Combining near-infrared fluorescence with Brainbow to visualize expression of specific genes within a multicolor context

Abstract

Fluorescent proteins are a powerful experimental tool, allowing the visualization of gene expression and cellular behaviors in a variety of systems. Multicolor combinations of fluorescent proteins, such as Brainbow, have expanded the range of possible research questions and are useful for distinguishing and tracking cells. The addition of a separately driven color, however, would allow researchers to report expression of a manipulated gene within the multicolor context to investigate mechanistic effects. A far-red or near-infrared protein could be particularly suitable in this context, as these can be distinguished spectrally from Brainbow. We investigated five far-red/near-infrared proteins in zebrafish: TagRFP657, mCardinal, miRFP670, iRFP670, and mIFP. Our results show that both mCardinal and iRFP670 are useful fluorescent proteins for zebrafish expression. We also introduce a new transgenic zebrafish line that expresses Brainbow under the control of the neuroD promoter. We demonstrate that mCardinal can be used to track the expression of a manipulated bone morphogenetic protein receptor within the Brainbow context. The overlay of near-infrared fluorescence onto a Brainbow background defines a clear strategy for future research questions that aim to manipulate or track the effects of specific genes within a population of cells that are delineated using multicolor approaches.

Inducible Mosaic Cell Labeling Provides Insights Into Pancreatic Islet Morphogenesis

“…In this work, we develop new tools for studying cell motility during islet morphogenesis. We show that a 5 kb neurod promoter, previously used for studies of the zebrafish nervous system (Mo and Nicolson, 2011;Cook et al, 2019), also directs robust expression to pancreatic islet cells. A neurod:memKate transgenic line highlights morphology of all endocrine cell types.…”

A More Open Approach Is Needed to Develop Cell-Based Fish Technology: It Starts with Zebrafish

“…Among the contents of the zebrafish toolkit, there are numerous fluorescence imaging techniques available to provide cell-based meat scientists with real-time optical cues on cell status and viability. 104 These make it easy to distinguish muscle cells from other cells. 105 With all these benefits, the expansive zebrafish toolkit has the potential to simplify and accelerate research and development and to facilitate eventual production of cell-based meat for consumption.…”

Development of an In Vivo Model to Study Clonal Lineage Relationships in Hematopoietic Cells Using Brainbow2.1/Confetti Mice

“…This could be due to stress hematopoiesis [41], strong selective pressure during progenitor homing or decreased cell viability of high Cre-expressing cells [27]. In several multifluorescent cell tracking methods, subcellular compartment labeling has proven to be useful for clonal analysis by imaging techniques [7,10,42]. Interestingly, the lack of iRV-Cre-GFP integration made it possible to detect R26R-Confetti nGFP via flow cytometry, without having to rely on confocal microscopy for nuclear localization.…”

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