title: Enhanced transfection of a bacterial plasmid into hybridoma cells by electroporation: application for the selection of hybrid hybridoma (quadroma) cell lines.
abstract: A procedure was investigated to transduce a bacterial plasmid containing a specific drug resistance marker (pSV2-neo), into a hybridoma cell line using electroporation. The effect of several buffers and the form of plasmid DNA (circular or linearized) on the stable transfection frequency were examined. When complete cell culture medium (DMEM) was used as electroporation buffer, we observed a two-fold increase in post-pulse viability and a ten- to thirty-fold increase in the transfection frequency of pSV2-neo, as compared with HEPES buffered 0.15 M sodium chloride. Supplementing DMEM with fetal bovine serum (DMEM + FBS) had some beneficial effect on post-pulse viability of the cells after electroporation, but did not markedly increase stable transfection frequency as compared with DMEM alone. Furthermore, with DMEM + FBS, the intact plasmid was transfected as effectively as linearized PSV2-neo. However, when using HEPES buffered saline, the transfection frequency of pSV2-neo increased two-fold after linearization as compared with intact plasmid. The drug resistance was used successfully as a marker for the selection of hybrid hybridoma (quadroma) cell lines after fusing two different hybridoma cell lines, producing anti-fibrin and anti-plasminogen activator antibodies respectively. The quadroma cells produced bispecific antibodies that are capable of accumulating plasminogen activator on a fibrin surface.
authors.affiliation: IVVO-TNO, Gaubius Laboratory, Leiden, The Netherlands.
authors.forename: R
authors.initials: R
authors.lastname: Bos
authors.forename: W
authors.initials: W
authors.lastname: Nieuwenhuizen
mesh_terms: D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D016923:Cell Death; D002459:Cell Fusion; D002469:Cell Separation / Q000379:methods*; D003470:Culture Media; D004558:Electric Stimulation; D004797:Enzyme-Linked Immunosorbent Assay; D005337:Fibrin / Q000276:immunology; D005819:Genetic Markers / Q000235:genetics; D006825:Hybridomas / Q000276:immunology; D010957:Plasmids; D010959:Tissue Plasminogen Activator / Q000276:immunology; D014162:Transfection / Q000235:genetics*
chemical_list: D000911:Antibodies, Monoclonal; D003470:Culture Media; D005819:Genetic Markers; D005337:Fibrin; D010959:Tissue Plasminogen Activator