RNA-Seq of pancreatic islets from whole body knockout of lncRNA Pax6os1 mice
Ontology highlight
ABSTRACT: A whole body knockout mouse was generated by deleting the 1st exon and the 620 bp immediately upstream of the gene encoding the long non-coding RNA, Pax6os1 via CRISPR.
Project description:RNA-Seq was performed on pancreatic islets from four transgenic mouse strains affecting LKB1 and AMPK. A conditional LKB1 knockout strain was generated. Double conditional knockouts for AMPK alpha1 and AMPK alpha2 were also generated. These conditional strains were crossed with RIP-Cre (driven by rat insulin promoter) or Ins1-Cre mice to generate LKB1 knockout and AMPK double knockout strains.
Project description:Zfp92, a repressive KRAB domain-containing zinc-finger protein, was identified by Gene Co-expression Network analysis to be an interesting candidate gene involved in endocrine specification and maturation. We examined the role of Zfp92, a KRAB-ZFP that is highly expressed in pancreatic islets of adult mice, by analyzing global Zfp92 knockout (KO) mice. Adult Zfp92 KO animals exhibited only mild changes in glucose homeostasis and no change in islet structure, although, male KO mice exhibited decreased growth, and female KO mice exhibited increased body fat accumulation on a high fat diet. We found that Zfp92 regulates a subset of transposable elements as well as Mafb, a transcription factor involved in islet development.
Project description:Gene expression differences between wild-type, pancreas-specific Hnf4alpha knockout (Hnf4a-pKO), heterozygous Hnf1alpha (Tcf1) (Hnf1aHET), and double mutant (Hnf4a-pKO;Hnf1aHET) mouse pancreatic islets were assesed using amplified RNA samples from islets of matched quality and endocrine purity, obtained from 60-75 day-old control and genetically modified male mice after 48 hours in culture. RNA was amplified using the Affymetrix GeneChIP two-cycle protocol and hybridized to Affymetrix GeneChip Mouse Genome 430 2.0 arrays [Mouse430_2].
Project description:Pancreatic islets were isolated from Hnf1a (Hepatocyte nuclear factor 1 alpha) knockout and wild-type mice and cultured ex vivo for two days in RPMI medium with 10% FBS before RNA extraction. The transcription profiles were obtained using Affymetrix GeneChip Mouse Genome 430A 2.0 arrays [Mouse430A_2].
Project description:Pancreatic islets were isolated from Hnf1a+/- (Hepatocyte nuclear factor 1 alpha) and wild-type mice and cultured ex vivo for two days in RPMI medium with 10% FBS before RNA extraction. The transcription profiles were obtained using Affymetrix GeneChip Mouse Genome 430 2.0 arrays [Mouse430_2].
Project description:Pancreas specific deletion of the Haster promoter region results in a variegated phenotype in pancreatic islets with overexpression or silencing of the Hnf1a gene. To determine the transcriptional consequence of the overexpression or silencing of Hnf1a is islet cells from the Haster pKO mice (Haster loxP/loxP;Pdx1-Cre), we performed scRNA-seq of pancreatic islets from control and adult female Haster pKO mice.
Project description:Pathways that stimulate β-cell regeneration remain of great clinical interest, yet effective therapeutic avenues that promote survival or reconstitution of β-cell mass remain elusive. Utilizing a mouse model with inducible β-cell apoptosis followed by adiponectin-mediated regeneration, we aimed to identify key molecules boosting β-cell viability. Within the regenerating pancreatic islets, we examined changes within the transcriptome, and observed an extensive upregulation of genes encoding proteins involved in lipid transport and metabolism. The most prominent targets were further confirmed by quantitative PCR and immunofluorescence. Among the upstream regulators predicted by pathway analysis of the transcriptome, we detected enhanced levels of two key transcription factors, HNF4α and PPARα. Enhanced leptin levels in circulation may also contribute to the anti-lipotoxic program in islets. In summary, our data suggest that improving local lipid metabolism as an important anti-lipotoxic phenomenon to boost β-cell regeneration, primarily mediated by adiponectin’s action on the β-cells directly as well as on the adipocyte. RNA profiles of pancreatic islets isolated from PANIC-ATTAT mice crossed with adiponectin wild-type (P-Adn+/+) or the overexpressing transgene (P-AdnTg/+) at 5 weeks after initial dimerizer administration.
Project description:M-NM-2-cell identity is determined by tightly regulated transcriptional networks that are modulated by extracellular cues, thereby ensuring M-NM-2-cell adaptation to the organismM-bM-^@M-^Ys insulin demands. We have observed in pancreatic islets that stimulatory glucose concentrations induced a gene profile that was similar to that of freshly isolated islets, indicating that glucose-elicited cues are involved in maintaining M-NM-2-cell identity. Low glucose induces the expression of ubiquitous genes involved in stress responses, nutrient sensing, and organelle biogenesis. By contrast, stimulatory glucose concentrations activate genes with a more restricted expression pattern (M-NM-2- and neuronal- cell identity). Consistently, glucose-induced genes are globally reduced in islets deficient with Hnf1a (MODY3), characterized by a deficient glucose metabolism. Of interest, a cell cycle gene module was the most enriched among the variable genes between intermediate and stimulatory glucose concentrations. Glucose regulation of the islet transcriptome was unexpectedly broadly maintained in islets from aged mice. However, the cell cycle gene module is selectively lost in old islets and the glucose activation of this module is not recovered even in the absence of the cell cycle inhibitor p16. We used microarrays to detail the global programme of gene expression regulated by glucose in young and aged pancreatic islets as well as freshly-isolated islets. Pancreatic islets from young and old mice were isolated and cultured at different glucose concentrations for RNA extraction and hybridization on Affymetrix microarrays. Islets were cultured at 3mM (G3), 5.5mM (G5), 11mM (G11) and 16mM (G16). Freshly-isolated islets (F) were also processed for RNA extraction . We also assessed the dynamic glucose regulation of gene expression at different time-points after an overnight at 3mM (T0): after 1h at 11mM (T1) and after 4h (T4).
Project description:Purpose: Myt3 is a putative pro-survival factor in pancreatic islets and is expressed from E18.5 onwards. The genes that Myt3 regulates and its role in islet-cell development, function and survival are not fully known. The purpose of this study was to determine the changes in gene expression following the suppression or over-expression of Myt3 to identify possible pathways that may be regulated by Myt3. Methods: Pancreatic islets were isolated from both male and female C57/B6 mice by collagenase digestion and were picked by hand. Hand-picked islets were plated onto 804G, a complete extracellular-matrix (ECM) produced by a rat bladder carcinoma cell line, treated tissue culture dishes and transduced with 1x106 pfu virus overnight. The next day media was changed to fresh RPMI and islets were cultured for 7 days. At the end of the culture period islets were dispersed and FACs sorted for GFP+ cells. Sorted cells from at least 5 experiments were pooled and RNA was isolated using Trizol reagent. 800ng of RNA was used in the Illumina TruSeq kit for library preparation. Results: Comparison of gene expression levels between control-transduced islets and either shMyt3- or Myt3-transduced islets resulted in the identification of 51 and 89 significantly altered genes respectively. Conclusions: Our study represents the first comprehensive analysis of genes that are potentially regulated by Myt3 and highlight potential pathways that are likely important for mediating Myt3's effects of islet development, function and survival. Isolated islets were transduced with adenoviruses, cultured for 7 days on ECM and FACS sorted. Cells from up to 5 experiments were pooled and sequenced.