Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Small RNA survey of mouse genome

ABSTRACT: Using criteria based on known examples, we identified approximately 1.7 million and 2.4 million sequences encoding putative miRNA precursor (stem-loop) structures in the mouse and human genomes, respectively, as well as large numbers of such structures in the genomes of fish, fruitfly and nematode worm. These predictions were then tested using high density custom microarrays containing modified oligonucleotides interrogated with purified small RNAs from different tissues. We found that 19% of 4,006 randomly chosen mouse predictions and 40% of 1,859 randomly chosen human predictions gave positive signals with small RNA isolated from whole 10-12 day embryos and a mixture of brain and testis, respectively, 84% of which hybridized to only one strand of the predicted stem-loop structure, whereas only 2% and 12% of equivalent random mouse and human predictions were positive. There was no difference in the validation rates between sequences exhibiting conservation and those that did not. High validation rates were also observed with predictions from fish, fly and worm genomes. Northern blot analysis of the array-positive mouse predictions confirmed that the vast majority of these sequences were detectable as small RNAs whose sizes ranged from 20-110 nucleotides. Taking into account false negative rates of the prediction and detection of known miRNAs, and assuming that this holds for other small RNAs, we estimate that there are 1 million small RNAs expressed in mouse and 3 million small RNAs in human. Keywords: genomic survey of small RNAs Stemloops were identified in the mouse and human genome using RNALfold with a window size of 110nt. Folding energy and other criteria were used to filter the structures into a set of miRNA-like stems. Stems were chosen randomly and probes designed against the stem regions. Stems were called present if one of the stem probes was more than 4 standard deviations above the signal distribution of mRNA degradation contamination probes designed against GAPDH and beta-actin.

ORGANISM(S): Mus musculus

SUBMITTER: John Mattick

PROVIDER: E-GEOD-7217 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background Clinical success of T cell receptor (TCR) gene therapy has previously been demonstrated for NY-ESO-1 TCR gene therapy. To increase numbers of cancer patients that can be treated with TCR gene therapy we aimed to identify a novel set of high-affinity cancer specific TCRs targeting different cancer testis (CT) antigens in prevalent HLA class I alleles. Methods In this study, we selected based on publicly available gene expression databases the most promising CT genes to target. From these selected genes we identified by HLA peptidomics the naturally processed and presented HLA class I peptides. With these peptide-HLA tetramers were generated, and by single cell sorting CT specific CD8+ T cells were selected, and expanded from the allo-HLA repertoire of healthy donors. By several functional assays high avidity CT-specific T cell clones with safe recognition pattern were selected. To evaluate the potential for clinical application in TCR gene therapy, TCRs were sequenced and transferred into peripheral blood derived CD8+ T cells. Results In total we identified, 7 novel CT-specific TCRs that effectively target MAGE-A1, MAGE-A3, MAGE-A6 and MAGE-A9 in the context of human leukocyte antigen(HLA) -A1, -A2, -A3, -B7, -B35 and -C7. TCR gene transfer into CD8⁺ T cells resulted in efficient cytokine production and cytotoxicity of variety of different tumor types without detectable cross-reactivity. In addition, major in vivo antitumor effects of MAGE-A1 specific TCR engineered CD8⁺ T cells was observed in an orthotopic xenograft model for established multiple myeloma, in which bone marrow located tumor cells were completely eradicated after T cell injection. Conclusion The identification of 7 novel CT-specific TCRs, reactive against CT antigens presented in a variety of different HLA class I alleles, allows selection of therapeutic TCRs for an increased number of cancer patients, and will improve development of personalized TCR gene therapy.

Project description:Developmental exposure of mouse fetuses to estrogens results in dose-dependent permanent effects on prostate morphology and function. Fetal prostatic mesenchyme cells express estrogen receptor alpha (ERα) and androgen receptors and convert stimuli from estrogens and androgens into signaling to regulate epithelial cell proliferation and differentiation. To obtain mechanistic insight into the role of different doses of estradiol (E2) in regulating mesenchymal cells, we examined E2-induced transcriptomal changes in primary cultures of fetal mouse prostate mesenchymal cells. Urogenital sinus mesenchyme cells were obtained from male mouse fetuses at gestation day 17 and exposed to 10 pM, 100 pM or 100 nM E2 in the presence of a physiological concentration of dihydrotestosterone (0.69 nM) for four days. Gene ontology studies suggested that low doses of E2 (10 pM and 100 pM) induce genes involved in cell adhesion, morphological tissue development, and sterol biosynthesis but suppress genes involved in growth factor signaling and cell adhesion. Genes showing inverted-U-shape dose responses (enhanced by E2 at 10 pM E2 but suppressed at 100 pM) were identified, and their enrichment in the glycolytic pathway was demonstrated. At the highest dose (100 nM), E2 induced genes enriched not only for cell adhesion but also steroid hormone signaling and metabolism, cytokines and their receptors, cell-to-cell communication, Wnt signaling, and TGF-β signaling. These results suggest that prostate mesenchymal cells may regulate epithelial cells through direct cell contacts when estrogen level is low whereas soluble growth factors might play significant roles when estrogen level is high. Primary culture urogenital sinus mesenchymal cells were isolated from prostate glands of gestation day 17 CD1 male mouse fetuses. Cells were then exposed to 10 pM or 100 pM 17beta-estradiol or vehicle (0.05% ethanol) for four days in the presence of 690 pM 5alpha-dihydrotestosterone. Total cellular RNA was then isolated for determination of transcriptomal profiles by Affymetrix mouse gene 1.0 ST array.

