小鼠体细胞核移植程序的研究

采用直接去核法、透明带切割法、PMM法3种核移植方法进行小鼠卵母细胞去核的研究。3种方法都未对卵母细胞核进行示踪,仅以第一极体作为参照进行去核,都属于盲吸法范畴,在去核率上没有显著差异。直接去核法对卵母细胞造成较大的伤害,去核操作过程中极易使卵母细胞膜破裂,发生崩解;透明带切割法分步操作使操作变得柔和,对卵母细胞的刺激减小,去核卵母细胞的存活率较高;PMM法靠脉冲电压在透明带上打孔进行辅助去核,脉冲参数稍大或压透明带太紧都极易在击破透明带的同时击破卵膜,使卵母细胞发生崩解。在构建重构胚的过程中,胞质内注射法较电融合法而言,程序简单,带入的供体胞质较少,构建重构胚的效率更高。

Study on Procedure of Mice Nuclear Transfer

Three procedures of nuclear transfer in mice were studied.They all referred the first polar body to orient for enucleating the nuclei,so the rates of nuclei enucleation were not different.Oocytes were extraordinarily sensitive to the conventional micromanipulation,which generally resulted in cell lysis.Cutting zona pellucida beforehand could make manipulation more softly to raise the live rate of manipulated oocytes.Pizeo-actuated micromanipulation was the best method for mice nuclear transfer,but it needed more delicate manipulation,which was not easy to master.Intracytoplasmic nuclei injection was more effective than cell electro-fusion method on reconstituting embryos of nuclear transfer.