Browse > Article

Development of Dual Reporter System of Mutant Dopamine 2 Receptor ($D_2R$) and Sodium Iodide Symporter (NIS) Transgenes  

Hwang, Do-Won (Program in Neuroscience, Seoul National University)
Lee, Dong-Soo (Program in Neuroscience, Seoul National University)
Kang, Joo-Hyun (Department of Nuclear Medicine, Seoul National University College of Medicine)
Chang, Young-Soo (Department of Nuclear Medicine, Seoul National University College of Medicine)
Kim, Yun-Hui (Program in Neuroscience, Seoul National University)
Jeong, Jae-Min (Department of Nuclear Medicine, Seoul National University College of Medicine)
Chung, June-Key (Department of Nuclear Medicine, Seoul National University College of Medicine)
Lee, Myung-Chul (Department of Nuclear Medicine, Seoul National University College of Medicine)
Publication Information
The Korean Journal of Nuclear Medicine / v.38, no.4, 2004 , pp. 294-299 More about this Journal
Abstract
Purpose: Both human NIS and mutant $D_2R$ transgenes are proposed as reporting system in transplanted cell tracking. Using hepatoma cell lines, we constructed a dual reporter system containing human sodium-iodide symporter (hNIS) and dopamine 2 receptor ($D_2R$) and compared its characteristics. Materials and Methods: The recombinant plasmid ($pIRES-hNIS/D_2R$) was constructed with IRES (internal ribosome entry site) under control of the CMV promoter $pIRES-hNIS/D_2R$ was transfected to human hepatoma SK-Hep1 cell line with lipofectamine. HEP-ND ($SK-Hep1-hNIS/D_2R$) cells stably expressing hNIS and $D_2R$ was established by selection with G418 for two weeks. RT-PCR was performed to investigate the expression of both hNIS and $D_2R$ genes. The expressions of hNIS and $D_2R$ were measured by $^{125}I$ uptake assays and receptor binding assays. Specific binding of $D_2R$ to $[^3H]spiperone$ was verified by Scatchard plot with (+) butaclamol as a specific inhibitor. $K_d\;and\;B_{max}$ values were estimated. The correlation between hNIS and $D_2R$ expression was compared by using each clone. Results: Similar quantities of hNIS and $D_2R$ genes were expressed on HEP-ND as RT-PCR assays. HEP-ND cells showed 30 to 40 fold higher radioiodine uptakes than those of parental SK-Hep1 cells. $^{125}I$ uptake in HEP-ND cells was completely inhibited by $KClO_4$, a NIS inhibitor Specific binding to HEP-ND cells was saturable and the $K_d\;and\;B_{max}$ values for HEP-ND cells were 2.92 nM, 745.25 fmol/mg protein and 2.91nM, 1323 fmole/mg protein in two clones, respectively. The radioiodine uptake by hNIS activity and $D_2R$ binding was highly correlated. Conclusion: We developed a dual positron and gamma imaging reporter system of hNIS and $D_2R$ in a stably transfected cell line. We expect that $D_2R$ and hNIS genes can complement mutually as a nuclear reporting system or that $D_2R$ can be used as reporter gene when hNIS gene were used as a treatment gene.
