In Vivo Imaging of Protein Interactions in the Germplasm with Bimolecular Fluorescent Complementation.

Abstract

Protein-protein interactions (PPIs) play a central role in all cellular processes. The discovery of green fluorescent protein (GFP) and split varieties, which are functionally reconstituted by complementation, led to the development of the bimolecular fluorescence complementation (BiFC) assay for the investigation of PPI in vivo. BiFC became a popular tool, as it is a convenient and quick technology to directly visualize PPI in a wide variety of living cells. In combination with the transparency of the early zebrafish embryo, it also permits detection of PPI in the context of an entire living organism, which performs all spatial and temporal regulations missing in in vitro systems like tissue culture. However, the application of BiFC in some research areas including the study of zebrafish is limited due to the lack of efficient and convenient BiFC expression vectors. Here, we describe the engineering of a novel set of Gateway®-adapted BiFC destination vectors to investigate PPI with all possible permutations for BiFC experiments. Moreover, we demonstrate the versatility of these destination vectors by confirming the interaction between zebrafish Bucky ball and RNA helicase Vasa in living embryos.