Primary study of a novel reporter gene/probe system human ER ligand binding domain/radiolabeled estradiol

Abstract

Objective To evaluate the feasibility of a new reporter gene/probe system,namely human ER ligand binding domain (hERL)/radionuclide labeled estradiol,and to provide basis for its monitoring gene and cell therapy from in vitro cellular uptake study and in vivo imaging experiment.Methods Recombinant plasmid pDC316- hERL -internal ribosome entry site-VEGF165 ( pDC316-hERL-IRES-VEGF165,or EIV) and recombinant Ad-EIV were constructed,which carried a reporter gene (hERL) and a therapeutic gene (VEGF165 ) through IRES.Adenovirus was used as a vector.MSCs were obtained from tibias and femurs of rat,and cultured normally.Ad-EIV and EIV coated with lipofectamine 2000 (Lipo-EIV) were transfected into MSCs.RT-PCR and Western blot were performed to detect the expression of hERL and VEGF165 from mRNA and protein level.The cellular uptake values of 125I labeled estradiol ( 125I-E2 ) were measured in Ad-EIV,Lipo-EIV and non-transfected MSCs at different incubation time ( 1,3,6,9,12 and 24 h).Ad-EIV transfected MSCs were injected into the left upper limb of rats,and non-transfected MSCs into the right upper limb as self-control.Micro PET/CT images were obtained after 1 d of transfection.Ttest and Pearson linear correlation analysis were used to analyze the data.Results After transfected with Ad-EIV,mRNA and protein expressions of hERL and VEGF165 in MSCs were increased with adenovirus multiplicity of infection ( MOI),and positive correlation could be seen ( r2 =0.953 and 0.966,both P ＜0.05).The expressions of mRNA and protein in Ad-EIV group were higher than those of Lipo-EIV transfected MSCs.Time-dependent accumulation of 125I-E2 was observed in the Ad-EIV group and Lipo-EIV group,and the highest uptake rates occurred at 24 h,with peak values of ( 10.94 ± 0.30) ％ and (8.93 ± O.18)％,respectively.Higher cellular uptakes could be seen at all time points in the Ad-EIV group than those of the Lipo-EIV group (t =4.132-16.168,all P ＜0.05).Moreover,the cellular uptakes with two vectors were significantly higher than those of non-transfected control cells at all the tested time points ( t =15.489- 26.560 for Ad-EIV and t =10.523 - 24.204 for Lipo-EIV,respectively,all P ＜ 0.05 ).Compared with the right upper limb,significantly higher radioactivity could be seen in the left side where Ad5-EIV transfected MSCs were injected.Conclusions Reporter gene hERL could be transfected into the cells via adenovirus and liposome as vectors.Its successful expression in cells could be detected and imaged by radiolabled estradiol.The reporter gene/probe system hERL/radiolabled estradiol might be used as a novel reporter gene/probe system for monitoring gene and cell therapy. Key words: Genes, reporter; Estrogen receptor ligand binding domain; Radionuclide imaging; Estradiol; Isotope labeling; Rats