Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression differences in mouse islets after isolation at different time points (0-48hr)

ABSTRACT: TGFbi (transforming growth factor-beta-induced) is a secreted protein and is capable of binding to both extracellular matrix (ECM) and cells. It thus acts as a bifunctional molecule enhancing ECM and cell interactions, a lack of which results in dysfunction of many cell types. In this study, we investigated the role of TGFbi in the function and survival of islets. Based on DNA microarray analysis followed by qPCR confirmation, the TGFbi gene showed drastic increases in expression in islets after culture. We demonstrated that recombinant TGFbi could preserve the integrity and enhance the function of cultured islets. Such a beneficial effect was mediated via signalling through FAK. Exogenous TGFbi was capable of sustaining high-level FAK phosphorylation in isolated islets, and FAK knockdown by siRNA in islets resulted in compromised islet function. TGFbi Tg islets showed better integrity and insulin release after in vitro culture. In vivo, b-cell proliferation was detectable in Tg but not wild type pancreata. At age above 12 months, Tg pancreata contained giant islets. Tg mice displayed better glucose tolerance than the controls. Tg islets were more potent in lowering blood glucose when transplanted into syngeneic mice with streptozotocin-induced diabetes, and these transplanted islets also underwent regeneration. Our results indicate that TGFbi is a vital trophic factor promoting islet survival, function and regeneration. At least some of its beneficial effect was mediated by signalling through FAK. Six independent batches of islets were isolated. For each batch, islets from 4 C57BL/6 mice were pooled, and were divided into 3 groups, which were harvested at 0 h, 24 h and 48 h after culture. Total RNA was extracted from the islets with QIAGEN RNeasy Micro Kits. After reverse transcription, cDNA served as a template for PCR-based cRNA amplification. It was then reverse-transcribed to produce second-generation cDNA, which was employed to generate biotin-labelled cRNA probes. The probes were hybridized on GeneChip Mouse Genome 430 2.0 Array (Affymetrix, Santa Clara, CA), which contains 39,000 transcripts. DNA microarray analysis was performed at the McGill University Genome Quebec Innovation Centre. For each time point, 6 chips were hybridized with cRNA from the 6 batches of isolated islets. For samples collected at 24 and 48 hours (6 batches for each time point), if the mean signal strength of a gene had above 4-fold difference over that of the 0 h samples, it was selected for reverse polymerase chain reaction (PCR) confirmation. Details of the experimental protocols and data analysis procedure can be found in http://www.affymetrix.com/support/downloads/manuals/expression_analysis_technical_manual.pdf .

ORGANISM(S): Mus musculus

SUBMITTER: bing han

PROVIDER: E-GEOD-27547 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The homeodomain transcription factor Nkx6.1 plays an important role in pancreatic islet Î²-cell development, but its effects on adult Î²-cell function, survival, and proliferation are not well understood. In the present study, we demonstrated that treatment of primary rat pancreatic islets with a cytomegalovirus promoter-driven recombinant adenovirus containing the Nkx6.1 cDNA (AdCMV-Nkx6.1) causes dramatic increases in [methyl-3H] thymidine and 5-bromo-2'-deoxyuridine (BrdU) incorporation and in the number of cells per islet relative to islets treated with a control adenovirus (AdCMV-Î²GAL), whereas suppression of Nkx6.1 expression reduces thymidine incorporation. Immunocytochemical studies reveal that >80% of BrdU-positive cells in AdCMV-Nkx6.1-treated islets are Î² cells. Microarray, real-time PCR, and immunoblot analyses reveal that overexpression of Nkx6.1 in rat islets causes concerted upregulation of a cadre of cell cycle control genes, including those encoding cyclins A, B, and E, and several regulatory kinases. Cyclin E is upregulated earlier than the other cyclins, and adenovirus-mediated overexpression of cyclin E is shown to be sufficient to activate islet cell proliferation. Moreover, chromatin immunoprecipitation assays demonstrate direct interaction of Nkx6.1 with the cyclin A2 and B1 genes. Overexpression of Nkx6.1 in rat islets caused a clear enhancement of glucose-stimulated insulin secretion (GSIS), whereas overexpression of Nkx6.1 in human islets caused an increase in the level of [3H]thymidine incorporation that was twice the control level, along with complete retention of GSIS. We conclude that Nkx6.1 is among the very rare factors capable of stimulating Î²-cell replication with retention or enhancement of function, properties that may be exploitable for expansion of Î²-cell mass in treatment of both major forms of diabetes. Keywords: Insulin secretion, islet biology, transcription factor, cell cycle regulation, diabetes We utilized a âsample x referenceâ experimental design strategy in which RNA extracted from rat pancreatic islets was hybridized to the microarray slide in the presence of labeled rat reference RNA (RRR, Stratagene, LaJolla, CA). Cultures were treated with adenoviruses expressing either the hamster form of Nkx6.1 or the beta-galactosidase enzyme. 5 biological replicates each representing independent islet isolations were used for microarray analysis. Briefly, five hundred nanograms of total RNA were used for gene expression profiling following reverse transcription and T-7 polymerase-mediated amplification/labeling with Cyanine-5. Labeled subject cRNA was co-hybridized to Operon rat 27K oligonucleotide arrays with equimolar amounts of Cyanine-3 labeled RRR. Slides were hybridized, washed, and scanned on a Gene Pix 5000 microarray scanner.

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Gene expression differences in mouse islets after isolation at different time points (0-48hr)

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