Electroporation of lymphoid cells: factors affecting the efficiency of transfection.
We have increased the efficiency of electroporation of lymphoid cells over fifty fold by optimising several biological and electrical parameters. Under optimised conditions, the electroporation efficiency was comparable to that reported for other cell types. Actively dividing cells were crucial for high transient transfection signal. The two most important electrical parameters were high capacitance (960 microF) and moderate decay constants in the range of 10-15 ms. The optimal field strength depended on the cell line, but was in the range 0.6-1 kV/cm. Administering the pulse in medium lacking serum gave higher efficiency than when isotonic salt solution was used and the transfection signal was depressed if cells and DNA were allowed to incubate for several minutes either before or after the pulse. Electroporation was carried out at room temperature and there was no advantage in using low temperatures (0-4 degrees C). When electroporated cells were grown in conditioned medium, the signal was enhanced about two fold depending on the source of the conditioned medium.
Anderson
M L
ML
Biochemistry Department, Glasgow University, U.K.
Spandidos
D A
DA
Coggins
J R
JR
D000818:Animals; D001402:B-Lymphocytes / Q000502:physiology*; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D002855:Chromatography, Thin Layer / Q000379:methods; D004247:DNA / Q000378:metabolism; D004553:Electric Conductivity; D008213:Lymphocyte Activation / Q000235:genetics; D051379:Mice; D013696:Temperature; D014162:Transfection
D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase