Specific Aim 2: To Determine If Gleevec Is Effective In

1404 WordsMay 2, 20176 Pages
Specific Aim 2: To determine if Gleevec is effective in inhibiting in vivo cell proliferation in TNBC cells with overexpressed Arg A recombinant plasmid containing a viral promoter driving Arg expression will be inserted into TNBC cells. Another empty recombinant plasmid will be inserted into different TNBC cells. Another plasmid containing a promoter driving Arg expression will be inserted into another group of TNBC cells. Soon after, an immunoblot will be run using these three different TNBC groups’ cells to ensure the proper transfer of plasmid. The proteins we will test for include Arg, Abl, actin, and Ras-MAPK. Actin is again serving as a positive control, while the expression of Ras-MAPK proteins can act as a quantitative…show more content…
Abl is known to mutate its kinase domain so that Gleevec cannot effectively inhibit it and cellular proliferation (Lovera et al., 2015). A western blot will be run to determine the extent this mutation will affect our experiment at the beginning and 4 months into our experiment. Some tumors from all three cell lines will have their cells lysed and run through the gel, which will test for both Abl and Abl-Gleevec compound, as well as a major substrate for Arg-Abl, ArgBP2. Results- The western blot containing the three different cell lines expressed an equal amount of actin expressed in all three lanes. This served as a control to ensure the plasmids were inserted correctly and there was no interference of any other gene. The lane with cells from mice group 1 showed darker bands on lines corresponding to the proteins Arg as opposed to the lane with mice group 2 cells. The bands corresponding to the protein Abl appeared to be the same on both lanes, demonstrating that Abl was not being accidentally overexpressed as well. These pieces of information elucidated to us that the Arg gene was being successfully overexpressed in line 1. The bands corresponding to Ras-MAPK were darker for mice group 1 than for group 2, demonstrating that this signal transduction pathway for cell proliferation has been downregulated with the overexpression Arg.

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