Study On Chromosome Preparation Of Single Blastomere From Mouse 4,8-Cell Stage Embryos

Chromosome is the material carrier of biological heredity, and displays the highstability and the species specificity in the biology during the generations. There isa common view that the karyotype and banding may provide basic data ofcytogenetics for origin, evolution and gene location at present.We used air-drying method to prepare chromosome specimen by 4- and 8-cell stage early embryo single blastomeres of Kunming mduse which werecultured in CZB medium, in the experiment. In order to determine the optimalconcentrations, and treatment durations of two kinds of spindle inhibitors——colchicine and vinblastine, we added different concentrations of them to CZB,respectively. Second, hypotonic treatment were carried out to pick out the besthypotonic fluid, thus we gained the ideal experimental parameter and evaluatedthe metaphases for four grades. The results were as follows:The first, there were significantly differences percent between 0.6μg/mlgroups and other three groups (0.2μg/ml groups, 0.4μg/ml groups and 0.8μg/mlgroups) in 2 hour groups; and there were significantly differences between0.4μg/ml groups and other three groups in 4 hour groups; and there weresignificantly differences between 0.6μg/ml groups and other three groups in 6hour groups; and there were significantly differences between 0.4μg/ml groupsand other three groups in 8 hour groups on the metaphases (P＜0.05), in theexperiment of the effect on the mouse 4- cell embryo stage single blastomere ofcolchicine with different concentrations and duration, by x~2 statistical analysis. Sowe selected the colchicine optimum parameters were 0.6μg/ml and 6 hour culturedduration.The second, there were no significantly differences between the four groupsin 2 hour groups; and there were significantly differences between 0.2μg/ml groups and other three groups in 4 hour groups; and there were no significantlydifferences between the four groups in 6 hour groups; and there were significantlydifferences between 0.4μg/ml groups and other three groups in 8 hour groups onthe metaphases (P＜0.05), in the experiment of the effect on the mouse 8-cellembryo stage single blastomere of colchicine with different concentrations andduration by x~2 statistical analysis. So we selected the colchicine optimumparameters were 0.2μg/ml and 6 hour cultured duration.The third, there were significantly differences percent between 10 hourgroups and other three groups (4 hour groups, 6 hour groups and 8 hour groups) in0.1μg/ml groups; and there were significantly differences between 8 hour groupsand 10 hour groups, but they had no significantly differences among other twogroups in 0.3μg/ml groups; and there were significantly differences between 10hour groups and 6 hour groups, but 10 hour groups had no significantlydifferences among other two groups in 0.5μg/ml groups; and there weresignificantly differences between 8 hour groups and other three groups in0.7μg/ml groups on the metaphases (P＜0.05), in the experiment of the effect onthe mouse 4- cell embryo stage single blastomere of vinblastine with differentconcentrations and duration, by x~2 statistical analysis. So we selected thevinblastine optimum parameters were 0.1μg/ml and 8 hour cultured duration.The fourth, there were significantly differences percent between 10 hourgroups and other three groups in 0.1μg/ml groups; and there were no significantlydifferences between 8 hour groups and 10 hour groups, but they had significantlydifferences among other two groups in 0.3μg/ml groups; and there weresignificantly differences between 10 hour groups and other three hour groups in0.5μg/ml groups; and there were significantly differences between 10 hour groupsand other three groups in 0.7μg/ml groups on the metaphases (P＜0.05), in theexperiment of the effect on the mouse 8- cell embryo stage single blastomere ofvinblastine with different concentrations and duration, by x~2 statistical analysis. Sowe selected the vinblastine optimum parameters were 0.1μg/ml and 8 hourcultured duration.The fifth, there were significantly differences between 0.45% sodium chloride groups and other two groups (0.5% sodium citrate groups and 0.35%potassium chloride groups) on the metaphases (P＜0.05), in the experiment of theeffect on the mouse 4-and 8-cell embryo stage single blastomere of differenthypotonic fluids, by x~2 statistical analysis.The sixth, there were no significantly differences between colchicine groupsand vinblastine groups on the metaphases, in the experiment of prepare mouse4-cell embryo stage single blastomere chromosome specimen by using optimumparameters; and there were significantly differences between colchicine groupsand vinblastine groups on the metaphases (P＜0.05), in the experiment of preparemouse 8-cell embryo stage single blastomere chromosome specimen by usingoptimum parameters.The end, the preparation quality of mouse 4-and 8-cell embryo stage singleblastomere specimen were 2.5, 2.7, 2.4, 1.9, respectively.