Shi Zhenhu, Yan Yelian, Zhu Ruiqing, Zhu Xinyue, Hu Kunlong, Yue Yingying, Xu Wenhuan, Xuan Mengqing, Gan Xinqi, Yang Zhiyuan, Zhang Yunhai, Cao Zubing
Anhui Province Key Laboratory of Local Livestock and Poultry, Genetic Resource Conservation and Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei, 230036, China.
Anhui Province Key Laboratory of Local Livestock and Poultry, Genetic Resource Conservation and Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei, 230036, China.
Theriogenology. 2025 Nov;247:117563. doi: 10.1016/j.theriogenology.2025.117563. Epub 2025 Jul 5.
Abnormal zygotic genome activation (ZGA) during the early development of somatic cell nuclear transfer (SCNT) embryos is one of the main reasons for the low cloning efficiency. The double homeobox (DUX) family, which includes important transcription factors in mammals, has been shown to play an important role in the ZGA process in mice. However, the role of DUXA, a member of the DUX family, in the early development of porcine somatic cloned embryos is unknown. Here, CRISPR/Cas9 gene editing and lentiviral infection technologies were used to construct stable DUXA knockout and overexpression cell lines for the production of SCNT embryos. Compared with that of wild-type (WT) SCNT embryos, the blastocyst rate of DUXA knockout embryos was significantly lower (P < 0.05), whereas the blastocyst rate of DUXA-overexpressing embryos was significantly greater (P < 0.05). Moreover, RT‒qPCR results revealed that DUXA knockout significantly reduced the expression levels of ZGA-related genes (TDG, SNAI1, RSRP1, TFAP2C, ZSCAN4, LEUTX, and KLF17) (P < 0.05). Additionally, in DUXA-overexpressing embryos, the mRNA levels of TDG, SNAI1, RSRP1, and TFAP2C significantly decreased (P < 0.05), whereas the ZSCAN4, LEUTX, and KLF17 mRNA levels increased (P < 0.05). These findings suggest that DUXA regulates the early development of porcine SCNT embryos by modulating the expression of ZGA-related genes. This research provides significant insights into the potential mechanisms of early embryo loss in porcine SCNT.
体细胞核移植（SCNT）胚胎早期发育过程中异常的合子基因组激活（ZGA）是克隆效率低下的主要原因之一。双同源盒（DUX）家族包含哺乳动物中的重要转录因子，已被证明在小鼠的ZGA过程中发挥重要作用。然而，DUX家族成员DUXA在猪体细胞核移植胚胎早期发育中的作用尚不清楚。在此，利用CRISPR/Cas9基因编辑和慢病毒感染技术构建了稳定的DUXA敲除和过表达细胞系，用于生产SCNT胚胎。与野生型（WT）SCNT胚胎相比，DUXA敲除胚胎的囊胚率显著降低（P<0.05），而DUXA过表达胚胎的囊胚率显著提高（P<0.05）。此外，RT-qPCR结果显示，DUXA敲除显著降低了ZGA相关基因（TDG、SNAI1、RSRP1、TFAP2C、ZSCAN4、LEUTX和KLF17）的表达水平（P<0.05）。此外，在DUXA过表达胚胎中，TDG、SNAI1、RSRP1和TFAP2C的mRNA水平显著降低（P<0.05），而ZSCAN4、LEUTX和KLF17的mRNA水平升高（P<0.05）。这些发现表明，DUXA通过调节ZGA相关基因的表达来调控猪SCNT胚胎的早期发育。本研究为猪SCNT胚胎早期丢失的潜在机制提供了重要见解。
