방사선의학 웹진

바로가기  >

Abstract
Purpose: In the programming of tumor-targeting bacteria, various therapeutic or reporter genes are expressed by different gene-triggering strategies. Previously, we engineered pJL87 plasmid with an inducible bacterial drug delivery system that simultaneously co-expressed two genes for therapy and imaging by a bidirectional tet promoter system only in response to the administration of exogenous doxycycline (Doxy). In this multi-cassette expression approach, tetA promoter (PtetA) was 100-fold higher in expression strength than tetR promoter (PtetR). In the present study, we developed pJH18 plasmid with novel Doxy-inducible gene expression system based on a tet promoter.

Procedures: In this system, Tet repressor (TetR) expressed by a weak constitutive promoter binds to tetO operator, resulting in the tight repression of gene expressions by PtetA and PtetR, and Doxy releases TetR from tetO to de-repress PtetA and PtetR.

Results: In Salmonella transformed with pJH18, the expression balance of bidirectional tet promoters in pJH18 was remarkably improved (PtetA:PtetR = 4~6:1) compared with that of pJL87 (PtetA:PtetR = 100:1) in the presence of Doxy. Also, the expression level by novel tet system was much higher in Salmonella transformed with pJH18 than in those with pJL87 (80-fold in rluc8 and 5-fold in clyA). Interestingly, pJH18 of the transformed Salmonella was much more stably maintained than pJL87 in antibiotic-free tumor-bearing mice (about 41-fold), because only pJH18 carries bom sequence with an essential role in preventing the plasmid-free population of programmed Salmonella from undergoing cell division.

Conclusions: Overall, doxycycline-induced co-expression of two proteins at similar expression levels, we exploited bioluminescence reporter proteins with preclinical but no clinical utility. Future validation with clinically compatible reporter systems, for example, suitable for radionuclide imaging, is necessary to develop this system further towards potential clinical application.

Affiliations

Dinh-Huy Nguyen  1   2 , Sung-Hwan You  1 , An-Trang Ngoc Vo  1   2 , Hien Thi-Thu Ngo  1   2 , Khuynh Van Nguyen  1   2 , Mai Thi-Quynh Duong  1   2 , Hyon E Choy  3 , Miryoung Song  4 , Yeongjin Hong  5 , Jung-Joon Min  6   7   8
1 Institute for Molecular Imaging and Theranostics, Chonnam National University Medical School, Gwangju, 61469, Republic of Korea.
2 Department of Molecular Medicine (BrainKorea21 Plus), Chonnam National University Graduate School, Gwangju, 61469, Republic of Korea.
3 Department of Microbiology, Chonnam National University Medical School, Gwangju, 61469, Republic of Korea.
4 Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Yongin, South Korea.
5 Department of Microbiology, Chonnam National University Medical School, Gwangju, 61469, Republic of Korea. yjhong@chonnam.ac.kr.
6 Institute for Molecular Imaging and Theranostics, Chonnam National University Medical School, Gwangju, 61469, Republic of Korea. jjmin@jnu.ac.kr.
7 Department of Molecular Medicine (BrainKorea21 Plus), Chonnam National University Graduate School, Gwangju, 61469, Republic of Korea. jjmin@jnu.ac.kr.
8 Department of Nuclear Medicine, Chonnam National University Medical School, Jeonnam, Gwangju, 61469, Republic of Korea. jjmin@jnu.ac.kr.