40 research outputs found
Inducible recombination is restricted to serotonergic neurons of adult TPH2-CreERT2/CAG-loxP.EGFP rats.
<p>(A) TPH2-CreERT2 rats were bred to CAG-loxP.EGFP rats to generate double-transgenic TPH2-CreERT2/CAG-loxP.EGFP rats. Under uninduced baseline conditions, the loxP-flanked lacZ minigene is expressed reflecting cell-type specific CAG-promoter activity. Upon Cre-mediated recombination (+ Tamoxifen), lacZ is replaced with the second reporter gene enhanced green fluorescent protein (EGFP). The appearance of EGFP serves as an indicator of Cre mediated recombination in double transgenic rats. TPH2-CreERT2/CAG-loxP.EGFP rats were daily injected with tamoxifen (40 mg/kg) or vehicle for five consecutive days starting between P60–90. Coronal sections show dual-label fluorescence immunohistochemistry for TPH/βgal (B,E,H,K) and TPH/GFP (C,F,I,L) in vehicle-treated rats (-Tx) and TPH/GFP in tamoxifen-treated (+Tx) rats (D,G,J,M). Colocalization is visualized at the level of caudal raphe nuclei (CR) (B–D), dorsal raphe nuclei (DR) (E–J) and median raphe nuclei (MR) (K–M) using confocal images. In vehicle-treated rats, TPH2-CreERT2/CAG-loxP.EGFP rats display strong basal, non-recombined βgal expression in TPH2+ 5-HT neurons (B,E,H,K) making these rats ideally suited to monitor tamoxifen-induced Cre-mediated recombination in 5-HT neurons. (C,F,I,L) Without tamoxifen treatment, background recombination, i.e. EGFP expression (arrows) hardly occurs. (D,G,J,M) After tamoxifen treatment, the majority of TPH+ 5-HT neurons in all raphe nuclei now show EGFP expression indicating Cre-mediated recombination in these neurons (GFP+/TPH+). Scale bars: 100 µm.</p
Cre expression is restricted to serotonergic neurons of the raphe nuclei.
<p>(A,C,E,G) DAB-immunohistochemistry with a Cre antibody of line #15 shows Cre staining in the brain stem and mid-brain, regions which contain serotonergic somata while extraserotonergic brain regions show no staining. (B,D,F,H) Coronal sections of dual-label fluorescence immunohistochemistry with Cre and TPH1 antibodies. The TPH1 antibody crossreacts with TPH2 and detects both isoenzymes. Colocalization of TPH1 and Cre confirms exclusive Cre expression in 5-HT neurons of the raphe nuclei. Caudal raphe nuclei (CR); dorsal raphe nuclei (DR); median raphe nuclei (MR). Scale bars: 100 µm.</p
Recombination efficacy and background recombination for rat TPH2-CreERT2 line #15.
<p>Caudal (CR), median (MR) and dorsal (DR) raphe nuclei were separately and jointly (total) calculated. Confidence-bounds (CI) were calculated using the Clopper-Pearson method based on significance level 95.0%.</p
Baseline βgal expression in the brain of CAG-loxP.EGFP Cre reporter rats.
