2001, Volume 3, Issue 11
Strategic Study of CAE >> 2001, Volume 3, Issue 11
Mouse Oocyte Enucleation with Surface Tension Assisted Method
Lab of Animal Embryonic Biotechnology, College of Animal Science, China Agricultural University, Beijing 100094, China
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Abstract
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.
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