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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]建立由视锥或视锥特异启动子驱动的永生化基因所组成的转基因小鼠肿瘤光感受器细胞系。[方法]分别以人GNA1和GNA2基因作为视锥和视杆特异基因，克隆其启动子区域。选择SV40大T(SV40LT)基因作为永生化基因。另外还使用了视黄酸结合蛋白基因(EIRBP)的增强子序列。构建了5‘-启动子-SV40LT-eIRBP-3’的转基因载体pGNA1和pGNA2。这些细胞被显微注射到小鼠受精卵中，产生转基因小鼠(TGM)。[结果]在含有pGNA2的TGMS中，可观察到视网膜变性，但眼内未发现肿瘤。相反，肿瘤发生在大脑中。从肿瘤的位置来看，它们被认为是起源于松果体。在眼球和…中检测到SV40LT的表达更多的脑瘤用免疫组织化学染色。对肿瘤进行分离培养。240d培养后克隆细胞，最终建立G2P-7细胞。G2P-7用荧光素酶方法鉴定了它们在不同基因启动子作用下表达外源基因的能力。Gnat2启动子活性高于Gnat1启动子。IRBP启动子在G2P-7中也有显著的活性。此外，松果体特异性色氨酸羟基酶基因启动子具有较高的活性，表明该细胞来源于松果体。[结论]我们建立了一株松果体肿瘤起源细胞系G2P-7，该细胞系表达视锥细胞特异性GNat2而不表达视杆细胞特异性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和治疗学会(2008年7月)和日本眼科学会(2009年4月)上。已经提交了一篇英文论文供出版。较少