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Development of a Dual Membrane Protein Reporter System Using Sodium Iodide Symporter and Mutant Dopamine D₂ Receptor Transgenes

¹ Programs in Neuroscience, Seoul National University, Seoul, Korea; and ² Department of Nuclear Medicine, Seoul National University College of Medicine, Seoul, Korea

Correspondence: For correspondence or reprints contact: Dong Soo Lee, MD, PhD, Department of Nuclear Medicine, Seoul National University Hospital, 28 Yongon-Dong, Jongno-Gu, Seoul 110-744, Korea. E-mail: dsl{at}plaza.snu.ac.kr

For noninvasive monitoring of cellular status by dual reporters, a dual membrane protein reporter system was developed and its in vivo applicability was examined. Human sodium iodide symporter (hNIS) and mutant dopamine D₂ receptor (D₂R) transgenes were chosen considering their complementarity. Methods: pIRES-hNIS/D₂R containing NIS and D₂R linked with an internal ribosomal entry site (IRES) was constructed and transfected into human hepatoma SK-Hep1 and rat glioma C6 cells. The cell lines stably expressing hNIS and D₂R (named SK-ND and C6-ND) were produced, which was confirmed by messenger RNA expression of reporter genes. The functional activities of hNIS and D₂R were measured by ¹²⁵I uptake assay and ³H-spiperone receptor-binding assays. A biodistribution study was performed on SK-ND tumor-bearing mice using ^99mTc-pertechnetate and ³H-spiperone. In vivo hNIS expression was examined using ^99mTc-pertechnetate -camera imaging and, D₂R expression was examined using a ³H-spiperone autoradiographic study. Results: ¹²⁵I uptake of SK-ND and C6-ND cell lines showed a maximum 97-fold and 43-fold increase, respectively, which were completely inhibited by KClO₄. Specific ³H-spiperone binding to SK-ND and C6-ND cell homogenates was observed, which were completely inhibited by (+)-butaclamol. Among the dual reporter gene–expressing cell lines, the activities of both reporters were inversely correlated with each other. Competition assay of hNIS-expressing cells by D₂R vector transfection and D₂R-expressing cells by hNIS vector transfection showed a dose-dependent decrease of hNIS and D₂R activities, respectively. In the biodistribution study, ^99mTc-pertechnetate accumulated 10-fold and ³H-spiperone accumulated 4-fold more in SK-ND tumors than that in parental SK tumors. In vivo imaging of ^99mTc-pertechnetate persisted until 5 wk after the cell graft in SK-ND tumors. Autoradiographic study of brain tissues from these mice also revealed an accumulation of ³H-spiperone in SK-ND tumors. Conclusion: We developed a dual membrane-bound positron and -imaging reporter system of hNIS and D₂R. We observed its reporting capability in vitro and in vivo and elucidated that these 2 membrane protein reporters competed with each other in their expression. Although we expect that hNIS and D₂R transgenes can complement each other as a dual reporter system, we suggest that one needs to validate the ratio of expression of the 2 membrane protein reporter transgenes for cellular status tracking.

Key Words: molecular nuclear imaging • reporter gene • dopamine D₂ receptor • human sodium iodide symporter

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