PUBLICATION

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

Authors
Perera, R.P., Dosch, R.
ID
ZDB-PUB-210220-44
Date
2021
Source
Methods in molecular biology (Clifton, N.J.)   2218: 303-317 (Chapter)
Registered Authors
Dosch, Roland
Keywords
Bimolecular fluorescence complementation (BiFC) assay, Bucky ball, Gateway vectors, Protein–protein interaction, Vasa, Zebrafish
MeSH Terms
  • Animals
  • Biological Assay/methods*
  • Embryo, Nonmammalian/metabolism
  • Fluorescence
  • Genetic Vectors/metabolism
  • Green Fluorescent Proteins/metabolism
  • Luminescent Proteins/metabolism
  • Microscopy, Fluorescence/methods*
  • Protein Interaction Mapping/methods
  • RNA Helicases/metabolism
  • Zebrafish/metabolism*
  • Zebrafish Proteins/metabolism*
PubMed
33606241 Full text @ Meth. Mol. Biol.
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.
Genes / Markers
Figures
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping