Transposon mutagenesis screen in Klebsiella pneumoniae identifies genetic determinants required for growth in human urine and serum
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PROVIDER: PRJEB76649 | ENA |
REPOSITORIES: ENA
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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:Transposon screen for PARylation targets
| PRJEB26343 | 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.
2013-08-28 | GSE46210 | GEO
Project description:Transposon mutagenesis screening of Newcastle disease virus (NDV)
| PRJNA575639 | ENA
Project description:Genotype directed anti-cancer therapies such BRAF inhibitor in BRAF mutant melanoma can show remarkable clinical efficacy but resistance limits their benefit. We show that a transposon activation screen efficiently identifies resistance genes to BRAF and captures a number of previously uncharacterized resistance mechanisms, including an E3 ubiquitin ligase NEDD4L and the Hippo pathway effector WWTR1 (TAZ). Resistance can be reversed by combining BRAF inhibition with tyrosine kinase inhibitors as observed previously for other resistance genes. Moreover, an integrative analysis of several gain- and loss-of-function genetic screens performed in the same context reveals smaller functional diversity of resistance mechanisms to MAPK inhibition than suggested by the broad range of resistance genes identified, implying common therapeutic strategies. A375 cells with lentiviral vector controls or WWTR1 cDNA plasmid.
2016-07-01 | E-GEOD-73470 | biostudies-arrayexpress
Project description:A PiggyBac transposon based screen in a murine pancreatic cancer model.This data has been described in the following article [doi:10.1038/ng.3164] and its further analysis can be freely submitted for publication. For information on the proper use of data shared by the Wellcome Trust Sanger Institute (including information on acknowledgement), please see http://www.sanger.ac.uk/datasharing/
2016-07-04 | E-ERAD-514 | biostudies-arrayexpress
Project description:Purpose:To identify resistance mechanisms for the chemotherapeutic drug fludarabine in chronic lymphocytic leukemia (CLL), as innate and acquired resistance to fludarabine-based chemotherapy represents a major challenge for long-term disease control. Methods: We employed piggyBac transposon-mediated mutagenesis, combined with next-generation sequencing, to identify genes that confer resistance to fludarabine in a human CLL cell line. Results: RNA-seq profiling of fludarabine-resistant cells suggested deregulated MAPK signaling as involved in mediating drug resistance in CLL.
2016-03-31 | GSE75844 | GEO
Project description:Purpose:To identify resistance mechanisms for the chemotherapeutic drug fludarabine in chronic lymphocytic leukemia (CLL), as innate and acquired resistance to fludarabine-based chemotherapy represents a major challenge for long-term disease control. Methods: We employed piggyBac transposon-mediated mutagenesis, combined with next-generation sequencing, to identify genes that confer resistance to fludarabine in a human CLL cell line. Results: RNA-seq profiling of fludarabine-resistant cells suggested deregulated MAPK signaling as involved in mediating drug resistance in CLL. To address if the fludarabine-resistant HG3 cells were transcriptionally different at a global level compared to their parental cells, we performed RNA-sequencing of three pairs of HG3 pools
2016-03-31 | E-GEOD-75844 | biostudies-arrayexpress
Project description:This deposition describes DNA sequences at integration sites in primary tumors and lung metastases from a sleeping-beauty transposon-mediated mutagenesis screen on Rb-deficient background in the mammary gland in MMTV-Cre:Rbf/f:T2/Onc3a:R26lsl_SB11 and MMTV-Cre:Rbf/f:T2/Onc3b:R26lsl_SB11 female mice. In these mice, Rb floxed (Rbf/f) alleles and a SB11 transposase, knocked into the ROSA26 locus (R26lsl_SB11), are mobilized via MMTV-Cre. Two different transgenic transposon concatemers, T2/Onc3a and T2/Onc3b, containing 11 and 28 copies on chromosomes 9 and 12 were used so that local hoping in the respective chromosomes can be discarded. These mice developed large primary tumors and macroscopic lung metastases; biopsies from the primary tumors and entire lung metastases were used to extract DNA. The DNA was subjected to sonication, ligation-mediated PCR with 79 bar coded primers, followed by next-generation DNA sequencing. We identified Primary-specific and lung Metastasis-specific gene-centric Common Integration Sites (gCIS) as well as shared gCIS, observed in both primary mammary tumors and lung metastases. Using sequence analysis of integration sites, we were able to demonstrate in multiple cases clonal relationship between primary lesions and metastases. The metastatic gCIS form specific hubs that may be prioritized for targeted therapy. For details, see reference below.
2023-05-17 | GSE232167 | GEO
Project description:Genotype directed anti-cancer therapies such BRAF inhibitor in BRAF mutant melanoma can show remarkable clinical efficacy but resistance limits their benefit. We show that a transposon activation screen efficiently identifies resistance genes to BRAF and captures a number of previously uncharacterized resistance mechanisms, including an E3 ubiquitin ligase NEDD4L and the Hippo pathway effector WWTR1 (TAZ). Resistance can be reversed by combining BRAF inhibition with tyrosine kinase inhibitors as observed previously for other resistance genes. Moreover, an integrative analysis of several gain- and loss-of-function genetic screens performed in the same context reveals smaller functional diversity of resistance mechanisms to MAPK inhibition than suggested by the broad range of resistance genes identified, implying common therapeutic strategies.
2016-07-01 | GSE73470 | GEO