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

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.