Characterization of the Ghost Fusion Method: a Method for Introducing Exogenous Substances into Cultured Cells
Cell Structure and Function
When erythrocytes were fused to cultured cells with HVJ, hemoglobin in erythrocytes was transferred to the cells. The hemoglobin became uniformly distributed in cells within 5 min, but components of the erythrocyte membranes did not become completely incorporated into recipient-cell membranes for about 2 h. Division of recipient cells was normal but did not start until one day after fusion. Human and guinea-pig erythrocytes had the highest fusion capacities of various animal erythrocytes
... erythrocytes tested. When these erythrocytes and iodinated bovine serum albumin (BSA) were dialyzed against hypotonic solution, BSA was trapped in erythrocyte ghosts within 30 min, at about 50 % of initial concentration in the dialysis bag. Of BSA trapped in ghosts, 80 % was found in the cytoplasmic fraction and 20 % associated with the membrane fraction. BSA was quite stable in these ghosts. Other proteins with various isoelectric points were also introduced into ghosts, and these proteins were transferred to recipient cells by virus-induced cell fusion of these ghosts. About 80 % of recipient cells in either monolayers or suspensions fused with the ghosts. Use of iodinated BSA showed that 0.25 % of the initial amount of BSA in the dialysis bag was introduced into recipient cells. Since we first reported a method for introducing an exogenous substance into cultured cells by fusion of the cells with erythrocyte ghosts loaded with the substance mediated by HVJ (Sendai virus) (the ghost fusion method) (5), various attempts to improve the method have been made in several laboratories (11, 15, 21) . Loyter et al. (11) and we (15) independently succeeded in introducing exogenous molecules into erythrocyte ghosts, without injuring the ability of the ghosts to fuse, by gradual hemolysis of the erythrocytes in a dialysis bag (22). Further basic information is needed, however, before this method can be used as a routine technique. The present experiments were carried out to clarify the following points : (1) the optimal conditions for introducing exogenous molecules into the ghosts, (2) the stability of protein molecules introduced into ghosts, and (3) the behavior of recipient cells after fusion with erythrocytes or erythrocyte ghosts. MATERIALS AND METHODS Cells and virus. A mutant of Ehrlich ascites tumor cells (ETC ) resistant to 8-azaguanine (16) 293 294 M. Yamaizumi et al. was propagated in the abdomen of ddO mice. The FL cell line, originating from normal human amniotic membrane (2) and a mutant of the mouse L cell line that is resistant to BudR (14) were cultured in Eagle's MEM supplemented with 10 % calf serum. Both cell line had a high plating efficiency (more than 70 %) and formed compact colonies. Fresh erythrocytes from various animals were collected in syringes with citrate and washed 3 times with PBS to remove elukocytes. To check their ability to fuse, the erythrocytes were washed once more with BSS-Ca (140 mM NaCl, 5.4 mM KCl, 0.34 mM Na2HPO4, 0.44 mM KH2PO4, and 2 mM CaCl2 buffered with 10 mM Tris-HCl at pH 7.6) by centrifugation at 2,000 rpm for 5 min, then suspended at a concentration of 1 % (v/v) in BSS-Ca. To introduce exogenous proteins into the erythrocyte ghosts, the erythrocytes finally washed with r-PBS (137 mM KCl, 2.7 mM NaCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, 4 mM MgCl2, pH 7.4) by centrifugation at 2,000 rpm for 5 min. The resulting erythrocyte pellet was used.