Diffusion loading conditions determine recovery of protein synthesis in electroporated P3X63Ag8 cells |
| |
Authors: | M R Michel M Elgizoli H Koblet C Kempf |
| |
Affiliation: | Institute for Hygiene and Medical Microbiology, University of Bern, Switzerland. |
| |
Abstract: | Using the suspension cell line P3X63Ag8 we have studied the impact of the composition of the diffusion medium on cellular protein synthesis under standard electroporation conditions in TBS-Na. This buffer contains the high saline concentration usually present in electroporation-mediated DNA transfection. Electroporation in the presence of TBS-Na resulted in an immediate shut-off of protein synthesis, even though both FITC-dextran (Mr 40 kD) and Semliki Forest virus core protein (Mr 33 kD) were incorporated efficiently into the cytoplasm across the electropores at 0 degrees C. Subsequent resealing of the pores was completed after a 5-min incubation at 37 degrees C. When compared with control cells, overall protein synthesis of electroporated cells recovered slowly to resume a 30% activity after 1 h of incubation at 37 degrees C. We have determined optimal conditions for diffusion loading (which necessitates the presence of ATP, GTP, amino acids, K+, Mg2+, and Ca2+) and resealing (in the presence of K+, Mg2+, and Ca2+), leading to a full and lasting recovery of protein synthesis within 5 min after pore closure. |
| |
Keywords: | |
|
|