Recoverin expression in the R28 retinal precursor cell line.
Recoverin expression in the R28 retinal precursor cell line.
复制标题
R28 视网膜前体细胞系中恢复蛋白的表达。
DOI：
10.1007/s11626-997-0091-5
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Imperato-Kalmar,EL
中科院分区：
文献类型：
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作者：
Seigel,GM;Mutchler,AL;Adamus,G;Imperato-Kalmar,EL
Dear Editor: In vitro studies of photoreceptor cell development have been limited by the availability of pure retinal cultures with unlimited lifes-pan. To date, human retinoblastoma tumor-derived cell lines have been most commonly used as models for the study of photoreceptor differentiation in vitro. The most studied of these include Y79 cells (Kyritsis et al., 1987) and WERI-RB1 cells (DiPolo and Farber, 1995). Such retinoblastoma cells are highly tumorigenic in vivo (del Cerro et al., 1993) and often show anomalous gene regulation that differs from that of the normal retina (Fong et al., 1988; DiPolo and Farber, 1995). Therefore, retinoblastoma-derived cells may not always be the best model system for the study of retinal cell growth and differentiation in vitro and have serious limitations for in vivo experimentation.As an alternative to transformed, tumorigenic retinal cell lines, this laboratory recently developed a 12S E1A-immortalized retinal cell culture (Seigel, 1996). The 12S portion of the E1A gene contains the immortalizing, but not the transforming functions of the gene (Whyte et al., 1988). The resulting retinal cell line R28 has been propagated in vitro for over 100 passages, exhibits nontransformed growth properties, and has a significant glial cell component (Seigel et al., 1996). In this study, we show that R28 cells exhibit immunoreactivity to recoverin, a calcium-binding protein expressed in photoreceptors (Wiechmann and Hammarback, 1993; Wiechmann et al., 1994) and Y79 retinoblastoma cells (Wiechman, 1996), which regulates rhodopsin phosphorylation (Kawamura et al., 1994; De Castro et al., 1995; Senin et al., 1995) and is hypothesized to play a role in cancer-associated retinopathy (Adamus et al., 1993; Polans et al., 1995). Because R28 cells represent a virtually unlimited supply of immortalized, nontumorigenic cells expressing recoverin, the cell line may provide a unique and useful way to study the regulation of recoverin gene expression and its integral role in photoreceptor differentiation and function in vitro and in vivo. For all experiments in this study, 12S E1A-immortalized retinal cells derived from PN6 Sprague-Dawley rats were used. Animals were used in accordance with the Association for Research in Vision and Ophthalmology's statement for the Use of Animals in Ophthalmic and Vision Research. Establishment of the cell culture, designated E1A-NR. 3, has been described previously (Seigel, 1996). Briefly, a'P2 12S E1A replication-defective retroviral vector was used to im-mortalize retinal tissue, which was then selected for 2 wk on the basis of neomycin resistance. Immortalized cells were maintained at 370 C in a 5% CO2 incubator under the following culture conditions: Dulbecco's modified Eagle's medium (DMEM+) with 10% calf se-rum, 1 X minimal essential medium (MEM) nonessential amino ac-ids (GIBCO Laboratories, NY), 1 X MEM vitamins (GIBCO), 0.37% sodium bicarbonate, 0.058% L-glutamine and 100 gLg/ml gentamicin. The R28 clone derived from a single cell was established by limiting dilution of E1A-NR. 3 in 96-well dishes. For cloning, medium
