Gene expression in the isolated islets of 10 day old, 4 wk old, 12 wk old, and 20 wk old Non-Obese Diabetic (NOD) mice compared to healthy age matched control NOD.B10 mice
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ABSTRACT: Gene expression in the islets of diseased NOD mice compared to congenic healthy NOD.B10 mice at various ages, during the progression of NOD disease. Islets were isolated from individual NOD and NOD.B10 mice by collagenase digestion. RNA was extracted using Trizol, combined with the RNeasy micro kit (Qiagen). RNA quality was assessed using the Agilent 2100 Bioanalyzer and the RNA 6000 Pico Reagent Kit (Agilent). Microarrays were performed using the Whole Mouse Genome Microarray Kit, 4M-CM-^W44K 2-color arrays (Agilent Technologies).
Project description:Gene expression in the islets of diseased NOD mice compared to congenic healthy NOD.B10 mice at various ages, during the progression of NOD disease.
Project description:Gene expression in the islets of NOD and NOD.B10 mice at 12 weeks of age, prior to the onset of destructive insulitis. The pancreata of NOD and NOD.B10 mice (n=6) were frozen and islets were isolated by laser capture microdissection. Cryosections (8 M-NM-<m) were cut and stained using the Arcturus HistoGene Frozen Section Staining kit (Applied Biosystems) and laser dissection was performed using the Leica AS LMD and the Leica IM 1000 Image Manager Basic software. Sections from at least 40 individual islets were collected and RNA was extracted using the RNeasy micro kit (Qiagen). RNA quality was assessed using the Agilent 2100 Bioanalyzer and the RNA 6000 Pico Reagent Kit (Agilent). Samples were preamplified using the TrueLabeling-PicoAMP kit (SA Biosciences), post-labeled with Cy5 and run against a Cy3-labeled mouse Universal RNA control (SA Biosciences). Microarrays were performed using the Whole Mouse Genome Microarray Kit, 4M-CM-^W44K 2-color arrays (Agilent Technologies).
Project description:These experiments were performed to identify differentially expressed genes in the pancreatic lymph nodes of hyperglycemic NOD mice with high levels of destructive insulitis compared to euglycemic mice with lower levels of insulitis. The pancreatic lymph nodes of 16-week-old hyperglycemic and euglycemic NOD mice were isolated and homogenized in Trizol reagent (Invitrogen). Total RNA was extracted from the aqueous phase using the RNeasy mini kit (Qiagen). RNA quality was assessed using the Agilent 2100 Bioanalyzer and the RNA 6000 Nano Reagent Kit (Agilent). Total RNA from the PLN of hyperglycemic mice (n=2) were labelled with Cy5 and run individually against a pool of Cy3-labeled total RNA isolated from euglycemic mice (n=2). Microarrays were performed using the Whole Mouse Genome Microarray Kit, 4M-CM-^W44K 2-color arrays (Agilent Technologies).
Project description:Beta-cells produce hybrid insulin peptides (HIPs) by linking insulin fragments to other peptides through peptide bonds. HIPs have unique amino acid sequences and are targeted by autoreactive T cells in type 1 diabetes (T1D). Individuals with recent-onset T1D have significantly higher levels of HIP-reactive T cells in their blood compared to non-diabetic control subjects. HIP-reactive T cells have also been found in the residual pancreatic islets of deceased T1D organ donors. In non-obese diabetic (NOD) mice, a major T1D animal model, several CD4 T cell clones that trigger diabetes have been shown to target HIPs. Through mass spectrometry, a subgroup of HIPs containing N-terminal amine groups of various peptides linked to aspartic acid residues of insulin C-peptide has been detected in NOD islets. Our research reveals that these HIPs form spontaneously in beta-cells via an aspartic anhydride intermediate mechanism. This process leads to the creation of a regular HIP with a standard peptide bond and a HIP-isomer (isoHIP) with an isopeptide bond linked to the carboxylic acid side-chain of the aspartic acid residue. Our mass spectrometric analyses confirmed the presence of both HIP isomers in murine islets, thereby validating the occurrence of this new reaction mechanism in beta-cells. The spontaneous formation of neoepitopes through the development of new peptide bonds within cells may contribute to the pathogenesis of T1D and other autoimmune diseases.
