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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 A Percent GFP Positive A METHODS 80 60 40 20 80 60 40 20 0 1 10 100 1000 Doxycycline Concentration (ng/µl) 4 Days Treatment C 80 60 40 20 0 120000 100000 80000 60000 40000 20000 0 30 100 Doxycycline Concentration (ng/µl) 1 Day 4 Day 30 100 Doxycycline Concentration (ng/µl) 1 Day 4 Day C 1 Day Treatment 4 Days Treatment Doxycycline Concentration Doxycycline Concentration D 100 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 1 0.8 0.6 0.4 0.2 0 1 10 100 1000 Doxycycline Concentration (ng/µl) 12000 Mean Intensity (Fluorescent Units) B 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 E 0 1 10 100 1000 Doxycycline Concentration (ng/µl) 1 Day Treatment A Figure 2: The activity of SG-TRE can increase with longer exposure to doxycycline. 100 100 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. 1 0.8 0.6 0.4 0.2 Figure 3: The activity of SG-TRE poorly correlates with vector copy number. 10000 8000 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 6000 4000 2000 0 0 1 10 100 1000 Doxycycline Concentration (ng/µl) 0 5 10 15 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.). 20 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.