传统育种方法由于代际间隔较长、选择精度和强度有限，难以满足日益增长的肉类和奶制品需求。尽管基因组选择（Genomic Selection，GS）显著缩短了代际间隔，但对于某些哺乳动物（如牛和羊），其代际间隔仍以年为单位，受到妊娠时间和性成熟周期等生物学限制。因此，提出了一种创新的育种策略——体外育种（In Vitro Breeding，IVB），旨在结合基因组选择和前沿生殖技术，加速家畜群体的遗传改良，随着基因编辑技术的不断进步，IVB逐渐更具优势。体外生产生殖细胞（体外配子重建）是IVB的关键流程，即从胚胎干细胞（Embryonic Stem Cells，ESCs）体外生成功能性生殖细胞。生殖细胞作为遗传信息的载体，在物种繁衍中发挥着关键作用，而原始生殖细胞（Primordial Germ Cells，PGCs）是所有生殖细胞的来源。ESCs是体外配子重建的起点细胞，其质量对于后续过程至关重要。然而，目前尚未建立稳定高效的绵羊胚胎干细胞体系，且其诱导分化效率较低，因此, 本研究旨在基于本实验室前期分离的绵羊ESCs，优化建立高效的绵羊ESCs培养体系，并探究其向PGCs分化的诱导条件。通过小分子化合物诱导或基因诱导，将绵羊ESCs体外诱导为原始生殖细胞样细胞，为实现绵羊配子的体外重建奠定基础。本研究不仅为IVB的应用提供了技术支持，也为加快绵羊品种改良进程提供了新的体外模型和解决方案。

本研究包括以下三部分：

试验一 绵羊ESCs稳定培养体系优化的研究

本试验旨在优化绵羊胚胎干细胞（sESCs）的培养体系，探究培养基中不同小分子组合和不同无饲养层培养方式对sESCs多能性及生长特性的影响。基于实验室前期分离的sESCs，本研究对比了去除LIF、Activin A或IWR-1，以及LCDM、FTW和CTFR体系对sESCs的多能性和生长形态的支持效果，分析了Rho激酶抑制剂Y27632对sESCs增殖和凋亡的影响，明确了Y27632在促进sESCs高效增殖的关键要作用，确定了适合sESCs的稳定培养基体系。通过在饲养层细胞（MEF）、基质胶、纤连蛋白、粘连蛋白、玻连蛋白和Geltrex基质上培养的sESCs的多能性和生长特性，发现在无饲养层培养基质中LN521对sESCs的支持效果更稳定，相比其他基质培养的sESCs具有更高的单克隆效率和更短的细胞倍增时间，并且已在体外支持sESCs传至30代仍然稳定。综上，本研究建立了稳定的sESCs无饲养层培养体系，为后续sESCs的诱导分化及应用研究提供了重要基础。

试验二 小分子化合物诱导绵羊ESCs分化为原始生殖细胞的研究

本试验旨在建立小分子化合物诱导sESCs分化为原始生殖细胞的诱导体系。在GK15培养基添加了LIF、EGF和SCF因子，以及BMP4或者BMP通路激活剂SB4，在2D或3D的培养环境中诱导sESCs向原始生殖细胞样细胞（sPGCLCs）分化。通过细胞免疫荧光、实时荧光定量PCR、蛋白免疫印迹等试验技术对诱导不同天数的sPGCLCs进行分子特征鉴定，利用SMART-seq分析BMP4和SB4诱导sPGCLCs的差异。结果表明，BMP4和SB4诱导的sPGCLCs均能表达PGC标记基因SOX17、NANOS3和PRDM1，且与甲基化相关的H3K4me3、H3K9me3、DNMT3A和DNMT3B蛋白表达下降，H3K27me3表达升高，符合PGC的甲基化重编程特征。转录组分析显示，BMP4和SB4诱导的sPGCLCs均进行了生殖细胞发育的生物过程，但BMP4组的sPGCLCs更倾向早期PGC，而SB4组的sPGCLCs则更倾向晚期PGC的特征。综上，本研究成功利用小分子化合物诱导sESCs分化为sPGCLCs，验证了诱导体系的有效性，并证实sESCs具有直接向PGCs分化的潜力，为后续通过基因激活方式诱导sESCs分化为PGCs提供了理论依据。

试验三 SOX17基因诱导绵羊ESCs分化为原始生殖细胞的研究

本试验旨在通过激活SOX17基因，或联合小分子化合物诱导的方式，于体外诱导sESCs分化为原始生殖细胞。首先构建了基于CRISPR/dCas9系统的SOX17基因激活工具，并采用脂质体转染、慢病毒转导及电穿孔等方法将其导入sESCs，检测SOX17基因表达水平得到最佳转染条件，比较单独激活SOX17进行诱导，单独使用小分子化合物诱导以及两种方法结合的方式进行诱导的细胞的形态变化和生殖细胞标记基因的表达水平，结果表明电转为最高效转染方法，三种诱导方法均能使PGCs标记基因表达上调，其中小分子诱导方法对细胞造成的影响最显著，本试验验证了SOX17在调控生殖细胞发育过程中的作用，为理解SOX17在绵羊生殖细胞命运调控中的分子机制提供了重要依据。同时探究了适用于sESCs的高效转染方法，为进一步在sESCs中通过基因编辑的方式挖掘更多基因的调控作用奠定基础。

