An efficient method for introducing macromolecules into living cells.
The hemagglutinin (HA) of influenza virus was used to obtain efficient and rapid bulk delivery of antibodies and horseradish peroxidase (HRP) into the cytoplasm of living tissue culture cells. By exploiting HA's efficient cell surface expression, its high affinity for erythrocytes, and its acid-dependent membrane fusion activity, a novel delivery method was developed. The approach is unique in that the mediator of both binding and fusion (the HA) is present on the surfaces of the target cells. A recently developed 3T3 cell line which permanently expresses HA, Madin-Darby canine kidney cells infected with influenza virus, and CV-1 cells infected with a simian virus 40 vector carrying the HA gene were used as recipient cells. Protein-loaded erythrocytes were bound to the HA on the cell surface and a brief drop in pH to 5.0 was used to trigger HA's fusion activity and hence delivery. About 3 to 8 erythrocytes fused per 3T3 and CV-1 cell, respectively, and 75-95% of the cells received IgG or HRP. Quantitative analysis showed that 1.8 X 10(8) molecules of HRP and 1.4 X 10(7) IgG molecules were delivered per CV-1 cell and 6.2 X 10(7) HRP molecules per 3T3 cell. Cell viability, as judged by methionine incorporation into protein and cell growth and division, was not impaired. Electron and fluorescence microscopy showed that the fused erythrocyte membranes remained as discrete domains in the cell's plasma membrane. The method is simple, reliable, and nonlytic. The ability to simultaneously and rapidly deliver impermeable substances into large numbers of cells will permit biochemical analysis of the fate and effect of a variety of delivered molecules.
Doxsey
S J
SJ
Sambrook
J
J
Helenius
A
A
White
J
J
D000818:Animals; D002459:Cell Fusion; D002460:Cell Line; D002470:Cell Survival; D002522:Chlorocebus aethiops; D004285:Dogs; D004912:Erythrocytes; D006389:Hemagglutinins, Viral; D006735:Horseradish Peroxidase / Q000008:administration & dosage; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000008:administration & dosage; D009980:Influenza A virus; D051379:Mice; D011506:Proteins / Q000008:administration & dosage*; D011991:Receptors, Virus; D013539:Simian virus 40
D006389:Hemagglutinins, Viral; D007074:Immunoglobulin G; D011506:Proteins; D011991:Receptors, Virus; D006735:Horseradish Peroxidase