在分子影像領域中，影像報導基因的發展是很重要的一環。具有多重影像報導基因之動物模型，能同時運用多種成像系統進行觀測並互相驗證。在本篇論文中，我們利用多種影像報導基因建立了兩個動物模型：1.成功結合三種不同影像報導基因，包括利用螢火蟲冷光酵素、綠螢光蛋白質、以及第一型單純疱疹病毒胸腺嘧啶激酶，可分別應用於光學系統及核子醫學成像系統於同一隻小鼠上造影。2.結合鐵蛋白與紅螢光蛋白質之融合影像報導基因，可應用於光學系統造影以及磁振造影系統。在第一個主題的系列實驗中，我們建立融合蛋白質帶有三種報導基因並且利用腦部專一表達之第一型纖維母細胞生長因子-1B 啟動子來驅動此融合影像報導基因。成功建立此融合影像報導基因轉殖小鼠。具有多項成像系統報導基因之小鼠將有助於對第一型纖維母細胞生長因子在胚胎發育及各種疾病過程中扮演角色之探討。除了在腦部的表現外，第一型纖維母細胞生長因子也於鼻咽、頭骨、脊椎以及睪丸處有訊號表達，主要表達在睪丸中的萊氏細胞。我們將此基因轉殖小鼠注射烷基化藥物Busulfan，此烷基化藥物已知用以破壞萊氏細胞以及中斷精子成熟過程。烷基化藥物的注射後，此基因轉殖小鼠之報導基因訊號隨之減少；隨著萊氏細胞的活性恢復，報導基因的訊號隨之增加，由此可知，第一型纖維母細胞生長因子-1B 啟動子之表現參與精子成熟過程。因此我們可利用此基因轉殖小鼠模型，建立此基因轉殖小鼠模型，可以利用其影像上之優勢，應用於追蹤腦部/睪丸細胞之增生、分化、遷移、以及發展之機制及過程。在第二個主題實驗中，我們成功建立鐵蛋白和紅螢光蛋白質之融合影像報導基因，因此我們可以在活體動物實驗中，利用光學系統和磁振造影系統分別對同一隻動物來進行造影。我們利用腺病毒來進行此融合蛋白質在小鼠腦部之傳遞及表達，接著進行造影。實驗結果顯示，此報導基因的確可以降低磁振造影T2時間，增加T2加權影像的對比度。在光學成像中，同樣的表達位置也呈現很強的紅螢光強度。此影像報導基因對於體內動態過程的呈現有很大的助益：像是利用此影像報導基因轉染的幹細胞或腫瘤細胞，我們可以利用磁振造影來偵測其在體內移動或轉移的過程。
影像報導基因在臨床前分子影像領域的發展，有愈來愈多的應用以及新穎的成像技術問世。在此論文中，我們檢視文獻中設計多重影像報導基因之原則，成功構築出兩個新穎之影像報導基因之融合蛋白質，並且成功應用於活體影像之造影。此研究可帶來給我們活體上或是體外細胞實驗中所展現的生物活性，同時，也讓我們了解及測試各個或合併之成像系統的限制及應用。再者，我們也可應用於長時間觀察報導基因的表現，觀察偵測啟動子在正常環境、疾病、或者反應外來刺激下影像報導基因改變之情形。影像報導基因之轉譯雖然有其屏障，但是融合各種影像報導基因的應用於未來是非常可期待的。

Reporter imaging is one of important areas in molecular imaging. Transgenic (Tg) mice expressing reporter genes take full advantage of imaging technologies in studies of live mice. In this thesis, two multimodality reporters were constructed, (1) a tri-fusion imaging reporter, containing fragments of firefly luciferase, enhanced green fluorescent protein, and herpes simplex virus type 1 thymidine kinase to allow fluorescent, bioluminescent and nuclear radioactive imaging in the same mice; (2) a ferritin and red fluorescent protein fusion reporter for magnetic resonance and fluorescent imaging. In the first study, we developed a tri-fusion reporter transgenic mouse under the control of human fibroblast growth factor 1B (FGF1B or F1B) promoter. As a multifunctional mitogen, FGF1 regulates distinct functions in multiple tissues including the proliferation of brain neural stem cells, in which FGF1 expression is activated by F1B promoter. This transgenic mouse could visualize F1B promoter activity in vivo through using multiple imaging platform. The surrogate marker expression of Tg mice revealed that F1B activity was not only in brain but presented also in nasopharynx, skull, spine, and testes, particularly in Leydig cells. Treating transgenic mice with alkylating agent Busulfan, which is known to eradicate Leydig cells and disrupt spermatogenesis, resulted in loss of reporter signals. The reporter expression recovered after the restoration of Leydig cells, indicating that F1B promoter activity is involved in male spermatogenesis. F1B tri-fusion reporter mouse model can be utilized for studies exploring the toxicity of chemicals hazardous to male reproduction. In the second study, we generated a ferritin and red fluorescent protein fusion reporter gene that enables the visualization of transgene expression in living animals by magnetic resonance imaging (MRI) and optical imaging. Delivery and expression of this fusion reporter in the mouse brain was achieved by using adenovirus before the detection by MRI and fluorescence imaging. The result demonstrated a T2 shortening effect by the fusion reporter and an increase of contrast in T2-weighted images at the sites co-localized with strong red fluorescence. This reporter is useful for visualizing dynamic processes such as the migration of reporter-transfected stem cells or metastasis of tumors using MRI with the added flexibility of combining optical tools such as fluorescence activated cell sorting and fluorescence microscopy.