Conventional breeding techniques have proven ineffective in meeting the escalating demand for meat and dairy products, primarily due to the extended generation intervals and the constrained precision and intensity of selection. Genomic Selection (GS) has been shown to reduce the intergenerational interval; however, for certain mammals (e.g., cattle and sheep), the intergenerational interval is still measured in years, which is biologically limited by gestation time and sexual maturity cycle. Consequently, an innovative breeding strategy, In Vitro Breeding (IVB), has been proposed to expedite the genetic enhancement of livestock populations by integrating genomic selection and state-of-the-art reproductive technologies. Notably, IVB has emerged as a progressively advantageous approach, given the ongoing advancements in gene editing technologies. The pivotal process within the IVB framework is in vitro gamete reconstruction, which entails the generation of functional germ cells from Embryonic Stem Cells (ESCs) within a laboratory setting.Germ cells are the source of all germ cells, and primordial germ cells (PGCs) play a key role in species reproduction as carriers of genetic information.ESCs are the source cells for in vitro germ cell reconstruction, and their quality is critical for the subsequent process. However, a stable and efficient sheep embryonic stem cell system has yet to be established, and its induced differentiation efficiency remains low. The objective of this study was twofold: first, to optimize the establishment of an efficient sheep ESC culture system based on sheep ESCs isolated in our laboratory, and second, to investigate the induction conditions for their differentiation into PGCs. The induction of primordial germ cell-like cells in vitro can be achieved through the use of small molecule compounds or gene induction techniques. This study aims to establish an effective in vitro system for the reconstruction of sheep gametes, which would be a significant advancement in the field. The findings of this study offer two significant contributions. They provide technical support for the application of IVB, and establish a novel in vitro model that can expedite the process of sheep breed improvement.

This study includes the following three parts:

Experiment 1: Study on the optimization of stable culture system for sheep ESCs

The objective of this experiment was to optimize the culture system of sheep embryonic stem cells (sESCs) and to investigate the effects of different culture conditions on their pluripotency and growth characteristics. Utilizing the pre-laboratory isolation of sESCs as a foundation, this study systematically compared the effects of removing LIF, Activin A, or IWR-1, as well as LCDM, FTW, and CTFR systems, on the support of pluripotency and growth morphology of sESCs. The study also involved the screening of optimal culture medium systems. The study further investigated the impact of the Rho-kinase inhibitor Y27632 on the proliferation and autophagy of sESCs. The results demonstrated that Y27632 plays a critical role in promoting the efficient proliferation of sESCs. Furthermore, the study characterized the pluripotency and growth characteristics of sESCs cultured on feeder layer cells (MEF), matrix adhesive, fibronectin, adherensin, hyaluronan, and Geltrex matrices. The results indicated that LN521 supported sESCs more effectively in feeder layer-free medium. Furthermore, sESCs cultivated in the feeder layer medium exhibited enhanced monoclonal efficiency and diminished cell multiplication compared to those cultured in alternative matrices. The study also demonstrated that LN521 maintained sESCs in vitro, ensuring their stability up to 30 generations. In summary, the present study has established a stable and efficient sESCs culture system, which provides a significant foundation for subsequent studies on the induction and differentiation of sESCs and their applications.

Experiment 2: Study on small molecule compounds inducing differentiation of sheep ESCs into primordial germ cells

The objective of this experiment was to establish an induction system for small molecule compounds to induce the differentiation of sESCs into primordial germ cells. The sESCs were induced to differentiate into primordial germ cell-like cells (sPGCLCs) in 2D or 3D culture environments in GK15 medium supplemented with LIF, EGF, and SCF factors, as well as BMP4 or the BMP pathway activator SB4. The sPGCLCs induced for different days were molecularly characterized by a variety of techniques, including cellular immunofluorescence, real-time fluorescence quantitative PCR, protein immunoblotting, and others. The differences in sPGCLCs induced by BMP4 and SB4 were analyzed by SMART-seq. The results demonstrated that both BMP4- and SB4-induced sPGCLCs expressed the PGC marker genes SOX17, NANOS3, and PRDM1. Furthermore, the expression of methylation-related H3K4me3, H3K9me3, DNMT3A, and DNMT3B proteins was decreased, and the expression of H3K27me3 was increased. These findings were consistent with the reprogramming characteristics of methylation in PGC. A subsequent analysis of the obtained data revealed that both BMP4- and SB4-induced sPGCLCs underwent the biological process of germ cell development. However, sPGCLCs in the BMP4 group exhibited a greater propensity to become early PGCs, while those in the SB4 group demonstrated a stronger tendency to manifest characteristics of late PGCs. In summary, this study successfully induced the differentiation of sESCs into sPGCLCs using small molecule compounds, thereby validating the induction system and confirming that sESCs possess the capacity to differentiate directly into PGCs. This provides a theoretical foundation for the subsequent induction of the differentiation of sESCs into PGCs by means of gene activation.

Experiment 3: Study on SOX17 gene induces differentiation of sheep ESCs into primordial germ cells

The objective of this experiment was to induce sESCs to differentiate into primary germ cells in vitro by activating the SOX17 gene, or in combination with small molecule compound induction. Initially, a SOX17 gene activation tool based on the CRISPR/dCas9 system was engineered and introduced into sESCs via liposome transfection, lentiviral transduction, and electroporation. The expression level of the SOX17 gene was then detected by real-time fluorescence quantitative PCR (qRT-PCR), and the optimal transfection conditions were identified to achieve efficient activation of SOX17. Subsequently, the morphological changes of cells and the expression levels of germ cell marker genes under activation of SOX17, induction by small molecule compounds, and combined induction of both were used to validate the role of SOX17 in regulating the development of germ cells. Concurrently, an efficient induction protocol was obtained, providing an important basis for understanding the molecular mechanism of SOX17 in the determination of the fate of the germ cells in sheep. This experiment explored an efficient transfection method applicable to sESCs and applied it to the activation of SOX17 gene and the induction of sESCs differentiation to primordial germ cells. This provides a reference for the efficient induction of germ cells in livestock animals in vitro and lays a foundation for further utilizing sESCs, an in vitro model, to tap the regulatory roles of more genes by means of gene editing.

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