<p>(A,B) X-Gal staining of sagittal sections shows ubiquitous βgal activity throughout the brain of adult CAG-loxP.EGFP rats (P90). (C–K) Dual-label fluorescence immunohistochemistry (IHC). (C–E) βgal/NeuN IHC of the cerebellum (C), cortex (D) and OB (E) shows strong colocalization of βgal with the neuronal marker NeuN. (F–H) βgal IHC with the serotonergic marker TPH2 (F), and the dopaminergic and noradrenergic marker tyrosine hydroxylase (TH) (G,H) shows abundant colocalization of βgal with 5-HT neurons in the dorsal raphe (F), with dopaminergic neurons in the ventral tegmental area and substantia nigra (G) and noradrenergic neurons in the locus coeruleus (H) confirming strong βgal expression in all monoaminergic neurons. (I,J) βgal/GAD67 IHC shows βgal expression in GABAergic neurons of the granular layer of the OB (I) and in the hippocampus (J). (K) βgal/GFAP IHC in the hippocampus shows infrequent βgal expression in glia. OB, olfactory bulb; DR, dorsal raphe nuclei; VTA, ventral tegmental area; SN, substantia nigra; LC, locus coeruleus; HC, hippocampus. Scale bars: 100 µm.</p
Transforming Growth Factor Beta Receptor 2 (TGFBR2) Changes Sialylation in the Microsatellite Unstable (MSI) Colorectal Cancer Cell Line HCT116
<div><p>Aberrant glycosylation is a common feature of many malignancies including colorectal cancers (CRCs). About 15% of CRC show the microsatellite instability (MSI) phenotype that is associated with a high frequency of biallelic frameshift mutations in the A10 coding mononucleotide microsatellite of the <i>transforming growth factor beta receptor 2</i> (<i>TGFBR2</i>) gene. If and how impaired TGFBR2 signaling in MSI CRC cells affects cell surface glycan pattern is largely unexplored. Here, we used the TGFBR2-deficient MSI colon carcinoma cell line HCT116 as a model system. Stable clones conferring doxycycline (dox)-inducible expression of a single copy wildtype <i>TGFBR2</i> transgene were generated by recombinase-mediated cassette exchange (RMCE). In two independent clones, dox-inducible expression of wildtype TGFBR2 protein and reconstitution of its signaling function was shown. Metabolic labeling experiments using the tritiated sialic acid precursor <i>N</i>-acetyl-D-mannosamine (ManNAc) revealed a significant decline (∼30%) of its incorporation into newly synthesized sialoglycoproteins in a TGFBR2-dependent manner. In particular, we detected a significant decrease of sialylated ß1-integrin upon reconstituted TGFBR2 signaling which did not influence ß1-integrin protein turnover. Notably, TGFBR2 reconstitution did not affect the transcript levels of any of the known human sialyltransferases when examined by real-time RT- PCR analysis. These results suggest that reconstituted TGFBR2 signaling in an isogenic MSI cell line model system can modulate sialylation of cell surface proteins like ß1-integrin. Moreover, our model system will be suitable to uncover the underlying molecular mechanisms of altered MSI tumor glycobiology.</p> </div
Generation of dox-inducible HCT116-TGFBR2 cell lines.
<p>(A) Schematic outline of recombination-mediated cassette exchange (RMCE). Retroviral transduction was performed using the proviral vector S2F-cLM2CG-FRT3 that contains a bidirectional dox-inducible promoter (P<sub>tet</sub>bi) allowing concurrent expression of two marker genes (<i>luciferase</i> and <i>mCherry</i>) in HCT116-mCherry clones. Expression cassettes are flanked by mutant (F3) and wildtype Flp-recombinase target sites (F) that allow directed cassette exchange via Flpo-recombinase. Retroviral packaging signal (Ψ<sup>+</sup>) and long terminal repeats (LTR) are indicated. (B) Characterization of viral integration sites by nrLAM-PCR and sequencing. In the upper part, clone-specific integration sites and affected genomic loci (open reading frame (orf); <i>Aldehyde dehydrogenase family 1 member L1</i> (<i>ALDH1L1</i>)) are depicted. In the lower part, 5′- and 3′- viral LTR DNA sequences (small letters) as well as the flanking genomic DNA sequences (capital letters) are shown.</p
Incorporation of <sup>3</sup>H-labeled monosaccharides.
<p>Radioactive labeling experiments were performed in the presence and absence of dox (0.5 µg/ml) and by exposure to TGF-ß1 (10 ng/ml) for 72h. (A) Incubation with <sup>3</sup>H-ManNAc resulted in a significant reduction of incorporated ManNAc in the TGFBR2 clones #5 and #22 but not in the parental HCT116-Tet-On cell line. (B) Incorporation of <sup>3</sup>H-L-fucose was slightly reduced in presence of dox in both TGFBR2 clones in contrast to HCT116-Tet-On cells. Values represent the mean of three independent experiments ±S.D.</p
Reconstitution of TGFBR2 signaling.
