Evaluations of transgene produced hemopexin using rat hepatocytes in tissue culture

Abstract

The objective of this study was to observe the production of transgene hpxn in JM2 cells grown in tissue culture. This research was aimed to provide insight into the production of rat hpxn and to provide a stepping-stone leading to other in vitro lab studies and in vivo animal trials. These studies are designed to gain new advances in the treatment of sickle cell disease development. Hpxn is a plasma glycoprotein that undergoes a conformational change while binding to circulating heme, a dangerous free radical containing macromolecule (Tolosano & Altruda, 2002). Hemolysis means rupture of heme containing blood cells (“Hemolysis”). Hemolysis increases heme concentrations in blood plasma (Delanghe & Langlosi, 2001). Hemolysis can be caused by a number of medical conditions including sickle cell disease (Ascenzi et al., 2005). Hpxn is highly variable between species (Ascenzi et al., 2005). Therefore, rat hepatocytes, JM2 cells, were necessary for this experiment to correctly assemble, fold, and glycosylate rat hpxn. The difference between the pORF5 wild type hpxn plasmid and the pORF5 missense hpxn plasmid is a four base pair change, which results in the production of different amino acids (“Missense mutation,” 2012). In two locations Carol Bruzzone previously changed two histadines to alanine in the pORF5 missense hpxn plasmid. These changes occurred at amino acids 79 and 149. pORF5 is an InvivoGen plasmid that is selectable in bacteria and functions in both bacterial and mammalian cells. The hypothesis of this experiment was that the pORF5 wild type hpxn plasmid transfected in tissue culture would produce a functional protein during glycosylation, while pORF5 missense hpxn plasmid transfected in tissue culture would yield a non-functional protein during glycosylation (“Missense mutation,” 2012). Graham Brown previously developed the activity assay utilized to determine the activity of the transgene produced hpxn. The assay was created based on hpxn activity research conducted by Ann Smith at the University of Kansas. During this assay, the absorbance of hpxn in the cell supernatant alone was measured, and then compared to the same sample with the addition of 10 μM hemin chloride. Theoretically the absorbance readings of the two conditions will differ if hpxn is present in the supernatant because hpxn and hemin chloride bind together.