Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and cardiac-specific Bmi1 deletion [mouse]
Ontology highlight
ABSTRACT: To explore the primary cause of Dilated Cardiomyopathy in Bmi1-null mice, we set out to identify differentially expressed genes by massively parallel sequencing of heart samples from Bmi1f/f;?MHCTM-Cretg/+ mice versus ?MHCTM-Cretg/+ control mice (17 weeks postinduction). Methods: Heart mRNA profiles of 17-weeks post-induction Bmi1f/f; MHCTM-Cretg/+ mice and MHCTM-Cretg/+ control mice were generated by deep sequencing, in triplicate, using Illumina GAIIx. Sequence reads were pre-processed with Cutadapt 1.2.1, to remove TruSeq adapters and mapped on the mouse transcriptome (Ensembl gene-build GRCm38.v70) using RSEM v1.2.3. The Bioconductor package EdgeR was used to normalize data with TMM and to test for differential expression of genes using GLM.
Project description:Ventricular chambers are essential for the rhythmic contraction and relaxation that occurs in every hearbeat throughout life. Congenital abnormalities in ventricular chamber formation cause severe human heart defects. How the early trabecular meshwork of myocardial fibres forms and subsequently develops into mature chambers is still poorly understood. Here we show that Notch signalling first connects chamber endocardium and myocardium to sustain trabeculation and later coordinates ventricular patterning and compaction with coronary vessel development to give rise to the mature chamber via a temporal sequence of ligand signalling determined by the glycosyltransferase Manic Fringe (Mfng). The early endocardial expression of Mfng favours Dll4-Notch1 signalling, Which induces trabeculation in the developing ventricle.Ventricular maturation and compaction in turn require Mfng and Dll4 downregulation in the endocardium, Which allows myocardial Jag1- And Jag2- Signalling to Notch1 in this tissue.Timely and spatial perturbation of this signalling equilibrium severely disrupts heart chamber formation. Our results open a new research avenue into the pathogenesis of cardiomyopathies. Dll4 and Notch1 conditional KOs using Nfact1 and/or Tie2 driven Cre expression: RNA was isolated from pooled whole hearts of 8 (Nfact1) or 9 (Tie2) E9.5 embryos per replicate. Dll4flox;Nfatc1-Cre and WT siblings (4 KO and 4 WT replicates), Notch1flox;Nfatc1-Cre and WT siblings (3 KO and 2 WT replicates), Dll4flox;Tie2-Cre and WT siblings (3 KO and 3 WT replicates). Jag1, Jag2 and Jag1Jag2 conditional KOs using cTnT driven Cre expression: RNA was isolated from pooled heart ventricles of 4 E15.5 embryos per replicate. Jag1flox;cTnT-Cre and WT siblings (3 KO and 3 WT replicates), Jag2flox;cTnT-Cre and WT siblings (3 KO and 2 WT replicates). Jag1flox;jag2flox;cTnT-Cre and WT siblings (3 KO and 2 WT replicates). MFng Gain Of Function using Tie2 driven Cre expression: RNA was isolated from pooled heart ventricles of 4 E15.5 embryos per replicate. MFng;Tie2-Cre and WT siblings (4 GOF and 4 WT replicates). For Dll4, Noth1 and Jag1 KOs, libraries were prepared using the standard Illumina TrueSeq RNASeq library preparation kit and sequenced in a GAIIx Illumina sequencer using a 75bp single end elongation protocol. For Jag2 and Jag1Jag2 KOs and MFng GOF libraries were prepared prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina and sequenced in a HiSeq2500 Illumina sequencer using a 61bp single end elongation protocol
Project description:Recently, it was described that mammalian cells are able to eliminate those with relative lower Myc levels in the epiblast through cell competition. We have described that cardiomyocytes during heart development are also able to complete eliminating cells with lower Myc levels. We have also shown that adult cardiomyocytes respond in the same way over long periods of time when cell competition is induced by overexpressing Myc in a mosaic fashion. We therefore have developed an RNASeq assay to further understand the mechanism of elimination of WT cells and the effect of mild Myc overexpression in cardiomyocytes. Myc overexpression in a mosaic fashion in adult cardiomyocytes, 2 hearts were analyzed and two wild type littermates were used as controls
Project description:This dataset covers the development of 7 organs (brain, cerebellum, heart, kidney, liver, ovary and testis) from embryonic day 10.5 to adulthood.
Project description:To explore the primary cause of Dilated Cardiomyopathy in heart samples from DCM-diagnosed patients who had undergone heart transplant (hDCM), we set out to identify differentially expressed genes by massively parallel sequencing of heart samples. Methods: Heart mRNA profiles from DCM-diagnosed patients who had undergone heart transplant (hDCM) were generated by deep sequencing, in triplicate, using Illumina GAIIx.
