9 research outputs found
The genomic locus of the TCRα LCR and transgene constructs.
<p>(<b>A</b>) Scale diagram of the TCRα/Dad1 genomic locus containing the TCRα LCR. (<b>B</b>) Diagrams (not drawn to scale) of the three heterologous TCRα LCR reporter transgenes used in these studies. Note that the hCD2 transgene is in the position of the TCRα gene with respect to the LCR sequences. In contrast, the HLA-B7 reporter gene is in the position of the Dad1 gene. Vertical arrows and numbers indicate the nine identified DNase I hypersensitive sites (HS) of the LCR. Horizontal arrows indicate the transcription orientation of the genes depicted. Solid boxes indicate exons. The asterisk denotes the placement of a premature stop codon.</p
Ectopic TCRα/hCD2 reporter gene activity is absent in B cells of TCRα/Dad1 dual-reporter gene BAC transgenic mice.
<p>(<b>A</b>) Real-time reverse transcriptase-mediated PCR experiments on RNA prepared from thymocytes and isolated B cells from individual TCRα/Dad1 dual-reporter BAC transgenic mice. Y-axis values indicate the relative hCD2-Cα reporter (open bars) and endogenous TCRα (filled bars) mRNA levels within an individual mouse (thymocyte level designated as 100%). hCD2-Cα reporter mRNA signals ranged from ∼10–20% of endogenous TCRα mRNA levels. The normalizing control mRNA was β-actin. Experiments on two separate individual mice per each of two independent BAC transgenic lines are shown. (<b>B</b>) The Dad1 promoter on the BAC is active in B cells. Real time RT-PCR detection of BAC-resident, Dad1 promoter-driven rCD2 reporter expression from B cells isolated from individual BAC transgenic mice. The data are normalized to endogenous Dad1 mRNA levels.</p
Ectopic TCRα LCR-driven upstream reporter gene (hCD2) expression is strongly suppressed by adding a second gene (HLAB7) downstream of the LCR.
<p>(<b>A</b>) Flow cytometry analyses of spleen lymphocytes from (at left) single-reporter hCD2-LCR (10 transgene copies) and (at right) two-reporter hCD2-LCR-B7 (3 transgene copies) transgenic mice. hCD2 levels are shown for spleen T cells (gated on Thy1+ cells) and spleen B cells (gated on CD19+ cells). Dashed curves indicate the non-transgenic spleen control signal. (<b>B</b>) Graph depicting mean fluorescence intensity (MFI) of hCD2 reporter protein staining in paired samples of thymocytes and isolated B cells (from the same mouse). Shown are three independent lines each of single-reporter hCD2-LCR and two reporter hCD2:1-8:B7 transgenic mice. MFI is normalized to thymocyte hCD2 expression (100%). In all cases, the substantial ectopic B cell expression of hCD2 seen from the single-reporter transgene is virtually eliminated in the presence of a second reporter gene downstream of the LCR.</p
Lymphoid organs express the highest levels of both hCD2 and HLA-B7 reporter transgenes.
<p>PhosphorImager analyses of northern blots of RNA prepared from the indicated tissues of the hCD2:1-8 (<b>A</b>), hCD2:1-8:B7 (<b>B</b>) or 1:8-B7 (<b>C</b>) transgenic mice. Reporter mRNA levels are quantified and normalized to 18S rRNA signal. Y-axis values represent the mean (+/− S.D.) expression levels relative to the thymus (designated as 100%) observed among three independent lines of mice bearing the indicated transgene.</p
HLA-B7 reporter mRNA expression in B cells is not suppressed by the presence of a second reporter gene upstream of the TCRα LCR.
