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Review
. 2020 Feb 19;31(2):293-302.
doi: 10.1021/acs.bioconjchem.9b00688. Epub 2019 Dec 23.

Beyond the Green Fluorescent Protein: Biomolecular Reporters for Anaerobic and Deep-Tissue Imaging

Harun F Ozbakir, Nolan T Anderson, Kang-Ching Fan, Arnab Mukherjee
Review

Beyond the Green Fluorescent Protein: Biomolecular Reporters for Anaerobic and Deep-Tissue Imaging

Harun F Ozbakir et al. Bioconjug Chem. .

Abstract

Fluorescence imaging represents cornerstone technology for studying biological function at the cellular and molecular levels. The technology's centerpiece is a prolific collection of genetic reporters based on the green fluorescent protein (GFP) and related analogs. More than two decades of protein engineering have endowed the GFP repertoire with an incredible assortment of fluorescent proteins, allowing scientists immense latitude in choosing reporters tailored to various cellular and environmental contexts. Nevertheless, GFP and derivative reporters have specific limitations that hinder their unrestricted use for molecular imaging. These challenges have inspired the development of new reporter proteins and imaging mechanisms. Here, we review how these developments are expanding the frontiers of reporter gene techniques to enable nondestructive studies of cell function in anaerobic environments and deep inside intact animals-two important biological contexts that are fundamentally incompatible with the use of GFP-based reporters.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1.
Figure 1.
Oxygen-independent biomolecular reporters. (A) Fluorescent proteins derived from LOV photoreceptors are characterized by 450 nm excitation and 495 nm emission. Anaerobically cultured E. coli cells expressing CreiLOV can be readily detected using fluorescence microscopy.(B) UnaG emits green fluorescence by associating with a heme end-product (biliverdin), which enables imaging of HeLa cells in 0.1% hypoxia (top panel). Under similar conditions, HeLa cells that express mCherry (bottom panel) are nonfluorescent. (C) Spinach2 is a small molecule dye-binding aptamer that exhibits oxygen-independent fluorescence similar to iLOV, a LOV-based fluorescent reporter. In contrast, GFP is nonfluorescent in anaerobic conditions and needs oxygen to activate fluorescence.
Figure 2.
Figure 2.
Reporter genes for MRI. (A) Genetically encoded ferritin enables migrating neuroblasts to be tracked in vivo, as indicated by the white arrow. (B) Charged polypeptides, in this case polylysine, can be used to visualize gene expression from a tumor specific promoter, using CEST. (C) Aquaporins produce diffusion weighted MRI contrast, which can be used to dynamically monitor doxycycline (dox) induced changes in tumor gene expression.
Figure 3.
Figure 3.
Oxygen-independent fluorescent proteins can be used in conjunction with MRI reporter genes to provide a unique molecular window into gut biology across cellular and organismal scales.
See this image and copyright information in PMC

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