Single-cell transfection by electroporation using an electrolyte/plasmid-filled capillary.
Single-cell transfection of adherent cells has been accomplished using single-cell electroporation (SCEP) with a pulled capillary. HEPES-buffered physiological saline solution containing pEGFP plasmid at a low concentration (0.16 approximately 0.78 microg/microL) filled a 15 cm long capillary with a tip opening of 2 microm. The electric field is applied to individual cells by bringing the tip close to the cell and subsequently applying one or two brief electric pulses. Many individual cells can thus be transfected with a small volume of plasmid-containing solution (approximately 1 microL). The extent of electroporation is determined by measuring the percentage loss of freely diffusing thiols (chiefly reduced glutathione) that have been derivatized with the fluorogenic ThioGlo 1. A mass transport model is used to fit the time-dependent fluorescence intensity decay in the target cells. The fits, which are excellent, yield the electroporation-induced fluorescence loss at steady state and the mass transfer rate through the electroporated cell membrane. Steady-state fluorescence loss ranged approximately from 0 to about 80% (based on the fluorescence intensity before electroporation). For the cells having a loss of thiol-ThioGlo 1 fluorescence intensity greater than 10% and mass transfer rate greater than 0.03 s(-1), EGFP fluorescence is observed after 24 h. The EGFP fluorescence is increased at 48 h. With a loss smaller than 10% and a mass transfer rate smaller than 0.03 s(-1), no EGFP fluorescence is detected. Thus, transfection success is closely related to the small molecule mass transport dynamics as indicated by the loss of fluorescence from thiol-ThioGlo 1 conjugates. The EGFP expression is weaker than bulk lipid-mediated transfection, as indicated by the EGFP fluorescence intensities. However, the success with the single-cell approach is considerably greater than lipid-mediated transfection.
Department of Chemistry, University of Pittsburgh, 219 Parkman Avenue, Pittsburgh, Pennsylvania 15260, USA.
Manyan
M
Wang
Owe
O
Orwar
Stephen G
SG
Weber
D045744:Cell Line, Tumor; D004573:Electrolytes / Q000737:chemistry*; D018274:Electroporation / Q000295:instrumentation* / Q000379:methods; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D014162:Transfection
D004573:Electrolytes