Electrofusion

Electrofusion is achieved using an electroporator, together with one of several methods of bringing cells into close membrane-membrane contact. Such methods include the natural adhesion of slime moulds (Neumann et al., 1980), the physical juxtapositioning of cells by manipulation with electrodes (Senda et al., 1979; Zimmermann and Scheurich, 1981), chemical agglutination (Weber et al., 1981; Chapel et al., 1986), sedimentation at high cell density to produce tight monolayers (Sowers, 1985) or multiple layers (Teissie and Rols, 1986), the formation of monolayers through cell growth (Tessie et al., 1982), immunological targeting (Lo et al., 1984; Lo and Tsong, 1989), and cell polarization induced by AC or magnetic fields with resulting mutual dielectrophoresis (Pohl, 1987; Schwan, 1989; Zimmermann et al., 1985).

Pohl (1951, 1958) presented a detailed description of the movement of neutral particles under the influence of nonuniform AC fields. In an electrical field, cells become polarized and the resulting cellular dipoles are attracted toward the oppositely charged electrodes. In a homogeneous field, the attractive forces acting on each side of a cell are balanced. A nonhomogeneous electric field may however, induce translational movement of the cell directed toward the pole at which the net attractive force is strongest. The electrical field will be homogeneous if the electrodes are perfectly flat, equidistant from one another and continuous across the opposite walls of the fusion chamber. When cells are introduced into the fusion chamber, however, localized nonhomogeneity in the field caused by the induced cellular dipoles will, if of sufficient strength, induce the translational movement of cells toward each other, the positive pole of one cell being attracted toward the negative pole of another. This phenomenon, termed mutual dielectrophoresis, leads to the formation of rows of cells, often referred to as pearl chains, along the lines of force. When cell-to-cell contact has been achieved, single or multiple high-intensity electrical pulses will induce membrane breakdown. If applied simultaneously, the AC and DC wave-forms will conflict and partially cancel each other. Therefore, the integration of the alignment (AC) field and fusion (DC) field is normally achieved by momentarily switching between the two outputs.

Several commercially produced instruments are available but frequently at a prohibitive cost. Furthermore, some biological systems require a degree of control and flexibility that cannot be obtained from standard, commercially manufactured units. Several authors have described electrofusion equipment of simple design that is inexpensive to construct (Watts and King, 1984; Zachrisson and Bornman, 1984; Mischke et al., 1986; Kramer et al., 1987). The facilities and parameters provided by such equipment are however, generally limited. In contrast, Jone et al., (1994) have reported the construction of an instrument that provides a considerable degree of flexibility and control over the fusion process. This apparatus has proven useful in the large-scale electrofusion of isolated higher plant protoplasts in somatic hybridization and in the electrotransfection of plant protoplasts resulting in the production of transgenic plants.