Reporter imaging in preclinical animal molecular imaging is rapidly evolving with new applications and development of novel imaging techniques. In this thesis, we examined literatures describing the design principles of multimodality reporters, generated two novel fusion reporters, and demonstrated in vivo imaging of reporter Tg mice. In particular, this research is improving our understanding of the biological activity, in vitro/in vivo functions as well as limitations of respective reporter genes, individually or in combination. Furthermore, measuring the changes of reporter genes as the result of promoter activation in normal development, in diseases, or responding to biological and physical stimuli has been feasible in longitudinal animal studies. Although there are barriers to translation of novel imaging reporters and techniques, the future outlook for multimodality reporter genes in research is promising.

1. Weissleder, R. and U. Mahmood, Molecular Imaging. Radiology, 2001. 219(2): p. 316-333.
2. James, M.L. and S.S. Gambhir, A molecular imaging primer: modalities, imaging agents, and applications. Physiol Rev, 2012. 92(2): p. 897-965.
3. Sigmund, C.D., Major approaches for generating and analyzing transgenic mice. An overview. Hypertension, 1993. 22(4): p. 599-607.
4. Gaj, T., C.A. Gersbach, and C.F. Barbas, 3rd, ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol, 2013. 31(7): p. 397-405.
5. Blasberg, R.G. and J.G. Tjuvajev, Molecular-genetic imaging: current and future perspectives. J Clin Invest, 2003. 111(11): p. 1620-9.
6. Rome, C., F. Couillaud, and C.T. Moonen, Gene expression and gene therapy imaging. Eur Radiol, 2007. 17(2): p. 305-19.
7. Kang, J.H. and J.K. Chung, Molecular-genetic imaging based on reporter gene expression. J Nucl Med, 2008. 49 Suppl 2: p. 164S-79S.
8. Kesarwala, A.H., et al., Second-generation triple reporter for bioluminescence, micro-positron emission tomography, and fluorescence imaging. Mol Imaging, 2006. 5(4): p. 465-74.
9. Kim, Y.J., et al., Multimodality imaging of lymphocytic migration using lentiviral-based transduction of a tri-fusion reporter gene. Mol Imaging Biol, 2004. 6(5): p. 331-40.
10. Ponomarev, V., et al., A novel triple-modality reporter gene for whole-body fluorescent, bioluminescent, and nuclear noninvasive imaging. Eur J Nucl Med Mol Imaging, 2004. 31(5): p. 740-51.
11. Ray, P., et al., Imaging tri-fusion multimodality reporter gene expression in living subjects. Cancer Res, 2004. 64(4): p. 1323-30.
12. Ornitz, D.M., et al., Receptor specificity of the fibroblast growth factor family. J Biol Chem, 1996. 271(25): p. 15292-7.
13. Zhang, X., et al., Receptor specificity of the fibroblast growth factor family. The complete mammalian FGF family. J Biol Chem, 2006. 281(23): p. 15694-700.
14. Wang, W.P., et al., Cloning of the gene coding for human class 1 heparin-binding growth factor and its expression in fetal tissues. Mol Cell Biol, 1989. 9(6): p. 2387-95.
15. Miller, D.L., et al., Compensation by fibroblast growth factor 1 (FGF1) does not account for the mild phenotypic defects observed in FGF2 null mice. Mol Cell Biol, 2000. 20(6): p. 2260-8.
16. Chen, G.J. and R. Forough, Fibroblast growth factors, fibroblast growth factor receptors, diseases, and drugs. Recent Pat Cardiovasc Drug Discov, 2006. 1(2): p. 211-24.
