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Katsuhiro Hosono, Kentaro Ohishi, Yoshitaka Yamaguchi, Jun Kudoh, Nobuyoshi Shimizu, Yoshihiro Hotta, Shinsei Minoshima; Establishment of A Mouse Cell Line using A Photoreceptor Gene-Specific Promoter and SV40 Large T antigen. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2253. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
We aimed at establishing mouse permanent cell lines that maintain characteristics of photoreceptor cells in order to use for studying the gene regulation on the onset of relevant eye diseases.
We attempted to make a transgenic mouse (TgM) by using the unique transgene construct consisting of three genetic elements including the promoter sequence of cone-specific transducin alpha gene GNAT2, the coding sequence of SV40 Large T antigen (LT) and the enhancer sequence of human RBP3 which is effective on the gene expression in rod and cones of eye.
The resulting TgMs developed brain tumors, which were believed to be of pineal gland origin. The TgMs also showed apoptosis-like degeneration in the photoreceptor cells. The tumor tissue was dispersed and the dissociated cells were kept in culture with periodical transfer for 240 days. After cloning by limiting dilution, a cell line named G2P-7 with a doubling time of 26.6 h was established. Analysis of gene expression by RT-PCR and DNA microarray revealed that G2P-7 cells express cone-specific Gnat2 but not rod-specific Gnat1. Furthermore, a luciferase reporter assay using the promoters of relevant genes were performed. The promoters of Gnat2 (cone-specific), Rbp3 (rod as well as cone-specific) and Tph1 (pineal gland-specific) showed significant promoter activity but Gnat1 (rod-specific) did not.
We established a mouse cell line, G2P-7, that shows the expression of cone-specific Gnat2 but not rod-specific Gnat1. It is considered that G2P-7 might maintain some characteristics of cone cells. This cell line would be useful for studying the gene regulation occurring in the photoreceptor cells and pineal gland.
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