Stably quad-fused reporter gene expressed homogenous cancer cell xenograft for in vivo optical and microPET imaging

Abstract

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Objectives: To construct a quad-fusion reporter gene (QFRG) system and obtain a homogenous cancer cell population which is a prerequisite step before cell-based therapy.

Methods: A quad-fused reporter plasmid, composed of Renilla luciferase (Rluc), DsRed monomer (DsRed), truncated HSV-1 thymidine kinase SR39 mutant (tTK) and Zeomycin resistant gene, was constructed. The non-small cell lung cancer cells (H1299) were transfected with this plasmid by Lipofectamine 2000 after DNA sequencing verification. The putative stably quad-fusion reporter expressed cells were obtained after zeomycin selection. The DsRed expression was examined by fluorescent microscope. The zeomycin-resistant, DsRed positive stable clones were further subjected to luciferase assay with coelenterazine substrate. The TK activity was confirmed by ³H-FEAU uptake. The final verified stably QFRG expressed cell clones were implanted on the SCID mice, and the in vivo optical imaging and microPET with ¹⁸F-FEAU were performed.

Results: The QFRG expressed H1299 cells grew well in zeomycin-containing culture medium and showed obvious DsRed expression and the luciferase activity in the transfected cells were confirmed by both in vitro and in vivo optical imagings. In vitro study of TK activity has showed a higher accumulation of ³H-FEAU in the QFRG transfected H1299 cells than wild-type cells. In vivo study revealed that the H1299 tumor xenograft has significant ¹⁸F-FEAU uptake.

Conclusions: We have successfully constructed a quad-fusion reporter plasmid in a homogenous cell population, of which the individual gene expression has been verified by zeomycin selection, in vitro fluorescent imaging of DsRed and in vivo imagings of Rluc and tTK expression.