Mus musculus
Ontology highlight
ABSTRACT:
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| DRR023875_1.fastq.gz | Fastqsanger.gz | |||
| DRR023875_2.fastq.gz | Fastqsanger.gz | |||
| DRR023876_1.fastq.gz | Fastqsanger.gz | |||
| DRR023876_2.fastq.gz | Fastqsanger.gz | |||
| DRR023877_1.fastq.gz | Fastqsanger.gz |
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Project description:DNA transposon-based gene delivery vectors represent a promising new branch of randomly integrating vector development for gene therapy. For the side-by-side evaluation of the piggyBac and Sleeping Beauty systems - the only DNA transposons currently employed in clinical trials - during therapeutic intervention, we treated the mouse model of Tyrosinemia type I. with liver-targeted gene delivery using both transposon vectors. For genome-wide mapping of transposon insertion sites we developed a new Next Generation Sequencing procedure called Streptavidin-Based Enrichment Sequencing, which allowed us to identify approximately 1 million integration sites for both systems. We revealed that a high proportion of piggyBac integrations are clustered in hot regions and found that they are frequently recurring at the same genomic positions among treated animals, indicating that the genome-wide distribution of Sleeping Beauty-generated integrations is closer to random. We also revealed that the piggyBac transposase protein exhibits prolonged activity, which predicts the risk of oncogenesis by generating chromosomal double-strand breaks. Safety concerns associated with prolonged transpositional activity draw attention to the importance of squeezing the active state of the transposase enzymes into a narrower time window.
2023-03-09 | GSE212895 | GEO
Project description:Streptococcus pneumoniae is a major cause of serious infections such as pneumonia and meningitis in both children and adults worldwide. Here, we describe the development of a high-throughput genome-wide technique, Genomic Array Footprinting (GAF), for the identification of genes essential for this bacterium at various stages during infection. GAF enables negative screens by means of a combination of transposon mutagenesis and microarray technology for the detection of transposon insertion sites. We tested several methods for the identification of transposon insertion sites and found that amplification of DNA adjacent to the insertion site by PCR resulted in non-reproducible results, even when combined with an adapter. However, restriction of genomic DNA followed directly by in vitro transcription circumvented these problems. Analysis of parallel reactions generated with this method on a large mariner transposon library, showed that it was highly reproducible and correctly identified essential genes. Comparison of a mariner library to one generated with the in vivo transposition plasmid pGh:ISS1, showed that both have an equal degree of saturation, but that 9% of the genome is preferentially mutated by either one. The usefulness of GAF was demonstrated in a screen for genes essential for survival of zinc stress. This identified a gene encoding a putative cation efflux transporter, and its deletion resulted in an inability to grow under high zinc conditions. In conclusion, we developed a fast, versatile, specific, and high-throughput method for the identification of conditionally essential genes in S. pneumoniae. Keywords: GAF Identification of transposon insertion sites
2007-06-26 | GSE6913 | GEO
Project description:The ability to chronicle transcription factor binding events throughout the development of an organism would facilitate mapping of transcriptional networks that control cell fate decisions. We describe a method for permanently recording protein-DNA interactions in mammalian cells. We endow transcription factors with the ability to deposit a transposon into the genome near to where they bind. The transposon becomes a M-bM-^@M-^\Calling CardM-bM-^@M-^] the transcription factor leaves behind to record its visit to the genome. The locations of the Calling Cards can be determined by massively-parallel DNA sequencing. We show that the transcription factor SP1 fused to the piggyBac transposase directs insertion of the piggyBac transposon near SP1 binding sites. The locations of transposon insertions are highly reproducible, and agree with sites of SP1-binding determined by ChIP-seq. Genes bound by SP1 are more likely to be expressed in the HCT116 cell line we used, and SP1-bound CpG islands show a strong preference to be unmethylated. This method has the potential to trace transcription factor binding throughout cellular and organismal development in a way that has heretofore not been possible. These data contain mapped insertion sites from the piggyBac (PB) transposon into HCT116 cell line and sequenced using an Illumina GAII analyzer. The first set contains the insertion sites of transposons mapped from the wild-type PB transposase and the second set contains the insertion sites of transposons mapped with the PB transposase fused to the transcription factor SP1. Other sequencing data present are ChIP-seq data for SP1 in the HCT116 cell line and RNA-seq data.
2011-12-31 | E-GEOD-34791 | biostudies-arrayexpress
Project description:Genome wide DNA methylation profiling of normal human embryonic stem cells, induced pluripotent stem cells, and somatic cells. The Illumina Infinium 27k Human DNA methylation Beadchip v1.2 was used to obtain DNA methylation profiles across approximately 27,000 CpGs in 5 normal hESCs, 15 hiPSCs (vector-containing and vector-free), and 5 somatic cells.
