Affiliation: University of Texas Southwestern Medical Center
Submitted by: Brandon Holmes
Posted Date: 01 Oct 2014
This assay was designed to detect small amounts of tau seeds in biological samples, such as brain homogenates from humans or rodent tauopathy models. HEK-293T cells stably expressing a human tau sequence were engineered to serve as biosensors of intracellular tau aggregation in a fluorescence resonance energy transfer (FRET)-based assay. Specifically, the biosensor cells express a tau repeat domain (RD) sequence containing the P301S mutation tagged to either cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP). At baseline, this tau protein is maintained in a soluble, monomeric form and is FRET negative. Upon introduction of tau seeds to the biosensor cells, which is aided by the co-application of phospholipids, the tau protein aggregates, and a FRET response can be sensitively and quantitatively measured using flow cytometry. This assay is compatible with multiple seed sources, including recombinant and synthetic tau seeds, as well as brain homogenate.
- Holmes BB, Furman JL, Mahan TE, Yamasaki TR, Mirbaha H, Eades WC, Belaygorod L, Cairns NJ, Holtzman DM, Diamond MI. Proteopathic tau seeding predicts tauopathy in vivo. Proc Natl Acad Sci U S A. 2014 Oct 14;111(41):E4376-85. Epub 2014 Sep 26 PubMed.
tau, seeding, prion, FRET