Electrofusion

Fusion is facilitated by the presence of Ca2+ (0.5 mM) and can be performed with very low numbers of cells and herein lies its advantage over fusion with PEG. It is also independent of cell type. It does require a pulse generator which can be carefully controlled and the fusion method of choice remains that using PEG. 

Although neither TK. cells nor HPRT- cells will grow separately in HAT medium it is found that mixed populations of these two mutant lines will grow in HAT medium (see Fig. 13.3). 

For cell communication to occur it is essential that the cells are in contact. Using autoradiography the diffusion of nucleotides is readily apparent only between cells in contact, whereas cells equidistant from a neighbour yet not in actual contact do not communicate. 

Cell communication may interfere with the selection of hybrids unless the cells are plated at sufficiently low densities that contact is made only occasionally. Alternatively, hybrids can be made using a cell line which does not communicate, and the L929 cell line is one which has lost this ability together with its HPRT- sub-line (A9) (Pitts, 1971). 

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Silicon Carbide. Sihcon carbide is made by the electrofusion of siUca sand and carbon. SiUcon carbide is hard, abrasion resistant, and has a high thermal conductivity. It is relatively stable but has a tendency to oxidize above 1400°C. The siUca thus formed affords some protection against further oxidation (see Carbides). 

Wojchowski, D.M., and Sytkowski, A.J. (1986) Hybridoma production by simplified avidin-mediated electrofusion./. Immunol. Meth. 90, 173-177. 

ISO 12176-2 2000 Plastics pipes and fittings - Equipment for fusion jointing polyethylene systems - Part 2 Electrofusion... 

Joints of all sizes may be made with heat fusion techniques. Fused joints may be made with either electrofusion or conventional heat fusion. Electrofusion joints are made with fittings which have embedded heating wires. Conventional fusion joints are made with special machines which trim the pipe ends, apply heat, and then force them together to form a bond. Consult the manufacturer regarding the sizes for which electrofusion fittings are available. 

Neumann E, Sowers AE, Jordan CA (1989) Electroporation and electrofusion in cell biology. Plemun Press, New York... 

Carrasco, A. R. D. G., J I., Rico, A., Ritter, E. (2000). Transfer of PLRV resistanee from Solanum verrucosum Schlechdt to potato (5. tuberosum L.) by protoplast electrofusion. Potato Research 43, 31 2. 

Fish, N., Karp, A., Jones, M. G. K. (1988). Production of somatic hybrids by electrofusion in Solanum. Theoretical and Applied Genetics, 76,260-266. 

Nickoloff, J.A. (Ed.) (1995). Methods in Molecular Biology, Vol. 48 Animal Cell Electroporation and Electrofusion Protocols. Human Press, pp. 3-38. 

Electrical breakdown of the membrane sets an upper limit on the fields that can be applied. Controlled electrical breakdown of the membrane is a fairly well understood process which is used in the techniques of electrofusion and electroporation but it is clearly undesirable in the devices under discussion. Surprisingly, fibroblasts can be cultivated for days with no apparent adverse effects in fields inducing trans-membrane potentials of 80% of the breakdown voltage (unpublished data). 

Another cell operation is to use a pulsed electric field to conduct electroporation or electrofusion. For instance, electrofusion of liposomes (5 pm dia.) and RBC has been conducted on a microchip (see Figure 8.40) [903]. 

Tomita, M. Tsong, T.Y. (1990). Selective production of hybridoma cells antigenic-based pre-selection of B lymphocytes for electrofusion with myeloma cells Biochim. Biophys. Acta, 1055, 199-206. 

FT-IR spectroscopy has been used in the investigation of welding crosslinked polyethylene pipes [23]. Three types of crosslinking systems were used namely, peroxide (PEXa), silane (PEXb) and electron beam (PEXc). Scholten and co-workers [23] observed that only PEXa pipes have a satisfactory electrofusion quality. The strength of electrofusion welds of PEXb and PEXc pipes is not acceptable. The most likely explanation for the differences in weld quality is related to the adhesion theory and more specifically to differences in composition. Figure 5.6 shows the infrared spectra of medium density polyethylene (MDPE), PEXa, PEXb and PEXc. 

A number of commercial machines are available and cuvettes may be bought or constructed from disposable spectrophotometer cuvettes into which aluminium electrodes are glued. They can be sterilised with 70% ethanol and then washed with sterile PBS. (This is also true for the disposable commercial cuvettes.) The same apparatus can also be used for cell fusion (electrofusion) (see 13.7.3 and Glassy, 1988). 

Although cells sometimes fuse spontaneously, the frequency of fusion can be dramatically increased by treatment with a number of reagents. Sendai virus used to be a very common fusogen (Watkins, 1971 Stadler and Adelbert, 1972) but is difficult to control. It has been largely replaced by lysolecithin or polyethyleneglycol (PEG) or by electrofusion. 

Although the mechanisms of electroporation, electrofusion, and electroinsertion are not known, biophysical data suggest that the primary field pulse effect is interfacial polarization by ion accumulation at the membrane surfaces. The resulting transmembrane electric field causes rearrangements of the lipids such that pores are formed1718. Electropores anneal slowly (over a period of minutes) when the pulse is switched off. 

Buchacher, A., Predl, R., Strutzenberger, K., Steinfellner, W., Trkola, A., Purtscher, M., Gruber, G., Tauer, C., Steindl, F., and Jungbauer, A. (1994). Generation of human monoclonal antibodies against HIV-1 proteins electrofusion and Epstein-Barr virus transformation for peripheral blood lymphocyte immortalization. AIDS Res. Hum. Retroviruses 10, 359-369. 

A research group at Goteborg University has demonstrated a microfluidic device for combinatorial electrofusion of liposomes and cells [94], Optical trapping was used to transport individual liposomes and cells through microchannels... 

Techniques, used by the gas and water industries to join and repair pipelines, include butt fusion, electrofusion socket welding and socket jointing (309). The study of bond strength testing for solvent joints in piping systems has noted extreme sensitivity to minor variations in sample preparation (259). 

Baumann, T., Kreis, W., Mehrle, W., Hampp, R. and Reinhard, E. (1990) Regeneration and characterization of protoplast-derived cell lines from Digitalis lanata Ehrh. and Digitalis purpurea L. suspension cultures after electrofusion under microgravity conditions, in Proceedings of the 4th European Symposium on Life Sciences. ESA Publications Div., ESTEC, Nordwijk, pp. 405-10. 

Neumann, E., Sowers, A.E., and Jordon, C.A., Electroporation and Electrofusion in Cell Biology, Plenum Press, New York, 1989. 

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