Establishment of a new promoter trapping vector using 2A peptide

• Published in: Biotechnology and bioprocess engineering 2024, 29(3), , pp.520-528
• Main Authors: Song, Eun Seon, Lee, Yun Haeng, So, Moon Kyoung, Kuk, Myeong Uk, Park, Ji Ho, Yoon, Jee Hee, Lee, Yoo Jin, Kim, Duyeol, So, Byeonghyeon, Byun, Youngjoo, Kwon, Hyung Wook, Park, Joon Tae
• Format: Article
• Language: English
• Subjects: Antibiotics; Biotechnology; Chemistry; Chemistry and Materials Science; Expression vectors; genetic vectors; Genomes; Genomic analysis; Industrial and Production Engineering; luciferase; Mammalian cells; mammals; Neomycin; Nucleotide sequence; peptides; promoter regions; Promoters; Research Paper; resistance genes; Sequence analysis; Trapping; 생물공학
• ISSN: 1226-8372; 1976-3816
• DOI: 10.1007/s12257-024-00096-4

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.