In vitro study of type Ⅰ intron-mediated dual reporter gene imaging for carcinoembryonic antigen / 中华核医学与分子影像杂志

ABSTRACT

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-l-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).Conelusions 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.