Expanding Transposase Technology to Regulatory Noncoding RNAs for Stable Expression in CHO Cells

Stable expression of regulatory non-coding RNAs enables targeted cell engineering in Chinese hamster ovary (CHO) cells but commonly relies on random integration, resulting in variable expression. Here, we establish a transposase-mediated platform for stable microRNA (miRNA) expression in CHO cells and demonstrate the first application of a DNA transposon system for long-term miRNA genome integration. Using miR-3096b-5p, we compared piggyBac-mediated integration with conventional random integration in antibody-producing CHO cells. Transposase-mediated integration enabled rapid generation of stable cell pools with higher fractions of GFP-expressing cells. Both integration strategies supported comparable growth, viability, and antibody production in batch cultures, indicating that transposase-mediated miRNA integration does not compromise bioprocess performance. At the molecular level, transposase-mediated integration resulted in significantly higher transgene copy numbers and markedly increased miRNA expression, translating into stronger and more consistent regulation of the target genes Fuk and Gmds. By combining high integration efficiency and expression stability with the regulatory potential of miRNAs, this approach expands the applicability of transposase systems beyond protein-coding transgenes and provides a robust platform for precise and durable post-transcriptional gene regulation in CHO cell engineering.