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Show OPTIMIZATION OF ADENOVIRUS-5 INTERACTION WITH HEPARIN SULFATE TO MAXIMIZE TRANSDUCTION OF HUMAN EMBRYONIC KIDNEY CELLS FOR GENE THERAPY Chuks C. Chimezie (Dr.D. Keith Roper) Department of Chemical Engineering, University of Utah To Optimize adenovirus interaction with heparin sulfate, human embryonic cells are inoculated in 5ml of DMEM supplemented with Alanine, Pyruvate, Glucose Glucose, Glutamine, Sodium Bi-carbonate and all these are added to an antibiotic. The cells are incubated in T-flask and split into four additional flasks when the cells reach 90% confluence on the surface of the flasks and the flasks are supplemented with fresh culture media to nurture new cell growth. The flasks are then infected with virus. Special care is taken when adenovirus is poured in to the cell and the cells that become infected with virus are placed inside the incubator. When 80-90% of the cells are infected they harvested and purified. In the next step the upper and lower band of the virus is collected. The high Performance liquid chromatography ( hplc) machine is used to determine to the presence of virus. Two columns are used in the hplc, first with the anion exchange column and then the heparin column. After conducting a series of experiments with the anion exchange column (aex) it is observed that a peak oc-curs at time equivalent to 20 minutes which shows the presence of adenovirus. When the experiment is conducted with a heparin column a peak is observed at 29 minutes which is also a virus. |