STUDIES ON THE TECHNIQUES FOR THE PREPARATION OF FISH MICROCELLS AND MINISEGREGANT CELLS

Using CAB-80 CAG-87 and CCRV-87 (these cell lines were devived from crucian carp blastula, caudal fin of crucian carp and caudal fin of Xingguo red carp respectively), we studied the preparation of fish microcells and minisegregant cells and the mechanism of their formation. In the preparation of microcells, observations were made on the process of micronucleation. The formation of micronuclei was the result of irregular division of interphase nuclei. We also studied the effects of colcemid concentration and treatment time on the proportions of micronucleated cells in the three cell lines. The proportion of micronucleation achieved was 40％ for CAB-80, and 30％ for CCRV-87 and CAG-87. High concentrations of colcemid or prolonged treatment time is toxic to the cells. The main procedures which can yield large quantities of fish minisegregant cells are as follows, 16-20 hours after incubation, colcemid is added to the cultures of CAB-80 cells at a final concentration of 0.3μg/ml. Mitotic cells are obtained by continuing incubation for another 10-14 hours. Then the mitotic-blocked ceils are collected and stored at 4℃ for 10 hours. At a temperature of 27℃ and a pH of 8.0, many cells display highly irregular patterns of division and about 50％ of them form minisegregant cells. Analysis of genetic material distribution and scanning electron microscopy showed that, in the formation of minisegregant cells, the process of nucleus change is the same as that of micronucleation, however, the mechanism of cytokinesis is different. Micronuclei are separated into many daughter cells due to the irregular cleavage of the cytoplasm.