TBS-brain extracts were applied to a HEK293 cell tau aggregation seeding assay(Holmes et al, 2014, Sanders et al, 2014), in which TauRDP301S-CFP and TauRDP301S-YFP are co-expressed intracellularly. The formation of intracellular fluorescent TauRDP301S aggregates leads to Foerster resonance energy transfer (FRET) activity between co-aggregated CFP and YFP-tags and correlates with the tau aggregation seeding activity of the applied brain extracts. After 24 hours, cells treated with extract (0.5 µg and 1.0 µg total protein per 96 well) from 9-month-old rTg4510 had significantly more intracellular YFP-positive (white arrows) aggregates compared to rTg4510-Mapt0/0 extracts; FRET activity of TauRDP301S aggregates appeared similar for both rTg4510 and rTg4510-Mapt0/0. WT and Mapt0/0 extracts never showed seeding activity. Addition of lipofectamine corrected for differences in cellular uptake of tau and led to similar differences in seeding activity between rTg4510 and rTg4510-Mapt0/0 mice. Two-tailed Student's T-test, mean±SEM ns, not significant, Insets 100 µm, Scale bars 50 µm. 