Advanced stable cell line development based on REMBAC (Rapid Efficient Manifold BACulovirus Transduction).

The BacMam platform is a scalable and efficient gene delivery system for mammalian cells, enabling the production of recombinant proteins or highly complex structures like bionanoparticles. This study represents the first comprehensive investigation to evaluate the performance of the BacMam platform in a common producer cell line HEK293-6E, on stable transgene expression mediated by the REMBAC (rapid efficient manifold baculovirus transduction) cassette. In this study, six transient high expression cassettes were adopted for stable gene expression and evaluated in HEK293-6E and Vero cells. The constructs included either the wild-type viral CMV promoter, a methylation-resistant mutant of the CMV promoter, or the mammalian EF-1α promoter. To further enhance expression, endogenous mammalian introns or viral long terminal repeats (LTRs) were included, along with β-globin insulators placed at both the 5' and 3' ends of the cassette. Additionally, the constructs were equipped with two antibiotic resistance genes, Hygromycin and G418, and a full-length Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), known to improve mRNA stability and enhance protein expression by modulating RNA structure [1].The results indicated that transduction of HEK293-6E cells achieved higher efficiency, resulted in more clones with a stably integrated expression cassette, and stronger protein expression than transduction of Vero cells. The inclusion of β-globin insulators significantly enhanced gene expression in HEK293-6E cells, while their effect in Vero cells was less pronounced. These findings highlight the importance of cell line selection and genetic design in optimizing recombinant protein production.