Inducible transposon mutagenesis in C. trachomatis
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ABSTRACT:
PROVIDER: PRJEB47970 | ENA |
REPOSITORIES: ENA
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Project description:Here, we report a CRISPR/Cas12k-transposon-assisted genome engineering (CTAGE) method that allows for high-throughput site-specific mutagenesis in microbial genomes. Exploiting the powerful CTAGE technique, we construct a site-specific transposon mutant library focusing on all the possible transcription factors (TFs) in Pseudomonas aeruginosa, enabling comprehensive identification of essential genes and new factors for antibiotic resistance.
2021-08-10 | GSE164797 | GEO
Project description:Transposon mutagenesis screening of Newcastle disease virus (NDV)
| PRJNA575639 | 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: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: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:Transposon mutagenesis screening of Sendai virus and mumps virus
| PRJNA515704 | ENA
Project description:Transposon mutagenesis of Burkholderia cepacia complex strain R-12632
| PRJEB40633 | 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