Over-expression of truncated FMRP with 295 amino acid residues: HEK293T cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Xianguo Fu
PROVIDER: E-GEOD-68668 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:We used microarrays to detail the global gene expression in stably transfected HEK 293T cells of the over-expression of truncated FMRP containing 295 amino acid residues, which were compared with control (stably transfected HEK 293T cells of empty lentiviral vector (pLEX-MCS).
2015-05-08 | GSE68668 | GEO
Project description:RNA microarray was used to analyze the differentialy expressed genes between stably transfected HEK293T cells of the overexpression of the new FMRP isoform with 297 amino acid and stably transfected HEK293T cells of empty lentiviral vector. we constructed the lentiviral vector of exons 1-9 together with the sequence of 140bp fragment of human FMR1 gene to overexpress the truncated FMRP with 297 amino acid, and transfected cells with the void plasmid pLEX-MCS were regarded as control group.
2017-07-26 | GSE101830 | GEO
Project description:Fragile X syndrome (FXS) is a rare disease but is the most common form of inherited intellectual disability and a leading cause of autism. FXS is due to the absence of the Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein mainly involved in translational control. Even if this molecular defect is known, no specific therapy is available for FXS. The first alteration observed in the brain of FXS patients and of Fmr1 KO mice, model for FXS, is represented by an abnormal dendritic morphology that is associated with an altered synaptic plasticity. These findings led to numerous studies focused on mature neurons. However, recently, an increasing body of evidence is pointing out the importance of FMRP in the early steps of brain development. Thus, with the purpose to decipher the earliest molecular events leading to FXS we developed a stem-cell-based disease model by knocking-down the expression of Fmr1 in mouse embryonic stem (ES) cells. To gain insights in the pathways that are affected when FMRP is repressed, we performed a gene expression profile analysis comparing total RNA from shFmr1 and shControl ES cells using whole genome mouse microarrays. Transcripts were clustered according to their Gene Ontology classification using the DAVID software. Surprisingly, the most altered functional category was Nervous system development and function, highlighting the role of FMRP also during the earliest steps of neural development. To study the precise role of FMRP in Embryonic stem (ES) cells, we used an RNAi-based loss-of-function strategy by transducing mouse ES cells with a lentivirus expressing GFP and a shRNA targeting the constitutive exon 1 of Fmr1 (shFmr1). A random shRNA (shCT) was used as a control sequence. Transduced cells were sorted by flow cytometry and established as non-clonal cell populations. RNA samples were harvested and profiling experiments were performed in â??dye-balanceâ?? as indicated for each replicate.
2016-07-04 | E-GEOD-71184 | biostudies-arrayexpress
Project description:Topoisomerases are crucial to solve DNA topological problems, but they have not been linked to RNA metabolism. Here we show that human topoisomerase 3M-NM-2 (Top3M-NM-2) is an RNA topoisomerase that biochemically and genetically interacts with FMRP, a protein deficient in Fragile X syndrome and known to regulate translation of mRNAs important for neuronal function and autism. Notably, the FMRP-Top3M-NM-2 interaction is abolished by a disease-associated FMRP mutation, and several human genetic studies link Top3M-NM-2 mutation to schizophrenia and intellectual disability. Top3M-NM-2 binds multiple mRNAs encoded by genes with neuronal functions related to schizophrenia and autism. Expression of one such gene, ptk2/FAK, is reduced in neuromuscular junctions of Top3M-NM-2 mutant flies. Synapse formation is defective in Top3M-NM-2 mutant flies and mice, as observed in FMRP mutant animals. Our