Systematic screening of stable expression loci in Chinese Hamster Ovary cells via orthogonal functional annotation analysis.

The development of engineered Chinese Hamster Ovary (CHO) cell lines capable of long-term, stable, and efficient recombinant protein expression is essential for industrial biologics manufacturing. Traditional random integration is hindered by epigenetic silencing and position effects, while current site-specific strategies are restricted by a limited stable library. To address this, we established a screening strategy based on two core criteria for ideal loci: 1) the capacity for sustained exogenous protein expression and 2) minimal impact on host cell physiology. By analyzing chromosomal location and expression data from 52 known stable loci, we identified 3,152 highly expressed genes. Subsequent Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses excluded genes essential for core growth and metabolism, yielding 252 candidate sites. These were further refined to 25 ideal candidate loci by evaluating their betweenness centrality in a protein-protein interaction (PPI) network, prioritizing hubs with potential genomic stability. After 40 generations of continuous culture, the Enhanced Green Fluorescent Protein (EGFP)-positive rates of DNA ligase 4-like protein (LIG4), L-threonine 3-dehydrogenase, mitochondrial (LOC100759874), and alpha-aminoadipic semialdehyde synthase, mitochondrial (AASS), remained above 95%. This study established an orthogonal functional enrichment screening strategy, which significantly improved the discovery efficiency of stable expression sites in CHO cells and laid a foundation for constructing highly stable recombinant cell lines suitable for industrial production.