17. Lee, D.C., et al., Isolation of neural stem/progenitor cells by using EGF/FGF1 and FGF1B promoter-driven green fluorescence from embryonic and adult mouse brains. Mol Cell Neurosci, 2009.
18. Nurcombe, V., et al., Developmental regulation of neural response to FGF-1 and FGF-2 by heparan sulfate proteoglycan. Science, 1993. 260(5104): p. 103-6.
19. Bartlett, P.F., et al., Regulation of neural stem cell differentiation in the forebrain. Immunol Cell Biol, 1998. 76(5): p. 414-8.
20. Jacob, A.L., et al., Fibroblast growth factor receptor 1 signaling in the osteo-chondrogenic cell lineage regulates sequential steps of osteoblast maturation. Dev Biol, 2006. 296(2): p. 315-28.
21. Fu, Y.M., et al., Acidic fibroblast growth factor in the developing rat embryo. J Cell Biol, 1991. 114(6): p. 1261-73.
22. Grunz, H., et al., Induction of mesodermal tissues by acidic and basic heparin binding growth factors. Cell Differ, 1988. 22(3): p. 183-9.
23. Payson, R.A., et al., Cloning of two novel forms of human acidic fibroblast growth factor (aFGF) mRNA. Nucleic Acids Res, 1993. 21(3): p. 489-95.
24. Myers, R.L., et al., Gene structure and differential expression of acidic fibroblast growth factor mRNA: identification and distribution of four different transcripts. Oncogene, 1993. 8(2): p. 341-9.
25. Myers, R.L., et al., Different fibroblast growth factor 1 (FGF-1) transcripts in neural tissues, glioblastomas and kidney carcinoma cell lines. Oncogene, 1995. 11(4): p. 785-9.
26. Myers, R.L., et al., Functional characterization of the brain-specific FGF-1 promoter, FGF-1.B. J Biol Chem, 1995. 270(14): p. 8257-66.
27. Chotani, M.A., et al., Human fibroblast growth factor 1 gene expression in vascular smooth muscle cells is modulated via an alternate promoter in response to serum and phorbol ester. Nucleic Acids Res, 1995. 23(3): p. 434-41.
28. Alam, K.Y., et al., Characterization of the 1B promoter of fibroblast growth factor 1 and its expression in the adult and developing mouse brain. J Biol Chem, 1996. 271(47): p. 30263-71.
29. Chiu, I.M., et al., Tumorigenesis in transgenic mice in which the SV40 T antigen is driven by the brain-specific FGF1 promoter. Oncogene, 2000. 19(54): p. 6229-39.
30. Weiss, W.A., et al., Neuropathology of genetically engineered mice: consensus report and recommendations from an international forum. Oncogene, 2002. 21(49): p. 7453-63.
31. Hsu, Y.C., et al., Brain-specific 1B promoter of FGF1 gene facilitates the isolation of neural stem/progenitor cells with self-renewal and multipotent capacities. Dev Dyn, 2009. 238(2): p. 302-14.
32. Chen, M.S., et al., Human FGF1 promoter is active in ependymal cells and dopaminergic neurons in the brains of F1B-GFP transgenic mice. Dev Neurobiol, 2015. 75(3): p. 232-48.
33. Lin, K.M., et al., Human breast tumor cells express multimodal imaging reporter genes. Mol Imaging Biol, 2008. 10(5): p. 253-63.
34. Alauddin, M.M., et al., Preclinical evaluation of the penciclovir analog 9-(4-[(18)F]fluoro-3-hydroxymethylbutyl)guanine for in vivo measurement of suicide gene expression with PET. J Nucl Med, 2001. 42(11): p. 1682-90.
35. Alauddin, M.M., P.S. Conti, and J.D. Fissekis, A general synthesis of 2'-deoxy-2'-[18F]fluoro-1-b-D-arabinofuranosyluracil and its 5-substituted nucleosides. J Label Compd Radiopharm, 2003. 46: p. 285-289.
36. Serganova, I., et al., Molecular imaging of temporal dynamics and spatial heterogeneity of hypoxia-inducible factor-1 signal transduction activity in tumors in living mice. Cancer Res, 2004. 64(17): p. 6101-8.
37. Qi, J., et al., High-resolution 3D Bayesian image reconstruction using the microPET small-animal scanner. Phys Med Biol, 1998. 43(4): p. 1001-13.
38. Ruangma, A., et al. Characterization of USC-MAP Image Reconstruction on Micropet-R4. in IEEE Nucl. Sci. Symp. Conf. 2004. Rome.
