Construction and Validation of Improved Triple Fusion Reporter Gene Vectors for Molecular Imaging of Living Subjects

Abstract

Multimodality imaging using several reporter genes and imaging technologies has become an increasingly important tool in determining the location(s), magnitude, and time variation of reporter gene expression in small animals. We have reported construction and validation of several triple fusion genes composed of a bioluminescent, a fluorescent, and a positron emission tomography (PET) reporter gene in cell culture and in living subjects. However, the bioluminescent and fluorescent components of fusion reporter proteins encoded by these vectors possess lesser activities when compared with the bioluminescent and fluorescent components of the nonfusions. In this study, we first created a mutant (mtfl) of a thermostable firefly luciferase (tfl) bearing the peroxisome localization signal to have greater cytoplasmic localization and improved access for its substrate, D-luciferin. Comparison between the three luciferases [mtfl, tfl, and firefly luciferase ( fl)] both in cell culture and in living mice revealed that mtfl possessed 6-to 10-fold (in vitro) and 2-fold (in vivo) higher activity than fl. The improved version of the triple fusion vector carrying mtfl as the bioluminescent reporter component showed significantly (P < 0.05) higher bioluminescence than the previous triple fusion vectors. Of the three different red fluorescent reporter genes ( jred, hcred, and mrfp1, isolated from jellyfish chromophore, coral Heteractis crispa, and coral Discosoma, respectively) evaluated, mrfp1 was able to preserve highest expression as a component of the triple fusion reporter gene for in vivo fluorescence imaging. A truncated version of wild-type herpes simplex virus 1 (HSV1) thymidine kinase gene (wttk) retained a higher expression level than the truncated mutant HSV1-sr39 TK (ttk) as the third reporter component of this improved triple fusion vector. Multimodality imaging of tumor-bearing mice using bioluminescence and microPET showed higher luciferase activity [(2.7 F 0.1 versus 1.9 F 0.1) Â (10 6 p/s/cm 2 /sr)] but similar level of fluorine-18-labeled 2 ¶-fluoro-2 ¶-deoxyarabinofuranosyl-5-ethyluracil (18F-FEAU) uptake (1.37 F 0.15 versus 1.37 F 0.2) percentage injected dose per gram] by mtfl-mrfp1-wttk-expressing tumors compared with the fl-mrfp1-wttkexpressing tumors. Both tumors showed 4-to 5-fold higher accumulation (P < 0.05) of 18F-FEAU than fluorine-18-labeled 9-(4-fluoro-3-hydroxymethylbutyl)guanine. This improved triple fusion reporter vector will enable high sensitivity detection of lower numbers of cells from living animals using the combined bioluminescence, fluorescence, and microPET imaging techniques. [Cancer Res 2007;67(7):3085-93]

Cancer stem cells from human breast tumors are involved in spontaneous metastases in orthotopic mouse models

“…To closely monitor BCSC fates and functions in mice, we labeled CD44 + cells from TN1 or TN2 tumors with dual-function reporters comprising the coding sequences of bioluminescent and fluorescent proteins (28)(29)(30)(31)43), which allow for both BLI in vivo and fluorescence imaging ex vivo, along with flow cytometry analyses. We generated several lentiviral constructs, comprising different reporter genes and promoter sequences, and assayed their utility in labeling BSCSs under a variety of conditions (multiplicity of infection, or MOI, of 5-100) (Fig.…”

“…Optical imaging approaches that use multifunctional reporter genes permit thorough analyses of disease models by linking in vivo and ex vivo assays and guiding the experimental design (27)(28)(29)(30)(31)(32). Such reporters include firefly luciferase (Luc) for whole-body tracking of cells via bioluminescence imaging (BLI) (33) and fluorescent proteins that facilitate intravital imaging and ex vivo analyses (e.g., fluorescence microscopy and flow cytometry).…”

BDNF Pretreatment of Human Embryonic-Derived Neural Stem Cells Improves Cell Survival and Functional Recovery after Transplantation in Hypoxic–Ischemic Stroke

“…Human embryonic-derived NSCs (SCR055; Millipore, Billerica, MA, USA) were transduced as previously described (30,34). In short, a bifusion construct containing firefly luciferase and enhanced green fluorescent protein was inserted into a lentiviral vector.…”

Semiautomated Radiosynthesis and Biological Evaluation of [18F]FEAU: A Novel PET Imaging Agent for HSV1-tk/sr39tk Reporter Gene Expression

“…Two stable clones of C6 cells expressing either HSV1-sr39 thymidine kinase (C6-sr39tk+) or a triple-fusion vector carrying a wild-type HSV1-thymidine kinase (C6-tk+) were utilized. C6, C6-tk+, and C6-sr39tk+ cells (5×10 5 ) were plated in 12 well dishes [23]. After 24 h, [ 18 F]FEAU or [ 18 F]FHBG (1 μCi) was added in each well and incubated at 37°C for 1 h. Cells were then washed three times with 1× phosphate-buffered saline and lysed in thymidine kinase lysis buffer, and the radioactivity of each well was counted in a Cobra II gamma counter (PerkinElmer).…”