The use of in situ hybridization to study the transgene pathway following cellular transfection with cationic phosphonolipids

Abstract

Gene therapy is a promising field of research and biotechnological development. Considering their safety and non-immunogenicity, cationic lipids are widely used for gene transfer in vitro and show promise for in vivo gene transfer applications. However, a better understanding of the mechanisms by which transfection occurs and the limiting steps in cellular transfer of foreign DNA are critical for significant improvements of gene transfer. In this work, we have traced the plasmid DNA into human hematopoietic cell line (K562) using the in situ hybridization method in order to define the main difficulties in transfection and to design new agents better adapted to cellular constraints. In this hematopoietic cell line, after showing the efficiency of our synthetic vectors and optimizing their formulation, we observed that only 5 h after transfection the nucleus to cytoplasm signal ratio was three to one, whereas at 24 h it was one to one. In parallel, the level of the reporter protein strongly increased between these times. Those results emphasize the rapidity of transfection and lead one to imagine chemical modifications adjusted to the environment.