Construction of a tumor-specific bioluminescent eukaryotic expression vector and analysis of its expressionin vitroandin vivo.
The aims of this study were to construct a tumor-specific bioluminescent eukaryotic vector driven by the hTERT gene promoter and to establish a stable HeLa cell line expressing a modified firefly luciferase gene. PhTERTp-luc and pGL4.17 (luc2/Neo) were digested withSacI andHindIII, respectively, and the recombinant vector phTERTp-luc-neo was generated by ligating the desired fragments. The expression of phTERTp-luc-neo was tested in a non-transformed cell line (MRC-5), and in telomerase-positive (HeLa, MCF-7 and 293T) and -negative (U2OS and SaOS) transformed cell lines using a luciferase assay. Results showed that the recombinant vector had higher luciferase activity in telomerase-positive transformed cell lines. PhTERTp-luc-neo was transfected into a HeLa cell line, selected by G418 and bioluminescence imaging, and a cell clone HeLa-luc that constitutively expressed both neomycin and luciferase was obtained. We also conducted experiments in animals to observe luciferase activityin vivousing stable cell lines that were subcutaneously implanted into BALB/c nude mice and tumor growth was monitored by bioluminescence imaging. The HeLa-luc cell line retained its oncogenicity and tumors were detected on the fifth day following implantation by bioluminescence imaging. This study has formed a basis for the study of the expression and regulation of hTERT and early tumor detection. It also provides a convenient, sensitive and reliable platform for cervical cancer research.
Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, Clinical Cancer Study Center and Key Laboratory of Tumor Biological Behavior, Wuhan, Hubei 430071, P.R. China.
Qian
Q
Chen
Fen
F
Li
Fuxiang
F
Zhou
Wenbo
W
Wang
Yu
Y
Xu
Wenjie
W
Sun
Yunfeng
Y
Zhou
hTERT; in vivo bioluminescence imaging; stable cell clone; telomerase; tumor-specific vector