Genome wide analysis of long term L1TD1 knockdown effects in NT2D1 cell line with inducible shRNA vectors
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE21275 | GEO | 2012/02/28
SECONDARY ACCESSION(S): PRJNA126577
REPOSITORIES: GEO
Similar Datasets
Project description:The purpose of this study was to analyze genome wide effects of long term L1TD1 silencing in embryonal carcinoma cell line (NT2D1) and to identify molecular level mechanismis of L1TD1 function and determine its possible role in pluripotency and self-renewal Six clones from NT2D1 cell line stably expressing TetR3 and L1TD1shRNA were analyzed. From each clone two samples were prepared: non-induced cells as control and doxycycline induced cells (+) to knockdown L1TD1, resulting altogether 12 samples. Samples were collected on day 6 after start of dox induction. One sample had to be excluded from the analysis because it did not cluster according to the sample groups.
2012-02-28 | E-GEOD-21275 | biostudies-arrayexpress
Project description:Pluripotency factors are known to be abnormally expressed cancer cells, which often correlate with poor prognosis. The molecular mechanisms by which pluripotency factors contribute to tumor aggressiveness are yet to be elucidated. The pluripotency marker L1TD1 interacts with other pluripotency factors and is a marker for human embryonic stem cells. In cancer, L1TD1 expression has been detected in solid tumors, including Embryonal tumors of the Central Nervous System. Previously, we reported that, in medulloblastoma, L1TD1 expression correlates with metastasis and shorter overall patient survival. Here, we use Affinity purification-mass spectrometry and global proteomics to map the L1TD1 protein interaction network and assess proteins regulated by L1TD1 expression, respectively. Interactome and global proteome analysis led to the enrichment of overlapping Biological Processes attributed to L1TD1 including Cell proliferation, Cell death and cell migration. We also used L1TD1 overexpressing tumor cell populations for in vitro validation of the omic data. L1TD1 overexpressing cells presented higher cell mobility, enhanced filopodial formation and distinct cell morphology. These cells were overall more proliferative and resistant to cisplatin treatment in vitro.
2024-02-09 | PXD040803 | Pride
Project description:RNA interference (RNAi) technology is a powerful methodology recently developed for the specific knockdown of targeted genes. RNAi is most commonly achieved either transiently by transfection of small interfering (si) RNA oligonucleotides, or stably using short hairpin (sh) RNA expressed from a DNA vector or virus. Much controversy has surrounded the development of rules for the design of effective siRNA oligonucleotides; and whether these rules apply to shRNA is not well characterized.To determine whether published algorithms for siRNA oligonucleotide design apply to shRNA, we constructed 27 shRNAs from 11 human genes expressed stably using retroviral vectors. We demonstrate an efficient method for preparing wild-type and mutant control shRNA vectors simultaneously using oligonucleotide hybrids. We show that sequencing through shRNA vectors can be problematic due to the intrinsic secondary structure of the hairpin, and we determine a strategy for effective sequencing by using a combination of modified BigDye chemistries and DNA relaxing agents. The efficacy of knockdown for the 27 shRNA vectors was evaluated against six published algorithms for siRNA oligonucleotide design. Our results show that none of the scoring algorithms can explain a significant percentage of variance in shRNA knockdown efficacy as assessed by linear regression analysis or ROC curve analysis. Application of a modification based on the stability of the 6 central bases of each shRNA provides fair-to-good predictions of knockdown efficacy for three of the algorithms. Analysis of an independent set of data from 38 shRNAs pooled from previous publications confirms these findings.The use of mixed oligonucleotide pairs provides a time and cost efficient method of producing wild type and mutant control shRNA vectors. The addition to sequencing reactions of a combination of mixed dITP/dGTP chemistries and DNA relaxing agents enables read through the intrinsic secondary structure of problematic shRNA vectors. Six published algorithms for siRNA oligonucleotide design that were tested in this study show little or no efficacy at predicting shRNA knockdown outcome. However, application of a modification based on the central shRNA stability should provide a useful improvement to the design of effective shRNA vectors.
| S-EPMC1409772 | biostudies-literature
Project description:Transcription factors are considered to be the master switches that determine the state and fate of cells. Sox2 is an important transcription factor necessary for the pluripotency and self-renewal of embryonic stem cells. Sox2 is generally considered to play a role in the initiation of transcription, but recent studies have shown that Sox2 may influence the alternative splicing of pluripotent genes with the assistance of proteins associated with RNA metabolism, suggesting that Sox2 may be regulated in a novel way during the maintenance of pluripotency in embryonic stem cells. Using mouse embryonic stem cell model, this project focused on Sox2 and proteins related to RNA metabolism to explore their mode of action and the mechanism of regulating the maintenance of pluripotency of embryonic stem cells.
2024-08-28 | GSE232524 | GEO
Project description:RNA interference through expression of short hairpin (sh)RNAs provides an efficient approach for gene function analysis in mouse genetics. Techniques allowing to control time and degree of gene silencing in vivo, however, are still lacking. Here we provide a generally applicable system for the temporal control of ubiquitous shRNA expression in mice. Depending on the dose of the inductor doxycycline, the knockdown efficiency reaches up to 90%. To demonstrate the feasibility of our tool, a mouse model of reversible insulin resistance was generated by expression of an insulin receptor (Insr)-specific shRNA. Upon induction, mice develop severe hyperglycemia within seven days. The onset and progression of the disease correlates with the concentration of doxycycline, and the phenotype returns to baseline shortly after withdrawal of the inductor. On a broad basis, this approach will enable new insights into gene function and molecular disease mechanisms.
