figshare
Browse
kbie_a_1684863_sm8509.docx (16.96 kB)

Effect of promoter, promoter mutation and enhancer on transgene expression mediated by episomal vectors in transfected HEK293, Chang liver and primary cells

Download (16.96 kB)
journal contribution
posted on 2019-10-31, 10:14 authored by Zhong-Jie Xu, Yan-Long Jia, Meng Wang, Dan-Dan Yi, Wei-Li Zhang, Xiao-Yin Wang, Jun-He Zhang

The episomal vector cannot integrate into the host cell chromosome, which has no potential risk in gene therapy. However, the low level of transgene expression driven by episomal vectors needs to be solved. In this study, we investigated the effects of enhancers, promoters and promoter variants on transgene expression levels driven by episomal vectors in HEK293, Chang liver and primary cells. Results showed that all eight cis-acting elements used could increase transfection efficiency and transient eGFP expression in transfected HEK293 and Chang liver cells. In stably transfected mammalian cells, the elongation factor-1 alpha (EF-1α) promoter and mutant-404 showed high and stable transgene expression. The mechanisms might be related to the type and quantity of transcription factor regulatory elements. Moreover, quantitative reverse transcription polymerase chain reaction analysis showed that mRNA expression levels were not directly proportional to protein expression levels. Furthermore, the EF-1α promoter conferred high transgene expression levels in primary cells, and the plasmid was also present in the episomal state. Taken together, these results provided valuable information for improving transgene expression with episomal vectors in mammalian cells.

Funding

This work was supported by the grant from the National Natural Science Foundation of China (No. U1804168, U1604193), the Plan of Scientific and Technological Innovation Team for University, Henan Province, China (No. 18IRTSTHN027), the Key Science and Technology Project of Henan (192102310149) and the Key Scientific Research Project of Universities of Henan Province (No. 18A350008).

History