Tossolini Ileana, Gugliotta Agustina, López Díaz Fernando, Kratje Ricardo, Prieto Claudio
UNL, CONICET, FBCB (School of Biochemistry and Biological Sciences), CBL (Biotechnological Center of Litoral), Ciudad Universitaria, Ruta Nacional 168 Km 472.4, C.C. 242. (S3000ZAA), Santa Fe, Argentina.
Regulatory Biology Laboratory, Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037, United States of America.
Plasmid. 2022 Jan-Mar;119-120:102620. doi: 10.1016/j.plasmid.2022.102620. Epub 2022 Feb 5.
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, new endogenous promoters were discovered 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.
对于在哺乳动物细胞中生产重组蛋白治疗药物而言，需要高基因表达率，因此通常使用强病毒衍生启动子。然而，它们通常会诱导细胞应激，并且可能易受表观遗传沉默的影响。内源性启动子能够在协调其活性与细胞及生物过程动态变化的同时，保持高表达水平，这可能有助于避免此类缺点。在这项工作中，基于高密度培养的CHO-K1细胞RNA测序数据中的高表达水平，发现了新的内源性启动子。选择Actb、Ctsz、Hmox1、Hspa5、Vim和Rps18基因的启动子来构建用于在哺乳动物细胞中生产重组蛋白的新表达载体。对计算机预测的启动子区域进行了实验验证，大多数启动子显示出与巨细胞病毒（CMV）相当或更高的转录活性。此外，还研究了降低培养温度后的稳定表达情况。所鉴定的内源性启动子（不包括Rps18）因其高强度、长期表达稳定性以及整合到宿主细胞调控网络中的特性，构成了CMV启动子的一个有前景的替代方案。这些启动子还可作为设计细胞工程策略和合成启动子以及进行工业细胞系开发的初始模板。