findings suggest that Top3M-NM-2 acts as an RNA topoisomerase and works with FMRP to promote expression of mRNAs critical for neurodevelopment and mental health. We have identified Top3M-NM-2 as the first RNA topoisomerase in eukaryotes. To study whether Top3M-NM-2 binds mRNAs in vivo and also to identify its genome-wide RNA targets, we performed HITS-CLIP (high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation; Licatalosi et al. Nature 456, 464-469, 2008). In this method, proteins are covalently crosslinked with bound RNAs in vivo, thus allowing specific mapping of the sites of interaction of the protein with the RNA. We cultured a HeLa cells stably-expressing HF-Top3M-NM-2 and irradiated the cells with UV to crosslink RNA and proteins in vivo. Following cell lysis, RNA was partially digested using various concentrations of RNase A. The RNA crosslinked to HF-Top3M-NM-2 was then immunoprecipitated with anti-Flag M2 argarose beads (Sigma). As a control, we also performed a mock immunoprecipitation using HeLa cells that do not express HF-Top3M-NM-2. The 3M-bM-^@M-^Y ends of the purified RNAs were ligated to a p32-labeled RNA adapter, fractionated by SDS-PAGE, and transferred to a nitrocellulose membrane. The p32-labeled RNA-Top3M-NM-2 complexes were detected by autoradiography and recovered from the membrane. The complexes were treated with Proteinase K to remove proteins. The RNA was ligated at their 5M-bM-^@M-^Yend to another RNA adaptor, and reverse-transcribed to cDNA. The cDNA obtained was fractionated a denaturing 6% TBE-Urea gel (InVitrogen), amplified by PCR, and subjected to high-throughput sequencing by Genome Analyzer II (Illumina). Sequenced tags were mapped to the human genome (hg18, NCBI/NIH) and to human RefSeq genes using ELAND program (Illumina).
2013-08-04 | E-GEOD-47502 | biostudies-arrayexpress
Project description:Goal of the experiment : Retroviral-mediated gene transfer of the THAP-zinc finger protein THAP1 inhibits endothelial cell proliferation through coordinated repression of critical cell cycle regulators and pRB-E2F target genes. Experimental design: To gain insight into the effects of THAP1 on endothelial cell growth regulatory pathways, we identified THAP1 target genes in primary human endothelial cells using oligonucleotide-based microarray technology. Human umbilical vein endothelial cells (HUVECs) were transduced with pMLV-MCS or pMLV-THAP1 retroviral expression vectors and, after 48h, cells were harvested for isolation of total RNA and preparation of Cy3- or Cy5-labeled cRNA probes, which were hybridized to DNA microarrays that contained 22 000 unique 60-nt oligonucleotide probes representing > 17 000 human genes. Independent microarray experiments were performed after one (#1xTHAP1) or two (#2xTHAP1) consecutive transductions using independent HUVEC primary cell cultures. Dye swap experiments were performed to eliminate the effect of dye bias, and for each gene probe, the data were subjected to statistical analysis to identify those probes for which a significant difference (p value < 0.01) in mean hybridization intensity was observed between HUVECs transduced with pMLV-MCS or pMLV-THAP1 retroviral expression vectors. Among the gene probes demonstrating a significant difference between the two conditions, we selected those whose expression varied in a similar manner in the two independent microarray experiments. . #2xTHAP1 experiment (Microarrays Code Bars 16011521022012 and 16011521022013): comparison of HUVEC-THAP1 and HUVEC-MCS after two consecutive retroviral transductions of HUVECs with pMLV-THAP1 or pMLV-MCS vectors (higher percentage of genes differentially expressed with p value < 0.01; higher folds) #1xTHAP1 experiment (Microarrays Code Bars 16011521025800 and 16011524025685): comparison of HUVEC-THAP1 and HUVEC-MCS after a single retroviral transduction of HUVECs with pMLV-THAP1 or pMLV-MCS vectors (lower percentage of genes differentially expressed with p value < 0.01; lower folds).
