title: Rational design of a triple reporter gene for multimodality molecular imaging.
abstract: Multimodality imaging using noncytotoxic triple fusion (TF) reporter genes is an important application for cell-based tracking, drug screening, and therapy. The firefly luciferase (fl), monomeric red fluorescence protein (mrfp), and truncated herpes simplex virus type 1 thymidine kinase SR39 mutant (ttksr39) were fused together to create TF reporter gene constructs with different order. The enzymatic activities of TF protein in vitro and in vivo were determined by luciferase reporter assay, H-FEAU cellular uptake experiment, bioluminescence imaging, and micropositron emission tomography (microPET). The TF construct expressed in H1299 cells possesses luciferase activity and red fluorescence. The tTKSR39 activity is preserved in TF protein and mediates high levels of H-FEAU accumulation and significant cell death from ganciclovir (GCV) prodrug activation. In living animals, the luciferase and tTKSR39 activities of TF protein have also been successfully validated by multimodality imaging systems. The red fluorescence signal is relatively weak for in vivo imaging but may expedite FACS-based selection of TF reporter expressing cells. We have developed an optimized triple fusion reporter construct DsRedm-fl-ttksr39 for more effective and sensitive in vivo animal imaging using fluorescence, bioluminescence, and PET imaging modalities, which may facilitate different fields of biomedical research and applications.
authors.affiliation: Department of Medical Imaging and Radiological Sciences, Kaohsiung Medical University, No. 100, Shih-Chuan 1st Road, Kaohsiung 80708, Taiwan.
authors.forename: Ya-Ju
authors.initials: YJ
authors.lastname: Hsieh
authors.affiliation: Molecular and Genetic Imaging Core/Taiwan Mouse Clinic, National Comprehensive Mouse Phenotyping and Drug Testing Center, No. 201, Section 2, Shih-Pai Road, Taipei 11217, Taiwan.
authors.forename: Luen
authors.identifier: 0000-0003-2616-2203
authors.initials: L
authors.lastname: Hwu
authors.affiliation: Molecular and Genetic Imaging Core/Taiwan Mouse Clinic, National Comprehensive Mouse Phenotyping and Drug Testing Center, No. 201, Section 2, Shih-Pai Road, Taipei 11217, Taiwan ; Institute of Clinical Medicine, National Yang-Ming University, No. 155, Section 2, Linong Street, Taipei 11221, Taiwan.
authors.forename: Chien-Chih
authors.initials: CC
authors.lastname: Ke
authors.affiliation: Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, No. 155, Section 2, Linong Street, Taipei 11221, Taiwan ; Department of Education and Research, Taipei City Hospital, No. 145, Zhengzhou Road, Datong District, Taipei 10341, Taiwan ; Biophotonic and Molecular Imaging Research Center, National Yang-Ming University, No. 155, Section 2, Linong Street, Taipei 11221, Taiwan.
authors.forename: Skye Hsin-Hsien
authors.initials: SH
authors.lastname: Yeh
authors.affiliation: Nuclear Medicine Department and PET-CT Center, Shunang Ho Hospital Ministry of Health and Welfare, Taipei Medical University, No. 291, Zhongzheng Road, Zhonghe District, New Taipei City 23561, Taiwan.
authors.forename: Chien-Feng
authors.initials: CF
authors.lastname: Lin
authors.affiliation: Molecular and Genetic Imaging Core/Taiwan Mouse Clinic, National Comprehensive Mouse Phenotyping and Drug Testing Center, No. 201, Section 2, Shih-Pai Road, Taipei 11217, Taiwan ; Center for Teaching and Learning Resources, Chinese Culture University, No. 55, Hwa-Kang Road, Taipei 11114, Taiwan.
authors.forename: Fu-Du
authors.initials: FD
authors.lastname: Chen
authors.affiliation: Molecular and Genetic Imaging Core/Taiwan Mouse Clinic, National Comprehensive Mouse Phenotyping and Drug Testing Center, No. 201, Section 2, Shih-Pai Road, Taipei 11217, Taiwan ; Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, No. 155, Section 2, Linong Street, Taipei 11221, Taiwan.
authors.forename: Hsin-Ell
authors.identifier: 0000-0003-4818-4921
authors.initials: HE
authors.lastname: Wang
authors.affiliation: Department of Electrical Engineering and Holistic Medical Device Development Center, Chung Yuan Christian University, No. 200, Chung Pei Road, Chung Li City 32023, Taiwan.
authors.forename: Kang-Ping
authors.initials: KP
authors.lastname: Lin
authors.affiliation: Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, No. 155, Section 2, Linong Street, Taipei 11221, Taiwan ; Department of Education and Research, Taipei City Hospital, No. 145, Zhengzhou Road, Datong District, Taipei 10341, Taiwan.
authors.forename: Ran-Chou
authors.initials: RC
authors.lastname: Chen
authors.affiliation: Molecular and Genetic Imaging Core/Taiwan Mouse Clinic, National Comprehensive Mouse Phenotyping and Drug Testing Center, No. 201, Section 2, Shih-Pai Road, Taipei 11217, Taiwan ; Institute of Clinical Medicine, National Yang-Ming University, No. 155, Section 2, Linong Street, Taipei 11221, Taiwan ; Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, No. 155, Section 2, Linong Street, Taipei 11221, Taiwan ; Biophotonic and Molecular Imaging Research Center, National Yang-Ming University, No. 155, Section 2, Linong Street, Taipei 11221, Taiwan ; National PET/Cyclotron Center and Department of Nuclear Medicine, Taipei Veterans General Hospital, No. 201, Section 2, Shih-Pai Road, Taipei 11217, Taiwan.
authors.forename: Ren-Shyan
authors.initials: RS
authors.lastname: Liu
mesh_terms: D000818:Animals; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D005455:Fluorescent Antibody Technique; D015774:Ganciclovir / Q000494:pharmacology; D017930:Genes, Reporter; D006801:Humans; D049409:Luciferases, Firefly / Q000378:metabolism; D008297:Male; D008819:Mice, Nude; D008856:Microscopy, Fluorescence; D057054:Molecular Imaging; D064847:Multimodal Imaging; D061848:Optical Imaging; D049268:Positron-Emission Tomography; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection
chemical_list: D011993:Recombinant Fusion Proteins; D049409:Luciferases, Firefly; D015774:Ganciclovir