Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Genome wide analysis of long term L1TD1 knockdown effects in NT2D1 cell line with inducible shRNA vectors

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

SUBMITTER: Nelly Rahkonen

PROVIDER: E-GEOD-21275 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

RNA-binding protein L1TD1 interacts with LIN28 via RNA and is required for human embryonic stem cell self-renewal and cancer cell proliferation.

Närvä Elisa E Rahkonen Nelly N Emani Maheswara Reddy MR Lund Riikka R Pursiheimo Juha-Pekka JP Nästi Juuso J Autio Reija R Rasool Omid O Denessiouk Konstantin K Lähdesmäki Harri H Rao Anjana A Lahesmaa Riitta R

Stem cells (Dayton, Ohio) 20120301 3

Human embryonic stem cells (hESC) have a unique capacity to self-renew and differentiate into all the cell types found in human body. Although the transcriptional regulators of pluripotency are well studied, the role of cytoplasmic regulators is still poorly characterized. Here, we report a new stem cell-specific RNA-binding protein L1TD1 (ECAT11, FLJ10884) required for hESC self-renewal and cancer cell proliferation. Depletion of L1TD1 results in immediate downregulation of OCT4 and NANOG. Furt ...[more]

PMID: 22162396

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Project description:Obesity and diabetes associated visual impairment and vascular dysfunctions are increasing reasons for vision loss. The detailed mechanisms in these diseases are still largely unknown, but mice models have been useful to study these mechanisms and explore the detailed effects of potential compounds. Such compounds usually have antioxidant and anti-inflammatory properties. These properties are found in anthocyanins and a major source of dietary anthocyanins in Nordic diet is bilberries (European wild blueberries, Vaccinium myrtillus). In this explorative study we show results with differentially expressed genes in retina using a high-fat diet (HFD) mouse model. Our findings displayed differential regulation of genes in pathways for apoptosis, inflammation and oxidative stress, especially systemic lupus erythematosus (SLE), mitogen activated protein kinase (MAPK) and glutathione metabolism. Mice fed with HFD had increased retinal gene expression of several crystallins, which was reduced in the retina of mice fed with bilberries. Bilberries seem to reduce the expression of genes in MAPK and to increase the expression of genes in glutathione metabolism pathway. All together despite minor effects in the mouse phenotype, a diet rich in bilberries may prevent the retinal gene expression changes in the early stages of obesity. Mice were fed ad libitum with normal control diet (NCD, 10% kcal fat), high-fat diet (HFD, 45% kcal fat), 5% (w/w) freeze-dried biberries (Vaccinium myrtillus) in NCD (NCD+BB) or HFD (HFD+BB) for 12 weeks. Diets were prepared in Research Diets Inc. Feed consumption and weight gain were measured during the feeding trial, and blood pressure and serum markers of obesity at the end. Retinas were collected and RNA extracted from all 24 mice samples, and pooled retinas from 4 mice per group were hybridized with standard Illumina protocols. The expression in retinas was analyzed using R, Pathvisio and DAVID to screen for differences between high-fat and berry induced changes to control diets and further bilberry induced changes to HFD up- or downregulated transcripts. diet: NCD, HFD, NCD+BB, HFD+BB

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.

Dataset Information

Genome wide analysis of long term L1TD1 knockdown effects in NT2D1 cell line with inducible shRNA vectors

Publications

RNA-binding protein L1TD1 interacts with LIN28 via RNA and is required for human embryonic stem cell self-renewal and cancer cell proliferation.

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