Electroporation of antibodies, DNA, and other macromolecules into cells: ahighly efficient method

While antibodies are a major extracellular tool of the highest specificity to answer important biomedical questions, the improvements in electroporati on discussed below may make it feasible to also use antibodies as an intrac ellular deletion tool to study (a) viruses inside the cell, (b) cancer cell s, (c) signal transduction, (d) genetics, (e) metabolism, and (f) other str uctures and mechanisms. Already, others have succeeded in depositing macrom olecules, including antibodies (Abs), and nucleic acids inside cells, using many techniques, including electroporation (EP). However, EP has limitatio ns that have precluded its widespread use, particularly its high kill rare for cells and the low percentage of cells that are able to incorporate macr omolecules. If these limitations could be overcome for Abs and nucleic acid s, then it would be practical to use them as highly specific probes for int racellular molecules. In our experiments using EP, we were able to largely prevent lethality for cells during EP by employing a commercially available cold-storage solution for organ transplants containing high K+ and Mg++ (V inSpan, Belzer UW cold-storage solution, DuPont Pharmaceuticals). This solu tion decreased cell death after standard EP by an average of 50% for a numb er of cell lines. Viability of WISH cells after EP approached 100%. In tran sfection studies, ViaSpan medium strongly increased both P3HR1 cell surviva l as well as the total number of cells transfected with DNA for green fluor escent protein (GFP). In additional experiments with Abs, we were able to s trongly increase the percent of cells that incorporated Ab by using two ser ial EPs. This enhanced the intracellular protection by Abs against viruses in Vero cells from 64% to a maximum of 98%. We were able to further simplif y the EP technique by using unpurified antiserum in place of purified IgG. Thus, this EP technique offers multiple advantages: simplicity, high cell v iability, high effectiveness, high specificity, rapid action, usefulness wi th adherent or non-adherent cells, and no requirement far purification of a ntibodies from antiserum. (C) 2000 Elsevier Science B.V. All rights reserve d.