Recombinant carcinoembryonic antigen as a reporter gene for molecular imaging

Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California Los Angeles, 700 Westwood Plaza, Los Angeles, CA 90095, USA.
European Journal of Nuclear Medicine (Impact Factor: 5.38). 09/2008; 36(1):104-14. DOI: 10.1007/s00259-008-0921-z
Source: PubMed


Reporter genes can provide a way of noninvasively assessing gene activity in vivo. However, current reporter gene strategies may be limited by the immunogenicity of foreign reporter proteins, endogenous expression, or unwanted biological activity. We have developed a reporter gene based on carcinoembryonic antigen (CEA), a human protein with limited normal tissue expression.
To construct a CEA reporter gene for PET, a CEA minigene (N-A3) was fused to the extracellular and transmembrane domains of the human Fc gamma RIIb receptor. The NA3-Fc gamma RIIb recombinant gene, driven by a CMV promoter, was transfected in Jurkat (human T cell leukemia) cells. Expression was analyzed by flow cytometry, immunohistochemistry (IHC), and microPET imaging.
Flow cytometry identified Jurkat clones stably expressing NA3-Fc gamma RIIb at low, medium, and high levels. High and medium NA3-Fc gamma RIIb expression could also be detected by Western blot. Reporter gene positive and negative Jurkat cells were used to establish xenografts in athymic mice. IHC showed staining of the tumor with high reporter gene expression; medium and low N-A3 expression was not detected. MicroPET imaging, using an anti-CEA (124)I-labeled single-chain Fv-Fc antibody fragment, demonstrated that only high N-A3 expression could be detected. Specific accumulation of activity was visualized at the N-A3 positive tumor as early as 4 h. MicroPET image quantitation showed tumor activity of 1.8 +/- 0.2, 15.2 +/- 1.3, and 4.6 +/- 1.2 percent injected dose per gram (%ID/g) at 4, 20, and 48 h, respectively. Biodistribution at 48 h demonstrated tumor uptake of 4.8 +/- 0.8%ID/g.
The CEA N-A3 minigene has the potential to be used as a reporter gene for imaging cells in vivo.

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    • "Reporter-positive tumors showed clear signal at 24 h (Fig. 5), and the tumor-to-background ratio, determined by ROI analysis of the images, was 2.8. In our previous study with the noninternalizing NA3-FcγRIIb reporter gene, we determined tumor-to-background ratio of 6.1 at 20 h in tumors, showing stronger IHC staining and expressing about 25,000 reporters per cell [9]. This observation suggests that even tumor masses with lower than 25,000 surface reporters (i.e., TR(1–99)-NA3 transfected Jurkat tumors) can still be detected by PET. "
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