Morphological evaluation of retinal ganglion cells expressing the L132C/T159C ChR2 mutant transgene in young adult cynomolgus monkeys  

作　　者：Wenyao Wang Yan Nan Zhuo-Hua Pan Mingliang Pu 

机构地区：[1]Department of Embryology/Anatomy,School of Basic Medical Scienees,Peking University,Beijing 100191,China [2]Department of Ophthalmolgy and Anatomy Cell Biology,Wayne State University School of Medicine,Detroit Michigan 48201,USA

出　　处：《Science China(Life Sciences)》2017年第11期1157-1167,共11页中国科学（生命科学英文版）

基　　金：supported by National Science Foundation of China(31571091 to Mingliang Pu),National Basic Research Program of China (2015CB351806 to Mingliang Pu);National Institutes of Health Grant (NIH) (EY17130 to Zhuo-Hua Pan);Dryer Foundation, the Ligon Research Center of Vision, and Research to Prevent Blindness to Department of Ophthalmology at Wayne State University

摘　　要：To characterize recombinant AAV2 （rAAV2）-mediated expression of L 132C/T 159C ChR2 mutant in retinal ganglion cells （RGCs） of young adult cynomolgus monkeys, rAAV2 vectors carrying a fusion construct of the ChR2 mutant and GFP （ChR2-GFP） were delivered to the vitreous chamber by intravitreal injection. Expression patterns of the ChR2 mutant in RGCs were examined by immunohistochemical methods three months after injection. The RNA-binding protein with multiple splicing （RBPMS） was used as an RGC specific marker to differentiate RGCs from other retinal neurons and non-neuronal cells. The numbers of RBPMS＋ and GFP＋ double-labeled RGCs in the central foveal varied with the eccentricity. The expression peaked within 100 p.m from the edge of the foveola and drastically decreased to a single superficial RGC layer approximately 300 ~tm from the edge. On average, the ratio of the double-labeled RGCs versus RBPMS＋ RGCs approached 0.324-0.15 （n=14 fields） at the central foveal region （0.1 to 0.53 mm）. We observed that the ratio reached 0.784-0.16 （n=21 fields） at peripheral retinal locations （eccentricity 〉7 mm）. This investigation demonstrates that RBPMS could serve as a valuable RGC specific marker for future investigations in this field.To characterize recombinant AAV2(rAAV2)-mediated expression of L132 C/T159 C ChR2 mutant in retinal ganglion cells(RGCs)of young adult cynomolgus monkeys, rAAV2 vectors carrying a fusion construct of the ChR2 mutant and GFP(ChR2-GFP) were delivered to the vitreous chamber by intravitreal injection. Expression patterns of the ChR2 mutant in RGCs were examined by immunohistochemical methods three months after injection. The RNA-binding protein with multiple splicing(RBPMS) was used as an RGC specific marker to differentiate RGCs from other retinal neurons and non-neuronal cells. The numbers of RBPMS+and GFP+ double-labeled RGCs in the central foveal varied with the eccentricity. The expression peaked within 100 μm from the edge of the foveola and drastically decreased to a single superficial RGC layer approximately 300 μm from the edge. On average,the ratio of the double-labeled RGCs versus RBPMS+ RGCs approached 0.32±0.15(n=14 fields) at the central foveal region(0.1 to 0.53 mm). We observed that the ratio reached 0.78±0.16(n=21 fields) at peripheral retinal locations(eccentricity >7 mm). This investigation demonstrates that RBPMS could serve as a valuable RGC specific marker for future investigations in this field.

关 键 词：ChR2 mutant transgene retinal ganglion cells cynomolgus monkey 

分 类 号：Q436[生物学—生理学]