Gibbons John, Arat Sezen, Rzucidlo Jacek, Miyoshi Kazuchika, Waltenburg Rachel, Respess Donald, Venable Alison, Stice Steve
Department of Animal and Dairy Sciences, University of Georgia, Athens, GA 30602, USA.
Biol Reprod. 2002 Apr;66(4):895-900. doi: 10.1095/biolreprod66.4.895.
Nuclear transfer to produce cattle is inefficient because 1) donor cells are not easily synchronized in the proper phase of the cell cycle, 2) the nucleus of these cells is not effectively reprogrammed, 3) the rate of attrition of late-term pregnancies is high, and 4) the health of early postnatal calves is compromised. The cyclin dependent kinase 2 inhibitor, roscovitine, was used to maximize cell cycle synchrony and to produce cells that responded more reliably to nuclear reprogramming. Roscovitine-treated adult bovine granulosa cells (82.4%) were more efficiently synchronized (P < 0.05) in the quiescent G0/G1 phase of the cell cycle than were serum-starved cells (76.7%). Although blastocyst development following nuclear transfer was elevated (P < 0.05) in the serum-starved group (21.1%) relative to the roscovitine-treated cells (11.8%), the number of cells in the blastocysts derived from roscovitine-treated cells was higher (P < 0.05) than those derived from the serum-starved group (roscovitine-treated group: 142.8 +/- 6.0 cells; serum-starved group: 86.8 +/- 14.5 cells). The resulting fetal and calf survival after embryo transfer was enhanced in the roscovitine-treated group (seven surviving calves from six pregnancies) compared with serum-starved controls (two calves born, one surviving beyond 60 days, from five pregnancies). Roscovitine culture can predictably synchronize the donor cell cycle and increase the nuclear reprogramming capacity of the cells, resulting in enhanced fetal and calf survival and increased cloning efficiency.
通过核移植来培育牛的效率很低,原因如下:1)供体细胞不容易在细胞周期的适当阶段同步化;2)这些细胞的细胞核没有得到有效的重编程;3)晚期妊娠的流产率很高;4)出生后早期小牛的健康受到损害。细胞周期蛋白依赖性激酶2抑制剂——罗斯考维汀,被用于使细胞周期同步化达到最大化,并产生对核重编程反应更可靠的细胞。与血清饥饿处理的细胞(76.7%)相比,用罗斯考维汀处理的成年牛颗粒细胞在细胞周期的静止G0/G1期更有效地实现了同步化(P < 0.05)(82.4%)。尽管相对于用罗斯考维汀处理的细胞(11.8%),血清饥饿组(21.1%)核移植后的囊胚发育有所提高(P < 0.05),但来自用罗斯考维汀处理细胞的囊胚中的细胞数量比血清饥饿组更高(P < 0.05)(用罗斯考维汀处理的组:142.8 ± 6.0个细胞;血清饥饿组:86.8 ± 14.5个细胞)。与血清饥饿对照组(5次妊娠产下2头牛犊,1头存活超过60天)相比,用罗斯考维汀处理的组胚胎移植后产生的胎儿和小牛存活率有所提高(6次妊娠产下7头存活小牛)。罗斯考维汀培养能够可预测地使供体细胞周期同步化,并提高细胞的核重编程能力,从而提高胎儿和小牛的存活率以及克隆效率。