Additional file 4: of MiR-210 promotes sensory hair cell formation in the organ of corti

Table of miRNAs normalized in reads-per-million (RPM). The number of reads of each miRNA was normalized by dividing the raw counts by the total number of million aligned reads per sample, i.e. reads per million (RPM). (XLSM 98 kb

Additional file 3: of MiR-210 promotes sensory hair cell formation in the organ of corti

Table of miRNA raw counts. Raw counts from sequencing of each of the small miRNA libraries. The number of sequences that correspond to each miRNA is listed. (XLSM 60 kb

Additional file 1: of MiR-210 promotes sensory hair cell formation in the organ of corti

Number of miRSeq reads and alignment statistics. (XLSM 8 kb

Normal T cell development, reduced splenic cellularity, and increased LN cell number in inducible Sgpl1-deficient mice.

<p>B and T cell subpopulations in tamoxifen-treated Sgpl1^Flox/FloxCre^+/− (open bars) and Sgpl1^Flox/Flox Cre^−/− mice (closed bars) (n = 4/group), were enumerated based on total live cell counts and cell proportions as established by flow cytometry. <i>A</i>, Thymus; <i>B, C</i>, spleen; <i>D, E</i> lymph nodes. In <i>C</i> and <i>E</i>, CD8 and CD4 T cells were analysed for co-expression of CD44 and CD62L to define populations of naive and memory T cells; the insert in <i>C</i> provides a gating example for naïve/memory type T cells.</p

Foxp3⁺ Treg are overrepresented in LN and spleen of in inducible Sgpl1-deficient mice.

<p>Two weeks after tamoxifen treatment, cells from blood, LN and spleen were stained for T cell markers and Foxp3. <i>A,</i> Mean percentage and <i>B</i>, absolute numbers of Foxp3⁺ cells among CD4⁺ T cells (n = 4/group).</p

Sphingolipid concentration in selected tissues of inducible Sgpl1-deficient mice.

<p>Two weeks after tamoxifen induction, tissues of Sgpl1^Flox/Flox Cre^+/− mice (open bars) and of Sgpl1^Flox/Flox Cre^−/− controls (filled bars) were obtained (n = 5/group). Tissues were extracted and sphingolipids were quantified by LC/MS. <i>A, B,</i> S1P; <i>C, D</i>, Sph; <i>E,</i> C16-ceramide. <i>A, C,</i> and <i>E</i> show absolute concentrations per weight of tissue; <i>B and D</i> show fold increase in inducible KO mice.</p

Generation of luciferase reporter mice (mR26CS-Luc).

<p>(A) Flp-RMCE into the modRosa26^FRT locus was performed, replacing the HygR in the modRosa26^FRT ES cells with the FRT/FRTwt-flanked sequence from the Flp-RMCE targeting plasmid. The Flp-RMCE targeting plasmid contains the pCAG promoter followed by a LoxP-flanked (floxed) STOP cassette (STOP2), the luciferase cDNA and a NeoR cassette, flanked as a group by FRT3/FRTwt sites. Successfully targeted ES cells were used to generate mR26CS-Luc mice. After crossing mR26CS-Luc mice with Cre mice, ubiquitous or tissue-restricted luciferase reporter expression can be obtained to monitor reporter expression in living mice upon Luciferin injection. Using Xenogen imaging, adult mR26CS-Luc/CMV-Cre mice show ubiquitous luciferase activity throughout the body (B), while mR26CS-Luc/AlbCre mice show luciferase activity restricted to the liver upon Luciferin injection (C). Luciferin-injected mR26SC-Luc control mice never showed luciferase activity.</p

Generation of mice with a modified Rosa26 locus (modRosa26^LoxP) and testing of different promoters.