39. Loening, A.M. and S.S. Gambhir, AMIDE: a free software tool for multimodality medical image analysis. Mol Imaging, 2003. 2(3): p. 131-7.
40. Deng, W.P., et al., Non-invasive in vivo imaging with radiolabelled FIAU for monitoring cancer gene therapy using herpes simplex virus type 1 thymidine kinase and ganciclovir. Eur J Nucl Med Mol Imaging, 2004. 31(1): p. 99-109.
41. Fu, H., J. DiRosario, and D.M. McCarty, Optimizing the mannitol-facilitated CNS entry of peripherally-delivered AAV2 vector, in Mol Therapy. 2006. p. S191.
42. Fu, H., et al., Significantly increased lifespan and improved behavioral performances by rAAV gene delivery in adult mucopolysaccharidosis IIIB mice. Gene Ther, 2007. 14(14): p. 1065-77.
43. Ge, R.S., et al., In search of rat stem Leydig cells: identification, isolation, and lineage-specific development. Proc Natl Acad Sci U S A, 2006. 103(8): p. 2719-24.
44. Tong, R., P. Ray, and S. Gambhir, The universal donor mouse: a mouse expressing a tri-fusion reporter gene in all tissues, in World Molecular Imaging Conference. 2008: Nice, France. p. 0911.
45. Mendis-Handagama, S.M. and H.B. Ariyaratne, Differentiation of the adult Leydig cell population in the postnatal testis. Biol Reprod, 2001. 65(3): p. 660-71.
46. Davidoff, M.S., et al., Progenitor cells of the testosterone-producing Leydig cells revealed. J Cell Biol, 2004. 167(5): p. 935-44.
47. Cohen, B., et al., Ferritin as an endogenous MRI reporter for noninvasive imaging of gene expression in C6 glioma tumors. Neoplasia, 2005. 7(2): p. 109-17.
48. Wilkinson, J.t., et al., Tissue-specific expression of ferritin H regulates cellular iron homoeostasis in vivo. Biochem J, 2006. 395(3): p. 501-7.
49. Gilad, A.A., et al., MRI reporter genes. J Nucl Med, 2008. 49(12): p. 1905-8.
50. Aung, W., et al., Visualization of in vivo electroporation-mediated transgene expression in experimental tumors by optical and magnetic resonance imaging. Gene Ther, 2009. 16(7): p. 830-9.
51. Kim, H.S., et al., In vivo imaging of tumor transduced with bimodal lentiviral vector encoding human ferritin and green fluorescent protein on a 1.5T clinical magnetic resonance scanner. Cancer Res, 2010. 70(18): p. 7315-24.
52. Choi, S.H., et al., Imaging and quantification of metastatic melanoma cells in lymph nodes with a ferritin MR reporter in living mice. NMR Biomed, 2011.
53. Yang, C., et al., MRI Reporter Genes for Noninvasive Molecular Imaging. Molecules, 2016. 21(5).
54. Vandsburger, M.H., et al., MRI reporter genes: applications for imaging of cell survival, proliferation, migration and differentiation. NMR Biomed, 2013. 26(7): p. 872-84.
55. Genove, G., et al., A new transgene reporter for in vivo magnetic resonance imaging. Nat Med, 2005. 11(4): p. 450-4.
56. Cohen, B., et al., MRI detection of transcriptional regulation of gene expression in transgenic mice. Nat Med, 2007. 13(4): p. 498-503.
57. Deans, A.E., et al., Cellular MRI contrast via coexpression of transferrin receptor and ferritin. Magn Reson Med, 2006. 56(1): p. 51-9.
58. Lobe, C.G., et al., Z/AP, a double reporter for cre-mediated recombination. Dev Biol, 1999. 208(2): p. 281-92.
59. Bernstein, M.A., K.F. King, and Z.J. Zhou, Handbook of MRI pulse sequences. 2004, Amsterdam ; Boston: Academic Press. 646.
60. Corsi, B., et al., Transient overexpression of human H- and L-ferritin chains in COS cells. Biochem J, 1998. 330 ( Pt 1): p. 315-20.
61. Picard, V., et al., Overexpression of the ferritin H subunit in cultured erythroid cells changes the intracellular iron distribution. Blood, 1996. 87(5): p. 2057-64.
62. Cozzi, A., et al., Overexpression of wild type and mutated human ferritin H-chain in HeLa cells: in vivo role of ferritin ferroxidase activity. J Biol Chem, 2000. 275(33): p. 25122-9.