Project description:MED1 (Mediator complex subunit 1) is expressed by human epidermal keratinocytes and functions as a coactivator of several transcription factors. To elucidate the role of MED1 in keratinocytes, we established keratinocyte-specific MED1-null (MED1epi-/-) mice using the K5Cre-LoxP system. To elucidate the mechanism(s) underlying abnormalities of keratinocytes derived from MED1epi-/- mice, we compared the gene expression patterns of MED1epi-/--derived keratinocytes with their wild type counterparts by microarray analysis. Generation of the MED1 conditional null mutation in epidermal keratinocytes and cell culture: MED1flox/flox mice (Jia et al, J Biol Chem 279:24427. 2004) were mated with K5Cre mice (Tarutani et al, Proc Natl Acad Sci U S A 94:7400. 1997) to generate F1 K5Cre+;MED1flox/+ mice. The K5Cre+;MED1flox/+ mice were then mated with MED1flox/flox mice to generate K5Cre+;MED1flox/flox mice (MED1epi-/-). Mice were screened for presence of the transgene by PCR using specific primers for the floxed MED1 gene and the human K5 gene according to the previous reports. Skin of newborn mice was excised after the mice were sacrificed with excessive anesthetic and were treated with dispase followed by trypsin to separate the epidermis from the dermis. Keratinocytes were seeded on type I collagen coated dishes, and were cultured in CnT07 conditioned culture medium (CELLnTEC, Bern, Switzerland). Keratinocytes were used for the experiment as a primary culture. All animal studies were conducted according to protocols approved by the Institutional Animal Care and Use Committee at Osaka University. Microarray analysis: For comprehensive comparison of gene expression patterns between keratinocytes derived from MED1epi-/- and from WT mice, microarray analysis was used. Total RNAs were extracted using an RNeasy kit (Qiagen, San Diego, CA). Then, 2μg total RNA was reverse transcribed to cDNA with T7 oligo d(T) primer (Affymetrix, Santa Clara, CA). The cDNA synthesis products were used for in vitro transcription reactions containing T7 RNA polymerase and biotinylated nucleotide analogue (pheudouridine base) cRNAs. The labeled cRNA products were then fragmented and loaded onto GeneChip(R) Mouse Genome 430 2.0 arrays (Affymetrix), and were hybridized according to the manufacturer’s protocol. Streptavidin-Phycoerythrin (Molecular Probes) was used as the fluorescent conjugate to detect hybridized target sequences. Raw intensity data from the GeneChip array were analyzed by GeneChip Operating Software (Affymetrix).

Project description:The haematopoietic cytokine thrombopoietin (Tpo) is the primary regulator of megakaryocyte and platelet numbers and is required for maintenance of the haematopoetic stem cell compartment. Tpo is a heavily glycosylated, hepatocyte-derived cytokine which functions by binding to its receptor (TpoR) on target cells and thereby activating intracellular signalling cascades that induce their proliferation and/or differentiation. In addition to its role in signal propagation, TpoR is expressed on the surface of platelets, where it contributes to regulation of Tpo levels by sequestering circulating cytokine. TpoR belongs to the homodimeric Class I cytokine receptor family but is unusual due to a duplication of the Cytokine binding Homology Region (CHR). Almost thirty years after initial discovery of TpoR, the structure of the human Tpo:TpoR interaction was recently reported. Here we determine the structure of extracellular portion of the murine Tpo:TpoR signalling complex using single particle cryo-EM. The structure reveals that Tpo:TpoR forms a largely symmetrical 1:2 complex. The cytokine cross-links the same site on the membrane-distal CHR of both receptor chains using opposing surfaces and with significantly different affinities. This orients the two membrane-proximal CHRs such that they contact one another adjacent to the plasma membrane. The potential cytokine-binding site in CHR2 is glycosylated and does not interact with Tpo. A large insertion in CHR1 that is unique to Tpo forms a partially structured loop that is disulphide bonded to CHR2 and, in one receptor chain, contacts cytokine. Biochemical analyses indicate that the glycosylated C-terminal domain of Tpo does not influence receptor binding. We demonstrate that the therapeutic TpoR agonist Romiplostim binds to the same site on the receptor as does cytokine. Our study characterises the Tpo/TpoR interaction structurally and biochemically to allow for the future development of potent TpoR agonists for therapeutic use.

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Small RNA survey of mouse genome

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