Wang Daqing, Liu Yiyi, Li Lu, Li Xin, Cheng Xin, Guo Zhihui, Cao Guifang, Zhang Yong
College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010011, China.
Animal Embryo and Developmental Engineering Key Laboratory of Higher Education, Institutions of Inner Mongolia Autonomous Region, Hohhot 010011, China.
Biology (Basel). 2025 Apr 28;14(5):484. doi: 10.3390/biology14050484.
The piggyBac+TET-on transposon induction system has a high efficiency in integrating exogenous genes in multiple cell types, can precisely integrate to reduce genomic damage, has a flexible gene expression regulation, and a strong genetic stability. When used in conjunction with somatic cell nuclear transfer experiments, it can precisely and effectively reveal the intrinsic mechanisms of early biological development. This study successfully reprogrammed black-boned sheep fibroblasts (SFs) into induced pluripotent stem cells (iPSCs) using the piggyBac+TET-on transposon system and investigated their impact on early embryonic development. Seven exogenous reprogramming factors (bovine OCT4, SOX2, KLF4, cMyc, porcine NANOG, Lin-28, and SV40 Large T) were delivered into SFs, successfully inducing iPSCs. A growth performance analysis revealed that iPSC clones exhibited a raised or flat morphology with clear edges, positive alkaline phosphatase staining, and normal karyotypes. The transcriptome analysis indicated a significant enrichment of iPSCs in oxidative phosphorylation and cell proliferation pathways, with an up-regulated expression of the ATP5B, SDHB, Bcl-2, CDK1, and Cyclin D1 genes and a down-regulated expression of BAX ( < 0.05). Somatic cell nuclear transfer experiments demonstrated that the cleavage rate (85% ± 2.12) and blastocyst rate (52% ± 2.11) of the iPSCs were significantly higher than those of the SFs ( < 0.05). The detection of trilineage marker genes confirmed that the expression levels of endoderm (DCN, NANOS3, FOXA2, FOXD3, SOX17), mesoderm (KDR, CD34, NFH), and ectoderm (NEUROD) markers in iPSCs were significantly higher than in SFs ( < 0.01). The findings demonstrate that black-boned sheep iPSCs possess pluripotency and the potential to differentiate into all three germ layers, revealing the mechanisms by which reprogrammed iPSCs influence early embryonic development and providing a critical foundation for research on sheep pluripotent stem cells.
piggyBac+TET-on转座子诱导系统在多种细胞类型中整合外源基因的效率很高，能精确整合以减少基因组损伤，具有灵活的基因表达调控能力和很强的遗传稳定性。当与体细胞核移植实验结合使用时，它能精确有效地揭示早期生物发育的内在机制。本研究利用piggyBac+TET-on转座子系统成功地将乌骨羊成纤维细胞（SFs）重编程为诱导多能干细胞（iPSCs），并研究了它们对早期胚胎发育的影响。将七个外源重编程因子（牛OCT4、SOX2、KLF4、cMyc、猪NANOG、Lin-28和SV40大T抗原）导入SFs，成功诱导出iPSCs。生长性能分析表明，iPSC克隆呈现凸起或扁平形态，边缘清晰，碱性磷酸酶染色呈阳性，核型正常。转录组分析表明，iPSCs在氧化磷酸化和细胞增殖途径中显著富集，ATP5B、SDHB、Bcl-2、CDK1和细胞周期蛋白D1基因表达上调，BAX表达下调（<0.05）。体细胞核移植实验表明，iPSCs的卵裂率（85%±2.12）和囊胚率（52%±2.11）显著高于SFs（<0.05）。三系标记基因检测证实，iPSCs中内胚层（DCN、NANOS3、FOXA2、FOXD3、SOX17）、中胚层（KDR、CD34、NFH）和外胚层（NEUROD）标记基因的表达水平显著高于SFs（<0.01）。这些发现表明，乌骨羊iPSCs具有多能性和分化为所有三个胚层的潜力，揭示了重编程的iPSCs影响早期胚胎发育的机制，为绵羊多能干细胞的研究提供了关键基础。