<p>(A) Phosphorylation of SMAD2 (pSMAD2) was detected by Western blot analysis. Treatment with dox (1 µg/ml) and TGF-ß1 (10 ng/ml) displayed higher levels of pSMAD2 in comparison to cells grown in the absence of dox. The parental HCT116 cell line served as negative control, whereas TGF-ß1 responsive HepG2 cells were used as a positive control. Total SMAD2 has been used as a loading control. (B) Target gene transcription of TGFBR2 signaling. Real-time RT-PCR experiments revealed dox-dependent <i>SMAD7</i> and <i>SERPINE</i> upregulation. Data are shown for HCT116-TGFBR2 clone #5 but also apply to clone #22 (data not shown). Values represent the means of three independent experiments ±S.D.</p
Cyclin A1 expression does not significantly influence PML-RARα-driven leukemogenesis.
<p>A. Kaplan-Meier survival curves of heterozygous PML-RARα-knockin mice with (SCL-cyclinA1-tg; n = 12) or without (control; n = 14) ectopic human cyclin A1 expression in the bone marrow. Although there was a trend towards an accelerated disease in the presence of ectopic cyclin A1 expression, latency and penetrance did not differ significantly between the two genotypes (p = 0.282, log-rank test). Also, the phenotype of acute myeloid leukemia was not changed by the presence of cyclin A1 (B). Shown here are examples of FACS analysis of bone marrow (upper panels) and spleen cells (lower panels) of diseased mice. Murine PML-RARα-driven leukemic blasts are characterized by the surface expression of CD34 and GR-1 which does not occur in non-leukemic mice. The number of CD34<sup>+</sup>/GR-1<sup>+</sup> cells in diseased mice did not alter significantly upon cyclin A1 expression. SCL, Stem Cell Leukemia enhancer driving tTA expression [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129147#pone.0129147.ref023" target="_blank">23</a>]; FI, fluorescent intensity; P/Rα-KI, PML-RARα-knockin mice. C. Kaplan-Meier survival curves of PML-RARα-knockin mice with wild type (PML-RARα-KI/cyclin A1<sup>+/+</sup>; n = 30) or cyclin A1-knockout (PML-RARα-KI/cyclin A1<sup>-/-</sup>; n = 35). Cyclin A1<sup>-/-</sup> mice without PML-RARα-expression did not develop a lethal phenotype (control/cyclinA1<sup>-/-</sup>; n = 3). Absence of murine cyclin A1 did not affect PML-RARα-driven leukemia. D. The PML-RARα-leukemic phenotype was not altered by the absence or presence of murine cyclin A1. May-Grünwald staining of blood smears showed the same distribution of leukemic blasts and high numbers of differentiated myeloid cells (upper panels). FACS analysis revealed comparable numbers of myeloid cells in the blood (CD11b<sup>+</sup>/GR-1<sup>+</sup>, lower panels).</p
Development of a cyclin A1-transgenic mouse model.
<p>A. Schematic overview about the constructs used to develop transgenic mouse lines. The driver mouse line SCL-tTA expresses the tetracycline-dependent transactivator protein (tTA) in hematopoietic stem cells under the control of the stem cell leukemia-factor enhancer [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129147#pone.0129147.ref023" target="_blank">23</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129147#pone.0129147.ref024" target="_blank">24</a>]. In the novel cyclin A1-bi-luciferase responder mouse line, the cDNA of cyclin A1 and luciferase as reporter gene were expressed in absence of tetracycline in parallel and inducibly under control of the bidirectional tTA-responsive promoter element Pbi-1. B. Bars indicate expression levels of human cyclin A1 expression as detected by qRT-PCR in bone marrow cells that were transduced with a retroviral tTA-containing construct and cultured with or without tetracycline in methylcellulose for 10 days (n = 2 for each sample). C. Three months old mice carrying either cyclin A1 alone (control) or together with the driver construct SCL-tTA (SCL-tTAxcyclinA1-tg; n = 3 for each genotype) were induced for seven weeks and investigated for luciferase activity and cyclin A1 mRNA in the bone marrow and spleen. High luciferase activity was only detectable in induced SCL-tTAxcyclinA1-tg bone marrow and spleen cells. Numbers indicate mean luciferase levels.</p