Casco-Robles Martin Miguel, Miura Tomoya, Chiba Chikafumi
Department of Life and Environmental Sciences, University of Tsukuba, Tennoudai 1-1-1, Tsukuba, Ibaraki, 305-8572, Japan,
Transgenic Res. 2015 Jun;24(3):463-73. doi: 10.1007/s11248-014-9857-1. Epub 2014 Dec 10.
The adult newt has the ability to regenerate the neural retina following injury, a process achieved primarily by the retinal pigment epithelium (RPE). To deliver exogenous genes to the RPE for genetic manipulation of regenerative events, we isolated the newt RPE65 promoter region by genome walking. First, we cloned the 2.8 kb RPE65 promoter from the newt, Cynops pyrrhogaster. Sequence analysis revealed several conserved regulatory elements described previously in mouse and human RPE65 promoters. Second, having previously established an I-SceI-mediated transgenic protocol for the newt, we used it here to examine the -657 bp proximal promoter of RPE65. The promoter assay used with F0 transgenic newts confirmed transgene expression of mCherry fluorescent protein in the RPE. Using bioinformatic tools and the TRANSFAC database, we identified a 340 bp CpG island located between -635 and -296 bp in the promoter; this region contains response elements for the microphthalmia-associated transcription factor known as MITF (CACGTG, CATGTG), and E-boxes (CANNTG). Sex-determining region box 9 (or SOX9) response element previously reported in the regulation of RPE genes (including RPE65) was also identified in the newt RPE65 promoter. Third, we identified DNA motif boxes in the newt RPE65 promoter that are conserved among other vertebrates. The newt RPE65 promoter is an invaluable tool for site-specific delivery of exogenous genes or genetic manipulation systems for the study of retinal regeneration in this animal.
成年蝾螈在受伤后具有再生神经视网膜的能力，这一过程主要由视网膜色素上皮（RPE）实现。为了将外源基因导入RPE以对再生事件进行基因操作，我们通过基因组步移分离了蝾螈RPE65启动子区域。首先，我们从蝾螈（日本红腹蝾螈）中克隆了2.8 kb的RPE65启动子。序列分析揭示了先前在小鼠和人类RPE65启动子中描述的几个保守调控元件。其次，我们之前已经为蝾螈建立了I-SceI介导的转基因方案，在此我们用它来检测RPE65的-657 bp近端启动子。对F0转基因蝾螈进行的启动子分析证实了mCherry荧光蛋白在RPE中的转基因表达。利用生物信息学工具和TRANSFAC数据库，我们在启动子中-635至-296 bp之间鉴定出一个340 bp的CpG岛；该区域包含小眼相关转录因子（称为MITF，其序列为CACGTG、CATGTG）的反应元件以及E盒（序列为CANNTG）。在蝾螈RPE65启动子中还鉴定出了先前报道的在RPE基因（包括RPE65）调控中起作用的性别决定区域框9（或SOX9）反应元件。第三，我们在蝾螈RPE65启动子中鉴定出了在其他脊椎动物中保守的DNA基序框。蝾螈RPE65启动子是用于在该动物中进行视网膜再生研究的外源基因或基因操作系统位点特异性递送的宝贵工具。
