Year: 2018
Type: article
Abstract: 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 C. elegans, we developed a comprehensive toolkit for C. elegans 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, Saccharomyces cerevisiae, and 2) a set of ten worm strains that each express a single, spectrally distinct fluorescent protein, under control of either the daf-21 or eft-3 promoters. We measured the in vivo emission spectrum (3 nm resolution) for each fluorescent protein in live C. elegans 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 C. elegans. Additionally, the transgenic C. elegans 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.
Source: Translational Medicine of Aging
Institution: University of Washington
Language: English
Subfield: Aging
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $350
Funders: Foundation for the National Institutes of Health, Office of Infrastructure and Environment, Cancer Genomics Centre, Branco Weiss Fellowship – Society in Science, National Institutes of Health, Israeli Centers for Research Excellence, National Institute on Aging, Office of Research Infrastructure Programs, National Institutes of Health
OpenAlex ID: W2802260035