Laboratory of Theriogenology, College of Veterinary Medicine, Chungnam National University, Daejeon, Republic of Korea.
Laboratory of Theriogenology, College of Veterinary Medicine, Chungnam National University, Daejeon, Republic of Korea; Research Institute of Veterinary Medicine, Chungnam National University, Daejeon, Republic of Korea.
Reprod Biol. 2022 Jun;22(2):100632. doi: 10.1016/j.repbio.2022.100632. Epub 2022 Mar 22.
Porcine cloning through somatic cell nuclear transfer (SCNT) has been widely used in biotechnology for generating animal disease models and genetically modified animals for xenotransplantation. Vitamin C is a multifunctional factor that reacts with several enzymes. In this study, we used porcine oocytes to investigate the effects of different concentrations of vitamin C on in vitro maturation (IVM), in vitro culture (IVC), and the derivation of nuclear transfer embryonic stem-like cells (NT-ESCs). We demonstrated that vitamin C promoted the cleavage and blastocyst rate of genetically modified cloned porcine embryos and improved the derivation of NT-ESCs. Vitamin C integrated into IVM and IVC enhanced cleavage and blastocyst formation (P < 0.05) in SCNT embryos. Glutathione level was increased, and reactive oxygen species levels were decreased (P < 0.05) due to vitamin C treatment. Vitamin C decreased the gene expression of apoptosis (BAX) and increased the expression of genes associated with nuclear reprogramming (NANOG, POU5F1, SOX2, c-Myc, Klf4, and TEAD4), antioxidation (SOD1), anti-apoptotic (Bcl2), and trophectoderm (CDX2). Moreover, vitamin C improved the attachment, derivation, and passaging of NT-ESCs, while the control group showed no outgrowths beyond the primary culture. In conclusion, supplementation of vitamin C at a dose of 50 µg/ml to the IVM and IVC culture media was appropriate to improve the outcomes of porcine IVM and IVC and for the derivation of NT-ESCs as a model to study the pre- and post-implantation embryonic development in cloned transgenic embryos. Therefore, we recommend the inclusion of vitamin C as a supplementary factor to IVM and IVC to improve porcine in vitro embryonic development.
猪体细胞核移植（SCNT）克隆已广泛应用于生物技术，用于生成动物疾病模型和用于异种移植的基因修饰动物。维生素 C 是一种多功能因子，可与几种酶发生反应。在这项研究中，我们使用猪卵母细胞来研究不同浓度的维生素 C 对体外成熟（IVM）、体外培养（IVC）和核移植胚胎干细胞样细胞（NT-ESCs）的衍生的影响。我们证明，维生素 C 促进了基因修饰克隆猪胚胎的卵裂和囊胚率，并提高了 NT-ESCs 的衍生效率。维生素 C 整合到 IVM 和 IVC 中增强了 SCNT 胚胎的卵裂和囊胚形成（P < 0.05）。由于维生素 C 的处理，谷胱甘肽水平增加，活性氧水平降低（P < 0.05）。维生素 C 降低了凋亡基因（BAX）的表达，增加了与核重编程相关的基因（NANOG、POU5F1、SOX2、c-Myc、Klf4 和 TEAD4）、抗氧化（SOD1）、抗凋亡（Bcl2）和滋养层（CDX2）的表达。此外，维生素 C 改善了 NT-ESCs 的附着、衍生和传代，而对照组在原代培养之外没有任何生长。总之，在 IVM 和 IVC 培养基中添加 50μg/ml 的维生素 C 剂量适合改善猪 IVM 和 IVC 的结果，并用于衍生 NT-ESCs，作为研究克隆转基因胚胎植入前和植入后胚胎发育的模型。因此，我们建议将维生素 C 作为补充因子添加到 IVM 和 IVC 中，以改善猪体外胚胎发育。
