摘要
就小鼠卵母细胞的卵龄对克隆胚的体外发育能力的影响进行了检测,以确定最佳的取卵时间,以及取卵后进行核移植操作的可耐受的时间。采用PMSG和hCG超排B6D2F1雌鼠。在体内老化实验中,分别于注射hCG后13,15,17,20h取卵用于核移植操作;在体外老化实验中,所有的卵均于注射hCG后13h取出并培养,然后在13,15,17,20h进行核移植操作。每个时间点的核移植操作在1h内完成,重构的胚胎培养1h后进行激活。结果显示:在体内老化实验中,注射hCG后13h取卵,获得的克隆囊胚发育率最高,为56.0%,15h取卵仍维持了其支持胚胎体外发育的能力,但17h及更长时间后取的卵,重构后的囊胚发育率显著下降(18.1%)。注射hCG后15h取的卵孤雌发育率最高(囊胚发育率为90.1%),并在17h仍维持较高的孤雌发育能力,在20h显著下降。在体外老化试验中,于注射hCG后13h取卵,分别于13,15,17h重构,都获得了较高的囊胚发育率(分别为57.7%,52.2%,46.3%),而在注射hCG后20h重构,囊胚发育率显著下降(14.8%)。体外老化的孤雌胚在注射hCG后22h激活时囊胚发育率显著下降。结果表明:卵母细胞在体内和体外老化都影响克隆胚的体外发育,最佳取卵时间为注射hCG后13h,取卵后立即用于重构可获得最佳囊胚发育率;体内、外老化卵支持重构胚较好发育的时间间隔是不同的,体内老化卵支持重构胚发育能力从注射hCG后17h开始下降,体外老化卵则发生在20h。这些结果可以指导操作,有利于在应用核移植进行其他基础研究时降低操作引起的误差。
The study analyzed the effects of postovulatory aging of mouse oocytes, both in vivo and in vitro, on the development of mouse somatic cell nuclear transfer embryos in vitro. Oocytes were collected from female B6D2F1 mice 13, 15, 17, 20 hours after hCG injection, respectively. Nuclear transfer micromanipulation was completed within one hour, and the reconstructed embryos were activated after one hour. At the same time, the parthenogenesis development was compared. For in vitro aging experiment, all oocytes were collected 13 hours after hCG injection,then nuclear transfer was performed 13, 15, 17, 20 hours after hCG injection. The results showed that the postovulatory aging of oocytes both in vivo and in vitro affected the development of the cloned embryos in vitro. The cloned blastocyst rate reached the highest when oocytes were collected 13 hours after hCG injection and being micromanipulated at once. The cloned blastocyst rate significantly decreased when oocytes were collected 17 hours after hCG injection and when oocytes were collected 13 hours after hCG injection and being micromanipulated 20 hours after hCG injection.These results provide us with some practicable nuclear transfer procedure rules for cloning to achieve better results and reduce the error by manipulation when nuclear transfer is used in the fundamental researches.
出处
《科技导报》
CAS
CSCD
2007年第3期28-33,共6页
Science & Technology Review
基金
国家自然科学基金项目(30300176
30400232)
国家自然科学基金国际合作重点项目
