Biotechnology and Bioprocess Engineering 2024; 29(3): 520-528  
Establishment of a new promoter trapping vector using 2A peptide
Eun Seon Song1 · Yun Haeng Lee1 · Moon Kyoung So1 · Myeong Uk Kuk1 · Ji Ho Park1 · Jee Hee Yoon1 · Yoo Jin Lee1 · Duyeol Kim1 · Byeonghyeon So1 · Youngjoo Byun3 · Hyung Wook Kwon1,2 · Joon Tae Park1,2
1 Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon 22012, Korea
2 Convergence Research Center for Insect Vectors, Incheon National University, Incheon 22012, Korea
3 College of Pharmacy, Korea University, Sejong 30019, Korea
Correspondence to: Hyung Wook Kwon
hwkwon@inu.ac.kr
Joon Tae Park
joontae.park@inu.ac.kr
Received: January 16, 2024; Revised: March 11, 2024; Accepted: March 19, 2024; Published online: April 24, 2024.
© The Korean Society for Biotechnology and Bioengineering. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Promoter trapping is a powerful tool for discovering promoters and uses promoter trapping vectors. However, the traditional trapping vector allows expression even if it does not integrate into the host cell genome, and even if it does integrate into the genome, it is more likely to integrate in a region other than the promoter region. In this study, to overcome the shortcomings of traditional trapping vectors, we used the bicistronic 2A system to link GFP and the neomycin resistance gene. Because this vector does not contain a promoter, simultaneous production of GFP and neomycin resistance protein requires integration into the promoter region. In fact, GFP expression was observed in more than 90% of the cell clones that survived in the medium containing antibiotics, confirming that the 2A system operates. The vector insertion location was confirmed through whole genome sequence analysis, and a 1-kb promoter candidate region was selected through promoter motif analysis. In fact, a 1-kb region inserted into a promoterless luciferase expression vector showed strong promoter activity, demonstrating its utility as a tool to find promoters. In summary, we constructed a novel promoter trapping vector using the 2A system and used it to discover the promoter with strong activity. This vector will increase the efficiency of promoter trapping, providing an opportunity to easily discover new promoters in mammalian cells.
Keywords: Promoter trapping · Bicistronic 2A system · Trapping vector

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