Wakayama Sayaka, Tanabe Yoshiaki, Nagatomo Hiroaki, Mizutani Eiji, Kishigami Satoshi, Wakayama Teruhiko
1 Advanced Biotechnology Center, University of Yamanashi , Kofu-shi, Yamanashi, Japan .
2 Faculty of Life and Environmental Sciences, University of Yamanashi , Kofu-shi, Yamanashi, Japan .
Cell Reprogram. 2016 Nov;18(6):382-389. doi: 10.1089/cell.2016.0026. Epub 2016 Sep 13.
Although animal cloning is becoming increasingly practicable, cloned embryos possess many abnormalities and so there has been a low success rate for producing live animals. This is most likely due to incomplete reprogramming of somatic cell nuclei before they start to develop as the donor nuclei are usually only exposed to the oocyte cytoplasm for 1-2 hours before reconstructed oocytes are activated to avoid oocyte aging. Therefore, in this study, we attempted to extend the exposure period of somatic cell nuclei to the oocyte cytoplasm to determine whether this could enhance reprogramming of donor nuclei. Donor nuclei were transferred into oocytes, following which pseudo-MII spindles (pMIIs) derived from these were extracted and injected into newly collected enucleated oocytes 24 hours after the first nuclear transfer (NT). These serial NT oocytes were then activated and their developmental potential was examined to full term. There was no obvious difference in the pMIIs of reconstructed oocytes at 6 and 24 hours after donor nucleus injection; however, in both of these, the chromosomes were more widely spread inside the spindle than in fresh intact oocytes. Furthermore, a few chromosomes remained in 25% and 47% of these enucleated oocytes at 6 and 24 hours after donor nucleus injection, respectively. When these pMIIs were injected into fresh enucleated oocytes, the developmental rate to blastocyst was significantly lower, but we could still obtain several healthy cloned offspring. Thus, serial NT at intervals of 24 hours using fresh oocytes is possible, but the success rate could not be improved due to loss of chromosomes during the second NT.
尽管动物克隆变得越来越可行，但克隆胚胎存在许多异常，因此活体动物的成功率一直很低。这很可能是由于体细胞核对在开始发育之前未完全重编程，因为在激活重构卵母细胞以避免卵母细胞老化之前，供体核通常仅在卵母细胞细胞质中暴露1-2小时。因此，在本研究中，我们试图延长体细胞核对在卵母细胞细胞质中的暴露时间，以确定这是否可以增强供体核的重编程。将供体核转移到卵母细胞中，然后在第一次核移植（NT）24小时后，从这些卵母细胞中提取伪MII纺锤体（pMII）并注射到新收集的去核卵母细胞中。然后激活这些系列NT卵母细胞，并检查它们直至足月的发育潜力。供体核注射后6小时和24小时重构卵母细胞的pMII没有明显差异；然而，在这两种情况下，纺锤体内的染色体比新鲜完整卵母细胞中的染色体散布得更广泛。此外，在供体核注射后6小时和24小时，分别有25%和47%的这些去核卵母细胞中残留有一些染色体。当将这些pMII注射到新鲜去核卵母细胞中时，发育到囊胚的比率显著降低，但我们仍然可以获得几只健康的克隆后代。因此，使用新鲜卵母细胞以24小时间隔进行系列NT是可行的，但由于第二次NT期间染色体的丢失，成功率无法提高。
