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Clonal analysis is important for many areas of hematopoietic stem cell research, including in vitro cell expansion, gene therapy, and cancer progression and treatment. A common approach to measure clonality of retrovirally transduced cells is to perform integration site analysis using Southern blotting or polymerase chain reaction-based methods. Although these methods are useful in principle, they generally provide a lowresolution, biased, and incomplete assessment of clonality. To overcome those limitations, we labeled retroviral vectors with random sequence tags or "barcodes." On integration, each vector introduces a unique, identifiable, and heritable mark into the host cell genome, allowing the clonal progeny of each cell to be tracked over time. By coupling the barcoding method to a sequencing-based detection system, we could identify major and minor clones in 2 distinct cell culture systems in vitro and in a long-term transplantation setting. In addition, we demonstrate how clonal analysis can be complemented with transgene expression and integration site analysis. This cellular barcoding tool permits a simple, sensitive assessment of clonality and holds great promise for future gene therapy protocols in humans, and any other applications when clonal tracking is important. (Blood. 2010;115(13):2610-2618

Cellular barcoding tool for clonal analysis in the hematopoietic system

Clonal analysis is important for many areas of hematopoietic stem cell research, including in vitro cell expansion, gene therapy, and cancer progression and treatment. A common approach to measure clonality of retrovirally transduced cells is to perform integration site analysis using Southern blotting or polymerase chain reaction-based methods. Although these methods are useful in principle, they generally provide a low-resolution, biased, and incomplete assessment of clonality. To overcome those limitations, we labeled retroviral vectors with random sequence tags or "barcodes." On integration, each vector introduces a unique, identifiable, and heritable mark into the host cell genome, allowing the clonal progeny of each cell to be tracked over time. By coupling the barcoding method to a sequencing-based detection system, we could identify major and minor clones in 2 distinct cell culture systems in vitro and in a long-term transplantation setting. In addition, we demonstrate how clonal analysis can be complemented with transgene expression and integration site analysis. This cellular barcoding tool permits a simple, sensitive assessment of clonality and holds great promise for future gene therapy protocols in humans, and any other applications when clonal tracking is important. (Blood. 2010;115(13):2610-2618

Study of Various Photomultiplier Tubes with Muon Beams And Cerenkov Light Produced in Electron Showers

The PMTs of the CMS Hadron Forward calorimeter were found to generate a large size signal when their windows were traversed by energetic charged particles. This signal, which is due to Cerenkov light production at the PMT window, could interfere with the calorimeter signal and mislead the measurements. In order to find a viable solution to this problem, the response of four different types of PMTs to muons traversing their windows at different orientations is measured at the H2 beam-line at CERN. Certain kinds of PMTs with thinner windows show significantly lower response to direct muon incidence. For the four anode PMT, a simple and powerful algorithm to identify such events and recover the PMT signal using the signals of the quadrants without window hits is also presented. For the measurement of PMT responses to Cerenkov light, the Hadron Forward calorimeter signal was mimicked by two different setups in electron beams and the PMT performances were compared with each other. Superior performance of particular PMTs was observed