An effective method to enhance transient gene expression by transfecting high-density CHO cells.

Chinese hamster ovary (CHO) cells are the predominant host for recombinant protein production. While transient gene expression (TGE) systems provide a faster, more cost-effective alternative to stable gene expression (SGE), their application is often hampered by low protein yields. Here, we optimized TGE systems based on high-density suspension transfection using 40 kDa polyethylenimine (PEI) by engineering vector regulatory elements (Kozak, polyA, introns, leader) and optimizing transfection conditions. Vector optimization led to a substantial enhancement in recombinant protein, with EGFP and SEAP levels increased by 5.33-fold and 4.13-fold, respectively (p < 0.05). By applying the optimized vector in combination with identified optimal conditions, a DNA concentration of 4 µg/mL, a DNA: PEI mass ratio of 1:5, and a repeated transfection strategy, we achieved a transfection efficiency of 76.34% while maintaining cell viability above 85%. Moreover, the combined optimization of vectors and transfection conditions robustly enhance the production of therapeutically relevant proteins, increased the expression of recombinant type III human collagen and human serum albumin by 4.86-fold and 5.50-fold, respectively (p < 0.05). In summary, high-density suspension transfection markedly boosts transient recombinant protein expression in CHO cells by improving transfection efficiency, expanding the population of recombinant cells, and maintaining high cell viability. This approach presents a promising and effective strategy for high yield transient transgene expression.