Hosaka K, Ohi S, Ando A, Kobayashi M, Sato K
Department of Applied Biological Science, Nihon University College of Bioresource Sciences.
Hum Cell. 2000 Dec;13(4):237-42.
In 1997, a cloned sheep "Dolly" was produced by nuclear transfer of somatic cell. The first birth of cloned mice derived from some somatic cells were succeeded in 1998. At present, it is shown that somatic cells, cumulus cells, fibroblasts and Sertoli cells can be used to the study of cloned animal as nuclear donor. In this study investigation was designed to compare with efficiency on the production of cloned embryos by using the microinjection and the electrofusion methods for nuclear transfer. Oocyte enucleation was performed with a micromanipulator. The oocyte was held by holding pipette, and was enucleated using a beveled pipette. Microinjection method: Cell's nucleus injection was carried out by piezo-micromanipulator. Cytochalasin B treated cumulus cell was aspirated into a injection pipette, and was broken its plasma membrane using the injection pipette. Then, the cumulus cell was injected into the enucleated ooplasm directly. Electrofusion method: The cell was aspirated into a beveled pipette, and then an aspirated cell was inserted into perivitelline space. Then, the pair of enucleated oocyte and cell was fused using electrical cell fusion apparatus. The reconstituted embryos were activated after nuclear transfer using St2+. Reconstituted embryos had been produced by the microinjection showed the embryonic development to over 8-cell stages. But, the rate of fragmentation of reconstituted embryos by the microinjection showed a little high rate in comparison with the electrofusion. When some reconstituted embryos by the microinjection were transplanted to pseudopregnant females' oviduct, 9 fetuses were observed at 14 days post coitum.
1997年，通过体细胞的核移植培育出了克隆羊“多莉”。1998年，首次成功培育出了源自某些体细胞的克隆小鼠。目前研究表明，体细胞、卵丘细胞、成纤维细胞和支持细胞都可作为核供体用于克隆动物的研究。本研究旨在比较使用显微注射和电融合方法进行核移植时克隆胚胎的生产效率。使用显微操作仪进行卵母细胞去核。用固定吸管固定卵母细胞，并用斜口吸管进行去核。显微注射法：通过压电显微操作仪进行细胞核注射。将用细胞松弛素B处理过的卵丘细胞吸入注射吸管，并用注射吸管破坏其质膜。然后，将卵丘细胞直接注入去核的卵细胞质中。电融合法：将细胞吸入斜口吸管，然后将吸入的细胞插入卵周隙。然后，使用细胞电融合仪将去核的卵母细胞和细胞对进行融合。核移植后使用St2+激活重构胚胎。通过显微注射产生的重构胚胎显示出胚胎发育至8细胞以上阶段。但是，与电融合相比，显微注射产生的重构胚胎的碎片化率略高。当将一些通过显微注射产生的重构胚胎移植到假孕雌性的输卵管中时，在交配后14天观察到9只胎儿。
