基于细胞内应变评估的卵母细胞去核方法及在机器人化克隆中的应用

Intracellular Strain Evaluation-Based Oocyte Enucleation and Its Application in Robotic Cloning

自1996年第一只克隆羊诞生,克隆技术就由于其在动物育种方面的巨大潜力而得到了大量关注。体细胞核移植技术是动物克隆的核心,其操作过程非常复杂,不可避免地造成细胞内损伤。通常来说,只有不到1%的重构胚胎能够发育成克隆动物,克隆的低成功率是制约其广泛应用的主要原因之一。为此,本文提出了一种基于细胞内应变评估的卵母细胞去核方法,来减少体细胞核移植中的潜在细胞内损伤,从而提高克隆成功率。首先,论文根据细胞内速度场计算细胞内应变,进而将内应变作为评价标准来优化去核操作;在此基础上,开发了一套机器人批量体细胞核移植系统,用于实现机器人化克隆。实验结果表明,本文方法将体细胞核移植后的胚胎囊胚率从10.0%提高到20.8%,并成功获得17头克隆小猪,这是世界首批由机器人完成克隆操作获得的克隆动物。对比手动核移植操作,本文将克隆成功率从平均0.73%提升到了2.50%,大幅提升了克隆效率。与此同时,基于细胞内应变的去核方法有望应用于其他生物操作,并建立一种通用的减少细胞内损伤的细胞操作规范。

Since the first cloned sheep was produced in 1996,cloning has attracted considerable attention because of its great potential in animal breeding.Somatic cell nuclear transfer(SCNT)is widely used for creating clones.However,SCNT is very complicated to manipulate and inevitably causes intracellular damage during manipulation.Typically,only less than 1%of reconstructed embryos develop into live cloned animals.This low success rate is considered to be the major limitation in the extensive application of cloning techniques.In this study,we proposed an intracellular strain evaluation-based oocyte enucleation method to reduce potential intracellular damage in SCNT.We first calculated the intracellular strain based on the intracellular velocity field and then used the intracellular strain as a criterion to improve the enucleation operation.We then developed a robotic batch SCNT system to apply this micromanipulation method to animal cloning.Experimental results showed that we increased the blastocyst rate from 10.0%to 20.8%,and we successfully produced 17 cloned piglets by robotic SCNT for the first time.The success rate of cloning was significantly increased compared to that of traditional methods(2.50%vs 0.73%on average).In addition to the cloning technique,the intracellular strain evaluation-based enucleation method is expected to be applicable to other biological operations and for establishing a universal cell manipulation protocol to reduce intracellular damage.