Sands Bryan, Burnaevskiy Nikolay, Yun Soo R, Crane Mathew M, Kaeberlein Matt, Mendenhall Alexander
Department of Pathology, University of Washington, Seattle, Washington, United States of America.
Transl Med Aging. 2018 Jan;2:1-10. doi: 10.1016/j.tma.2018.01.001. Epub 2018 Apr 13.
One way scientists can observe and quantify processes in living cells is to engineer the genomes of animals to express multiple fluorescent proteins and then quantify those signals by various imaging techniques. To allow our laboratories to confidently quantify mixed (overlapping) fluorescent signals for our studies in the basic biology of gene expression and aging in , we developed a comprehensive toolkit for that we describe here. The Toolkit consists of two components: 1) a series of vectors for DNA assembly by homologous recombination (HR) in the yeast, and 2) a set of ten worm strains that each express a single, spectrally distinct fluorescent protein, under control of either the or promoters. We measured the emission spectrum (3nm resolution) for each fluorescent protein in live and showed that we can use those pure spectra to unmix overlapping fluorescent signals in spectral images of intestine cells. Seven of ten fluorescent proteins had signals that appeared to be localized in vesicular/elliptical foci or tubules in the hypodermis. We conducted fluorescence recovery after photobleaching (FRAP) experiments and showed that these structures have recovery kinetics more consistent with freely diffusing protein than aggregates (Q35::YFP). This toolkit will allow researchers to quickly and efficiently generate mutlti-fragment DNA assemblies for genome editing in Additionally, the transgenic and the measured emission spectra should serve as a resource for scientists seeking to perform, or test their ability to perform, multidimensional (multi-color) imaging experiments.
科学家观察和量化活细胞中过程的一种方法是改造动物基因组以表达多种荧光蛋白，然后通过各种成像技术对这些信号进行量化。为了使我们的实验室能够自信地量化用于基因表达和衰老基础生物学研究中的混合（重叠）荧光信号，我们开发了一套全面的工具包并在此进行描述。该工具包由两个部分组成：1）一系列用于在酵母中通过同源重组（HR）进行DNA组装的载体，以及2）一组十种线虫品系，每种品系在肌动蛋白或肌球蛋白启动子的控制下表达一种光谱上不同的荧光蛋白。我们测量了活线虫中每种荧光蛋白的发射光谱（3纳米分辨率），并表明我们可以使用这些纯光谱来解析肠道细胞光谱图像中重叠的荧光信号。十种荧光蛋白中的七种具有似乎定位于皮下的囊泡/椭圆形病灶或小管中的信号。我们进行了光漂白后荧光恢复（FRAP）实验，结果表明这些结构的恢复动力学与自由扩散的蛋白质比聚集体（Q35::YFP）更一致。该工具包将使研究人员能够快速有效地生成用于线虫基因组编辑的多片段DNA组装体。此外，转基因线虫和测得的发射光谱应为寻求进行或测试其进行多维（多色）成像实验能力的科学家提供资源。
