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Membrane Sandwich Electroporation for in Vitro Gene Delivery to Embryonic Stem Cells and Embryonic Bodies

Zhengzheng Fei1, Shengnian Wang1, Brian E. Henslee1, and Ly James Lee2. (1) Department of Chemical and Biomolecular Engineering, The Ohio State University, 140 West 19th Avenue, Columbus, OH 43210, (2) Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH 43210

Genetic manipulation of cultured Embryonic Stem (ES) cells provides great potential for ex vivo gene therapy. Little progress has been made due to the difficulties involved in successful transfection. Viral transduction of ES cells is very efficient, but safety issues, such as immune and inflammatory responses, have hampered their clinical uses in humans. Electroporation is one of the most popular non-viral gene transfer methods for ES cell transfection. However, cell viability is still an issue due to the high electric voltage used and the nonuniform electric field strength distribution generated during the process. Our previous work demonstrated a new membrane sandwich electroporation (MSE) approach using NIH 3T3 fibroblasts and plasmids GFP and SEAP as model materials. In the MSE method, the focused electric field enhances cell permeabilization at a low electric voltage, leading to high cell viability; more important, the sandwich membrane configuration is able to provide better gene confinement near the cell surface, facilitating gene delivery into the cells. We have tested individual ES cells using our MSE method and got very encouraging results. Our on-going effort is to investigate the transfection of embryonic bodies with controlled size by a modified MSE system. Embryonic Bodies experience very low transfection efficiencies using current electroporation techniques.