Large-scale production of yeast hybrids by electrofusion

Abstract

Large‐scale production of electrically fused yeast protoplasts from Saccharomyces cerevisiae AH 22[pADH 040‐2] and S. cerevisiae AH215 was achieved by the use of the so‐called helical fusion chamber. Both strains were of the same mating type a and carried the following auxotrophic markers: his4 in the case of AH22 and leu2, his3 in the case of AH215. AH22 also is a carrier of the plasmid pADH 040‐2. This plasmid confers the leu2 gene of yeast and the β‐lactamase gene from Escherichia coli, and this feature enables quick detection of plasmid‐positive cells. After dielectrophoresis (275 V/cm, 800 kHz) fusion was induced by two field pulses (10 kV/cm, 10 μs duration) applied at an interval of 0.5 s. 50 to 60 hybrids per run were isolated after regeneration on selection medium.

Effect of n-alcohols on the electrotransformation and permeability of Saccharomyces cerevisiae

“…The strain was cultivated in YEP [1% yeast extract (Difco), 2°7o peptone (Difco), 2% glucose] medium to a mean exponential phase. The reversion rate of leu2 is less than 10 -9 (Schnettler et al 1984).…”

“…They are easily cultivated and divide rapidly, so that in a comparatively short time they yield a great number of cells. This makes them suitable for investigating electroinduced events in biomembranes (Schnettler et al 1984;Schnettler and Zimmermann 1985;Tsoneva et al 1989;Salek et al 1990;Tomov and Tsoneva 1992). In the last few years intact yeast cells have been successfully transformed by applying electrical pulses (Hashimoto et al 1985;Hood and Stachow 1990;Rech et al 1989;Meilhoc et al 1990).…”

Electrical breakdown, electropermeabilization and electrofusion

“…Recently Schnettler and Zimmermann [(90), see also (181)] considerably improved the electrofusion technique of yeast cells, so that now large numbers of yeast hybrids can be produced with the aid of this technique. The work o f these authors simultaneously led to the formulation 226 U. Zimmermann of advanced protocols for the efficient and reproducible production of hybridoma cells by means of electrofusion (91).…”

“…Instead of the simple two electrode wire chamber used by Halfmann et al (178)(179)(180). Schnettler and Zimmermann (90,181)used the helical chamber (Fig. 26) for electrofusion and showed that about 60 hybrids could be obtained per experimental run when electrofusion was performed in isotonic sorbitol solution (181).…”

The Generation of Ig-Secreting UC 729-6 Derived Human Hybridomas by Electrofusion

“…This recently developed method has been shown to be effective for obtaining viable hybrids of plant (2,3), yeast (4), and mammalian (5-9) origin, including interkingdom hybridization (10).…”