In vitro study of type I intron-mediated dual reporter gene imaging for carcinoembryonic antigen

Objective To develop a specific trans-splicing intron ribozyme type Ⅰ-mediated dual reporter gene system (Rib53-Fluc-tk) for targeting CEA. Methods The 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. Results The 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). Conclusions A 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