Affinity Purification of Intraflagellar Transport (IFT) Proteins in Mice Using Endogenous Streptavidin/FLAG Tags.

Abstract

Biological complexity is achieved through elaborate interactions between relatively few individual components. Affinity purification (AP) has allowed these networks of protein-protein interactions that regulate key biological processes to be interrogated systematically. In order to perform these studies at the required scale, easily transfectable immortalized cell lines have typically been used. Gene-editing now affords the systematic creation of isogenic mouse models carrying endogenous tags for affinity proteomics. This may allow protein-protein interactions to be characterized in the appropriate tissue for a particular biological process or disease phenotype under physiological conditions, and for interaction landscapes to be compared across tissues. Here we demonstrate application to intraflagellar transport (IFT) proteins, which are WD40-domain-containing proteins that are essential for the formation and function of all types of cilia. We describe a method to generate mice with an endogenous C-terminal streptavidin/FLAG tag, using Ift80 as an example, and demonstrate the successful implementation of AP in this model. This method can easily be adapted for N- and C-terminal tagging of many other proteins in vivo.