Immunospecific vesicle targeting facilitates microinjection into lymphocytes.

Abstract

Antibody-directed targeting of vesicles to cells dramatically enhances polyethylene glycol-mediated fusion and microinjection. Sealed erythrocyte ghosts, containing fluorescent bovine serum albumin, were targeted to murine spleen and thymus cells, and to lymphocyte, monocyte, and fibroblast cell lines. In all cases, targeted cell populations showed substantial levels of microinjection, whereas populations treated with the fusogen in the absence of targeting were not significantly microinjected. To achieve attachment of vesicles to selected cells, the cells were first labeled with biotin-modified antibody then treated with sealed ghosts prepared from avidin-coupled erythrocytes. This procedure should prove useful when the injection of specific cell populations is desired, or with cell types such as lymphocytes that are difficult to fuse, or when the use of limited reagents necessitates high injection efficiencies.Microinjection allows the introduction into cells of biologically interesting molecules so that their properties and effects on cell function can be studied. Although microinjection protocols have been used to study a number of important questions in cell biology (1-4), their applications have been restricted by inherent limitations. Mechanical injection (5) can be used to introduce discrete volumes of reagent into cells, but it is limited by the number and type of cells that can be treated (1). Vesicle-mediated protocols, using fusogens such as polyethylene glycol (PEG) or Sendai virus, can be used to inject large numbers of cells (1), but fusion efficiencies are generally low and selectivity is lacking.We, as others (6), have reasoned that bringing two membranes into close apposition should increase the probability that they will fuse, although contact per se between two membranes does not generally lead to fusion (7). PEG-mediated microinjection can be enhanced by agglutinating vesicles to cells with phytohemagglutinin (6, 8). Our laboratory has developed protocols for the efficient, antibody-directed targeting of vesicles to cell surface antigens (9). In this paper, we describe the use of vesicle targeting with PEG-mediated fusion to promote the microinjection of selected cell types, particularly of lymphocytes and lymphoid cell lines. The frequency of vesicle-lymphocyte fusions in these experiments is about 104 times higher than values generally observed for PEG-mediated lymphocyte fusion. MATERIALS AND METHODSAntibodies. Goat anti-mouse immunoglobulin (anti-MIg) was raised as described (10). Anti-mouse cell surface (anti-MCS) was prepared by injecting rabbits with DBA/2 spleen and lymph node cells in complete Freund's adjuvant. Rabbit anti-mouse Fab fragment (anti-MFab) was generously donated by A. Good (University of California, Berkeley). All three antisera were passed over DEAE-Sephacel (Pharmacia). Fab fragment of antiMIg (11) and the affinity-purified F(ab')2 fragment of anti-MFab (9) were prepared as described. Two monoclonal antibodies were obtained from Becton Dickinson: mouse ant...

Glass beads load macromolecules into living cells

“…Cells can be loaded by causing them to fuse with a suitable membrane-delimited vehicle (usually a red blood cell ghost or liposome) that has itself been loaded with macromolecules (Doxseye/a/. 1985; Godfrey et al 1983;Pagano & Weinstein, 1978;Schlegel & Rechsteiner, 1975). Cells can be reversibly permeabilized to macromolecules by electric shocks, a technique used predominantly for transfection (Neumann et al 1982).…”

Muscle cell components dictate hepatocyte gene expression and the distribution of the Golgi apparatus in heterokaryons.

“…These results raise the intriguing possibility that there may be one or more muscle components or trans-acting factors capable of targeting the MTOC and Golgi to the nuclear periphery, independent of the centrioles. By liposome or red blood cell-mediated fusion, osmotic lysis of pinocytic vesicles, or cellular microinjection (Capecchi 1980;Szoka et al 1981;Okada and Rechsteiner 1982;Godfrey et al 1983), it should now be possible to introduce muscle macromolecules into the responsive hepatocytes described here and test this hypothesis directly.…”

A method for incorporating macromolecules into adherent cells.

“…Two problems are associated with cell loading by fusion with red blood cells (17,7) or liposomes (15). First, the membrane of the loaded cell is contaminated with red blood cell or liposome membrane.…”