Effects of induction current and other factors on large-scale electrofusion for pronuclear transplantation of mouse eggs.
In this paper, we describe the procedure of large-scale and efficient electrofusion for pronuclear transplantation in mouse eggs and the tolerance of the eggs for electric stimulus, assessed in vitro and in vivo development. The fusion chamber was arranged in parallel by dielectrodes (30-mm length, 1-mm width, and 2-mm height), and 0.3 M mannitol in distilled water was used as a fusion solution. The agglutination cleavage of enucleated eggs with karyoplast was easily orientated in parallel with electrodes by alternating current between 100 and 500 kHz at 2 and 10 V/mm. Immediately after the orientation, a direct current of 150 V/mm was given for 200 microseconds twice and repeated three times to induce fusion of the enucleated eggs with karyoplast. More than five eggs, at least, can be submitted to electrofusion at the same time. The eggs that were not fused were treated again in the same manner. The proportion of eggs fused with karyoplast was increased by preincubation in M16 medium prior to submitting them to the electrofusion. When the eggs were incubated for 60 min, 80% of them were fused with karyoplast by the first electric treatment; in contrast, only 19% of the eggs were fused if they were submitted to electrofusion directly. It was found that between the CD-1 and F1 strains there was a difference in tolerance of the eggs to electric stimulus and that this was depend on the nuclei but not on cytoplasm.(ABSTRACT TRUNCATED AT 250 WORDS)
Kono
T
T
National Institute of Animal Industry, Ibaraki, Japan.
Tsunoda
Y
Y
D000818:Animals; D001755:Blastocyst / Q000502:physiology; D001757:Blastomeres / Q000502:physiology; D046508:Culture Techniques; D004558:Electric Stimulation; D004560:Electricity; D004624:Embryo Transfer; D005260:Female; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D053652:Nuclear Transfer Techniques; D010063:Ovum / Q000648:ultrastructure*