School of Science Division of Life Science 26 Development of an Algorithm to Analyse Live-cell Imaging Data Supervisor: POON Randy Yat Choi / LIFS Student: QI Qiuyuan / MATH-SF Course: UROP 1100, Fall Histone H2B-GFP fusion proteins have long been a stable DNA marker in cell cycle research, enabling live visualization of chromosome dynamics in cell lines. However, observing those Histone H2B-GFP-marked cells consumes significant time and effort. In my UROP1000 program, I and Peter Fu Wai Kuwn have finished developing a seemingly promising system that utilizes image tracking and H2B-GFP signals to identify Mphase cells, thus reflecting cells’ behavior under drugs in terms of mitosis. The focus of this report is to do large-scale tests that give a full picture of such systems’ accuracy and precision under various circumstances. A small test was first done on the previous Live Cell Imaging(LCL) where we identified the crucial impact of the time resolution(X min/frame) on the accuracy of the system’s counting of mitosis period. In the later larger tests, we encountered the problem caused by prolonged mitosis and signal fading caused by unstable H2B-GFP inheritance across generations. All these problems show such a system requires further improvement before practicing. In Depth Characterization of Novel Cell Cycle Regulators in Cancer Cells Supervisor: POON Randy Yat Choi / LIFS Student: WANG Shuying / BCB-IRE Course: UROP 1100, Spring Two experiments are described in this report: the generation of cell line RPE-1 2-6-3 H2B-mRFP, and the fusion of HtTB2 and HeLa H2B-mRFP cells by polyethylene glycol (PEG). The Sleeping Beauty (SB) transposon system was utilized in the cell line generation. The first trial was successful and an RPE-1 2-6-3 cell line stably expressing H2B-mRFP gene was obtained. Yet the generated cell line was soon lost due to contamination and consequently, a second trial was performed. The second trial, however, failed to show any sign of effective transfection. Limited trials of cell fusion were performed with undesirable outcomes. Two major issues were identified: no sign of cell fusion was observed, and fluorescence signals were lost over time. The causes of contamination and failure of transfection and fusion are examined with approaches for enhancement proposed.
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