Shaaban S A, Crognale M A, Calderone J B, Huang J, Jacobs G H, Deeb S S
Department of Medicine, University of Washington, Seattle 98195, USA.
Invest Ophthalmol Vis Sci. 1998 May;39(6):1036-43.
Changes in retinal photopigments represent a fundamental step in the evolution of visual systems, in that addition of new pigment types or alterations in the spectral absorption properties of existing pigments modify visual capacities and thus open new visual worlds. To provide a tool that would allow direct examination of the changes caused by the presence of novel photopigments, this study was designed to determine whether a gene encoding a human cone photopigment introduced into the mouse genome would be expressed in a cone-specific manner and would support phototransduction.
Mice transgenic for the human long wavelength-sensitive (L) photopigment were generated by microinjection of fertilized mouse eggs. RNA expression in different tissues was monitored by reverse transcription-polymerase chain reaction analysis. Photopigment protein was localized in retinal cross sections and wholemounts by antibody staining. Light transduction of the cone photopigments was assessed by flicker photometric electroretinography (ERG).
The human transgene was expressed specifically in the mouse cones in quantities comparable to those of the mouse middle wavelength-sensitive (M) pigment gene. Immunocytochemical analysis showed that the human L pigment was abundantly synthesized in most mouse cones, was translocated to the outer segments, and caused no detectable cone degeneration. Electroretinographic spectral sensitivity analysis showed that the human L pigment was efficient in eliciting an electrical response. The degree of expression of the transgene in the two founders correlated well with the spectral responsivity of the ERG.
The human L photopigment transduces light efficiently in mouse cones, implying that all protein domains necessary for efficient interaction with intracellular transport and signal transduction machineries in mouse cones have been conserved through evolution. The expression of the human L photopigment gene in both classes of cone of the mouse retina indicates that the transgene did not have the regulatory elements necessary for restricting its expression to mouse M cones or that such elements are not recognized in mouse UV-sensitive cones.
视网膜光色素的变化是视觉系统进化中的一个基本步骤，因为新色素类型的增加或现有色素光谱吸收特性的改变会改变视觉能力，从而开启新的视觉世界。为了提供一种能够直接检测新型光色素存在所引起变化的工具，本研究旨在确定引入小鼠基因组的编码人视锥光色素的基因是否会以视锥特异性方式表达并支持光转导。
通过显微注射受精的小鼠卵产生转人长波长敏感（L）光色素基因的小鼠。通过逆转录 - 聚合酶链反应分析监测不同组织中的RNA表达。通过抗体染色将光色素蛋白定位在视网膜横切片和整装片中。通过闪烁光度视网膜电图（ERG）评估视锥光色素的光转导。
人转基因在小鼠视锥细胞中特异性表达，其数量与小鼠中波长敏感（M）色素基因的数量相当。免疫细胞化学分析表明，人L色素在大多数小鼠视锥细胞中大量合成，转运到外段，并且未引起可检测到的视锥细胞退化。视网膜电图光谱敏感性分析表明，人L色素能够有效地引发电反应。两个转基因创始人中转基因的表达程度与ERG的光谱响应性密切相关。
人L光色素在小鼠视锥细胞中能有效地转导光，这意味着与小鼠视锥细胞内的细胞内运输和信号转导机制有效相互作用所需的所有蛋白质结构域在进化过程中都得到了保留。人L光色素基因在小鼠视网膜的两类视锥细胞中均有表达，这表明该转基因没有将其表达限制在小鼠M视锥细胞所需的调控元件，或者这些元件在小鼠紫外线敏感视锥细胞中未被识别。
