Doxycycline-induced transcription of the GFP gene.

<p><b>A</b>. Quantification of doxycycline-induced GFP transcription in GFP(CAG)₈₉ and GFP(CAG)₀ cell lines. Cells were induced with doxycycline for three days prior to analysis. Two independent primer sets, both specific for GFP exon 2, were used to amplify GFP transcripts by quantitative RT-PCR. For each primer set, the individual results were internally normalized to β-actin and then to the levels of GFP transcripts in uninduced GFP(CAG)₀ cells. The values for the uninduced levels of GFP transcripts in GFP(CAG)₈₉ cells are indicated. The increase over uninduced levels in GFP(CAG)₈₉ cells was 76-fold for primer set 1 and 98-fold for primer set 2. <b>B</b>. Transcription-induced changes in numbers of GFP+ cells in GFP(CAG)₈₉ cells. Cells were treated with doxycycline for 0, 24, or 48 hours prior to analysis for GFP+ cells by flow cytometry, using the High gate indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113952#pone-0113952-g004" target="_blank">Figure 4A</a> to define the population of GFP+ cells. The frequencies of GFP+ cells at 0, 24, and 48 hours, respectively, were 0.02±0.013%, 0.20±0.02%, and 0.37±0.01%, as determined by counting three samples of 50,000 cells. C. Kinetics of induction of GFP expression in GFP(CAG)₀ cells. Doxycycline (2 µg/mL) was added at time 0 to wells of 6-well plates containing 100,000 GFP(CAG)₀ cells. Individual wells were harvested at the indicated times and analyzed by flow cytometry, using the High gate indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113952#pone-0113952-g004" target="_blank">Figure 4A</a> to define the population of GFP+ cells.</p

GFP fluorescence in GFP(CAG)₈₉ cells and in GFP(CAG)₀ cells before and after addition of doxycycline.

<p><b>A</b>. Fluorescence microscopy. Brightfield and fluorescent images of the same cell populations are shown. <b>B</b>. Flow cytometry. In each cell line, with or without added doxycycline, 50,000 cells were analyzed by flow cytometry and plotted as a histogram. Although not shown, unmodified T-REx cells—in the presence or absence of doxycycline—give a distribution that is indistinguishable from uninduced GFP(CAG)₈₉ cells.</p

Relationship between GFP fluorescence intensity and CAG repeat tract length.

<p><b>A</b>. Isolation of cells from different parts of the distribution of fluorescence intensity. Induced GFP(CAG)₈₉ cells were sorted according to the indicated gates, single cells were grown into colonies, and their CAG tracts were amplified by PCR and sequenced. <b>B</b>. Distribution of tract lengths in cells sorted by fluorescence intensity. <b>C</b>. Fluorescence intensity of cells with different CAG tract lengths.</p