Journal Article
Enhanced design of pCMViR-TSC plasmid vector for sustainably high cargo gene expression in mammalian cells
Masakiyo Sakaguchi; Rie Kinoshita; Nahoko Tomonobu; Yoshihiko Sakaguchi; Junichiro Futami; Akira Yamauchi; Hitoshi Murata; Ken-ichi Yamamoto; Tetta Takahashi; Yuma Gohara; Toshiki Ochi; Fan Jiang; Ni Luh Gede Yoni Komalasari; Youyi Chen; I Made Winarsa Ruma; I Wayan Sumardika; Jin Zhou; Tomoko Honjo; Futoshi Kuribayashi; Kazumi Sagayama; Shinichi Toyooka; Eisaku Kondo; Yusuke Inoue
In Vitro Cellular & Developmental Biology - Animal · Vol. 60, Issue 10 · pp. 1215-1227 · 2024
Abstract
The first-generation pCMViR-TSC, implemented through the promoter sandwich rule, yields 10- to 100-fold higher gene expression than the standard plasmid used with the CMV (cytomegalovirus) or CAG promoter. However, the vector’s shortcomings limit its utility to transient expression only, as it is not suitable for establishing stable transformants in mammalian cells. To overcome this weakness, we here introduce the improved plasmid vector pSAKA-4B, derived from pCMViR-TSC as a second-generation chromosome-insertable vector. This vector facilitates the linear entry of the expression unit into the TTAA site of DNA universally with transposase assistance. The vector is helpful for the indefinite expression of our target gene. The new vector system is proven here to be efficient in establishing stable transformants with a high likelihood of positive clones that exhibit significantly elevated expression levels of the delivered foreign gene. This system, alongside the first-generation vector, is therefore instrumental for diverse basic research endeavors concerning genes, proteins, cells, and animals, and potentially for clinical applications such as gene therapy.