Transcription profiling of mouse Emx2 knock-out urogenital epithelium
Ontology highlight
ABSTRACT: Series of samples studying effect of knock out Emx2 in urogenital epithelium of mouse embryos at E10.5. Experiment Overall Design: The epithelial cells of the gonadal primordium were obtained by Laser Microdissection System. The specimens prepared from three individuals were mixed as one pool. There are three experimental replicates in each genotype, 3-pools of wild-type and 3-pools of Emx2 KO mouse.
Project description:The thalamus of the brain acts as a relay station; taking inputs from several parts of the brain and then sending the information to the cortex and vice versa. It is also the structure know to affected in several brain developmental disorders such as schizophrenia, autism spectrum disorders, bipolar disorders etc. Upon in situ hybridisation one can visualise the expression of the transcription factor Tcf7l2 to be highest in prosomeric regions of the thalamus throughout development. With this information in mind we set out to find out, if the expression of Tcf7l2 is essential for the identity of the thalamic structure. Therefore, Tcf7l2 was knocked (KO) out using Cre+mice at embryonic stage E18.5 and postnatal adult stage P60. The E18.5 Tcf7l2 KO is a total knockout and the P60 Tcf7l2 KO is neuron-specific knockout. Total RNA was extracted and sent for sequencing using Illumina 2500. The data obtained was aligned by HISAT2 alignment tool, and the excon read counts were gathered using htseq counts, and expression normalization and differential gene expression was analysed using DESeq2.
Project description:Mice lacking the function of the PcG protein CBX2 (also known as M33) show defects in gonadal, adrenal, and splenic development. In particular, XY knockout mice develop ovaries but not testes, and the gonads are hypoplastic in both sexes. To clarify the molecular basis of the defects, RNA prepared from both wild type and KO gonads were subjected to microarray analyses. Mouse embryonic gonads of Cbx2 knockout (KO) were sectioned and recovered at E11.5 for RNA extraction and hybridization on Affymetrix microarrays. Four different genotypes of embryos at E11.5 (XY WT, XX WT, XY Cbx2 KO, and XX Cbx2 KO) were frozen in OCT compound without fixation. They were sectioned to 30 μm slices and stained with hematoxylin. The developing gonadal cells localized by GATA4 immunostaining and morphology were prepared using a Laser Microdissection System. The specimens prepared from two or three individuals were combined into one group, and three groups were prepared for each genotype.
Project description:Calcium Dependent Protein Kinases (CDPKs) play important function in calcium signaling at various stage of Plasmodium falciparum. CDPK7 is one such kinase, which plays a crucial role in the development of Plasmodium falciparum parasite. In order to understand the function of PfCDPK7, we performed comparative phosphoproteomic analysis of PfCDPK7 knockout mutant and wild-type lines. In brief, proteins from PfCDPK7 knockout mutant and wild-type parasites were isolated, digested with trypsin and labelled with iTRAQ tags. Phosphopeptides were then enriched from iTRAQ labeled peptides and subjected to LC-MS/MS analysis. This led to the identification of putative substrates of PfCDPK7 in the parasites. A comparative proteomic analysis was performed to measure the protein abundance changes in the PfCDPK7 mutant parasites.
Project description:Yeast strains (wild type, Naa10 KO and Naa20 KO, resp: wt, natA-delta and natB-delta) were profiled for full proteome analysis by SWATH MS
Project description:Label-free quantitative affinity purification experiments were performed to identify the protein composition of pericentriolar satellites in the wild-type and two acentrosomal (stil KO; cep152 KO) DT40 cell lines.
