The hydrodynamic-based transfection procedure consists in rapidly injecting plasmid DNA in a large volume of saline via tail vein of mice. This leads to a high expression level in liver and in particular in hepatocytes. Its direct use in clinical trials of gene therapy is maybe not possible, but it may provide a new tool for gene function analysis in the context of the whole animal. Just as for other transfection techniques, the exact mechanism of the hydrodynamic procedure is not known.
In the present study, we tried to obtain some information about the mechanism of transfection, in the hope of a better understanding of the process. Therefore we have followed the uptake and intracellular pathway of 35S-labelled plasmid DNA in the liver after a conventional or hydrodynamic injection and tried to establish a possible correlation between these results and the level of expression. Our results clearly show that after a hydrodynamic injection of luciferase expressing DNA, a high level of luciferase activity was observed in liver and, to the same extent, in hepatocytes isolated immediately after the injection. The distributions of radioactivity by centrifugation methods in total liver and isolated hepatocytes show that a major part of the DNA remained bound to the outer face of the plasma membrane for a relatively long time. When hepatocytes were incubated with pancreatic DNAse immediately after their isolation leading to nearly a complete hydrolysis of the DNA, luciferase expression was not changed. Hepatocytes isolated very few minutes after injection exhibited an expression level as high as observed in total liver, meaning that the DNA responsible for transfection becomes very rapidly associated with hepatocytes prior to isolation. These results suggest thus that the DNA internalised and leading to transfection at short time represents only a small part of the total DNA and that the major part of the DNA remaining at the outer face of the plasma membrane would not ..
|Date of Award
|Michel Jadot (Supervisor), Simone De Coninck (Co-Supervisor), Michel Herin (Jury) & André Piront (Jury)