Comparative assessment of fluorescent proteins for in vivo imaging in an animal model system
Comparative assessment of fluorescent proteins for in vivo imaging in an animal model system
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DOI：
10.1091/mbc.e16-01-0063
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发表时间：
2016-11-07
影响因子：
3.3
通讯作者：
Goldstein, Bob
中科院分区：
文献类型：
--
作者：
Heppert, Jennifer K.;Dickinson, Daniel J.;Goldstein, Bob
荧光蛋白标签是用于可视化基因产物和分析其体内动力学的基本工具。基因组编辑的最新进展加速了荧光蛋白标签精确插入到不同生物体的基因组中。这些进展扩大了体内成像实验的潜力，并促进了新的、明亮的、光稳定的荧光蛋白的实验。大多数不同荧光蛋白的亮度和光稳定性的定量比较都是在体外进行的，从控制它们在细胞或生物体中的表现的生物变量中去除。为了解决差距，我们在动物模型系统中定量评估荧光蛋白的体内特性。我们产生了在胚胎中表达绿色、黄色或红色荧光蛋白的转基因秀丽隐杆线虫菌株，并在相同条件下对表达不同荧光蛋白的胚胎进行成像以进行直接比较。我们发现mNeonGreen在体内并不像体外数据预测的那样明亮，但对于特定类型的实验来说，它是一种比GFP更好的标记，我们报告了最佳的红色荧光蛋白。这些结果确定了理想的荧光蛋白成像在体内在C。elegans胚胎，并提出了良好的候选荧光蛋白，以测试在其他动物模型系统的体内成像实验。
Fluorescent protein tags are fundamental tools used to visualize gene products and analyze their dynamics in vivo. Recent advances in genome editing have expedited the precise insertion of fluorescent protein tags into the genomes of diverse organisms. These advances expand the potential of in vivo imaging experiments and facilitate experimentation with new, bright, photostable fluorescent proteins. Most quantitative comparisons of the brightness and photostability of different fluorescent proteins have been made in vitro, removed from biological variables that govern their performance in cells or organisms. To address the gap, we quantitatively assessed fluorescent protein properties in vivo in an animal model system. We generated transgenic Caenorhabditis elegans strains expressing green, yellow, or red fluorescent proteins in embryos and imaged embryos expressing different fluorescent proteins under the same conditions for direct comparison. We found that mNeonGreen was not as bright in vivo as predicted based on in vitro data but is a better tag than GFP for specific kinds of experiments, and we report on optimal red fluorescent proteins. These results identify ideal fluorescent proteins for imaging in vivo in C. elegans embryos and suggest good candidate fluorescent proteins to test in other animal model systems for in vivo imaging experiments.