Electroporation of extraneous proteins into CHO cells: increased efficacy by utilizing centrifugal force and microsecond electrical pulses.
A novel electroporation system employing an oscillating electric pulse and centrifugal force was used to introduce extraneous proteins into CHO cells. Following the electrical pulse, the compression and subsequent rebound induced by the centrifugal acceleration and deceleration, respectively, enhanced protein uptake, presumably by a hydrodynamic pumping of extracellular solutions through the permeabilized membrane. Protein uptake was quantitated by measuring the amount of radiolabeled, extraneous, CHO proteins introduced into unlabeled CHO cells. The amount of protein introduced into electroporated CHO cells was enhanced up to four-fold by a combination of electric pulse and centrifugal force compared to that introduced by electric pulse only. The optimum gradient of centrifugal force (GCF, temporal change of centrifugal force) was 590 and -470 g/s during acceleration and deceleration, respectively. The optimum electric field was 5 kV/cm with a 30-microsecond pulse length. At this optimum electroporation condition, approximately 5 pg of proteins (up to 200 kDa molecular weight) were introduced per CHO cell. These same settings also permitted electroporation of other membrane impermeable substances including propidium iodide and ethidium bromide. Introduction of extraneous materials into the cytoplasm during electroporation was confirmed by the ability of anti alpha-tubulin to stain the microtubules and propidium iodide and ethidium bromide to stain the nuclei. Cells electroporated with optimum device settings exhibited no significant decrease in clonogenic survival.
Kim
D
D
Department of Radiation Oncology, William Beaumont Hospital, Royal Oak, Michigan 48073.
Lee
Y J
YJ
Rausch
C M
CM
Borrelli
M J
MJ
D000818:Animals; D001345:Autoradiography / Q000379:methods; D016466:CHO Cells; D002470:Cell Survival; D002498:Centrifugation / Q000379:methods; D006224:Cricetinae; D004558:Electric Stimulation / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D014162:Transfection
D011506:Proteins; D013462:Sulfur Radioisotopes; D008715:Methionine