Establishment of immortalized culture-cells derived from cone and rod photoreceptors and construction of in vitro model system of retinal diseases

视锥细胞和视杆细胞永生化培养细胞的建立及视网膜疾病体外模型体系的构建

• 批准号: 17390468
• 负责人: MINOSHIMA Shinsei
• 金额: $ 10.73万
• 依托单位: Hamamatsu University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17390468/
• 关键词: 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

[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 … More 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

[目的]我们的目的是建立来自转基因小鼠诱导的肿瘤的细胞系，其由杆或锥体特异性基因启动子驱动的免疫基因的转化一致性。 SV40大T（SV40LT）基因被选为免疫化基因。还使用了类维生素性结合蛋白基因（EIRBP）的增强子序列。构建了转基因（PGNAT1和PGNAT2），例如5'Promoter-SV40LT-EIRBP-3'。将它们显微注射到小鼠Zygotes中以产生转基因小鼠（TGM）。使用了TGM的眼睛或其他组织中发生的肿瘤。[结果]在具有PGNAT2的TGM中，观察到残留变性，但在眼睛中未发现肿瘤。相反，肿瘤是在大脑中发展的。从肿瘤的位置来看，它们被认为起源于松果体。在眼睛中检测到SV40LT的表达，并且……更多的带有免疫染色的脑肿瘤。分离并培养肿瘤。 240天培养后，将细胞克隆，最后建立了G2P-7细胞。 G2P-7的特征是它们在使用荧光素酶方法的各种基因启动子表达外源基因表达的能力中。 GNAT2启动子的活性高于GNAT1。 IRBP启动子还揭示了G2P-7的显着活性。此外，松果体特异性的多面角羟化酶基因的启动子具有较高的活性，表明该细胞源自松果腺。[结论]我们建立了松果体肿瘤肿瘤的细胞系G2P-7，在哪种锥体特异性的GNAT2中，但在其中表达了圆锥体特异性的GNAT2，但rod-drod-drod-od-tixific gnat1不是。 G2P-7对于光感受器和松果体基因的表达分析很有用。一份英文论文已提交出版。较少的

项目成果

Mutation View/KMcamcerDB : a database for cancer gene mutations.

Mutation View/KMcamcerDB：癌症基因突变数据库。

• 发表时间: 2007
• 期刊: Cancer Science 98
• 作者: Shimizu;N.
• 通讯作者: N.

Stable minihairpin structures forming at minisatellite DNA isolated from yellow fin sea Acanthopagrus latus.

从黄鳍海棘螈中分离出的小卫星 DNA 形成稳定的小发夹结构。

• 期刊: Comparative Biochemistry and Physiology (in press)
• 作者: Kato;M.
• 通讯作者: M.

Association between glaucoma and gene polymorphism of endothelin type A receptor.

青光眼与内皮素A型受体基因多态性的关联。

• 发表时间: 2005
• 期刊: Molecular Vision 11
• 作者: Yasuaki Harabuchi;et.al;原渕保明;Takeo Fukuchi;Takeo Fukuchi;Haruki Abe;Haruki Abe;澤田 英子;阿部春樹;白柏 基宏;福地 健郎;Fumie Hayama;Takeo Fukuchi;Motohiro Shirakashi;Takeo Fukuchi;Hideko Sawada;Haruki Abe;Haruki Abe;Haruki Abe;Haruki Abe;阿部春樹;阿部春樹;白柏 基宏;澤田 英子;Tetsuya Togano;Takeo Fukuchi;Masaaki Seki;Kiyoshi Yaoeda;Motohiro Shirakashi;Takeo Fukuchi;K.Hashimoto;Haruki Abe;Masaaki Seki;福地 健郎;阿部 春樹;阿部 春樹;白柏 基宏;原 浩昭;白柏 基宏;上田 潤;上田 潤;福地 健郎;福島 淳志;白柏 基宏;葉山 文恵;福地 健郎;八百枝 潔;Tetsuya Togano;Takeo Fukuchi;Masaaki Seki;Kiyoshi Yaoeda;Motohiro Shirakashi;Takeo Fukuchi
• 通讯作者: Takeo Fukuchi