Supporting information

Enhancing recombinant protein content, like lactoferrin (LTF), in livestock milk through transgenic methods presents significant market opportunities. This study aimed to identify integration sites and regulatory elements for stable lactoferrin expression in goat mammary epithelial cells (GMECs). Utilizing ATAC-seq and RNA-seq on mammary epithelial cells derived from lactation and dry period mammary, we mapped genome-wide chromatin accessibility in the mammary gland. Through a comprehensive review of the literature, analysis of chromatin structural variations, and screening of gene safe harbors, we identified 15 candidate integration sites with high accessibility. Comparison of reporter gene expression showed that the FAM13A locus outperformed others, yielding luciferase expression and fluorescence intensity 3.5 times higher than ROSA26 while ensuring long-term transgene stability. Furthermore, We identified a bidirectional promoter element from the SF3B1-COQ10B housekeeping gene, a new ubiquitous chromatin-opening element (UCOE) derived from goat genes. This UCOE enhances transgene integration efficiency by 5.9 times and improves transgene expression and stability compared to low GC content regulatory sequences. Using somatic cell nuclear transfer and embryo transfer with the UCOE and human lactoferrin (hLTF) expression cassette integrated into the ROSA26 locus, we successfully produced transgenic lambs, while targeting the FAM13A locus led to developmental issues. Our findings indicate that high chromatin accessibility at the integration site significantly enhances transgene expression, suggesting that UCOE elements with elevated GC content can be effectively utilized to improve transgene expression and stability. This study represented the pioneering identification and characterization of the UCOE element in dairy goats and elucidated the significant impact of the GC content of exogenous genes on the integration of large DNA fragments and their subsequent expression. The findings established a foundational framework for breeding dairy goats with enhanced lactoferrin production and lay the groundwork for the development of high-expression mammary gland bioreactors.

通过转基因技术提升家畜乳汁中重组蛋白（如乳铁蛋白(lactoferrin, LTF)）的含量，具备可观的市场前景。本研究旨在筛选可在山羊乳腺上皮细胞(goat mammary epithelial cells, GMECs)中稳定表达乳铁蛋白的整合位点与调控元件。本研究针对泌乳期与干奶期的乳腺上皮细胞开展转座酶可及性测序(ATAC-seq)与转录组测序(RNA-seq)，绘制了全基因组范围内的乳腺染色质可及性图谱。通过系统性文献调研、染色质结构变异分析以及基因安全港筛选，我们共筛选得到15个高可及性的候选整合位点。报告基因表达对比实验显示，FAM13A位点的表现优于其余位点：其荧光素酶表达量与荧光强度较ROSA26位点提升3.5倍，同时可保证转基因的长期稳定性。此外，本研究从SF3B1-COQ10B持家基因中筛选得到一段双向启动子元件，这是一种源自山羊基因组的新型通用染色质开放元件(ubiquitous chromatin-opening element, UCOE)。相较于低GC含量的调控序列，该UCOE可将转基因整合效率提升5.9倍，并优化转基因的表达水平与稳定性。将整合有该UCOE与人乳铁蛋白(human lactoferrin, hLTF)表达盒的载体靶向整合至ROSA26位点后，通过体细胞核移植与胚胎移植技术，我们成功获得了转基因羔羊；而靶向整合至FAM13A位点则出现了发育异常问题。本研究结果表明，整合位点的高染色质可及性可显著提升转基因表达水平，同时提示高GC含量的UCOE元件可有效用于优化转基因的表达与稳定性。本研究首次在奶山羊中鉴定并表征了UCOE元件，阐明了外源基因GC含量对大片段DNA整合及其后续表达的显著影响。本研究成果为培育高乳铁蛋白表达奶山羊提供了理论基础，同时为开发高表达乳腺生物反应器奠定了技术框架。