Electroporation of antibodies, DNA, and other macromolecules into cells: a highly efficient method.
While antibodies are a major extracellular tool of the highest specificity to answer important biomedical questions, the improvements in electroporation discussed below may make it feasible to also use antibodies as an intracellular deletion tool to study (a) viruses inside the cell, (b) cancer cells, (c) signal transduction, (d) genetics, (e) metabolism, and (f) other structures and mechanisms. Already, others have succeeded in depositing macromolecules, including antibodies (Abs), and nucleic acids inside cells, using many techniques, including electroporation (EP). However, EP has limitations that have precluded its widespread use, particularly its high kill rate for cells and the low percentage of cells that are able to incorporate macromolecules. If these limitations could be overcome for Abs and nucleic acids, then it would be practical to use them as highly specific probes for intracellular 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(++) (ViaSpan, Belzer UW cold-storage solution, DuPont Pharmaceuticals). This solution decreased cell death after standard EP by an average of 50% for a number of cell lines. Viability of WISH cells after EP approached 100%. In transfection studies, ViaSpan medium strongly increased both P3HR1 cell survival as well as the total number of cells transfected with DNA for green fluorescent protein (GFP). In additional experiments with Abs, we were able to strongly increase the percent of cells that incorporated Ab by using two serial 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 simplify the EP technique by using unpurified antiserum in place of purified IgG. Thus, this EP technique offers multiple advantages: simplicity, high cell viability, high effectiveness, high specificity, rapid action, usefulness with adherent or non-adherent cells, and no requirement for purification of antibodies from antiserum.
Baron
S
S
Department of Microbiology & Immunology, University of Texas Medical Branch, Galveston, TX 77555-1019, USA. sabaron@utmb.edu
Poast
J
J
Rizzo
D
D
McFarland
E
E
Kieff
E
E
D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D002460:Cell Line; D002470:Cell Survival; D003470:Culture Media; D004247:DNA; D004553:Electric Conductivity; D018274:Electroporation / Q000379:methods* / Q000592:standards; D049452:Green Fluorescent Proteins; D006801:Humans; D007424:Intracellular Fluid / Q000276:immunology; D008164:Luminescent Proteins; D046911:Macromolecular Substances; D011817:Rabbits; D013997:Time Factors
D000914:Antibodies, Viral; D003470:Culture Media; D008164:Luminescent Proteins; D046911:Macromolecular Substances; D049452:Green Fluorescent Proteins; D004247:DNA