Characterization of GAP43-Gfp fusion protein in PC12 and NIH3T3 stable transfectant cells

Abstract

Nerve regeneration is a physiological process in which damaged axons regrow after they have been injured. In the fully grown mammalian Central Nervous System (CNS), nerve regeneration is almost nonexistent. Researchers are conducting experiments and trying to understand the mechanisms of nerve regeneration by manipulating neuronal cells at cellular and molecular levels to develop therapies for patients with nerve damage. To study the mechanism of nerve regeneration, our laboratory has been focused on studying genes and proteins that are activated and expressed during and post axonal damage. Researchers had found that GAP-43, a protein with an apparent molecular weight of 43 kilodalton (kD) by electrophoresis, is overexpressed in axonal growth cones during neuronal development and nerve regeneration. Studies led researchers to understand that GAP-43 plays a key role in axonal regeneration, particularly in neurite growth and in axonal pathfinding. Our hypothesis is that sustained high level expression of the intrinsic protein GAP-43 enhances neuritogenesis and nerve regeneration. A plasmid encoding a chimeric protein in which the GAP-43 protein is fused with a fluorescent GFP molecule was used to express a GAP43-GFP fusion protein, which allowed to detect and measure expression levels in transfected cells. The plasmid with the recombinant hGAP43-tGFP gene expressing GAP43-GFP fusion protein was transfected into PC12 cells and stable transfectant cells were selected and grown in the laboratory. Over time, the stable transfectant cells expressing GAP43-GFP showed a significant loss of expression, which was observed through fluorescent microscopy and Western blot analysis. When comparing cells expressing GFP versus GAP43-GFP, the latter showed a much lower level of expression of the fluorescent protein. To understand better the rate of loss of expression of GAP43-GFP, experiments were carried by transfecting PC12 and NIH3T3 cells and collecting cell lysates to conduct Western blot analysis of levels of expression