Kishigami Satoshi, Mizutani Eiji, Ohta Hiroshi, Hikichi Takafusa, Thuan Nguyen Van, Wakayama Sayaka, Bui Hong-Thuy, Wakayama Teruhiko
Laboratory for Genomic Reprogramming, Center for Developmental Biology, RIKEN Kobe, 2-2-3 Minatojima-minamimachi, Kobe 650-0047, Japan.
Biochem Biophys Res Commun. 2006 Feb 3;340(1):183-9. doi: 10.1016/j.bbrc.2005.11.164. Epub 2005 Dec 9.
The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) is believed to be associated with epigenetic errors including abnormal DNA hypermethylation. Recently, we elucidated by using round spermatids that, after nuclear transfer, treatment of zygotes with trichostatin A (TSA), an inhibitor of histone deacetylase, can remarkably reduce abnormal DNA hypermethylation depending on the origins of transferred nuclei and their genomic regions [S. Kishigami, N. Van Thuan, T. Hikichi, H. Ohta, S. Wakayama. E. Mizutani, T. Wakayama, Epigenetic abnormalities of the mouse paternal zygotic genome associated with microinsemination of round spermatids, Dev. Biol. (2005) in press]. Here, we found that 5-50 nM TSA-treatment for 10 h following oocyte activation resulted in more efficient in vitro development of somatic cloned embryos to the blastocyst stage from 2- to 5-fold depending on the donor cells including tail tip cells, spleen cells, neural stem cells, and cumulus cells. This TSA-treatment also led to more than 5-fold increase in success rate of mouse cloning from cumulus cells without obvious abnormality but failed to improve ES cloning success. Further, we succeeded in establishment of nuclear transfer-embryonic stem (NT-ES) cells from TSA-treated cloned blastocyst at a rate three times higher than those from untreated cloned blastocysts. Thus, our data indicate that TSA-treatment after SCNT in mice can dramatically improve the practical application of current cloning techniques.
通过体细胞核移植（SCNT）进行动物克隆的成功率较低，据信这与包括异常DNA高甲基化在内的表观遗传错误有关。最近，我们通过使用圆形精子细胞阐明，核移植后，用组蛋白脱乙酰酶抑制剂曲古抑菌素A（TSA）处理合子，可以根据移植细胞核的来源及其基因组区域显著减少异常DNA高甲基化[S. Kishigami, N. Van Thuan, T. Hikichi, H. Ohta, S. Wakayama. E. Mizutani, T. Wakayama, 与圆形精子细胞显微授精相关的小鼠父本合子基因组的表观遗传异常，《发育生物学》（2005年，待发表）]。在此，我们发现，卵母细胞激活后用5 - 50 nM TSA处理10小时，能使体细胞克隆胚胎在体外更有效地发育至囊胚阶段，根据供体细胞（包括尾尖细胞、脾细胞、神经干细胞和卵丘细胞）的不同，效率提高2至5倍。这种TSA处理还使来自卵丘细胞的小鼠克隆成功率提高了5倍以上，且无明显异常，但未能提高胚胎干细胞克隆的成功率。此外，我们成功地从经TSA处理的克隆囊胚中建立了核移植胚胎干细胞（NT - ES），其成功率是未经处理的克隆囊胚的三倍。因此，我们的数据表明，小鼠体细胞核移植后进行TSA处理可以显著改善当前克隆技术的实际应用。
