Genome-wide analysis of gene transcription in myocardium of mice transgenically expressing human cardiolipin synthase 1 (hCLS1) in cardiac tissue
ABSTRACT: Analysis of the transcriptome of cardiac tissue from mice transgenically expressing human cardiolipin synthesis. The hypothesis tested was that cardiac specific transgenic expression of cardiolipin synthase alters myocardial lipidomic flux resulting in compensatory metabolic gene transcriptional changes that will attenuate pathological environmental and dietary insults on bioenergetics. Total RNA obtained from cardiac tissue from transgenic cardiac specific expressing human cardiolipin synthase 1 (hCLS1) mouse model at 4 months of age compared to wildtype littermates
Project description:The fatty acid synthase (FASN) is the major fat synthesizing enzyme. FASN is an indispensable enzyme because mice with genetic deletion of Fasn are not viable. Apart from its physiological function, previous studies indicated that FASN could also exert a pathophysiological role, in the heart, because patients with heart failure showed up-reguation of FASN. To investigate the in vivo function of FASN up-regulation in the heart, we generated mice with myocardium-specific expression of FASN under control of the alpha-MHC promoter. Two different founder lines were generated with high and low FASN over-expression. Microarray gene expression profiling of heart tissue was performed of heart tissue from transgenic mice with high and low FASN expression Overall design: Microarray gene expression profiling was performed with heart tissue isolated from three study groups: (i) Transgenic mice with high cardiac FASN expression, (ii) transgenic mice with low cardiac FASN expression, and (iii) B6 control mice.
Project description:The fatty acid synthase (FASN) is the major fat synthesizing enzyme. FASN is an indispensable enzyme because mice with genetic deletion of Fasn are not viable. Apart from its physiological function, previous studies indicated that FASN could also exert a pathophysiological role, in the heart, because patients with heart failure showed up-reguation of FASN. To investigate the in vivo function of FASN up-regulation in the heart, we generated mice with myocardium-specific expression of FASN under control of the alpha-MHC promoter. Two different founder lines were generated with high and low FASN over-expression. Microarray gene expression profiling of heart tissue was performed of heart tissue from transgenic mice with high and low FASN expression Microarray gene expression profiling was performed with heart tissue isolated from three study groups: (i) Transgenic mice with high cardiac FASN expression, (ii) transgenic mice with low cardiac FASN expression, and (iii) B6 control mice.
Project description:Analysis of the transcriptome of cardiac tissue from mice trangenically engineered with a doxycycline inducible Tafazzin shRNA knockdown to mimic the loss of function of Tafazzin in Barth syndrome. ShRNA induced knockdown mice were compared to wildtype littermates also fed the doxycyline diet. The hypothesis was to investigate adaptive metabolic and compensatory gene transcriptional changes in myocardium of the mouse model of Barth syndrome Total RNA obtained from 2 month old cardiac tissue from the doxycycline inducible shRNA Tafazzin knockdown mouse model of Barth syndrome compared to wildtype littermates also fed 625 mg/Kg doxycycline diet
Project description:Purpose: Core 3 derived glycans, a major type of O-glycan expressed by normal epithelial cells of the gastrointestinal tract, are downregulated during malignancy, because of loss of expression of functional β3-N-acetylglucosaminyltransferase-6 (core 3 synthase). We investigated the expression of core 3 synthase in normal pancreas and pancreatic cancer and evaluated the biological effects of re-expressing core 3 synthase in pancreatic cancer cells that had lost expression. Experimental Design: We determined that pancreatic tumors and tumor cell lines have lost expression of core 3 synthase. We therefore re-expressed in human pancreatic cancer cells (Capan-2 and FG) to investigate the contribution of core 3 glycans to malignant progression. Results: Pancreatic cancer cells expressing core 3 synthase showed reduced in vitro cell proliferation, migration and invasion compared with vector control cells. Expression of core 3 O-glycans induced altered expression of β1 integrin, decreased activation of focal adhesion kinase, led to the down regulation of expression of several genes including REG1α and FGFR3, and altered lamellipodia formation. The addition of a GlcNAc residue by core 3 synthase leads to the extension of the tumor associated Tn structure on MUC1. Orthotopic injection of FG cells expressing core 3 synthase into the pancreas of nude mice produced significantly smaller tumors and decreased metastasis to the surrounding tissues compared to vector control FG cells. Conclusions: These findings indicate that expression of core 3 derived O-glycans in pancreatic cancer cells suppresses tumor growth and metastasis through modulation of glycosylation of mucins and other cell surface and extracellular matrix proteins. Two-condition experiment, Core 3 synthase stable expression (C3) vs. vector control (PLVX) cells. Biological replicates: 3 Core 3 synthase stable expression, 3 vector control, independently grown and harvested. One replicate per array.
