Establishment of A Mouse Cell Line using A Photoreceptor Gene-Specific Promoter and SV40 Large T antigen

Abstract

Purpose: : 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.

Methods: : 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.

Results: : 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.

Conclusions: : 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.

Keywords: gene/expression • retinal culture • photoreceptors