Handmade somatic cell cloning in cattle: Analysis of factors contributing to high efficiency in vitro
Handmade somatic cell cloning in cattle: Analysis of factors contributing to high efficiency in vitro
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DOI：
10.1095/biolreprod.102.008771
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发表时间：
2003-02-01
影响因子：
3.6
通讯作者：
Callesen, H
中科院分区：
文献类型：
--
作者：
Vajta, G;Lewis, IM;Callesen, H
体细胞克隆的广泛应用受到生物和技术问题的阻碍，这些问题包括需要熟练劳动力的复杂和耗时的程序。最近，无区技术已经发表，对微操纵器有限制或没有要求。本工作的目的是优化无微操作器（即手工）过程的某些步骤，分析发育囊胚的形态，并解释所观察到的高效率的因素。优化程序包括选择合适的去核培养基，融合时对的取向，融合时间和培养条件。由于这些改进的步骤，以每个重构胚胎的囊胚数和每个工作小时的囊胚数来衡量的体外效率是迄今为止所描述的最高的。本实验所用牛血清在体外胚胎发育方面优于其他蛋白质来源。对这种效应的一种可能解释是，与市售的胎牛血清相比，牛血清的有丝分裂活性相当高。通过倒置显微镜、内细胞质量-滋养层鉴别染色和透射电镜对囊胚进行形态学分析，显示囊胚的平均质量较高。将两个核移植胚胎聚集形成的单个囊胚移植到受者体内后，获得了较高的初始妊娠率。改进的手工体细胞核移植方法作为一种简单、廉价、高效的替代传统体细胞核移植的技术，有望成为一种有用的技术。
Widespread application of somatic cell cloning has been hampered by biological and technical problems, which include complicated and time-consuming procedures requiring skilled labor. Recently, zona-free techniques have been published with limited or no requirement for micromanipulators. The purpose of the present work was to optimize certain steps of the micromanipulator-free (i.e., handmade) procedure, to analyze the morphology of the developing blastocysts, and to explain factors involved in the high efficiencies observed. Optimization of the procedure included selection of the appropriate medium for enucleation, orientation of pairs at fusion, timing of fusion, and culture conditions. As a result of these improved steps, in vitro efficiency as measured by blastocysts per reconstructed embryo and blastocysts per working hour was among the highest described so far. The cattle serum used in our experiments was superior to other protein sources for in vitro embryo development. One possible explanation of this effect is the considerable mitogenic activity of the cattle serum compared with that of commercially available fetal calf serum. Morphological analysis of blastocysts by inverted microscopy, inner cell mass-trophoblast differential staining, and transmission electron microscopy revealed high average quality. A high initial pregnancy rate was achieved after the transfer of single blastocysts derived by aggregation of two nuclear transfer embryos into recipients. The improved handmade somatic cell nuclear transfer method may become a useful technology as a simple, inexpensive, and efficient alternative to traditional somatic cell nuclear transfer.