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.