Evaluation of the electroinjection method for introducing proteins into living cells.
The introduction of impermeant probes such as antibodies and other proteins into living cells without compromising physiological function is an important approach for studying cellular regulatory mechanisms. Many techniques including direct microinjection, liposome-mediated delivery, fusion of red cell ghosts, and osmotic lysis of pinocytic vesicles have been used to introduce proteins into intact cells. We have used a modification of the voltage-discharge technique to introduce antibodies and other proteins into living physiologically responsive pheochromocytoma and other cultured cells. In this technique, called electroinjection, a single discharge of relatively low field strength is used to transiently permeabilize the plasma membrane. Our experiments demonstrate that electroinjection permits the introduction of large amounts (microM) of probe into 2-5 x 10(6) cells simultaneously without compromising cell viability or physiological responsiveness when performed under carefully defined conditions. They also demonstrate that electroinjection results in a single population of loaded cells and that protein incorporation is a function of field strength, capacitance, molecular weight of the protein, and the concentration of the protein in the electroinjection buffer. Interestingly, a significant fraction of the protein electroinjected into cells is trapped in the plasma membrane when cells are shocked at high capacitance. These results demonstrate that electroinjection appears to be an efficient method for loading exogenous proteins into cells while maintaining the integrity of the physiological properties of the cell.
Wilson
A K
AK
Department of Physiology and Biophysics, College of Medicine, University of Illinois, Chicago 60612.
Horwitz
J
J
De Lanerolle
P
P
D000310:Adrenal Gland Neoplasms; D000818:Animals; D000906:Antibodies / Q000008:administration & dosage*; D002395:Catecholamines / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival; D004594:Electrophysiology / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives*; D005452:Fluoresceins / Q000008:administration & dosage / Q000378:metabolism*; D015759:Ionomycin / Q000494:pharmacology; D008845:Microinjections / Q000379:methods*; D010673:Pheochromocytoma; D051381:Rats; D012710:Serum Albumin, Bovine / Q000008:administration & dosage / Q000378:metabolism*
D000906:Antibodies; D002395:Catecholamines; D005452:Fluoresceins; C039217:fluorescein isothiocyanate bovine serum albumin; D012710:Serum Albumin, Bovine; D015759:Ionomycin; D016650:Fluorescein-5-isothiocyanate