Spatially and temporally controlled gene transfer by electroporation into adherent cells on plasmid DNA-loaded electrodes.
Functional characterization of human genes is one of the most challenging tasks in current genomics. Owing to a large number of newly discovered genes, high-throughput methodologies are greatly needed to express in parallel each gene in living cells. To develop a method that allows efficient transfection of plasmids into adherent cells in spatial- and temporal-specific manners, we studied electric pulse-triggered gene transfer using a plasmid-loaded electrode. A plasmid was loaded on a gold electrode surface having an adsorbed layer of poly(ethyleneimine), and cells were then plated directly onto this modified surface. The plasmid was detached from the electrode by applying a short electric pulse and introduced into the cells cultured on the electrode, resulting in efficient gene expression, even in primary cultured cells. The location of transfected cells could be restricted within a small area on a micropatterned electrode, showing the versatility of the method for spatially controlled transfection. Plasmid transfection could also be performed in a temporally controlled manner without a marked loss of the efficiency when an electric pulse was applied within 3 days after cell plating. The method described here will provide an efficient means to transfer multiple genes, in parallel, into cultured mammalian cells for high-throughput reverse genetics research.
Yamauchi
Fumio
F
Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan.
Kato
Koichi
K
Iwata
Hiroo
H
D000818:Animals; D002448:Cell Adhesion; D002460:Cell Line; D004247:DNA / Q000737:chemistry; D018274:Electroporation / Q000379:methods*; D006801:Humans; D008839:Microelectrodes; D008970:Molecular Weight; D010957:Plasmids; D011094:Polyethyleneimine / Q000737:chemistry; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors; D014162:Transfection / Q000379:methods*
D011094:Polyethyleneimine; D004247:DNA