Project description:We have previously generated ABIN1[D485N] knock-in mice that appear relatively normal for 2 months after birth, but develop large spleen at 3 months, which is followed by the appearance of high levels of antibodies against self DNA and self-nuclear antigens and severe inflammation of the kidney and other tissues after 4-5 months, hall marks of lupus like autoimmunity. Autoimmunity in ABIN1[D485N] mice appears to be caused by hyper activation of pathogen sensing Toll-like Receptor (TLR) pathway, because the phenotype of the ABIN1[D485N] mice is completely suppressed by crossing them to mice that are either lacking or expressing functionally defective proteins, which play key role in TLR signalling. MyD88 KO, IRAK4 or IRAK1 kinase-inactive knock-in mice. However, cell type(s) required to drive lupus, and how this contributes to the development of the pathology, is unknown. Recently, we have identified atypical myeloid (CD11b+ve) populations in the blood, lungs liver, kidney and spleen of ABIN1[D485N] mice. Importantly, these cells are detectable at four weeks, at least two months before auto-antibodies are detected in the serum. More detailed flow cytometry analysis of these cells revealed that, in the blood, the majority were CD115+ve and these cells could be further subdivided into Ly6C+ve and Ly6C-ve, which are characteristics of the inflammatory and patrolling monocytes, respectively. While in the lungs and spleen of WT mice the Ly6C-ve CD115+ve (patrolling monocytes) are almost absent, in the ABIN1[D485N] mice this population is more than 20-fold increased. There is also increase in the number of neutrophil population (CD11b+ GR-1hi) in the spleen of the ABIN1[D485N] mice. Although we have shown that the formation of these atypical myeloid cells proceeds the development of auto-antibodies and organ inflammation in the ABIN1[D485] mice, the signalling pathway leading to formation of these cells and their role in autoimmunity is unknown. We therefore undertook RNAseq analysis to characterise these cells in more detail. The atypical CD11b+ve cells present in spleen of ABIN1[D485N] mice are heterogeneous with three major populations, neutrophils (CD11b+ GR-1hi), Ly6C+veCD115+ve cells and Ly6C-veCD115+ve. How these cell populations in ABIN1[D485N] mice relate to one another, and whether they share a common precursor is unknown. In order to profile the cells, the neutrophils, Ly6C+veCD115+ve cells and Ly6C-veCD115+ve populations were purified by cell sorting and RNAseq analysis carried out. The RNA seq analysis was carried out with at least four replicate samples from each cell populations from ABIN1[D485N] and WT controls.
Project description:We carried out RNA-seq of cerebellar tissues of control and prion-inoculated BL6 mice at two time points: 56 days post-inoculation (early disease) and 182 days post-inoculation (terminal disease).
Project description:The histone variant H2A.B3 (H2A.Bbd/H2A.Lap1) has a tissue-specific expression pattern and is found to be most highly expressed during spermatogenesis. In order to investigate the role of H2A.B3 in chromatin packaging and regulation of spermatogenesis we created a line of TALEN-edited FVBN mice in which a deletion was introduced into the single exon of the H2AFB3 gene. The deletion was confirmed by Sanger sequencing and off-target effects investigated using exome sequencing. The deletion is visible in the RNA-Seq data of KO mouse, and the deleterious effect on the H2A.B3 protein has been confirmed using immunohistochemistry.
Project description:The circadian clock controls a wide variety of metabolic and homeostatic processes in a number of tissues, including the kidney. However, the role of the renal circadian clocks remains largely unknown. To address this question we performed transcriptomic analysis in mice with inducible and conditional ablation of the circadian clock system in the renal tubular cells (Bmal1lox/lox/Pax8-rtTA/LC1 mice). Deep sequencing of the renal transcriptome revealed significant changes in the expression of genes related to metabolic pathways and organic anion transport. In parallel, kidneys from Bmal1lox/lox/Pax8-rtTA/LC1 mice exhibited a significant decrease in the NAD+/NADH ratio suggesting an increased anaerobic glycolysis and/or decreased mitochondrial function. In-depth analysis of two selected pathways revealed (i) a significant increase in plasma urea levels correlating with increased renal arginase 2 (Arg2) activity, hyperargininemia and increase of the kidney arginine content; (ii) a significantly increased plasma creatinine concentration and reduced capacity of the kidney to secrete anionic drugs (furosemide), paralleled by a ~80% decrease in the expression levels of organic anion transporter OAT3 (SLC22a8). Collectively, these results indicate that the renal circadian clocks control a variety of metabolic/homeostatic processes at both the intra-renal and systemic levels and are involved in drug disposition. Mice with a specific ablation of the Arntl gene encoding BMAL1 in the renal tubular cells were compared to wild-type littermate at ZT4 and ZT16 (ZT â Zeitgeber time units; ZT0 is the time of light on and ZT12 is the time of light off).
Project description:Determination of differentially expressed genes in the proximal colon and distal ileum tissue in MR1 and IL-17A deficiency at steady-state. Tissue from naïve mice was harvested, total RNA extracted and subjected to RNASeq analysis.
Project description:This experiment sought to understand the transcriptomic changes that occur in the larval zebrafish heart following injury. 600 hearts were laser injured at 3 days post fertilisation, extracted 48 hours later and pooled into three groups of 200. RNA was extracted from the whole heart(s) and sent for sequencing along with 3 groups of 200 uninjured hearts, extracted and processed identically. RNA sequencing, quality control and alignment was performed by the commercial company GENEWIZ.