Establishment of immortalized culture-cells derived from cone and rod photoreceptors and construction of in vitro model system of retinal diseases
| Project/Area Number |
17390468
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Ophthalmology
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| Research Institution | Hamamatsu University School of Medicine |
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Principal Investigator |
MINOSHIMA Shinsei Hamamatsu University School of Medicine, Department of Medical Photobiology Photon Medical Research Center, Professor (90181966)
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| Co-Investigator(Kenkyū-buntansha) |
HOTTA Yoshihiro Hamamatsu University School of Medicine, Department of Ophthalmology, Professor (90173608)
KITAGAWA Masatoshi Hamamatsu University School of Medicine, Department of Biochemistry 1, Professor (50294971)
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| Project Period (FY) |
2005 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥16,760,000 (Direct Cost: ¥15,500,000、Indirect Cost: ¥1,260,000)
Fiscal Year 2007: ¥5,460,000 (Direct Cost: ¥4,200,000、Indirect Cost: ¥1,260,000)
Fiscal Year 2006: ¥4,100,000 (Direct Cost: ¥4,100,000)
Fiscal Year 2005: ¥7,200,000 (Direct Cost: ¥7,200,000)
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| Keywords | Photoreceotor / cone / rod / cultured cell / tumor / transducine / transgenic mice / SV40 Large T antigen / 培養系 / 色覚オプシン / ロドプシン |
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| Research Abstract |
[Purpose] We aimed to establish cell lines of photoreceptors from tumors induced in transgenic mice with a transgene consisting of an immortalizing gene driven by a rod-or cone-specific gene promoter.
[Methods] Human GNAT1 and GNAT2 genes were employed as the cone and rod specific genes, respectively, and their promoter regions were cloned. SV40 Large T (SV40LT) gene was selected as an immortalizing gene. The enhancer sequence of retinoid-binding protein gene (eIRBP) was also used. Transgenes (pGNAT1 and pGNAT2) were constructed such as 5'-Promoter-SV40LT-eIRBP-3'. These were microinjected into the mouse zygotes to produce transgenic mice (TgMs). Tumors developed in the eyes or other tissues of TgMs were used.
[Results] In TgMs with pGNAT2, retinal degeneration was observed but no tumor was found in eyes. Instead, tumors were developed in brain. Judging from the position of the tumors, they were considered to originate from pineal gland. Expression of SV40LT was detected in the eyes and
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brain tumors with immuno staining. The tumors were isolated and cultured. After 240-days culturing, cells were cloned and finally G2P-7 cells were established. G2P-7 was characterized for their capability in the expression of exogenously introduced genes using various gene promoters with a luciferase method. Gnat2 promoter showed higher activity than Gnat1. Irbp promoter also revealed a significant activity in G2P-7. Furthermore, the promoter of pineal gland-specific tryptophan hydroxylase gene showed a high activity, indicating this cell was derived from pineal gland.
[Conclusion] We established a pineal gland tumor-originated cell line G2P-7, in which cone-specific Gnat2 is expressed but rod-specific Gnat1 is not. G2P-7 will be useful for the expression analysis of photoreceptor and pineal gland genes.
[Additional] These results were presented in The Japanese Society for Gene Diagnosis and Therapy (2008 Jul) and Japanese Ophthalmological Society (2009 Apr). An English paper has been submitted for publication. Less
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Research Products
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