| S-EPMC1874634 | biostudies-literature
Project description:shRNA expression is an established technique to transiently or permanently deplete cells of a particular mRNA/protein. In functional analyses of oncogenic pathways it can thus be used as an alternative to pharmacologic inhibitors, or as a means to address otherwise undruggable targets. Here we describe and functionally validate a simple reiterative cloning system to generate concatenated multi-shRNA expression plasmids. The multi-shRNA expression cassette can eventually be subcloned into any suitably designed vector for the stable transfection of cells, here tested with derivatives of the Sleeping Beauty transposon vector for stable transfection of multiple myeloma cell lines at the lowest biosafety level. We finally test inducible versions of such multi-cassette knockdown vectors and show their efficacy for the induced concerted knockdown of all four components of the MEK/MAPK-module in the Ras/MAPK pathway. The described vector system(s) should be useful for functional knockdown analyses in a wide array of cell line models.
| S-EPMC6195277 | biostudies-literature
Project description:HIV-1 is a complex retrovirus that uses host machinery to promote its replication. Understanding cellular proteins involved in the multistep process of HIV-1 infection may result in the discovery of more adapted and effective therapeutic targets. Kinases and phosphatases are a druggable class of proteins critically involved in regulation of signal pathways of eukaryotic cells. Here, we focused on the discovery of kinases and phosphatases that are essential for HIV-1 replication but dispensable for cell viability. We performed an iterative screen in Jurkat T-cells with a short-hairpin-RNA (shRNA) library highly enriched for human kinases and phosphatases. We identified 14 new proteins essential for HIV-1 replication that do not affect cell viability. These proteins are described to be involved in MAPK, JNK and ERK pathways, vesicular traffic and DNA repair. Moreover, we show that the proteins under study are important in an early step of HIV-1 infection before viral integration, whereas some of them affect viral transcription/translation. This study brings new insights for the complex interplay of HIV-1/host cell and opens new possibilities for antiviral strategies.
| S-EPMC2822867 | biostudies-literature
Project description:Oncogenic signaling promotes tumor invasion and metastasis, in part, by increasing the expression of tri- and tetra- branched N-glycans. The branched N-glycans bind to galectins forming a multivalent lattice that enhances cell surface residency of growth factor receptors, and focal adhesion turnover. N-acetylglucosaminyltransferase I (MGAT1), the first branching enzyme in the pathway, is required for the addition of all subsequent branches. Here we have introduced MGAT1 shRNA into human HeLa cervical and PC-3-Yellow prostate tumor cells lines, generating cell lines with reduced transcript, enzyme activity and branched N-glycans at the cell surface. MGAT1 knockdown inhibited HeLa cell migration and invasion, but did not alter cell proliferation rates. Swainsonine, an inhibitor of α-mannosidase II immediately downstream of MGAT1, also inhibited cell invasion and was not additive with MGAT1 shRNA, consistent with a common mechanism of action. Focal adhesion and microfilament organization in MGAT1 knockdown cells also indicate a less motile phenotype. In vivo, MGAT1 knockdown in the PC-3-Yellow orthotopic prostate cancer xenograft model significantly decreased primary tumor growth and the incidence of lung metastases. Our results demonstrate that blocking MGAT1 is a potential target for anti-cancer therapy.
| S-EPMC3434202 | biostudies-literature
Project description:BackgroundShort hairpin RNA (shRNA) encoded within an expression vector has proven an effective means of harnessing the RNA interference (RNAi) pathway in mammalian cells. A survey of the literature revealed that shRNA vector construction can be hindered by high mutation rates and the ensuing sequencing is often problematic. Current options for constructing shRNA vectors include the use of annealed complementary oligonucleotides (74 % of surveyed studies), a PCR approach using hairpin containing primers (22 %) and primer extension of hairpin templates (4 %).ResultsWe considered primer extension the most attractive method in terms of cost. However, in initial experiments we encountered a mutation frequency of 50 % compared to a reported 20-40 % for other strategies. By modifying the technique to be an isothermal reaction using the DNA polymerase Phi29, we reduced the error rate to 10 %, making primer extension the most efficient and cost-effective approach tested. We also found that inclusion of a restriction site in the loop could be exploited for confirming construct integrity by automated sequencing, while maintaining intended gene suppression.ConclusionIn this study we detail simple improvements for constructing and sequencing shRNA that overcome current limitations. We also compare the advantages of our solutions against proposed alternatives. Our technical modifications will be of tangible benefit to researchers looking for a more efficient and reliable shRNA construction process.
| S-EPMC1343552 | biostudies-literature
Project description:Orphan nuclear receptor Esrrb is vital in maintaining ES cells and like Oct4, Sox2 and Nanog is essential for self-renewal and pluripotency. Esrrb functions in somatic cells via LBD/AF-2-dependent coactivator recruitment to target genes. Here we show that in ES cells coactivator recruitment is similarly required and identify Ncoa3 as the Esrrb coactivator needed for activation of its target genes. Ncoa3 is essential for self-renewal and the induction of pluripotency in reprogramming, and genome-wide analysis of Ncoa3 binding reveals extensive overlap with Esrrb and pluripotency factors along with marks of active genes. Mechanistically, we show Ncoa3 is specifically required to bridge RNApol2 to Esrrb. We thus identify a new member of the ES pluripotency network and describe Esrrb and Ncoa3 as key factors linking core pluripotency factors to the general transcription machinery. Three biological replicates each for control scrambled shRNA and Ncoa3 shRNA transfected E14 mouse ESCs. The global gene expression profiles of Ncoa3 knockdown cells were compared to control scrambled shRNA knockdown cells 4 days post-transfection.
2012-10-02 | E-GEOD-40192 | biostudies-arrayexpress