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.
