表面张力辅助去核法对小鼠卵母细胞的去核研究

Mouse Oocyte Enucleation with Surface Tension Assisted Method

论述了一种新的小鼠卵母细胞去核方法─—表面张力辅助去核法 (STA) ,并同时与其他 3种去核方法 (简称A法、B法、C法 )进行比较。STA法是将卵母细胞核在固定管的辅助下 ,借助液体表面张力通过透明带上的破口被挤出。经STA法去核的卵母细胞质体用于电融合介导的小鼠耳成纤维细胞核移植实验 ,结果有76 1%细胞与细胞质体融合形成重组胚 ,85 4%的重组胚形成原核 ,49 4%卵裂形成 2

In the present experiment a new simple method Surface Tension Aided (STA) for mouse oocyte enucleation is employed and is compared with three other ones. In method STA, the chromosome spindle was squeezed out by the surface tension of the drop edge with the help of micropipette through a slit made in advance in zona pellucida. In method A, the nucleus was aspirated out through a plat end, 25 μm micropipette via the slit. In method B, the nucleus was aspirated out through a 10μm micropipette directly. In method C, the nucleus was assumed just under the first polar body and the enucleating procedure was carried out by aspirated one third of cytoplasm beneath the polarbody. The manipulation time in method A (3 min/oocyte) was significantly longer than that in method STA (1.33min/oocyte), method B (1.30 min/oocyte) and method C (1.41 min/oocyte); The cytoplasm loss in method C (28.4 %) was significantly higher than those in the other three methods. In methods STA, A and B, very small amount (approximately 5 %) of cytoplasm was lost. The accuracy rate of method C (35.3 %) was significantly lower than those in the other three methods, the accuracy rate of method STA, A and B was above 95 % and there is no significant differences among these methods. Some of the cytoplasts produced by STA were used for mouse ear fibroblast cell nuclear transfer by electrofusion. Majority (76.1 %) of the cell cytoplast pairs fused to form reconstructed embryos, 85.4 % of reconstituted embryos developed to form pronuclei and 49.4 % of them clove to form 2 cell embryos. [