Project description:41K whole genome oligo-microarrays (Agilent Technologies) were used to characterize age-dependent changes in gene expression in pancreatic lymph nodes (PLN) obtained from up to 7 individual NOD mice at 6 different time points (10 days (n=7), and 4 weeks (n=6), 8 weeks (n=4), 12 weeks (n=7), 16 weeks (n=6), and 20 weeks of age (n=5)), compared to NOD.B10 tissue controls. "Milestone Genes" are genes whose expression was significantly changed (approximately 3 fold) as the result of splicing or changes in transcript level. Milestone Genes were identified among genes within type one diabetes (T1D) susceptibility regions (Idd). Milestone Genes showing uniform patterns of changes in expression at various time points were identified. Potential T1D candidate genes were identified among Milestone Genes within Idd regions and/or hierarchical clusters. These studies identified tissue- and age-specific changes in gene expression that may play an important role in the inductive or destructive events of T1D. NOD/LtJ (NOD), NOD.B10Sn-H2b/J (NOD.B10) female mice of multiple ages were used for this study. Six Groups of NOD mice were sacrificed at 10 days, and 4, 8, 12, 16, and 20 weeks of age and pancreatic lymph nodes (PLN) were removed and prepared for mRNA analysis. Two Groups of 10 NOD.B10 mice [10 days (n=10) + 20 weeks of age (n=10)] were used as a tissue specific control. Gene (mRNA) expression was analyzed using the 41K Whole Mouse Genome Oligo Microarray Kit (Agilent Technologies), and gene expression data are processed (cy3: NOD, cy5: NOD.B10) and reported as normalized expression ratios: Log 10 (NOD processed signal / NOD.B10 processed signal) (Log ratio). 10days of age; PLN_10d (n=7), 4 weeks of age; PLN_4w (n=6), 8 weeks of age; PLN_8w (n=4), 12 weeks of age; PLN_12w (n=7), 16 weeks of age; PLN_16w (n=6), 20 weeks of age; PLN_20w (n=5)
Project description:41K whole genome oligo-microarrays (Agilent Technologies) were used to characterize age-dependent changes in gene expression in peripheral blood cells (PBC) obtained from up to 8 individual NOD mice at 6 different time points (10 days (n=8), and 4 weeks (n=3), 8 weeks (n=7), 12 weeks (n=8), 16 weeks (n=7), and 20 weeks of age (n=4)), compared to NOD.B10 tissue controls. "Milestone Genes" are genes whose expression was significantly changed (approximately 3 fold) as the result of splicing or changes in transcript level. Milestone Genes were identified among genes within type one diabetes (T1D) susceptibility regions (Idd). Milestone Genes showing uniform patterns of changes in expression at various time points were identified. Potential T1D candidate genes were identified among Milestone Genes within Idd regions and/or hierarchical clusters. These studies identified tissue- and age-specific changes in gene expression that may play an important role in the inductive or destructive events of T1D. NOD/LtJ (NOD), NOD.B10Sn-H2b/J (NOD.B10) female mice of multiple ages were used for this study. Six Groups of NOD mice were sacrificed at 10 days, and 4, 8, 12, 16, and 20 weeks of age and peripheral blood cells (PBC) were removed and prepared for mRNA analysis. Two Groups of 10 NOD.B10 mice [10 days (n=10) + 20 weeks of age (n=10)] were used as a tissue specific control. Gene (mRNA) expression was analyzed using the 41K Whole Mouse Genome Oligo Microarray Kit (Agilent Technologies), and gene expression data are processed (cy3: NOD, cy5: NOD.B10) and reported as normalized expression ratios: Log 10 (NOD processed signal / NOD.B10 processed signal) (Log ratio). 10days of age; PBC_10d (n=8), 4 weeks of age; PBC_4w (n=3), 8 weeks of age; PBC_8w (n=7), 12 weeks of age; PBC_12w (n=8), 16 weeks of age; PBC_16w (n=7), 20 weeks of age; PBC_20w (n=4)
Project description:Fluoride (F) is used in dentistry, in therapeutic doses, to prevent dental caries. Recently, animal studies have suggested that in low doses, F might reduce glucose and be beneficial in the prophylaxis of diabetes, but the involved mechanisms are still unknown. The present study evaluated changes in the pancreatic islets of NOD mice exposed to low dose of F, using morphological, immunohistochemical and proteomic tools.