2014-01-01 | GSE42043 | GEO
Project description:Transposable genetic elements are ubiquitous, yet their presence or absence at any given position within a genome can vary between individual cells, tissues, or strains. Transposable elements have profound impacts on host genomes by altering gene expression, assisting in genomic rearrangements, causing insertional mutations, and serving as sources of phenotypic variation. Characterizing a genome?s full complement of transposons requires whole genome sequencing, precluding simple studies of the impact of transposition on interindividual variation. Here, we describe a global mapping approach for identifying transposon locations in any genome, using a combination of transposon-specific DNA extraction and microarray- based comparative hybridization analysis. We use this approach to map the repertoire of endogenous transposons in different laboratory strains of Saccharomyces cerevisiae and demonstrate that transposons are a source of extensive genomic variation. We also apply this method to mapping bacterial transposon insertion sites in a yeast genomic library. This unique whole genome view of transposon location will facilitate our exploration of transposon dynamics, as well as defining bases for individual differences and adaptive potential. Keywords: transposon mapping
2006-11-15 | GSE6278 | GEO
Project description:Streptococcus pneumoniae is a major cause of serious infections such as pneumonia and meningitis in both children and adults worldwide. Here, we describe the development of a high-throughput genome-wide technique, Genomic Array Footprinting (GAF), for the identification of genes essential for this bacterium at various stages during infection. GAF enables negative screens by means of a combination of transposon mutagenesis and microarray technology for the detection of transposon insertion sites. We tested several methods for the identification of transposon insertion sites and found that amplification of DNA adjacent to the insertion site by PCR resulted in non-reproducible results, even when combined with an adapter. However, restriction of genomic DNA followed directly by in vitro transcription circumvented these problems. Analysis of parallel reactions generated with this method on a large mariner transposon library, showed that it was highly reproducible and correctly identified essential genes. Comparison of a mariner library to one generated with the in vivo transposition plasmid pGh:ISS1, showed that both have an equal degree of saturation, but that 9% of the genome is preferentially mutated by either one. The usefulness of GAF was demonstrated in a screen for genes essential for survival of zinc stress. This identified a gene encoding a putative cation efflux transporter, and its deletion resulted in an inability to grow under high zinc conditions. In conclusion, we developed a fast, versatile, specific, and high-throughput method for the identification of conditionally essential genes in S. pneumoniae. Keywords: GAF pGHost vs mariner
2007-06-26 | GSE6912 | GEO
Project description:Streptococcus pneumoniae is a major cause of serious infections such as pneumonia and meningitis in both children and adults worldwide. Here, we describe the development of a high-throughput genome-wide technique, Genomic Array Footprinting (GAF), for the identification of genes essential for this bacterium at various stages during infection. GAF enables negative screens by means of a combination of transposon mutagenesis and microarray technology for the detection of transposon insertion sites. We tested several methods for the identification of transposon insertion sites and found that amplification of DNA adjacent to the insertion site by PCR resulted in non-reproducible results, even when combined with an adapter. However, restriction of genomic DNA followed directly by in vitro transcription circumvented these problems. Analysis of parallel reactions generated with this method on a large mariner transposon library, showed that it was highly reproducible and correctly identified essential genes. Comparison of a mariner library to one generated with the in vivo transposition plasmid pGh:ISS1, showed that both have an equal degree of saturation, but that 9% of the genome is preferentially mutated by either one. The usefulness of GAF was demonstrated in a screen for genes essential for survival of zinc stress. This identified a gene encoding a putative cation efflux transporter, and its deletion resulted in an inability to grow under high zinc conditions. In conclusion, we developed a fast, versatile, specific, and high-throughput method for the identification of conditionally essential genes in S. pneumoniae. Keywords: GAF zinc
2007-06-26 | GSE6911 | GEO
Project description:SB transposon insertion sites in mouse BaF3 cells
| PRJNA306511 | ENA
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:Transcription factors direct gene expression, and so there is much interest in mapping their genome-wide binding locations. M-BM- Current methods do not allow for the multiplexed analysis of TF binding, and this limits their throughput. We describe a novel method for determining the genomic target genes of multiple transcription factors simultaneously. DNA-binding proteins are endowed with the ability to direct transposon insertions into the genome near to where they bind. The transposon becomes a M-bM-^@M-^\Calling CardM-bM-^@M-^] marking the visit of the DNA-binding protein to that location. A unique sequence M-bM-^@M-^\barcodeM-bM-^@M-^] in the transposon matches it to the DNA-binding protein that directed its insertion. The sequences of the DNA flanking the transposon (which reveal where in the genome the transposon landed) and the barcode within the transposon (which identifies the TF that put it there) are determined by massively-parallel DNA sequencing. To demonstrate the methodM-bM-^@M-^Ys feasibility, we determined the genomic targets of eight transcription factors in a single experiment. The Calling Card method promises to significantly reduce the cost and labor needed to determine the genomic targets of many transcription factors in different environmental conditions and genetic backgrounds. These data contain Ty5 insertion sites mapped by an Illumina GAII analyzer in the S. cerevisiae genome for the background strain without any Sir4 present (1 run), in strains expressing Sir4-tagged copies of three well-characterized TFs: Gal4, Leu3, and Gcn4 (1 run each), and a multiplex of eight Sir4-tagged TFs pooled in a single experiment (2 biological replicates), and insertions from the Thi2-Sir4 fusion expressed from its native locus in two conditions (1 run each). The format of each insertions file is [chromosome number] [position of genomic base] [direction of insertion] [number of reads at that position]. Raw sequencing data comes in two varieties. Paired-end data contains a 5 bp barcode at the beginning of read #2. Single-end data contains a 2 bp barcode on the beggining of read #1.
2011-02-18 | E-GEOD-27381 | biostudies-arrayexpress