Project description:Gene expression was compared between a Tet-OFF transfomant expressing thymidylate synthase trasgene and its parental human colorectal cancer cell line, DLD-1, or the transformant exposed to doxycycline.
Project description:Aims: Cardiomyocyte-specific nitric oxide synthase 3 (NOS3) overexpression reduces left ventricular (LV) remodelling after myocardial infarction in mice, but its effect on sustained LV pressure-overload remains incompletely understood. We investigated LV structural and functional adaptation to elevated afterload in mice with cardiomyocyte-restricted NOS3 overexpression (NOS3TG) and wild type littermates (WT). Methods and Results: Hemodynamic indices, cardiac hypertrophy and interstitial fibrosis were measured 10 weeks after transverse aortic constriction (TAC). After 10 weeks TAC, NOS3TG had better preserved systolic function (maximum rates of pressure development normalized to maximal pressure 77±6 versus 65±2 ms-1, P=0.05), reduced heart weight-body weight ratio (HW/BW, 5.0±0.3 versus 5.8±0.1, P<0.05), and cardiomyocyte width than WT (14.9±0.4 vs 16.7±0.2 ?m, P<0.05). After 10 weeks TAC, a 44k cDNA chip-based microarray analysis was validated using real time PCR and revealed significantly altered expression pattern of genes involved in cellular growth, matrix remodelling, and inflammation between genotypes. Conclusions: Cardiomyocyte-restricted NOS3 overexpression attenuates TAC-induced hypertrophy via autocrine inhibition of cardiomyocyte cell growth, but does not mitigate myocardial fibrosis. The subsequent diastolic dysfunction suggests that inhibition of matrix producing cells during hypertrophic stress is necessary to prevent functional and structural deterioration of the pressure-overloaded heart. Left ventricular mRNA expression profiles were compared between alpha-myosin heavy chain driven nitric oxide synthase 3 (alpha-MHC-NOS3) transgenic and wild type (WT) littermate mice at baseline and 10 weeks after transversal aortic constrcition-induced pressure-overload. Biological repeats: n=4, two males and two females, for each group and condition. Transgenic mice were backcrossed for seven generations (F7) to a C57Bl/6 N background and age and weight matched animals were used for microarray experiments.
Project description:The raf kinase inhibitor protein, RKIP, is up-regulated on cadiac biopsy specimens of heart failure patients. To investigate the in vivo role of an increased cardiac content of RKIP, we generated transgenic mice with myocardium-specific expression of RKIP (PEBP1; phosphatidylethanolamine binding protein 1) under control of the alpha-MHC promoter in B6 (C57BL/6J) background. Because RKIP is a dual-specific GRK2 and Raf kinase inhibitor, RKIP-transgenic mice were compared to transgenic mice with myocardium-specific expression of the GRK2 inhibitor, GRK2-K220R, which is a kinase-inactive GRK2 (ADRBK1) mutant with dominant-negative function. In frame of our studies, we found that RKIP-transgenic mice developed signs of heart failure and cardiotoxic lipid load at an age of 8 months. In contrast, transgenic expression of GRK2-K220R improved cardiac function and protected against chronic pressure overload-induced symptoms of heart failure. To identify genes related to cardiotoxic lipid load, we further determined the cardiac gene expression profile of Tg-SCD1 mice with mypcardium-specific expression of SCD1 (stearoyl-CoA desaturase-1) and Tg-UCP1 mice with heart-specific expression of UCP1 (uncoupling protein 1). Overall design: Whole genome microarray gene expression profiling was performed of hearts from aged, 8-month-old Tg-RKIP mice, Tg-GRK2-K220R mice, Tg-SCD1 mice, and Tg-UCP1 mice. All transgenic mice had B6 background, and the control group consisted of age-matched non-transgenic B6 mice. Two biological replicates were made of each group, and total cardiac RNA of three mice was pooled for one gene chip.
Project description:We report the application of sequencing-by-synthesis technology for high-throughput profiling of small RNAs involved in Chalcone synthase A (CHS-A) sense cosuppression in petunia. Sense cosuppression is a classical form of eukaryotic post-transcriptional gene silencing. It was first reported in transgenic petunia, where a transgene overexpressing the host Chalcone Synthase-A (CHS-A) gene caused the degradation of the homologous transcripts and the loss of flower pigmentation. Though sense cosuppression is recognized as an RNA silencing mechanism, little evidence has been yet provided demonstrating its association with the generation of individual small interfering RNAs (siRNAs) that are the assumed determinants. In this work, the deep sequencing of small RNAs in cosuppressed transgenic petunia and WT petunia respectively allowed for the identication of siRNAs that vastly predominate in the silenced flower and guide prominent cleavage events in CHS-A mRNA. Examination of 2 small RNA populations from WT and cosuppressed petunia petals respectively