延边黄牛体细胞克隆囊胚染色体标本制备的研究

Study on Preparation of Chromosomes of High Quality from Blastocysts of Somatic Cell Cloning in Yanbian Yellow Cattle

[目的]探讨适合延边黄牛体细胞克隆胚胎染色体标本制备的方法。[方法]利用延边黄牛体细胞克隆产生的囊胚制备染色体标本,采用传统的细胞遗传学方法,将延边黄牛体细胞克隆囊胚分别在含有0.11、.0和10.0μg/ml浓度秋水仙素的CR1aa培养液中继续培养4或6 h,以使细胞分裂停留在中期。处理后的囊胚移入柠檬酸钠低渗液中,室温下处理15～20 min。将低渗后的单个胚胎转移至载玻片上,固定并用体积分数为25%的Giemsa溶液染色1～5 h,蒸馏水冲洗脱色,于室温干燥。显微镜下计算胚胎细胞数和有丝分裂指数以及标本制备质量。[结果]结果表明,1.0μg/ml秋水仙素处理6 h后的染色体标本制备效果较为理想。[结论]该研究为制备延边黄牛体细胞克隆囊胚染色体标本提供适宜的试验参数。

[Objective]The purpose was to find out a suitable method for preparation of chromosomes from embryos of somatic cell cloning in Yanbian Yellow Cattle.[Method] The blastocysts of somatic cell cloning in Yanbian Yellow Cattle were subjected to cytogenetic analysis.The blastocysts of somatic cell cloning in Yanbian Yellow Cattle were cultured in CR1aa containing 0.1、1.0 and 10.0 μg/ml colchicine for a further 4 or 6 h,respectively.Each blastocyst was treated with sodium citrate at room temperature for 15～20 min and fixed on slides.The slides were stained in a φ=25% Giemsa solution for 1～5 h,and taken off the color with distilled water,and then dried in air.Cell number and mitotic index were counted,and chromosome spreads were observed under a compound microscope.[Result]The result indicated that the treatment of 1.0 μg/ml for 6 h was better than others.[Conclusion]This research will provide feasible experimential parameters for preparation of chromosomes from embryos of somatic cell cloning in Yanbian Yellow Cattle.