A protocol for adult somatic cell nuclear transfer in medaka fish (Oryzias latipes) with a high rate of viable clone formation.
A protocol for adult somatic cell nuclear transfer in medaka fish (Oryzias latipes) with a high rate of viable clone formation.
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青鳉鱼 (Oryzias latipes) 成体体细胞核移植方案，具有高存活克隆形成率。
DOI：
10.1089/cell.2013.0004
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发表时间：
2013
影响因子：
1.6
通讯作者：
Y. Wakamatsu
中科院分区：
文献类型：
--
作者：
E. Bubenshchikova;E. Kaftanovskaya;T. Adachi;H. Hashimoto;M. Kinoshita;Y. Wakamatsu
在此之前，我们成功地产生了完全长大，克隆青（日本水稻鱼，青）使用供体核从成年尾鳍组织和nonenucleated受体卵的原代培养细胞进行热休克处理，以诱导二倍体化的核。然而，利用这种方法形成克隆的机制尚不清楚，并且成体克隆形成率不足以用于基础和应用科学研究。为了深入了解机制并提高这种克隆形成方法的成功率，我们测试了两种不同的核转移方案。在一个方案中，供体细胞核的转移时间发生了变化，而在另一个方案中，供体细胞的大小发生了变化;每个方案都是基于我们最初的方法。最终，我们获得了一个意想不到的高成人克隆形成率使用的协议，不同的供体核转移的时间。具体来说，17%发育到囊胚阶段的移植物最终发育成成年克隆。这种方法的成功率是使用原始方法获得的成功率的13倍。分析这种高成功率的克隆形成的原因将有助于阐明这种方法发生的克隆形成的机制。
Previously, we successfully generated fully grown, cloned medaka (the Japanese rice fish, Oryzias latipes) using donor nuclei from primary culture cells of adult caudal fin tissue and nonenucleated recipient eggs that were heat shock-treated to induce diploidization of the nuclei. However, the mechanism of clone formation using this method is unknown, and the rate of adult clone formation is not high enough for studies in basic and applied sciences. To gain insight into the mechanism and increase the success rate of this method of clone formation, we tested two distinct nuclear transfer protocols. In one protocol, the timing of transfer of donor nuclei was changed, and in the other, the size of the donor cells was changed; each protocol was based on our original methodology. Ultimately, we obtained an unexpectedly high rate of adult clone formation using the protocol that differed with respect to the timing of donor nuclei transfer. Specifically, 17% of the transplants that developed to the blastula stage ultimately developed into adult clones. The success rate with this method was 13 times higher than that obtained using the original method. Analyses focusing on the reasons for this high success rate of clone formation will help to elucidate the mechanism of clone formation that occurs with this method.