PUBLICATION

Generation of Transgenic Lines of Zebrafish Expressing Fluorescently Tagged CCM Proteins to Study Their Function and Subcellular Localization Within the Vasculature

Authors
Donat, S., Abdelilah-Seyfried, S.
ID
ZDB-PUB-200612-7
Date
2020
Source
Methods in molecular biology (Clifton, N.J.)   2152: 207-224 (Chapter)
Registered Authors
Abdelilah-Seyfried, Salim
Keywords
Cerebral cavernous malformations, Endocardium, Endothelium, GFP, Gateway cloning, Zebrafish, mCherry
MeSH Terms
  • Animals
  • Animals, Genetically Modified*
  • Biomarkers
  • Cloning, Molecular
  • Endocardium/metabolism
  • Endothelial Cells/metabolism
  • Endothelium/metabolism
  • Gene Expression*
  • Genes, Reporter
  • Hemangioma, Cavernous, Central Nervous System/genetics*
  • Hemangioma, Cavernous, Central Nervous System/metabolism*
  • Humans
  • Mice, Knockout
  • Microtubule-Associated Proteins/genetics*
  • Microtubule-Associated Proteins/metabolism*
  • Mutation
  • Protein Transport
  • Recombinant Fusion Proteins/genetics
  • Zebrafish
PubMed
32524555 Full text @ Meth. Mol. Biol.
Abstract
Our knowledge of the structure, localization, and interaction partners of cerebral cavernous malformations (CCM) proteins is mainly based on cell culture studies that lack the physiology of a three-dimensional multi-tissue environment. Uncovering the subcellular localization and the dynamic behavior of CCM proteins is an important aspect of characterizing the endothelial cell biology of CCM scaffold formation and for describing interactions with other protein complexes. However, the generation of specific antibodies to locate CCM scaffolds within cells has been challenging. To overcome the lack of functional antibodies, here, we describe the methodology involved in the generation of a construct for the expression of a fluorescently labeled CCM fusion construct and in the establishment of a transgenic zebrafish reporter line. The transgenic expression of fluorescently labeled CCM proteins within the developing zebrafish vasculature makes it possible to study the detailed subcellular localization and the dynamics of CCM proteins in vivo.
Genes / Markers
Figures
Expression
Phenotype
Mutation and Transgenics
Human Disease / Model Data
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping
Errata and Notes