New Transgenic Lines for Localization of GFP-Tagged Proteins by Electron Microscopy

Localization of uncharacterized proteins of interest by electron microscopy is often challenging, especially so when attempted in vivo. Immunogold labeling (the standard technique) is inherently dependent on robust cross-reacting antibodies, the availability of which is limited, especially to those working in nonmammalian models such as zebrafish.

Ariotti et al. have developed a new method to localize any GFP-tagged protein at the ultrastructural level using existing zebrafish GFP lines (Fig. 1). ¹ They fused a sequence encoding a peroxidase known as “APEX” ² with a small, genetically encoded, anti-GFP antibody known as a “GBP-nanobody,” ³ and generated both ubiquitous and heat-shock-inducible transgenic lines. Remarkably, APEX-GBP binds GFP with high affinity that when an APEX-GBP line is crossed with a GFP-fusion line, in vivo protein localization can be examined by electron microscopy using a simple diaminobenzidine reaction.

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FIG. 1.

APEX-GBP zebrafish can be used for ultrastructural localization of GFP-tagged proteins in vivo. (A) GFP-protein-of-interest transgenic line is crossed with an APEX-GBP expressing line. (B) APEX-GBP binds with high affinity to the GFP-tagged protein. Localization is visualized with a DAB reaction after fixation. (C) Confocal projection of DMD-citrine fish. (D) Dystrophin localization is visualized by black DAB reaction product at the myoseptum (arrows). (E) Control embryos show no DAB reaction product. Color images available online at www.liebertpub.com/zeb