Lü Wenli, Yang Hua, Xu Hui, Zhang Yanli
Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing 210095, Jiangsu, China.
College of Animal Science & Technology, Nanjing Agricultural University, Nanjing 210095, Jiangsu, China.
Sheng Wu Gong Cheng Xue Bao. 2025 Jul 25;41(7):2707-2718. doi: 10.13345/j.cjb.250136.
The CRISPR/dCas9 system is a gene editing tool that has proven to be highly efficient and precise. By utilizing transcriptional activators, such as VP64, p65, and Rta, the system can effectively and stably activate target genes. Sox17, a transcription factor belonging to the SOX family, plays a crucial role in the differentiation of the germ layers and the determination of cell fates during the early stages of embryonic development. Sheep embryonic stem cells (sESCs) are characterized by their capacity for self-renewal and multidirectional differentiation, serving as a significant model for studying the mechanisms of cell differentiation during early embryonic development. However, the importing of exogenous genes into sESCs is challenging due to their unique growth characteristics. The objective of this study was to investigate the conditions necessary for successfully activating in sESCs. To this end, we employed the CRISPR/dCas9 system along with liposome transfection, lentivirus invasion, and electroporation to activate in sESCs. The expression of was then determined by fluorescence quantitative PCR, on the basis of which the performance of different transfection methods was compared. The results indicated that the electroporation group had the best transfection effect and the highest expression among the three transfection methods. The efficient and stable gene activation protocol will provide a reference for embryonic stem cell research in other species, especially livestock animals, and lay the foundation for the subsequent study of gene function and realization of precise cell fate regulation by regulating gene expression in sheep embryonic stem cells.
CRISPR/dCas9系统是一种已被证明高效且精确的基因编辑工具。通过利用转录激活因子，如VP64、p65和Rta，该系统能够有效且稳定地激活靶基因。Sox17是一种属于SOX家族的转录因子，在胚胎发育早期的胚层分化和细胞命运决定中起着关键作用。绵羊胚胎干细胞（sESCs）具有自我更新和多向分化的能力，是研究早期胚胎发育过程中细胞分化机制的重要模型。然而，由于其独特的生长特性，将外源基因导入sESCs具有挑战性。本研究的目的是探究在sESCs中成功激活[具体基因未提及]所需的条件。为此，我们采用CRISPR/dCas9系统结合脂质体转染、慢病毒侵染和电穿孔来激活sESCs中的[具体基因未提及]。然后通过荧光定量PCR测定[具体基因未提及]的表达，在此基础上比较不同转染方法的性能。结果表明，在三种转染方法中，电穿孔组具有最佳的转染效果和最高的[具体基因未提及]表达。高效稳定的基因激活方案将为其他物种，尤其是家畜动物的胚胎干细胞研究提供参考，并为后续通过调控绵羊胚胎干细胞中的基因表达来研究基因功能和实现精确的细胞命运调控奠定基础。
