Enhanced design of pCMViR-TSC plasmid vector for sustainably high cargo gene expression in mammalian cells
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.
Authors
- Nahoko Tomonobu (ORCID: https://orcid.org/0000-0002-1121-3912)
- Shinichi Toyooka (ORCID: https://orcid.org/0000-0002-7588-6745)
- Masakiyo Sakaguchi (ORCID: https://orcid.org/0000-0002-0566-4872)
- Fan Jiang (ORCID: https://orcid.org/0000-0002-9257-206X)
- Eisaku Kondo (ORCID: https://orcid.org/0000-0002-2939-036X)
- Rie Kinoshita (ORCID: https://orcid.org/0000-0003-1977-7920)
- I Wayan Sumardika (ORCID: https://orcid.org/0000-0002-4709-9816)
- Toshiki Ochi (ORCID: https://orcid.org/0000-0001-8440-6144)
- Yusuke Inoue (ORCID: https://orcid.org/0000-0003-4298-9737)
- Futoshi Kuribayashi (ORCID: https://orcid.org/0000-0002-3416-3583)
- Akira Yamauchi (ORCID: https://orcid.org/0000-0001-9205-6922)
- I Made Winarsa Ruma (ORCID: https://orcid.org/0000-0002-9468-1104)
- Kazumi Sagayama
- Junichiro Futami (ORCID: https://orcid.org/0000-0002-0020-2855)
- Yuma Gohara
- Yoshihiko Sakaguchi (ORCID: https://orcid.org/0000-0002-2699-4619)
- Youyi Chen (ORCID: https://orcid.org/0000-0001-9932-3966)
- Ni Luh Gede Yoni Komalasari (ORCID: https://orcid.org/0000-0003-0275-1839)
- Hitoshi Murata (ORCID: https://orcid.org/0000-0002-0581-0323)
- Tetta Takahashi
- Tomoko Honjo (ORCID: https://orcid.org/0009-0000-2552-2794)
- Jin Zhou
- Ken-Ichi Yamamoto
Institutions
- Udayana University (ID)
- Kansai Medical University (JP)
- Okayama University (JP)
- Gunma University (JP)
- Kawasaki Medical School (JP)
- Dalian University of Technology (CN)
- Okayama University Hospital (JP)
- Liaoning Cancer Hospital & Institute (CN)
- First Affiliated Hospital Zhejiang University (CN)
- Zhejiang University (CN)
- Tokushima Bunri University (JP)
Publication Details
- Journal
- In Vitro Cellular & Developmental Biology - Animal
- Published
- 2024-11-21
- DOI
- https://doi.org/10.1007/s11626-024-00992-2
- Citations
- 1
- Primary Topic
- RNA Interference and Gene Delivery
- Type
- article
- Field-Weighted Citation Impact
- 0.23
Funders
- Nagase Science Technology Foundation
- Okayama University
- Japan Society for the Promotion of Science