Hall Vanessa J, Ruddock Nancy T, Cooney Melissa A, Korfiatis Natasha A, Tecirlioglu R Tayfur, Downie Shara, Williamson Mark, French Andrew J
Monash University, Monash Institute of Reproduction and Development, Centre for Early Human Development, 27-31 Wright Street, Clayton, Vic. 3168, Australia.
Theriogenology. 2006 Jan 20;65(2):424-40. doi: 10.1016/j.theriogenology.2005.05.042. Epub 2005 Jun 23.
The efficiency of generating cloned animals following somatic cell nuclear transfer appears to have reached a plateau, despite ongoing research to improve developmental outcomes. A major limitation appears in the restricted nature of the adult/donor cell to de-differentiate to form a totipotent nucleus. Serial nuclear transfer, a modified cloning technique, has increased the developmental competence of amphibian, murine and porcine cloned embryos. This procedure involves a second nuclear transfer step; pronuclear-like cloned nuclei are transferred into pronuclear stage zygotic cytoplasts. The present study reports on the development of a serial nuclear transfer technique in the bovine, based on a zona-free method (hand-made cloning), resulting in the birth of a cloned calf. Comparisons were made between embryos produced by hand-made cloning and serial nuclear transfer. There were no differences between in vitro development or differential cell counts in the blastocysts produced. Transfer of 16 serial hand-made cloned blastocysts resulted in the production of one healthy calf (6%), whereas hand-made cloning resulted in the birth of 1 calf from 23 transferred blastocysts (4%). One serial nuclear transfer pre-term fetus had renal and hepatic abnormalities (previously observed in clones from this cell line). Although it may not be as beneficial in the bovine as in other species, normal placentation (size, placentomes and umbilicus) was encouraging. Refinement of this technique may help to identify species-specific differences in zygotic competence that affect reprogramming of donor cell nuclei and that may improve efficiency.
尽管一直在进行研究以改善发育结果，但体细胞核移植后产生克隆动物的效率似乎已达到一个平台期。一个主要限制在于成年/供体细胞去分化形成全能细胞核的能力有限。连续核移植是一种改良的克隆技术，已提高了两栖类、鼠类和猪类克隆胚胎的发育能力。该程序涉及第二个核移植步骤；将类原核克隆细胞核转移到原核期合子细胞质体中。本研究报告了基于无透明带方法（手工克隆）在牛中开发的连续核移植技术，并由此诞生了一头克隆小牛。对通过手工克隆和连续核移植产生的胚胎进行了比较。所产生的囊胚在体外发育或差异细胞计数方面没有差异。16个连续手工克隆囊胚的移植产生了一头健康小牛（6%），而手工克隆23个移植囊胚产生了1头小牛（4%）。一头连续核移植早产胎儿有肾脏和肝脏异常（此前在该细胞系的克隆中观察到）。尽管它在牛中的益处可能不如在其他物种中那么大，但正常的胎盘形成（大小、胎盘小叶和脐带）令人鼓舞。改进这项技术可能有助于识别影响供体细胞核对重编程的合子能力方面的物种特异性差异，并可能提高效率。