Keywords
dopamine 2 receptor; human sodium iodide symporter; SK-Hep1 cell line;
Citations & Related Records
연도 인용수 순위
  • Reference
1 Herschman HR, MacLaren DC, Iyer M, Namavari M, Bobinski K, Green LA, et al. Seeing is believing: non-invasive, quantitative and repetitive imaging of reporter gene expression in living animals, using positron emission tomography. Neurosci Res 2000;15:699-705
2 Min JJ, Chung JK, Lee YJ, Shin JH, Yeo JS, Jeong JM, et al. In vitro and in vivo characteristics of a human colon cancer cell line, SNU-C5N, expressing sodium-iodide symporter. Nucl Med Biol 2002;29:537-45   DOI   ScienceOn
3 Barrio JR, Satyamurthy N, Huang SC, Keen RE, Nissenson CH, Hoffman JM, et al. 3-(2'-[$^{18}F$]fluoroethyl)spiperone: in vivo biochemical and kinetic characterization in rodents, nonhuman primates, and humans. J Cereb Blood Flow Metab 1989;9:830-9   DOI   PUBMED
4 Bahn MM, Huang SC, Hawkins RA, Satyamurthy N, Hoffman JM, Barrio JR, et al. Models for in vivo kinetic interactions of dopamine D2-neuroreceptors and 3-(2'-[$^{18}F$]fluoroethyl)spiperone examined with positron emission tomography. J Cereb Blood Flow Metab 1989;9:840-9   DOI   PUBMED
5 Chung JK. Sodium iodide symporter: its role in nuclear medicine. J Nucl Med 2002;43:1188-200   PUBMED
6 Shin JH, Chung JK, Kang JH, Lee YJ, Kim KI, Kim CW. Feasibility of sodium/iodide symporter gene as a new imaging reporter gene: comparison with HSV1-tk. Eur J Nucl Med Mol Imaging 2004;31:425-32   DOI   PUBMED
7 Palacios JM, Niehoff DS, Kuhar MJ. [$^3H$]spiperone binding sites in brain: Autoradiographic localization of multiple receptors. Brain Research 1981;213:277-89
8 MacLaren DC, Gambhir SS, Satyamurthy N, Barrio JR, Sharfstein S, Toyokuni T,etal. Repetitive, non-invasive imaging of the dopamine $D_2$ receptor as a reporter gene in living animals. Gene Thrapy 1999;6:785-91   DOI   ScienceOn
9 Gurtu V, Yan G, Zhang G. IRES bicistronic expression vectors for efficient creation of stable mammalian cell lines. Biochem Biophys Res Commun 1996;229:295-8   DOI   ScienceOn
10 Adonai N, Nguyen KN, Walsh J, Iyer M, Toyokuni T, Phelps ME, et al. Ex vivo cell labeling with $^{64}Cu$-pyruvaldehyde-bis(N4-methylthiosemicarbazone) for imaging cell trafficking in mice with positron-emission tomography. Proc Natl Acad Sci U S A 2002;99:3030-5   DOI   ScienceOn
11 Vile JM, D'Souza UM, Strange PG. [$^3H$]nemonapride and [$^3H$]spiperone label equivalent numbers of D2 and D3 dopamine receptors in a range of tissues and under different conditions. J Neurochem 1995;64:940-3   DOI   PUBMED   ScienceOn
12 Yaghoubi SS, Wu L, Liang Q, Toyokuni T, Barrio JR, Namavari M, et al. Direct correlation between positron emission tomographic images of two reporter genes delivered by two distinct adenoviral vectors. Gene Thrapy 2001;8:1072-80   DOI   ScienceOn
13 Woodward R, Coley C, Daniell S, Naylor LH, Strange PG. Investigation of the role of conserved serine residues in the long form of the rat D2 dopamine receptor using site-directed mutagenesis. J Neurochem 1996;66:394-402   DOI   PUBMED   ScienceOn
14 Yu Y, Annala AJ, Barrio JR, Toyokuni T, Satyamurthy N, Namavari M, et al. Quantification of target gene expression by imaging reporter gene expression in living animals. Nature Medicine 2000;6:933-7   DOI   ScienceOn
15 Dai G, Levy O, Carrasco N. Cloning and characterization of the thyroid iodide transporter. Nature 1996;379:458-60   DOI   ScienceOn
16 Gambhir SS, Barrio JR, Herschman HR, Phelps ME. Assays for noninvasive imaging of reporter gene expression. Nucl Med Biol 1999;26:481-90   DOI   ScienceOn
17 Missale C, Nash SR, Robinson SW, Jaber M, Caron MG. Dopamine receptors: from structure to function. Physiol Rev 1998;78:189-225   DOI   PUBMED
18 Liang Q, Satyamurthy N, Barrio JR, Toyokuni T, Phelps MP. Noninvasive, quantitative imaging in living animals of a mutant dopamine $D_2$ receptor reporter gene in which ligand binding is uncoupled from signal transduction. Gene Thrapy 2001;8:1490-98   DOI   ScienceOn
19 Bunzow JR, Van Tol HH, Grandy DK, Albert P, Salon J, Christie M,etal. Cloning and expression of a rat D2 receptor cDNA. Nature 1988;336:783-7   DOI   ScienceOn
20 Sun X, Annala AJ, Yaghoubi SS, Barrio JR, Nguyen KN, Toyokuni T, et al. Quantitative imaging of gene induction in living animals. Gene Thrapy 2001;8:1572-9   DOI   ScienceOn
21 Satyamurthy N, Barrio JR, Bida GT, Huang SC, Mazziotta JC, Phelps ME. 3-(2'-[$^{18}F$]fluoroethyl)spiperone, a potent dopamine antagonist: synthesis, structural analysis and in-vivo utilization in humans. Int J Rad Appl Instrum 1990;41:113-29   DOI   ScienceOn