ABSTRACT: For this project, we explored the genetic determinants of the heart development condition termed patent foramen ovale (PFO) using quantitative trait loci (QTL) mapping and genomics/transcriptomics analyses. Two mice strains were chosen that exhibit highly divergent phenotypes associated with PFO, 129T2/SvEms and QSi5. In this experiment, we performed whole genome sequencing (WGS) on genomic DNA extracted from liver specimens of 129T2/SvEms or QSi5 mice in order to identify genomic variants that may contribute to the phenotypes associated with PFO.
Project description:The nasal epithelium represents the first line of defense against inhaled pathogens, allergens, and irritants and plays a key role in the pathogenesis of a spectrum of acute and chronic airways diseases. Despite age-dependent clinical phenotypes triggered by these noxious stimuli, little is known about how ageing affects the structure and function of the airway epithelium that is crucial for lung homeostasis and host defense. The aim of this study was therefore to determine age-related differences in structural and functional properties of primary nasal epithelial cultures from healthy children and non-smoking elderly people. To achieve this goal, highly differentiated nasal epithelial cultures were established from nasal brushes at air-liquid-interface and used to study epithelial cell type composition, mucin (MUC5AC and MUC5B) expression and ion transport properties. Further, we determined age-dependent molecular signatures using global proteomic analysis. We found lower numeric densities of ciliated cells and higher levels of MUC5AC expression in cultures from children vs. elderly people. Bioelectric studies showed no differences in basal ion transport properties, ENaC-mediated sodium absorption or CFTR-mediated chloride transport, but detected decreased calcium-activated TMEM16A-mediated chloride secretory responses in cultures from children vs. elderly people. Proteome analysis identified distinct age-dependent molecular signatures associated with ciliation and mucin biosynthesis, as well as other pathways implicated in ageing. Our data identified intrinsic, age-related differences in structure and function of the nasal epithelium and provide a basis for further studies on the role of these findings in age-dependent airways disease phenotypes observed with a spectrum of respiratory infections and other noxious stimuli.
Project description:A 45h time-course RNA-seq study was performed to analyse the different circadian phenotypes of human colorectal cancer cell line HCT116 WT, HCT116 ARNTL Knockout, HCT116 PER2 Knockout and HCT116 NR1D1 Knockout cells. Samples were taken every 3h starting from 9h after cell synchronization for a period of 45h resulting in 16 time-points for each cell line.
Project description:WGBS was performed on: 1) untreated SH-SY5Y human neuroblastoma cells (day 0) 2) vincristine-treated SH-SY5Y human neuroblastoma cells (7 days of treatment - day 7) 3) vincristine-treated SH-SY5Y human neuroblastoma cells (7 days of treatment followed by 7 days of recovery - day 14)
Project description:The combination of multi-omic techniques, e.g. genomics, transcriptomics, proteomics, metabolomics and epigenomics has revolutionised studies in medical research. These are employed to support biomarker discovery, better understand molecular pathways and identify novel drug targets. Despite concerted efforts in integrating omic datasets, there is an absence for the integration of all four biomolecules in a single extraction protocol. Here, we demonstrate for the first time a novel, minimally destructive integrated protocol for the simultaneous extraction of artificially degraded DNA, proteins, lipids and metabolites from pig brain samples. We used an MTBE-based approach to separate lipids and metabolites, followed by subsequent isolation of DNA and proteins. We have validated this protocol against standalone extraction protocols and show comparable or higher yield of all four biomolecules. This integrated protocol is key towards facilitating preservation of irreplaceable samples while promoting downstream analyses and successful data integration by removing bias from univariate dataset noise and varied distribution characteristics. Keywords: deg
Project description:Basement membranes (BMs) are ubiquitous extracellular matrices whose composition remains elusive, limiting our understanding of BM regulation and function. By developing a bioinformatic and in vivo discovery pipeline, we define a network of over 220 human proteins localized to BMs. More than 100 BM-network genes associate with human phenotypes and by screening over 63,000 germline genomic sequences from families with rare disorders, we discovered novel predicted loss-of-function variants in MATN2. Biochemical analyses suggest that MATN2 interacts with many BM proteins, and we found that depletion of MATN2 affected levels of core BM components in human podocyte-derived extracellular matrix.