2006-09-11 | E-TABM-24 | biostudies-arrayexpress
Project description:To investigate the regulation of mRNA by 3'end unridylation, we performed RNA sequencing on HEK 293T cells and its TENT3A/B knockout strains (ΔTENT3A/B). We performed transcriptomic analysis comparing mRNA abundance in HEK 293T cells and ΔTENT3A/B
2022-11-23 | GSE214609 | GEO
Project description:To investigate the regulation of mRNA by 3'end unridylation, we performed RNA sequencing on HEK 293T cells and its TENT3A/B knockout strains (ΔTENT3A/B). We performed transcriptomic analysis comparing mRNA abundance in HEK 293T cells and ΔTENT3A/B
2022-11-23 | GSE214608 | GEO
Project description:Small hepatocyte-like progenitor cells (SHPCs) are hepatocytic progenitor cells that transiently form clusters in rat livers treated with retrorsine and with 70% partial hepatectomy (PH). We previously reported that transplantation of Thy1+ cells derived from D-galactosamine-treated livers promotes SHPC expansion, resulting in the acceleration of liver regeneration. Extracellular vesicles (EVs) produced by Thy1+ cells act on sinusoidal endothelial cells (SECs) and Kupffer cells to secrete IL17B and IL25, respectively, resulting in SHPC activation through IL17 receptor B (RB) signaling. Our aim is to identify factors in Thy1-EVs that activate IL17RB signaling. Thy1+ cells isolated from rats with D-galactosamine-induced liver injury were cultured for one week. Although some liver stem/progenitor cells proliferated into colonies, others maintained as mesenchymal cells (MCs). Thy1-MCs or Thy1-liver stem/progenitor cells were transplanted into retrorsine/PHtreated livers to examine their effects on SHPCs. SHs isolated from adult rat livers were used to validate factors regulating growth induction. The number and size of SHPCs remarkably increased in livers transplanted with Thy1-MCs. Comprehensive analysis of Thy1-MC-EVs revealed that miR-199a-5p, CINC-2, and MCP-1 are candidates for stimulating SHPC growth. Administration of the miR-199a-5p mimic, and not CINC-2, promoted SH growth. SECs treated with CINC-2 induced IL17b expression and their conditioned medium promoted SH growth. Thy1-MC transplantation may accelerate liver regeneration due to SHPCs expansion, which is stimulated by CINC-2/IL17RB signaling and miR-199a-5p.
2023-04-07 | PXD039384 | Pride
Project description:Elf5 (or ESE-2) is an ETS transcription factor that is abundantly expressed in the mammary epithelium, where it plays a critical role in dictating cell fate and lineage choices. These changes are in part mediated by alterations in the expression and activity of critical components of the Jak/Stat pathway. While the biological function of Elf5 in mammary gland development has been well characterized, its role in breast cancer remains to be elucidated. Here we show that loss of Elf5 leads to features associated with epithelial-mesenchymal transition (EMT) in the mouse mammary gland during pregnancy and lactation. These cellular changes in Elf5-null mammary epithelia are also reflected at the molecular level by the global enrichment of EMT-related gene signatures. ELF5 is expressed in higher level in weakly metastatic breast cancer cells that retained epithelial features compared to highly metastatic cells with mesenchymal features. ELF5 knockdown in T47D breast cancer cells resulted in EMT and increased migration. Conversely, ectopic expression of Elf5 revert mesenchymal-like MDA-MB-231 cells and its lung-tropic variant LM2 to an epithelial phenotype, with reduced migration, invasion and lung metastatic abilities. Finally, we showed that Elf5 binds directly to the promoter of the EMT transcriptional factor Snail2 (Slug) and repress its expression. Taken together, these data established a novel function for Elf5 in inhibiting EMT in normal mammary epithelium and in breast cancer through direct targeting of Snail2. This SuperSeries is composed of the following subset Series: GSE32143: LM2 cell: infected with lentivirus to stably express Elf5 vs GFP GSE32144: MDA-MB-231 cell: infected with lentivirus to stably express mut-Elf5 vs WT-Elf5
2012-09-20 | E-GEOD-32150 | biostudies-arrayexpress
Project description:Identification of EWSR1 and EWS-FLI1 transcript variants associated with the silencing of HNRNPH1 in TC32 Ewing sarcoma and 293T human embryonic kidney (HEK-293T) cells.
2019-09-20 | GSE119974 | GEO