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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

基本信息

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

项目摘要

[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】利用一种由杆状或锥状特异性基因启动子驱动的永生化基因组成的转基因小鼠，在肿瘤诱导下建立光感受器细胞系。【方法】以人GNAT1和GNAT2基因分别作为锥特异基因和杆状特异基因，克隆其启动子区域。选择SV40大T （SV40LT）基因作为永生化基因。还使用了类维甲酸结合蛋白基因（eIRBP）增强子序列。构建了5′-Promoter-SV40LT-eIRBP-3′等转基因基因（pGNAT1和pGNAT2）。这些物质被微注射到小鼠受精卵中，产生转基因小鼠（TgMs）。在TgMs的眼睛或其他组织中形成的肿瘤被使用。【结果】含pGNAT2的TgMs患者有视网膜变性，但未见眼部肿瘤。相反，肿瘤在大脑中发展。从肿瘤的位置判断，它们被认为起源于松果体。SV40LT在眼睛和脑肿瘤组织中均有表达。分离培养肿瘤。培养240 d后克隆细胞，最终建立G2P-7细胞。G2P-7的特点是能够利用荧光素酶法表达外源引入的各种基因启动子。Gnat2启动子活性高于Gnat1。Irbp启动子在G2P-7中也显示出显著的活性。此外，松果体特异性色氨酸羟化酶基因启动子表现出高活性，表明该细胞来源于松果体。[结论]建立了松果体肿瘤源性细胞系gp2p -7，其中表达锥特异性Gnat2，而不表达杆状特异性Gnat1。G2P-7将用于光感受器和松果体基因的表达分析。[附加]这些结果发表在日本基因诊断与治疗学会（2008年7月）和日本眼科学会（2009年4月）。一篇英文论文已提交出版。少