Single Cell Electroporation and DNA dynamics: from bulk to micro/nanofluidics: NANOEP

Programme(s): FP7-PEOPLE - Specific programme "People" implementing the Seventh Framework Programme of the European Community for research, technological development and demonstration activities (2007 to 2013)

Topic(s): FP7-PEOPLE-2012-CIG - Marie-Curie Action: "Career Integration Grants"

Call for proposal: FP7-PEOPLE-2012-CIG

Funding Scheme: MC-CIG - Support for training and career development of researcher (CIG)

Grant agreement ID: 334419

Objective

Electroporation or electropermeabilization is the electrical disruption of a cell’s membrane to introduce foreign DNA, RNAs, drugs, proteins, or other therapies into the living cells. This technique is one of the most popular non-viral gene transfer methods for both in vitro and in vivo application. However, this method is suffering from several limitations such as: i) low transfection efficiency, ii) poor cell viability, and iii) random uptake. Multiple applications of electroporation in biotechnology and medicine rely on trial and error optimization of treatment conditions rather than knowledge of pores, dynamics and electrotransfer of DNA, due to the limited spatial and temporal resolution of traditional tools. More interesting, the phenomena behind electro-mediated membrane permeabilization to plasmid DNA (~4.5 kbp) have been shown to be significantly more complex than those for small molecules. Therefore, successful DNA electrotransfer into cells depends not only on cell permeabilization (or poration) but also on the way plasmid DNA interacts with the membrane and, once into the cell, migrates toward the nuclei.

The main aim of this proposal to understand and control the transport of DNA in electroporation treatment such that stable, safe and efficient gene transfection can be achieved. Here I will use single molecule techniques (such as AFM, FRET, confocal and optical tweerers) with high spatio-temporal resolution to investigate the process of electroporation at different length (from bulk to micro/nano) and time scale (ranging from micro-seconds to hours). In addition, these unique tools will be integrated with novel micro/nanofluidics to investigate the electroporation and electrokinetic flow of DNA at micro/nano-scale. I will observe all the processes in real time with unprecedented details and quantitatively examine the kinetics of pore formation/resealing and cytoplasmic trafficking of DNA.