Role of the human transcription factor T in chordoma growth

在实验室成立的第一年，我们已经获得了运行所需的基本设备，如微量离心机，计算机，蛋白质凝胶和蛋白质印迹设备，PCR热循环仪，细胞培养用品，以及日常实验室操作的额外用品。 我们已经签署了必要的材料转让协议，以获得已发表的脉络膜细胞系。我们已经获得，扩大，并冷冻等分的第一个只有两个公布的脉络膜细胞系称为UCH-1。保持培养物连续生长用于实验。我们已经测试了该细胞系的集落形成，这是测试电离辐射效应的先决条件。不幸的是，该细胞系不能形成足够的集落;因此，我们获得了能形成集落的衍生细胞系UCH-1 N。我们已经在实验室里确认了菌落的形成。最后，我们已安排转让最近出版的第二条生产线。 在第一个细胞系中，使用蛋白质印迹和免疫沉淀，我们已经证实了高水平表达的人T。我们已经初步优化了Western印迹和免疫沉淀人T的抗体条件，并开始优化定量RT-PCR条件。 我们最近获得了用于cDNA微阵列分析的微阵列材料，并将在不久的将来处理天然脉络膜细胞系和人参考cDNA的样品。我们将在不久的将来进行天然UCH-1细胞的激酶组阵列分析。我们优化了用聚凝胺转导抗T shRNA逆转录病毒颗粒的条件，并选择嘌呤霉素抗性菌落，以获得稳定表达shRNA的细胞系。 我们已经优化了用于分析上述细胞系的测定，以确定正常条件下的代谢水平。将水平与通过shRNA下调T的条件下的细胞系进行比较。

In the first year since establishing the laboratory, we have acquired the basic equipment required for operation, such as microcentrifuges, computers, protein gel and Western blotting equipment, PCR thermal cycler, cell culture supplies, as well as additional supplies for routine laboratory operation. We have executed the Materials Transfer Agreements necessary for us to obtain the published chordoma cell lines. We have obtained, expanded, and frozen aliquots of the first of only two published chordoma cell line called UCH-1. Cultures were kept for continuous growth for experiments. We have tested this cell line for colony formation, which is a prerequisite for testing the effect of ionizing radiation. Unfortunately, the cell line does not form adequate colonies; thus, we have obtained a derivative cell line UCH-1N that does form colonies. We have confirmed colony formation in our lab. Finally, we have arranged for transfer of the second recently published line. In the first cell line, using western blotting and immunoprecipitation, we have confirmed high-level expression of human T. We have eimpirically optimized antibody conditions for Western blotting and immunoprecipitating human T, and are beginning to optimize quantitative RT-PCR conditions. We have recently obtained the microarray supplies for cDNA microarray analysis, and will process the samples for native chordoma cell line and human reference cDNA in the near future. We will be performing kinome array analysis of native UCH-1 cells in the very near future. We have optimized conditions for transduction of anti-T shRNA retroviral particles with polybrene and selection of puromycin-resistant colonies in order to obtain cell lines stably expressing shRNA's. We have optimized assays for analysis of the aforementioned cell lines to determine metabolic levels under normal conditions. Levels will be compared to cell lines under conditions of T downregulation by shRNA.