<p>PhosphorImager analyses of representative northern blot (bottom right inset) analyzing levels of spleen B cell expression of hCD2 and HLA-B7 mRNA relative to those observed in thymus (designated as 100% for all lines) in three independent lines (10, 17, 22) of hCD2:1-8:B7 transgenic mice. Reporter signals are normalized to 18S loading control signal. HLA-B7 expression levels in B cells (B), relative to thymus (T), from this dual-reporter transgene construct is similar to that seen in single-reporter 1-8:B7 transgenic mice (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015527#pone-0015527-g005" target="_blank">Fig. 5</a>). Levels of hCD2 mRNA from the dual-reporter hCD2:1-8:B7 transgene are as low, relative to thymus, as the hCD2 protein signals detected in flow cytometry (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015527#pone-0015527-g004" target="_blank">Fig. 4</a>).</p
The TCRα LCR drives significant mRNA expression levels of a 3′-linked HLA-B7 reporter gene in B cells.
<p>(<b>A</b>) Northern blot analyses of RNA prepared from thymocytes and isolated spleen B cells from three independent lines of 1:8-B7 transgenic mice. Thy  =  Thymus, SpB  =  Spleen B cells, NTG  =  non-transgenic. (<b>B</b>) Graph depicting PhosporImager analyses of HLA-B7 reporter expression levels normalized to 18S rRNA. Y-axis values are expressed relative to thymus mRNA levels (designated as 100%).</p
Increased T cell-selectivity of epigenetic hCD2 promoter chromatin activation in the dual-reporter transgene context.
<p>(<b>A</b>) Chromatin immunoprecipitation assay on thymocytes and isolated spleen B cells detecting the trimethyl-lysine 4 epigenetic mark on Histone H3 from single reporter (hCD2:1-8 line 4) and dual-reporter (hCD2:1-8:B7 line 10) transgenic mice. Y-axis values represent the ratio of percent H3K4me3 marks obtained at the hCD2 promoter to that detected at the endogenous GAPDH promoter, an internal standard used here as a normalizing control. (<b>B</b>) Confirmation of ChIP results using distinct, independent lines of transgenic mice bearing single reporter (hCD2:1-8 line 29) and dual reporter (hCD2:1-8:B7 line 22) transgenes. For both experiments, the Y-axis values are derived from the formula hCD2 [H3K4me3 – IgG/input]/GAPDH [H3K4me3 – IgG/input].</p
Integration site-independent expression of two reporter transgenes concurrently flanking the TCRα LCR.
<p>(<b>A&C</b>) Northern blot analyses of thymocyte mRNA from hCD2 and HLA-B7 reporter trasngenes linked concurrently (or individually) to opposite sides of the TCRα LCR. Transgenic line numbers are indicated. NTG  =  non-transgenic. (<b>B&D</b>) PhosporImager analyses of northern blot experiments. The graphs depict the mean (+/− S.E.) of three representative experiments. Y-axis values indicate the percent of the maximum transgenic hCD2 mRNA signal per copy (normalized to endogenous TCRα mRNA signal) observed in each experiment. The copy number-related (within a narrow 2–3 fold range) characteristic of full LCR activity is orientation-independent and conferred upon both reporter genes in single-reporter and dual-reporter transgene contexts.</p
A TCRα/Dad1 reporter BAC transgene harbors an active Dad1 promoter.
<p>(<b>A</b>) Diagram of the TCRα/Dad1 dual-reporter BAC construct. VαhCD2 refers to the TCRα V-region promoter driving a human cytoplasmic tail-less human CD2 cDNA. The rCD2 refers to the BAC-resident Dad1 promoter driven rCD2 reporter cDNA. The TCRα constant region exons (Cα), HS of the TCRα LCR (small arrows) and Dad1 exons are shown. The Not I sites shown are ∼78-kb apart. (<b>B</b>) Representative northern blot analysis of rCD2 reporter expression from BAC transgenic line 86 in the indicated organs. The 18S signal is used as a loading control. (<b>C</b>) PhosphorImager analyses of northern blot experiments examining rCD2 reporter gene expression in the indicated organs of individuals representing two independent BAC transgenic mouse lines. The distribution of mRNA levels is expressed relative to the levels observed in the thymus.</p