The discovery of how to utilize CRISPR (clustered, regularly interspaced, short, palindromic repeats)-Cas (CRISPR-associated) systems for genome modification has accelerated development of the field of genome editing, especially in large animals such as pigs. The low efficiency of somatic cell nuclear transfer (SCNT) is now becoming a major obstacle in the production of genome-edited animals via cell-mediated approaches and improving efficacy of this technique is crucial. In this study, we propose a few simple modifications to a zona-free SCNT protocol that are effective to produce numerous high-quality blastocysts. To refine the SCNT protocol we modified the following steps/factors: 1) culture medium for SCNT embryos, 2) chemical treatment to prevent precocious activation of the manipulated/reconstructed oocytes and 3) donor cell serum starvation treatment. Although changes in each of these steps only resulted in small improvements, the combination of all modifications altogether significantly enhanced developmental competence of SCNT embryos. Our modified method yielded approximately three times greater blastocyst formation rates. Moreover, resulting blastocysts had roughly twice as many cells as compared to blastocysts produced by the conventional SCNT method. With these significant in vitro improvements, our refined SCNT method is potentially suited for use in the production of genome edited pigs.

中文翻译：

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体细胞核移植方法的组合改进可改善猪胚胎发育

如何利用 CRISPR（成簇、规则间隔、短、回文重复）-Cas（CRISPR 相关）系统进行基因组修饰的发现加速了基因组编辑领域的发展，尤其是在猪等大型动物中。体细胞核转移 (SCNT) 的低效率现在正成为通过细胞介导的方法生产基因组编辑动物的主要障碍，提高该技术的功效至关重要。在这项研究中，我们建议对无带 SCNT 协议进行一些简单的修改，这些协议可有效产生大量高质量的囊胚。为了改进 SCNT 协议，我们修改了以下步骤/因素：1) SCNT 胚胎的培养基，2) 化学处理，以防止操纵/重建卵母细胞的过早激活和 3) 供体细胞血清饥饿处理。尽管这些步骤中的每一步的变化都只会带来很小的改进，但所有修改的组合显着增强了 SCNT 胚胎的发育能力。我们改进的方法产生了大约三倍的囊胚形成率。此外，与通过传统 SCNT 方法产生的囊胚相比，所得囊胚的细胞数量大约是其两倍。凭借这些显着的体外改进，我们改进的 SCNT 方法可能适用于基因组编辑猪的生产。所有修饰的组合显着增强了 SCNT 胚胎的发育能力。我们改进的方法产生了大约三倍的囊胚形成率。此外，与通过传统 SCNT 方法产生的囊胚相比，所得囊胚的细胞数量大约是其两倍。凭借这些显着的体外改进，我们改进的 SCNT 方法可能适用于基因组编辑猪的生产。所有修饰的组合显着增强了 SCNT 胚胎的发育能力。我们改进的方法产生了大约三倍的囊胚形成率。此外，与通过传统 SCNT 方法产生的囊胚相比，所得囊胚的细胞数量大约是其两倍。凭借这些显着的体外改进，我们改进的 SCNT 方法可能适用于基因组编辑猪的生产。