Optimizing electroporation conditions in primary and other difficult-to-transfect cells.
Electroporation is a valuable tool for nucleic acid delivery because it can be used for a wide variety of cell types. Many scientists are shifting toward the use of cell types that are more relevant to in vivo applications, including primary cells, which are considered difficult to transfect. The ability to electroporate these cell types with nucleic acid molecules of interest at a relatively high efficiency while maintaining cell viability is essential for elucidating the pathway(s) in which a gene product is involved. We present data demonstrating that by optimizing electroporation parameters, nucleic acid molecules can be delivered in a highly efficient manner. We display transfection results for primary and difficult-to-transfect cell types including human primary fibroblasts, human umbilical vein endothelial cells, Jurkat cells, and two neuroblastoma cell lines [SK-N-SH (human) and Neuro-2A (mouse)] with plasmid DNAs and siRNAs. Our data demonstrate that by determining proper electroporation conditions, glyceraldehyde phosphate dehydrogenase mRNA was silenced in Jurkat cells when compared with negative control siRNA electroporations as early as 4 h post-transfection. Other experiments demonstrated that optimized electroporation conditions using a fluorescently labeled transfection control siRNA resulted in 75% transfection efficiency for Neuro-2A, 93% for human primary fibroblasts, and 94% for HUVEC cells, as analyzed by flow cytometry.
Jordan
Elizabeth T
ET
Bio-Rad Laboratories, Hercules, CA 94547, USA. liz_jordan@bio-rad.com
Collins
Michelle
M
Terefe
Joseph
J
Ugozzoli
Luis
L
Rubio
Teresa
T
D000818:Animals; D001709:Biotechnology; D002460:Cell Line; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D018274:Electroporation / Q000379:methods*; D015870:Gene Expression; D006801:Humans; D019169:Jurkat Cells; D051379:Mice; D009447:Neuroblastoma / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection / Q000379:methods*
D034741:RNA, Small Interfering