Generation and characteristics of transgenic rabbit model of retinal degeneration

本研究的目的是获得一只表达突变视紫红质基因的兔，并利用视网膜电图仪(ERG)、组织学和免疫组织化学方法对进行性视网膜变性进行特征分析。NZW兔BAC文库中鉴定出含有视紫红质基因的兔DNA克隆。视紫红质基因Pro347Leu突变在E.col.这些细菌被显微注射到受精卵中。经Southern印迹分析鉴定转基因阳性动物。用实时定量RT-PCR定性检测转基因基因在视网膜组织中的表达水平。记录不同刺激强度诱发的暗视和明视ERG。视网膜石蜡切片苏木精-伊红染色。同时进行RhodSpin和凝集素免疫组织化学及电子显微镜检查。确定了10个转基因阳性的创始人，其中6个将转基因传递给了后代。在6个转基因株系中，转基因的表达水平在7-80%之间，并与转基因拷贝数和视网膜变性的速度大致相关。在第7行，视杆状ERG a波在12个月龄时几乎消失，但锥形a波保留了60%的对照。视网膜组织学显示光感受器进行性丧失，外节缩短，12周时ONL仅表现出1-2个规律。这是首个转基因兔视网膜变性模型。视网膜变性的速度取决于转基因的表达水平。由于兔的眼睛大，易于操作，这些动物将成为研究视网膜变性的病理生理和治疗的有用模型。

Purpose of the present research was to generate a rabbit that expresses a mutant rhodopsin gene, and to characterize the progressive retinal degeneration using electroretinography(ERG), histology, and immunohistochemistry. The rabbit DNA clone that contains a rhodopsin gene was identified in a NZW rabbit BAC library. The Pro347Leu mutation of the rhodopsin gene was introduced homologous recombination in E. col. and these BACs were microinjected into fertilized eggs. Transgene-positive animals were identified by Southern blot analysis. The expression level of transgene in the retinal tissue was measured by qualitative real-time RT-PCR. Scotopic and photopic ERGs elicited by different stimulus intensities were recorded. Paraffin retinal sections were stained with hematoxylin and eosin. Rhodspin and lectin immunohistochemistory, and electronmicroscopy was also performed. Ten transgene-positive founders were identified, six of which passed transgenes to offsprings. The expression level of the transgene ranged from 7-80% for six transgenic lines, and was roughly correlated with transgene copy number and speed of retinal degeneration. In line 7, the rod ERG a-wave was nearly extinguished by 12-month-old, but the cone a-wave retained 60% of controls. Retinal histology demonstrated a progressive loss of photoreceptors and a shortening of the outer segments, and ONL at visual streak showed only 1-2 laws by 12 weeks. This is the first transgenic rabbit model of retinal degeneration. The speed of retinal degeneration was dependant on the expression level of the transgene. Because the rabbits have large eyes and are easy to handle, these animals will be a useful model to stud the pathophysiology and treatment of retinal degeneration