Agilent 2100 bioanalyzer is easy to
use and requires a short setup
time, while results for GFP
expression are comparable to data
obtained using conventional flow
cytometry. The cell assay chip is
disposable, can accommodate up
to 6 samples, and requires as little
as 20,000 cells per sample.
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