Transcript Doxycycline Concentration (ng/µl) Median Intensity

The behavior of a randomly integrated inducible system in human embryonic stem cells
Aman
1
Nihal ,
David
2
Vereide ,
2,3
Thomson
and James
1Madison
West High School, 30 Ash Street, Madison, WI
2Morgridge Institute for Research, 330 North Orchard Street, Madison, WI
3Department of Cell and Regenerative Biology, University of Wisconsin-Madison
INTRODUCTION
RESULTS
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Percent GFP Positive
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METHODS
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Doxycycline Concentration (ng/µl)
4 Days Treatment
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Doxycycline Concentration (ng/µl)
1 Day
4 Day
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Doxycycline Concentration (ng/µl)
1 Day
4 Day
C
1 Day Treatment
4 Days Treatment
Doxycycline Concentration
Doxycycline Concentration
D
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Mean Intensity
(Fluorescent Units)
Percent GFP Positive
B
B
F
A. Shown is the relationship between the
concentration of doxycycline in the medium and
the average percent of cells positive for GFP. The
cells were analyzed after one or four days of
exposure to doxycycline. Note how the amount of
doxycycline required to minimally activate SG-TRE
(above 3 ng/mL) is comparable at both time points.
For each concentration, the results of nine clonal
lines for one day exposure and three clonal lines
for four day exposure are shown (average ± S.D.).
A. Cells from A, presented as bar graphs for two
concentrations of doxycycline only. Note how
longer treatment causes a modest increase in the
number of GFP-positive cells and a stronger but
quite variable increase in the average intensity of
each positive cell, for doses just prior to saturation
(≤ 100 ng/mL). Thus, concentrations above 100
ng/mL often resulted in signals beyond the
detectable range (see C) for cells treated for four
days. For each time point, the results of three
clonal lines are shown (average ± S.D.).
B. These histograms compare the intensities of a
single clonal line (Clone 4, part of the above
compilations) at different concentrations of
doxycycline after one day (left) and four days
(right) treatment. The x-axis represents increasing
fluorescence intensity. Cells from each
concentration are depicted on separate rows.
These histograms reveal that GFP intensity tends to
be greater in the four day samples than in the one
day samples; in fact, the intensities of the four day
samples with high amounts of doxycycline are so
much greater that they go off the scale.
Intensity (Fluorescent Units)
Abbreviations: SG-TRE, second generation tetracycline-regulated promoter;
GFP, green fluorescent protein; EF1α, elongation factor 1α promoter;
IRES, internal ribosome entry site; PURO, puromycin resistance; FACS, fluorescence
activated cell sorting.
Phase contrast
GFP
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Doxycycline Concentration (ng/µl)
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Mean Intensity
(Fluorescent Units)
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Mean Intensity/
Max. Mean Intesity
A. The SG-TRE promoter containing binding sites for rtTA. When rtTA binds, which
occurs in the presence of doxycycline, GFP expression is allowed.
B. The rtTA-IRES-PURO vector to constitutively produce the rtTA necessary for the
SG-TRE promoter to function.
C. RNA of the rtTA-IRES-PURO vector. Arrows indicate where ribosomes will begin
translation; the IRES functions as an additional translational start site. The RNA
will produce rtTA for the control of the SG-TRE cassette and puromycin, a
selective agent.
D. Electroporation (the use of electricity to create holes in the cell membrane). The
holes allow the DNA vectors (green circles) and RNA (red lines, encoding
hyPBase, an enzyme that integrates the vectors into the genomic DNA) to enter
the cell. The cell membrane quickly repairs itself.
E. Colony formation. Cells were seeded in six well plates as individual cells, and
those that took up the vectors were selected with puromycin. The arrow
represents a pipette used to pick a colony of cells formed from a single cell.
Isolated colonies were further cultured until there were enough cells
(approximately one million cells) for analysis as in F, and harvested to isolate
genomic DNA.
F. Doxycycline titration. Clonal cell lines established in E were treated with varying
doses of doxycycline. The green and blue arrow represents the pipette used to
deliver the doxycycline to twelve well plates. Once treated for the desired
number of days, the cells were analyzed for GFP expression by FACS.
Median Intensity/
Max. Median Intensity
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Doxycycline Concentration (ng/µl)
1 Day Treatment
A
Figure 2: The activity of SG-TRE can increase
with longer exposure to doxycycline.
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Percent GFP Positive
 An inducible gene system is a biological system in which the expression of a gene
depends on the presence of a certain molecule (its inducer).
 Inducible gene systems can be tightly regulated, and therefore allow scientists to
control the expression and expression level of gene(s) of interest, thus enabling the
careful study of the specific role of gene(s) in cells.
 We were interested in determining how a particular inducible gene system (SGTRE) functioned in human embryonic stem (ES) cells.
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Figure 3: The activity of SG-TRE poorly correlates with vector copy number.
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Shown is the relationship between the number of integrated GFP expression vectors within each clonal line and
the intensity of GFP (measured at 30 ng/uL after 1 day treatment). There appears to be little correlation
between the two variables, perhaps an indication of a phenomenon known as silencing, in which cells over time
block the expression of a certain gene from occurring.
R² = 0.1178
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Doxycycline Concentration (ng/µl)
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Integrated Copy Number
CONCLUSIONS
Figure 1: The SG-TRE functions dose-dependently in human ES cells.
A. Phase contrast or fluorescent images of cells at increasing concentrations of doxycycline.
B. Shown is the relationship between the concentration of doxycycline in the medium and the average
mean or median intensity of GFP fluorescence. These data were collected to determine the
behavior of SG-TRE in human ES cells. Cells were analyzed after exposure to the indicated amounts
of doxycycline for one day. These graphs reveal the level of doxycycline required for a response as
well as a dose-dependent relationship between doxycycline and GFP expression. In nature we often
see a standard bell curve with the plurality of data points in the center of the curve. A characteristic
of the perfect bell curve is that the median and mean values are the same. By comparing those
values here, one can determine how well the observed induction of GFP approximates an ideal
induction. For each concentration, the results of nine clonal lines are shown (average ± S.D.).
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 We found a dose-dependent relationship between the concentration of doxycycline and gene expression from SG-TRE. In certain inducible systems there is not a dose-dependent
relationship, but rather complete expression of a gene once a certain dose is introduced to the system (“on or off”). The fact that the SG-TRE system is dose-dependent in human ES
cells indicates that scientists can use the system to tailor the expression of genes of interest.
 We also determined that the activity of SG-TRE increases with longer exposure to doxycycline. This observation will be an important factor for experiments in which the induced
gene is expressed for durations greater than one day.
 Finally, we found a poor correlation between the copy number of vectors introduced to the system and the activity of SG-TRE, an effect likely due to gene silencing. This result
underscores the importance of having multiple integrated vectors, so that at least one is operational.