Microelectroporation technology for genetic engineering

Electroporation, in which electrical pulses are applied across biological cells to permeabilize the cell membranes, is widely used in biotechnology to introduce macromolecules, in particular gene constructs, into biological cells. Conventional electroporation is performed on a large population of cells and employs an empirical trial-and-error procedure, which limits its effectiveness. The fundamental nature of electroporation is not well understood. To improve the effectiveness of electroporation and to develop a tool for studying the fundamental biophysics of electroporation, micro-electromechanical (MEMS) technology was used to develop a chip that can incorporate individual cells in the chip circuit and electroporate the cells under controlled conditions. The control of electroporation in individual cells is made possible by the fact that electroporated cells allow flow of ions across the cell membranes and this flow can be measured and used as a feedback. This single cell level electroporation method is referred as to “micro-electroporation”.; In this thesis the fundamental principles of microelectroporation are discussed first. Then the microfabrication procedure for the microelectroporation chip is described. The properties of the chip are examined through experiments with different types of cells as well as mathematical analysis. It is demonstrated that the chip can be used to induce reversible electroporation in individual cells under controlled conditions and that the electrical parameters of electroporation can be determined.; Subsequently, a membrane impermeant nucleic acid stain (YOYO-1) was used with fluorescence intensity analysis to demonstrate how microelectroporation technology can be used to regulate cross membrane transport of macromolecules by means of adjusting the strength and length of electroporation pulses.; The ability of the microelectroporation technology to genetically manipulate individual cells was demonstrated by inserting an EGFP gene construct into a prostate cancer cell that resided in the chip. The engineered cell was incubated in the chip and successfully expressed the gene.; Finally, the microelectroporation technology made it possible to discover that there is a significant difference between membrane currents of live and dead cells. This suggested that measuring cell membrane current with the microelectroporation chip could provide an instantaneous electrical means to detect cell viability.; In summary, the dissertation introduces microelectroporation technology and demonstrates its potential in biotechnology, particularly genetic engineering.