For the production of recombinant protein therapeutics in mammalian cells, a high rate of gene expression is desired and hence strong viral-derived promoters are commonly used. However, they usually induce cellular stress and can be susceptible to epigenetic silencing. Endogenous promoters, which coordinates their activity with cellular and bioprocess dynamics while at the same time they maintain high expression levels, may help to avoid such drawbacks. In this work, endogenous promoters were identified based on high expression levels in RNA-seq data of CHO-K1 cells cultured in high density. The promoters of Actb, Ctsz, Hmox1, Hspa5, Vim and Rps18 genes were selected for generating new expression vectors for the production of recombinant proteins in mammalian cells. The in silico-derived promoter regions were experimentally verified and the majority showed transcriptional activity comparable or higher than CMV. Also, stable expression following a reduction of culture temperature was investigated. The characterized endogenous promoters (excluding Rps18) constitute a promising alternative to CMV promoter due to their high strength, long-term expression stability and integration into the regulatory network of the host cell. These promoters may also comprise an initial panel for designing cell engineering strategies and synthetic promoters, as well as for industrial cell line development.

Equine chorionic gonadotropin (eCG) is a heterodimeric glycoprotein hormone produced by pregnant mares that has been used to improve reproduction activity in different types of livestock. Several strategies to produce the hormone in a recombinant way have been reported; nevertheless, none approach has been able to produce a recombinant eCG (reCG) with significant in vivo bioactivity or in sufficient quantities for commercial purposes. For this reason, the only current product available on the market consists of partially purified preparations from serum of pregnant mares (PMSG). Herein, we describe a highly efficient process based on third-generation lentiviral vectors as delivery method for the production of reCG in suspension CHO-K1 cells, with productivities above 20 IU.106 cell-1.d-1 and 70% purification yields after one purification step. Importantly, reCG not only demonstrated biological activity in bovine cattle but also this bioactivity appeared to be higher than PMSG, since 140 IU of reCG were needed to exert the same biologic effect in an ovulation synchronization protocol compared to 400 IU of PMSG. The results obtained show that the developed strategy represents an attractive option to produce reCG and constitutes an auspicious alternative for the replacement of animals as a source of PMSG.