<p>(A) Scheme depicting the generation of the modRosa26^LoxP in ES cells. A Stop sequence and a HygR selection cassette flanked by heterospecific LoxP sites (Lox511 and LoxP) were targeted to the Rosa26 locus between exons 1 and 2 by homologous recombination. After successful recombination, the Stop cassette is located downstream of the endogenous Rosa26 promoter. (B) Cre-RMCE into the modRosa26^LoxP locus. In the Cre-RMCE targeting plasmid, a promoter, EGFP and an FRT-flanked neomycin selection cassette (NeoR) were flanked by heterospecific LoxP sites (Lox511 and LoxP) as a group. Cre-RMCE was used to replace the HygR in the modRosa26^LoxP ES cells with the Lox511/LoxP-flanked sequence in the RMCE targeting plasmid. A pCAG, CMV or EF1α promoter driving EGFP was introduced. Insertion of EGFP without any promoter (NoP) controls for functional shielding of the integration site from the endogenous Rosa26 promoter. Binding regions for TaqMan genotyping primers (1, 3) and probe (2), primers for checking integration into the modRosa26^LoxP locus (4–7) and the Southern hybridization probe, as well as the BamHI sites used for Southern blot analysis are indicated. (C) Southern blot analysis on genomic DNA-derived ES cell lines shows specific integration into the modRosa26^LoxP locus. A 2.4-kb BamHI fragment was detected using a Neo probe. mR26-pCAG-EGFP, mR26-CMV-EGFP, mR26-EF1α-EGFP and mR26-NoP-EGFP ES cells show successful targeting of the modRosa26^LoxP locus, without additional integrations at random sites. Wt ES cells show no signal. (D) Modified ES cells showing strong EGFP fluorescence. The pCAG, EF1α and CMV promoters drive strong EGFP expression in the modRosa26^LoxP in vitro. ES cells without promoter but with EGFP inserted into the modRosa26^LoxP locus do not show EGFP fluorescence, indicating functional shielding of the integration site from the endogenous Rosa26 promoter. (E) FACS analysis in mR26-NoP-EGFP ES cells showed no EGFP fluorescence when compared to wt ES cells. mR26-EF1α-EGFP ES cells showed the highest EGFP fluorescence, followed by mR26-pCAG-EGFP, ActB and mR26-CMV-EGFP ES cells.</p

Histology of inducible Sgpl1-deficient mice in EAE.

<p>For histological analysis thoracic sections of spinal cord tissue from Sgpl1^Flox/Flox Cre^+/− and Sgpl1^Flox/Flox Cre^−/− mice undergoing EAE (day 24) were stained (<i>A</i>) with H&E to visualize CNS-invading cells (scale bar is 500 µm, arrows highlight areas of inflammation); <i>B</i>, for CD3⁺ T cells (scale bar is 500 µm, rectangles indicate area of magnification, where scale bar represents 100 µm); and <i>C</i>, with solochrome to assess the integrity of the myelin sheath (scale bar is 500 µm, arrows highlight areas of beginning demyeliniation).</p

Tissue-restricted gene expression from the modRosa26^FRT locus.

<p>A) Scheme depicting the generation of the modRosa26^FRT locus in ES cells. A Stop sequence and a HygR selection cassette flanked by heterospecific FRT sites (FRT3 and FRTwt) were targeted to the Rosa26 locus between exons 1 and 2 by homologous recombination. After successful recombination, the Stop cassette is located downstream of the endogenous Rosa26 promoter (B) Flp-RMCE was performed by introducing the VeCad or αSMA promoter driving Cre recombinase into modRosa26^FRT ES cells, which were then used to generate transgenic mice (mR26-VeCad-Cre or mR26-αSMA-Cre mice). The primers and probe for genotyping are indicated (1–3). Both lines were crossed with ActB-EGFP reporter mice to monitor Cre expression, yielding mR26-VeCad-Cre/ActB-EGFP and mR26-αSMA-Cre/ActB-EGFP mice. (C) Immunostaining for EGFP in mR26-αSMA-Cre/ActB-EGFP mice shows αSMA promoter activity almost exclusively in smooth muscle cells, as seen in lung, stomach and intestine. Few EGFP stained intestinal epithelial cells indicate ectopic SMA promoter activity (asterisks) (D) Immunostaining for EGFP in mR26-VeCad-Cre/ActB-EGFP mice reveals VeCad promoter activity mostly restricted to endothelial cells, as seen in lung, brain and kidney. Some EGFP stained epithelial cells in kidney also indicate ectopic VeCad-Cre expression (asterisks).</p