Project description:shRNAs were assessed for off-target effects by comparing the gene expression profiles of cells that they had been infected into. shRNAs designed with the shERWOOD algorithm and house in the ultramir microRNA scafold were found to have very little off targeting. Purpose: A major detriment to RNAi is off-targeting. We wished to assess the level of off targeting of microRNA (ultramiR) housed shERWOOD shRNAs as compared to similar shRNAs in the TRC collection. Methods: 5 shRNAs targeting each of two genes were infected into the 4T1 cell line. For each gene one shRNA was selected from the TRC collection and one based on the shERWOOD algorithm. For each gene, the exrpession profiles of the corresponding shRNA infected cells were compared using RNAseq. Conclusions: Highly similar profiles were observed between shERWOOD selected shRNAs. TRC shRNAs produced profiles indicative of off-targeting.
Project description:LncRNAs represent a major transcriptional output of the human genome, but the function of many of these elements is unknown. For chromatin-localized lncRNAs, identification of genomic binding sites of these lncRNAs provides one opportunity to characterize their function. In this experiment, we have used capture hybridization analysis of RNA targets with high-throughput sequencing (CHART-seq) to identify the binding sites of the lncRNA LINC00899 in HeLa cells. LINC00899 is of interest as its depletion results in mitotic delay, suggesting a role in mitotic progression. This experiment contains 5 replicate batches where each batch contains a sample with antisense capture oligonucleotides (COs) to hybridize to and pull down the lncRNA transcript; an input control for the antisense pulldown; a control sample with sense COs, which should not hybridize to and pull down the lncRNA transcript; and an input control for the sense pulldown. All samples in the same batch were generated at the same time, and each pulldown (antisense or sense) was performed on chromatin obtained from independent cell cultures. All samples were subjected to high-throughput paired-end sequencing across two lanes.
Project description:To investigate the role of RAD21 in the transcriptional regulation of global gene expression at early stage of colorectal cancer developments, we peformed the genome-wide analysis to map genomic regions bound by Rad21 in normal small testinal crypts and tumors (adenomas) harvested from Apc Min/+ mice using ChIP-seq. ChIP-seq naalysis identified high confidence RAD21 binding sites unique to normal crypts or adenomas, as well as those common to both tissues. We further performed RNA-seq to profile the changes in gene expression from normal WT crypts to adenomas at the very early stage of adenomagenesis in the context of Rad21 heterozygous loss. mRNA profiles of normal small intestinal crypts (WT) and adenomas from Apc Min/+ and Apc Min/+:Rad21+/- double mutant mouse; Mapping of Rad21 genomic binding sites in normal intestinal crypts (WT) and Apc Min/+ adenomas
Project description:Effect of induced Methylation on 3D genome organisation in yeast. Hi-C experiment were performed on yeast expressing or not the 4 murine DNMTs (DNMT1, 3a, 3b and 3L).
Project description:Synthetic biology holds immense promise to tackle key problems we are facing, for instance in resource use, environmental health, and human health care. However, comprehensive safety measures are needed to deploy genetically engineered microorganisms in open-environment applications. Intrinsic, genetically encoded biocontainment systems, which control cell survival based on environmental cues, can solve this issue. Here, we describe a genetic biocontainment system based on conditional stability of essential proteins. We used a yeast-adapted destabilizing domain degron, which can be stabilized by estradiol addition (ERdd). Leveraging the yeast GFP collection and lab automation platforms, we ERdd-tagged 775 essential genes and screened for strains with estradiol dependent growth. Three genes, SPC110, DIS3 and RRP46, were found to be particularly suitable targets. Respective strains showed no growth defect in the presence of estradiol and strong growth inhibition in its absence. SPC110-ERdd offered the most stringent containment, with an escape frequency of 7.0x10-8, and full growth restoration at 100 nM estradiol. By systematically analyzing the containment escapees, we identified the non-essential C-terminal region of SPC110 as target for escape mutations. Its removal decreased the escape frequency with a single ERdd tag further to 4.9x10-9. Combining SPC110-ERdd with a second ERdd tag on either DIS3 or RRP46 resulted in escape frequencies below the detection limit of the used assay (<2x10-10). Being based on conditional protein stability, this approach is mechanistically orthogonal to previously reported intrinsic biocontainment systems. It thus can be readily combined with other systems, for instance ones based on transcriptional or translational control of essential gene expression, to achieve multiplexed, extremely stringent control over the survival of engineered organisms.