Engineering DNA transfer into Cells by Precision i.. (E-DNA-T-PEP)
Engineering DNA transfer into Cells by Precision in Electroporation
Date du début: 1 oct. 2013,
Date de fin: 30 sept. 2018
"The proposal aims to understand and control the transport of DNA in electroporation process at the molecular/subcellular level such that more efficient and safer non-viral gene delivery can be achieved. The introduction of naked DNA into living cell via non-viral routes is the safest approach in gene therapy. Electroporation is the electrical disruption of biological membranes to introduce naked DNA into the cell. Due to our lack of information about fundamentals of electropores formation and DNA electrotransfer, electroporation methods still suffer from low transfection efficiency, random uptake and excessive cell damage.The main barriers to achieving this goal are: i) understanding the creation of electropores at molecular level; ii) understanding the underlying mechanism of DNA transport across the membrane of a cell during and after electric pulses and iii) controlling the electrotransfer of DNA through these pores into a cell at molecular level. It is almost impossible to overcome these barriers based on our current rudimentary understanding of cell electroporation.The successful outcome of this project will significantly aid the development of gene delivery into living cells, which will lead to electroporation-based therapies in the near future.To this end, I will apply a multidisciplinary approach, combining disciplines as physical chemistry, transport phenomena, DNA dynamics, biophysics and cell biology. To unveil the entire electroporation process, innovatively I will employ the integrated atomic force microscopy with micro/nanofluidics to visualize the evolution of pore size/density at the membrane level. Furthermore, to understand the DNA electrotransfer, I will study how DNA interacts with electropores and moves through them using optical tweezers and single-molecule FRET. Finally, I will dissect the role of cytoskeleton on the transport of DNA, by mapping out the relationship between the viscoelasticity of cell and location of DNA inside the cell."
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