Next Generation Sequencing of wild-type HEK293 cells compared to STAT5b knock-out HEK293 cells using Crispr/Cas9
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA732661 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:During kidney diseases, diverse tissue-specific pathways can regulate kidney injury and prognosis. In our study, we focused on STAT5, a member of the Janus Kinase/ Signal Transducer and Activator of Transcription (JAK/STAT) pathway, classically described in immune cells which has been recently described in intrinsic kidney cells. STAT5 activation is detected in both human podocytes and tubular cells in FSGS and ATI, respectively. Additionally, STAT5 deficiency in either glomerular or tubular epithelium aggravates the functional and structural alterations in a range of experimental models of glomerular or tubular disease. Using RNA sequencing, we show here that STAT5b inactivation in HEK-293 cells resulted in dysregulation of multiple metabolic pathways, including glycolysis and oxidative phosphorylation. In conclusion, activating renal epithelial STAT5 represents a new therapeutic avenue with the potential for a range of beneficial effects in kidney disease.
2021-06-01 | GSE175508 | GEO
Project description:G-protein-coupled receptors (GPCRs) are pivotal cell membrane proteins that sense extracellular molecules and activate cellular responses. The G-protein α subunit i (Gαi) family represents the most common GPCR-coupling partner and consists of eight subunits with distinct signaling properties. However, analyzing the coupling pattern has been challenging owing to endogenous expression of the Gαi subunits in virtually all cell lines. Here, we generate a HEK293 cell line lacking all Gαi subunits, which enables the measurement of GPCR-Gαi coupling upon transient re-expression of a specific Gαi subunit. We profile Gαi-coupling selectivity across 11 GPCRs by measuring ligand-induced inhibitory activity for cAMP accumulation. The coupling profiles are then classified into three clusters, representing those preferentially coupled to Gαz, those to Gαo, and those with unapparent selectivity. These results indicate that individual Gαi-coupled GPCRs fine-tune Gαi signaling by exerting coupling preference at the Gαi-subunit level.
| S-EPMC9884212 | biostudies-literature
Project description:Bacterial Cas9 nuclease induces site-specific DNA breaks using small gRNA as guides. Cas9 has been successfully introduced into Drosophila for genome editing. Here, we improve the versatility of this method by developing a transgenic system that expresses Cas9 in the Drosophila germline. Using this system, we induced inheritable knock-out mutations by injecting only the gRNA into embryos, achieved highly efficient mutagenesis by expressing gRNA from the promoter of a novel non-coding RNA gene, and recovered homologous recombination-based knock-in of a fluorescent marker at a rate of 4.5% by co-injecting gRNA with a circular DNA donor.
| S-EPMC4025491 | biostudies-literature
Project description:Genetic STAT5B immunodysregulatory disorder
| PRJNA416163 | ENA
Project description:Genetic STAT5B immunodysregulatory disorder
| PRJNA416162 | ENA
Project description:CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging in primary NK cells. For example, transgene delivery using lentiviral or retroviral transduction resulted in a limited yield of genetically-engineered NK cells due to substantial procedure-associated NK cell apoptosis. We describe here a DNA-free method for genome editing of human primary and expanded NK cells using Cas9 ribonucleoprotein complexes (Cas9/RNPs). This method allowed efficient knockout of the TGFBR2 and HPRT1 genes in NK cells. RT-PCR data showed a significant decrease in gene expression level, and a cytotoxicity assay of a representative cell product suggested that the RNP-modified NK cells became less sensitive to TGF?. Genetically modified cells could be expanded post-electroporation by stimulation with irradiated mbIL21-expressing feeder cells.
| S-EPMC6101749 | biostudies-literature
Project description:The Acyl-CoA-binding domain-containing protein (ACBD3) plays multiple roles across the cell. Although generally associated with the Golgi apparatus, it operates also in mitochondria. In steroidogenic cells, ACBD3 is an important part of a multiprotein complex transporting cholesterol into mitochondria. Balance in mitochondrial cholesterol is essential for proper mitochondrial protein biosynthesis, among others. We generated ACBD3 knock-out (ACBD3-KO) HEK293 and HeLa cells and characterized the impact of protein absence on mitochondria, Golgi, and lipid profile. In ACBD3-KO cells, cholesterol level and mitochondrial structure and functions are not altered, demonstrating that an alternative pathway of cholesterol transport into mitochondria exists. However, ACBD3-KO cells exhibit enlarged Golgi area with absence of stacks and ribbon-like formation, confirming the importance of ACBD3 in Golgi stacking. The glycosylation of the LAMP2 glycoprotein was not affected by the altered Golgi structure. Moreover, decreased sphingomyelins together with normal ceramides and sphingomyelin synthase activity reveal the importance of ACBD3 in ceramide transport from ER to Golgi.
| S-EPMC8303370 | biostudies-literature
Project description:Here we report both TET1-WT and catalytically dead mutant TET1 (TET1-MUT) directly interact with STAT5B in PDX2 B-ALL cells. To study the landscape of TET1 and STAT5B binding in whole genomic DNA and test if the STAT5B binding signal is affected by TET1, we conduct the ChIP-seq assay with flag antibody (TET1-WT, TET1-MUT) and STAT5B antibody (sgNS or sgTET1) respectively. The results showed that TET1 bound peaks overlap with STAT5B very well and the tag density of STAT5B on target genes decreases in TET1-impeded cells. We conclude that TET1 recruit STAT5B to the promoters of its target genes and further promote the target genes’ transcriptions.
2023-02-13 | GSE190852 | GEO
Project description:A series of dual-channel gene expression profiles obtained using Rosetta/Merck Mouse TOE 75k microarrays was used to examine the sex-dependent and STAT5b-dependent differences in gene expression in adult mouse liver. This series is comprised of 4 pools of 3 randomly chosen independent wildtype male and female mouse liver cDNA samples and 4 pools of 3 randomly chosen independent STAT5b-deficient male and female mouse liver cDNA samples, totaling 16 pools. The pools were paired randomly to generate 4 comparisons of M-WT:F-WT, M-WT:M-KO, F-KO:F-WT, and F-KO:M-KO. Comparison of the set of sex-dependent genes with the set of genes responsive to the loss of STAT5b in males shows that 75% of the sex-specific genes were also regulated by STAT5b in males. Only 20% of the sex-specific genes retained sex-specificity in the absence of STAT5b, indicating a large role for STAT5b in sex-specific liver gene expression. Keywords: genetic knockout and sex response
2006-07-26 | GSE5072 | GEO