High yields of stable transformants by hypo-osmolar plasmid electroinjection.
Electrotransfection of mouse L cells and macrophages suspended in strongly hypo-osmolar solutions gave high yields of stable transformants which significantly exceeded the clone number obtained under iso-osmolar conditions. The cells survived these extremely low osmolarities for 1 h without any apparent deterioration of cellular or membrane functions. Highest yields were obtained in buffered 75 mosmol solutions containing 30 mM KCl and an appropriate amount of inositol provided that the strength of the breakdown pulse was matched to the dramatic increase in cell volume at low osmolarity. The absolute clone number depended on the post-incubation time in the hypo-osmolar solution after application of a single breakdown pulse at 4 degrees C. The absolute number of transformants was maximum when post-incubation was restricted to 2 min. Towards longer incubation times the absolute number decreased even though the relative clone number was similar. This was because of a corresponding decrease of the number of viable cells. It is conceivable that enhanced DNA uptake in hypo-osmolar solutions is faciliated by an overall slight (and reversible) increase in membrane permeability generated by the osmotically created tension in the membrane of the swollen cells.
Däumler
R
R
Lehrstuhl für Biotechnologie, Universität Würzburg, F.R.G.
Zimmermann
U
U
D000818:Animals; D002463:Cell Membrane Permeability; D007739:L Cells; D008264:Macrophages; D051379:Mice; D009994:Osmolar Concentration; D010957:Plasmids; D014162:Transfection