Project description:Fibrosis is defined as an abnormal matrix remodeling and loss of tissue homeostasis due to excessive synthesis and accumulation of extracellular matrix proteins in tissues. At present, there is no effective therapy for organ fibrosis. Previous studies demonstrated that aged plasminogen activator inhibitor-1(PAI-1) knockout mice develop spontaneously cardiac-selective fibrosis without affecting any other organs including kidney. Therefore, the PAI-1 knockout model of cardiac fibrosis provides an excellent opportunity to find the igniter(s) of cardiac fibrosis and its status in unaffected organs. We hypothesized that differential expressions of profibrotic and antifibrotic genes in PAI-1 knockout hearts and unaffected organs lead to cardiac selective fibrosis. In order to address this prediction, we have used a genome-wide gene expression profiling of transcripts derived from aged PAI-1 knockout hearts and kidneys. The variations of global gene expression profiling were compared within four groups: wildtype heart vs. knockout heart; wildtype kidney vs. knockout kidney; knockout heart vs. knockout kidney and wildtype heart vs. wildtype kidney. Analysis of illumina-based microarray data revealed that several genes involved in different biological processes such as immune system processing, response to stress, cytokine signaling, cell proliferation, adhesion, migration, matrix organization and transcriptional regulation were affected in hearts and kidneys by the absence of PAI-1, a potent inhibitor of urokinase- and tissue-type plasminogen activator. Importantly, the expressions of a number of genes, involved in profibrotic pathways were upregulated or downregulated in PAI-1 knockout hearts compared to wildtype hearts and PAI-1 knockout kidneys. To our knowledge, this is the first comprehensive report on the influence of PAI-1 on global gene expression profiling in the heart and kidney and its implication in several biological processes including fibrogenesis. Total RNA was extracted from hearts and kidneys derived from three PAI-1 knockout (12- month old) and three wild-type mice (12-month old) using RNeasy Fibrous Tissue Mini Kit (Qiagen, Valencia, CA) following the manufacturer’s instructions. The quality of RNA (RNA Integrity, RIN) in all 12 samples (3 wildtype hearts; 3 PAI-1 KO hearts; 3 wildtype kidneys; and 3 PAI-1 KO kidneys) was checked using the bioanalyzer. We have used a genome-wide gene expression profiling of transcripts derived from aged PAI-1 knockout hearts and kidneys. The variations of global gene expression profiling were compared within four groups: wildtype heart vs. knockout heart; wildtype kidney vs. knockout kidney; knockout heart vs. knockout kidney and wildtype heart vs. wildtype kidney.
Project description:The oxidative phosphorylation (OXPHOS) system localized in the inner mitochondrial membrane secures production of the majority of ATP in mammalian organisms. Individual OXPHOS complexes form supramolecular assemblies termed supercomplexes. The complexes are linked not only by their function but also by interdependence of individual complex biogenesis or maintenance. For instance, cytochrome c oxidase (cIV, COX) or cytochrome bc1 complex (cIII) deficiencies affect the level of fully assembled NADH dehydrogenase (cI) in monomeric as well as supercomplex forms. It was hypothesized that cI is affected at the level of enzyme assembly as well as at the level of cI stability and maintenance. However, the true nature of interdependency between cI and cIV is not fully understood yet. We used a HEK293 cellular model where the COX4 subunit was completely knocked out, serving as an ideal system to study interdependency of cI and cIV, as early phases of cIV assembly process are disrupted. Total absence of cIV was accompanied by profound deficiency of cI, documented by decrease in the levels of cI subunits and significantly reduced amount of assembled cI. Supercomplexes assembled from cI, cIII and cIV were missing in COX4 KO due to loss of cIV and decrease in cI amount. Pulse-chase metabolic labelling of mtDNA-encoded proteins uncovered a decrease in the translation of cIV and cI subunits. Moreover, partial impairment of mitochondrial protein synthesis correlated with decreased content of mitochondrial ribosomal proteins. In addition, complexome profiling revealed accumulation of cI assembly intermediates indicating that cI biogenesis, rather than stability, was the affected process. We propose, that impairment of mitochondrial protein synthesis caused by cIV deficiency represents one of the mechanisms which may couple biogenesis of cI and cIV.
Project description:zebrafish embryos proteome for slbp2 KO F3 and wild type at 2.5hpf and 3.5hpf, three replicates for every sample. There are 12 samples in total. Then we analyse the different expressed genes and hope to find out clue which result in serious phenotype of slbp2 KO F3.
Project description:We have analyzed gene expression in cone photoreceptors isolated from wild type and C-DGCR8 (DiGeorge Syndrome Critical Region Gene 8) KO mice at five different time points to get a mechanistic inside into the altered molecular pathways after microRNAs depletion. Cones were isolated by FACS from wild type and C-DGCR8 KO mice expressing Cre recombinase postnatally specifically in cones (D4-Cre) and bearing conditional null Dgcr8 allele. Experiments were performed in duplicates from different time points (P30, P40, P50, P60, and P90). Total RNA was extracted and next-generation RNA sequencing was performed.