Basic Science Investigation
In vitro study of type Ⅰ intron-mediated dual reporter gene imaging for carcinoembryonic antigen
Yajing Zhang, Jizheng Chen, Xuemei Gao, Xue Hu, Xiao Zhang, Jiaqian Liang, Zairong Gao
Published 2015-08-25
Cite as Chin J Nucl Med Mol Imaging, 2015, 35(4): 298-302. DOI: 10.3760/cma.j.issn.2095-2848.2015.04.015
Abstract
ObjectiveTo develop a specific trans-splicing intron ribozyme type Ⅰ-mediated dual reporter gene system (Rib53-Fluc-tk) for targeting CEA.
MethodsThe novel CEA-targeting trans-splicing ribozyme with the downstream reporter system (Rib53-Fluc-tk) was constructed by genetic engineering technology. The trans-splicing reaction product was evaluated using the 131I-5-iodo-2'-fluro-1-beta-D-arabinofuranosy-luracil (FIAU) cellular uptake rates and the bioluminescence. Two-sample t test, the analysis of variance and the least significant difference (LSD) t test was performed for data analysis.
ResultsThe sequence of Rib53-Fluc-tk was proved by gene-sequencing test. Human MCF-7 breast cancer cells showed a high ratio of firefly luciferase/renilla luciferase (0.64±0.10, n=4). A 520 bp band of product existed, which matched with the predicted size using RNA from cells transfected with Rib53-Fluc-tk in MCF-7. Signals were detected by bioluminescence in human embryonic kidney 293T cells co-transfected with Rib53-Fluc-tk and pCDNA3.1-CEA. The labelling rate of 131I-FIAU was (64.02±4.79)% (n=3). The radiochemical purity was (95.96±1.07)% (n=3), and the stability of the radiocompound remained high in human serum at least for 24 h. The uptake of 131I-FIAU in 293T cells transfected with Rib53-Fluc-tk was (0.31±0.01)% (n=4), while it increased with the incubation time in 293T cells co-transfected with pCDNA3.1-CEA and Rib53-Fluc-tk and reached (1.40±0.06)% at 4.5 h (F=1 007.29, t=136.34, both P<0.01).
ConclusionsA novel and specific reporter gene in the cellular level was established. Taking advantage of trans-splicing reaction of the ribozyme, it could improve the specificity of the reporter gene imaging.
Key words:
Genes, reporter; Arabinofuranosyluracil; Carcinoembryonic antigen; Bioluminescence imaging
Contributor Information
Yajing Zhang
Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei Province Key Laboratory of Molecular Imaging, Wuhan 430022, China
Wuhan Institute of Virology, Chinese Academy of Science, Wuhan 430071, China
Jizheng Chen
Wuhan Institute of Virology, Chinese Academy of Science, Wuhan 430071, China
Xuemei Gao
Xue Hu
Xiao Zhang
Jiaqian Liang
Zairong Gao