Mus musculus
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA198037 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR830791.fastq.gz | Fastqsanger.gz | |||
| SRR830792.fastq.gz | Fastqsanger.gz | |||
| SRR830793.fastq.gz | Fastqsanger.gz | |||
| SRR830794.fastq.gz | Fastqsanger.gz | |||
| SRR830795.fastq.gz | Fastqsanger.gz |
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Project description:Somatic transposon mutagenesis in mice is an efficient strategy to investigate the genetic mechanisms of tumorigenesis. The identification of tumor driving transposon insertions traditionally requires the generation of large tumor cohorts to obtain information about common insertion sites. Tumor driving insertions are also characterized by their clonal expansion in tumor tissue, a phenomenon that is facilitated by the slow and evolving transformation process of transposon mutagenesis. We describe here an improved approach for the detection of tumor driving insertions that assesses the clonal expansion of insertions by quantifying the relative proportion of sequence reads obtained in individual tumors. To this end, we have developed a protocol for insertion site sequencing that utilizes acoustic shearing of tumor DNA and Illumina sequencing. We analyzed various solid tumors generated by PiggyBac mutagenesis and for each tumor >10^6 reads corresponding to >10^4 insertion sites were obtained. In each tumor, 9 to 25 insertions stood out by their enriched sequence read frequencies when compared to frequencies obtained from tail DNA controls. These enriched insertions are potential clonally expanded tumor driving insertions, and thus identify candidate cancer genes. The candidate cancer genes of our study comprised many established cancer genes, but also novel candidate genes such as Mastermind-like1 (Mamld1) and Diacylglycerolkinase delta (Dgkd). We show that clonal expansion analysis by high-throughput sequencing is a robust approach for the identification of candidate cancer genes in insertional mutagenesis screens on the level of individual tumors. Solid tumors in mice were generated by somatic transposon mutagenesis with a PiggyBac transposon system. Insertion sites of transposons in 11 tumors and 6 non-cancerous tail controls were determined by Illumina high-throughput sequencing. Insertions were determined both on 5' and 3' sides of the transposon (PB5 and PB3, respectively). Quantitative analysis of read numbers revealed enrichment of certain insertions in tumors, but not in controls, and these enriched insertions identify candidate cancer genes.
2013-08-28 | E-GEOD-46210 | biostudies-arrayexpress
Project description:Somatic transposon mutagenesis in mice is an efficient strategy to investigate the genetic mechanisms of tumorigenesis. The identification of tumor driving transposon insertions traditionally requires the generation of large tumor cohorts to obtain information about common insertion sites. Tumor driving insertions are also characterized by their clonal expansion in tumor tissue, a phenomenon that is facilitated by the slow and evolving transformation process of transposon mutagenesis. We describe here an improved approach for the detection of tumor driving insertions that assesses the clonal expansion of insertions by quantifying the relative proportion of sequence reads obtained in individual tumors. To this end, we have developed a protocol for insertion site sequencing that utilizes acoustic shearing of tumor DNA and Illumina sequencing. We analyzed various solid tumors generated by PiggyBac mutagenesis and for each tumor >10^6 reads corresponding to >10^4 insertion sites were obtained. In each tumor, 9 to 25 insertions stood out by their enriched sequence read frequencies when compared to frequencies obtained from tail DNA controls. These enriched insertions are potential clonally expanded tumor driving insertions, and thus identify candidate cancer genes. The candidate cancer genes of our study comprised many established cancer genes, but also novel candidate genes such as Mastermind-like1 (Mamld1) and Diacylglycerolkinase delta (Dgkd). We show that clonal expansion analysis by high-throughput sequencing is a robust approach for the identification of candidate cancer genes in insertional mutagenesis screens on the level of individual tumors.
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Project description:A Sleeping Beauty (SB) transposon forward genetic screen was performed to identify the genes that promote osteosarcoma (OS) development and metastasis. Mutagenesis induced OS in wild type mice and accelerated it on a Trp53 deficient background. Analysis of tumors demonstrated that Trp53 deficiency is correlated with genomic instability, which was virtually absent in tumors induced by SB mutagenesis alone. Metastases developed in a subset of animals and in nearly all cases were clonal related to primary tumors. Over 200 candidate genes were identified, many of which are altered in human cancers including OS. Signaling pathways enriched for candidate genes were also identified and a subset of these pathways and genes were functionally validated and represent new targets for OS treatment. Bisulphite converted DNA from the 21 diagnosis osteosarcoma patients and 3 hOB cell line replicates were hybridised to the Illumina Infinium 450K Human Methylation Beadchip.
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Project description:U2OS cells were co-transfected with PiggyBac (PB) transposon plasmid pPB-SB-CMV-puro-SD3 and the transposase p-hyPBASE. The modified PiggyBac (PB) transposon, which has a constitutively active CMV promoter which can stimulate or disrupt expression of neighboring genes, depending on insertional orientation. For each library, between 10e7-10e8 cells were transfected, cultured with the addition of 3 µg/ml puromycin for one week to select cells that had incorporated the transposon, and then used for screens of resistance to alpha-toxin-induced cell death with minimal further expansion. Mutagenized cells were treated twice with alpha-toxin (500 ng/ml) for 48h to ensure the selection of only highly resistant cells. Genomic DNA (gDNA) was isolated from 5-10 X 10e6 cells and transposon insertion sites were mapped using high throughput sequencing.
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2015-04-07 | GSE58770 | GEO
Project description:Using piggyBac transposon based insertional mutagenesis, we have generated a haploid neural cell library harboring genome-wide mutations for genetic screening, and identified Park2 gene as a proof-of-concept in screening for Mn2+-mediated toxicity.
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Project description:U2OS cells were co-transfected with PiggyBac (PB) transposon plasmid pPB-SB-CMV-puro-SD3 and the transposase p-hyPBASE. The modified PiggyBac (PB) transposon, which has a constitutively active CMV promoter which can stimulate or disrupt expression of neighboring genes, depending on insertional orientation. For each library, between 107-108 cells were transfected, cultured with the addition of 2 µg/ml puromycin for one week to select cells that had incorporated the transposon, and then used for screens of virus resistance with minimal further expansion. Mutagenized cells were challenged with replication-competent recombinant EboGP-VSV at MOI of 1 or 10 and propagated for up to 3 weeks with regular media changes to select virus-resistant colonies. After virus resistant cells were selected, genomic DNA (gDNA) was isolated from 5-10 X 106 cells and transposon insertion sites were mapped using high throughput sequencing.
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Project description:We mapped Sleeping Beauty (SB) and piggyBac (PB) insertions in primary human CD4+ T cells and compared their insertion profiles with those of the MLV retrovirus and the HIV lentivirus with respect to proximity to genes, TSSs, CpG islands, DNase I hypersensitive sites, repetitive elements, chromatin marks and transcriptional status of genes. Examination of two transposon systems in one cell type.
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