Institute of Molecular Physiology, Johannes-Gutenberg University, Mainz, Germany.
National Eye Institute, NIH, Bethesda, MD, United States of America.
PLoS One. 2018 Nov 15;13(11):e0207222. doi: 10.1371/journal.pone.0207222. eCollection 2018.
The retinal pigment epithelium (RPE) is an epithelial monolayer in the back of the vertebrate eye. RPE dysfunction is associated with retinal degeneration and blindness. In order to fully understand how dysregulation affects visual function, RPE-specific gene knockouts are indispensable. Since the currently available RPE-specific Cre recombinases show lack of specificity or poor recombination, we sought to generate an alternative. We generated a tamoxifen-inducible RPE-specific Cre transgenic mouse line under transcriptional control of an RPE-specific Tyrosinase enhancer. We characterized the Cre-mediated recombinant expression by crossing our RPE-Tyrosinase-CreErT2 mouse line with the tdTomato reporter line, Ai14. Detected fluorescence was quantified via high-content image analysis. Recombination was predominantly observed in the RPE and adjacent ciliary body. RPE flatmount preparations revealed a high level of recombination in adult mice (47.25-69.48%). Regional analysis of dorsal, ventral, nasal and temporal areas did not show significant changes in recombination. However, recombination was higher in the central RPE compared to the periphery. Higher levels of Cre-mediated recombinant expression was observed in embryonic RPE (~83%). Compared to other RPE-specific Cre transgenic mouse lines, this newly generated RPE-Tyrosinase-CreErT2 line shows a more uniform and higher level of recombination with the advantage to initiate recombination in both, prenatal and postnatal animals. This line can serve as a valuable tool for researches exploring the role of individual gene functions, in both developing and differentiated RPE.
视网膜色素上皮 (RPE) 是脊椎动物眼睛后部的单层上皮细胞。RPE 功能障碍与视网膜变性和失明有关。为了充分了解失调如何影响视觉功能,RPE 特异性基因敲除是必不可少的。由于目前可用的 RPE 特异性 Cre 重组酶显示缺乏特异性或重组效率差,我们试图生成替代物。我们在 Tyrosinase 增强子的转录控制下生成了一种可诱导的 RPE 特异性 Cre 转基因小鼠系。我们通过将我们的 RPE-Tyrosinase-CreErT2 小鼠系与 tdTomato 报告系 Ai14 杂交,来表征 Cre 介导的重组表达。通过高内涵图像分析检测荧光定量。重组主要发生在 RPE 和相邻的睫状体。RPE 平面培养物显示成年小鼠中重组水平较高(47.25-69.48%)。对背侧、腹侧、鼻侧和颞侧区域的区域分析未显示重组有显著变化。然而,中央 RPE 的重组水平高于周边。在胚胎 RPE 中观察到更高水平的 Cre 介导的重组表达(~83%)。与其他 RPE 特异性 Cre 转基因小鼠系相比,这种新生成的 RPE-Tyrosinase-CreErT2 系显示出更均匀和更高水平的重组,并且具有在产前和产后动物中启动重组的优势。该系可作为研究个体基因功能在发育中和分化的 RPE 中的作用的有价值的工具。