Project description:In order to identify cellular factors that influence the efficiency of AAV-HR mediated TI, we performed an unbiased genome-wide screening in a library of near haploid human cells (HAP1) mutagenized by retroviral insertions.
Project description:Recently, gene-trap mutagenesis in near haploid human cells has been used to disrupt genes and identify novel host-pathogen interactions and elucidate mechanisms of drug action. Building on this technology, we here report the generation of a human gene-trap mutant collection of individual clones, which currently covers over a quarter of the expressed genome. Strand specific RNA-Seq of ribosomal RNA depleted total cellular RNA to measure transcript abundance and to detect fusion transcripts in KBM7 cells at standard culture conditions.
Project description:Recently, gene-trap mutagenesis in near haploid human cells has been used to disrupt genes and identify novel host-pathogen interactions and elucidate mechanisms of drug action. Building on this technology, we here report the generation of a human gene-trap mutant collection of individual clones, which currently covers over a quarter of the expressed genome.
Project description:To identify regulators of exit from the naïve pluripotent state we performed an enhancer trap screen under sensitized conditions. Briefly, haploid mouse ESCs with a Puromycin resistance gene under the control of Oct4 regulatory elements were mutagenized using viruses harboring enhancer trap constructs and subjected to differentiation in the presence of 3uM CHIR with concomitant selection for Oct4 expression. Selected cells were harvested after 16, 20, and 23 days and the extracted genomic DNA, together with genomic DNA of cells taken before selection, subjected to inverse PCR and sequencing. This allowed for mapping of enhancer trap integration sites and identifying genes whose targeting is enriched during selection.
Project description:This phase II trial is studying how well VEGF Trap works in treating patients with previously treated metastatic colorectal cancer. VEGF Trap may stop the growth of colorectal cancer by blocking blood flow to the tumor.
Project description:One hundred million HAP1 Tet-On-MLKLTE/SD cells were mutagenized with the GFP gene trap retrovirus, as described (PMID: 21623355). The selection experiment was performed by inducing MLKLTE/SD expression with 200 ng/mL DOX over five days and subsequently allowing resistant colonies to grow for four days after withdrawing DOX. Resistant colonies were then pooled together and genomic DNA was extracted from the pool (Qiagen, 51306). Retroviral gene trap insertion sites were amplified using a linear amplification (LAM) PCR protocol as described previously (PMID: 21623355), sequenced using the Genome Analyzer (Illumina).
Project description:In fibroblasts, p65-dependent genes can be sub-divided, depending on whether they are Trap-80-dependent or -independent. To examine the generality of this grouping, we performed a microarray analysis of wild-type and Trap-80 knock-down fibroblasts, before and after stimulation of NF-kappaB activity using TNF-alpha. RNA was extracted from three independent cultures of wild-type and Trap-80 knock-down fibroblasts, before and after stimulation for 1 hour with 5ng/ml TNF-alpha. The unstimulated and stimulated wild-type samples, and the stimulated Trap-80 knock-down samples, were used for microarray analysis.
Project description:Natural genetic variation between two mouse strains was used as a natural mutagenesis screen to test various aspects of the proposed model for enhancer selection and function.