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Noncytotoxic orange and red/green derivatives of DsRed-Express2 for whole-cell labeling.

ABSTRACT: Whole-cell labeling is a common application of fluorescent proteins (FPs), but many red and orange FPs exhibit cytotoxicity that limits their use as whole-cell labels. Recently, a tetrameric red FP called DsRed-Express2 was engineered for enhanced solubility and was shown to be noncytotoxic in bacterial and mammalian cells. Our goal was to create derivatives of this protein with different spectral properties.Building on previous studies of DsRed mutants, we created two DsRed-Express2 derivatives: E2-Orange, an orange FP, and E2-Red/Green, a dual-color FP with both red and green emission. We show that these new FPs retain the low cytotoxicity of DsRed-Express2. In addition, we show that these new FPs are useful as second or third colors for flow cytometry and fluorescence microscopy.E2-Orange and E2-Red/Green will facilitate the production of healthy, stably fluorescent cell lines and transgenic organisms for multi-color labeling studies.

SUBMITTER: Strack RL

PROVIDER: S-EPMC2678115 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Noncytotoxic orange and red/green derivatives of DsRed-Express2 for whole-cell labeling.

Strack Rita L RL Bhattacharyya Dibyendu D Glick Benjamin S BS Keenan Robert J RJ

BMC biotechnology 20090403

<h4>Background</h4>Whole-cell labeling is a common application of fluorescent proteins (FPs), but many red and orange FPs exhibit cytotoxicity that limits their use as whole-cell labels. Recently, a tetrameric red FP called DsRed-Express2 was engineered for enhanced solubility and was shown to be noncytotoxic in bacterial and mammalian cells. Our goal was to create derivatives of this protein with different spectral properties.<h4>Results</h4>Building on previous studies of DsRed mutants, we cre ...[more]

PMID: 19344508

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Project description:BackgroundThe use of genetically-encoded fluorescent proteins has revolutionized the fields of cell and developmental biology and in doing so redefined our understanding of the dynamic morphogenetic processes that shape the embryo. With the advent of more accessible and sophisticated imaging technologies as well as an abundance of fluorescent proteins with different spectral characteristics, the dynamic processes taking place in situ in living cells and tissues can now be probed. Photomodulatable fluorescent proteins are one of the emerging classes of genetically-encoded fluorescent proteins.ResultsWe have compared PA-GFP, PS-CFP2, Kaede and KikGR four readily available and commonly used photomodulatable fluorescent proteins for use in ES cells and mice. Our results suggest that the green-to-red photoconvertible fluorescent protein, Kikume Green-Red (KikGR), is most suitable for cell labeling and lineage studies in ES cells and mice because it is developmentally neutral, bright and undergoes rapid and complete photoconversion. We have generated transgenic ES cell lines and strains of mice exhibiting robust widespread expression of KikGR. By efficient photoconversion of KikGR we labeled subpopulations of ES cells in culture, and groups of cells within ex utero cultured mouse embryos. Red fluorescent photoconverted cells and their progeny could be followed for extended periods of time.ConclusionTransgenic ES cells and mice exhibiting widespread readily detectable expression of KikGR are indistinguishable from their wild type counterparts and are amenable to efficient photoconversion. They represent novel tools for non-invasive selective labeling specific cell populations and live imaging cell dynamics and cell fate. Genetically-encoded photomodulatable proteins such as KikGR represent emergent attractive alternatives to commonly used vital dyes, tissue grafts and genetic methods for investigating dynamic behaviors of individual cells, collective cell dynamics and fate mapping applications.

Dataset Information

Noncytotoxic orange and red/green derivatives of DsRed-Express2 for whole-cell labeling.

Publications

Noncytotoxic orange and red/green derivatives of DsRed-Express2 for whole-cell labeling.

Similar Datasets

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