Generation of transgenic mice
Transgenic animals were created from transgene vectors based on the tetPrP.Xho vector [21, 22]. First, we constructed a cDNA that sequentially encodes tau-ubiquitin-luciferase as a contiguous gene (Fig. 1). The tau gene is a human cDNA for 0N4R tau that encodes the P301L mutation. This cDNA has been expressed in mice previously [23]. The cDNA for ubiquitin is derived from human and the cDNA for luciferase is derived from firefly (Phontinus pyralis). The fused cDNA was generated by polymerase chain reaction (PCR)-based methods that amplified each of these genes independently using primers that produced novel restriction endonuclease sites to enable stitching the gene together to create a single open-reading-frame for a poly-protein that we abbreviated as TUL.
After linearizing the vector plasmid with Not1 enzymes that separate the bacterial plasmid sequences from the promoter element and tetPrP.Xho vector sequences, the transgene DNA fragment was purified by agarose gel electrophoresis and injected into fertilized embryos from FVB/NJ mice in the University of Florida Mouse Modeling Core. From these injections, two founders were identified by PCR of the genomic DNA extracted from tail biopsy by standard methods of proteinase K digestion, high salt precipitation, and ethanol precipitation. The PCR protocol used was as follows: ddH2O 20.7 μL, 10X PCR buffer (w/MgCl2) 2.5 μL, 5 mM dNTPs 0.5 μL, 50 μM PrP-AS (antisense primer -5′-CCA AGC CTA GAC CAC GAG AAT GC-3′), 50 μM TUL-S (sense primer – 5′-CAA ATT GTA ACT CGA TCA GGC CCC TGG GGC GGT-3′), Taq DNA Polymerase (New England Biolabs, Ipswich, MA, USA) 0.1 μL, Tail DNA 1 μL. The PCR program used was as follows: 94 °C for 30 s, 55 °C for 60 s, 72 °C for 3 m, 35 cycles, followed by 72 °C for 10 min then hold at 10 °C.
One of these founders efficiently transmitted the transgene to offspring, producing a line termed Y-74. Mice from this line were crossed to mice that express the tet-Transactivator (tTA) under the CamKIIα promoter [24] [B6.Cg-Tg(Camk2a-tTA)1Mmay/Dbo, Jackson Laboratories, Bar Harbor, ME, USA], which had been backcrossed to FVB/NJ mice twice. Mice bigenic for CamKII-tTA and tet.TUL were expected to express the transgene exclusively in the forebrain.
All procedures involving mice were reviewed and approved by the University of Florida Animal Care and Use Committee. The mice described here are available through the Jackson Laboratories.
Immunoblotting
To detect transgenic human tau expression in brain, mice were deeply anesthetized and then perfused with ice-cold PBS. The brains were then quickly removed and the forebrains were dissected and flash frozen on dry ice. To homogenize the brain, the tissue was sonicated, using an ultrasonic probe (Misonix microson ultrasonic cell disrupter XL, Farmingdale, NY USA) at an intensity setting of 2. Each brain was sonicated three times for 10 s each in 1× PBS (phosphate-buffered saline) containing 1:100 v/v protease inhibitor cocktail (Sigma, St. Louis, MO, USA). The tissue homogenate was centrifuged at 17,000 × g for 5 min to produce a clarified brain homogenate. The protein concentrations of the resulting homogenates were then determined by bicinchoninic acid assay, as described by the manufacturer (Pierce Biotechnology/Thermo Fisher, Waltham, MA, USA). Fifty micrograms of each brain homogenate were mixed with 2× Laemmli sample buffer and boiled for 5 min. Sample buffers contained β-mercaptoethanol to reduce any disulfide bonds and the samples were electrophoresed in 4–20 % Tris-Glycine sodium dodecyl sulfate-polyacrylamide gels (Invitrogen Life Technologies, Grand Island, NY, USA). Following transfer to nitrocellulose membranes (Amersham/GE Healthcare Life Sciences, Pittsburgh, PA, USA), the membranes were blocked in 5 % milk in PBS-T (1X PBS, 0.1 % Tween-20) for 1 h, then incubated for 2 h at room temperature with the mouse monoclonal antibody Tau 13 (Covance, Princeton, NJ, USA) at 1:4000 in PBS-T and 5 % milk. The membrane was then washed with PBS-T, then incubated for 1 h at room temperature with a goat-mouse secondary antibody (KPL, Gaithersburg, MD, USA) at 1:5000 in PBS-T and 5 % milk before developing with enhanced chemiluminescence reagents (Thermo Scientific Inc., Rockford, IL, USA) and visualizing with a FluorChem E imager (Protein Simple, Santa Clara, CA, USA).
Imaging
For in vivo imaging of the bigenic tTA/TUL mice, we used the Perkin Elmer IVIS Spectrum In Vivo System (Xenogen Biosciences Perkin Elmer, Waltham, MA, USA). Mice were placed under deep anesthesia and were first imaged prior to injection with luciferin. Mice were then injected with 100 μL of 30 mg/mL XenoLight Rediject D-luciferin Ultra (Xenogen Biosciences Perkin Elmer, Waltham, MA, USA). Images were obtained on the IVIS imager from 6 min after injection (peak signal was previously determined at approximately 12 min after injection of luciferin). During every scan, images were taken at the same exposure setting (determined by the IVIS imager during the first scan) and multiple images were captured to insure that the peak signal had been reached. To quantify the bioluminescence for each animal, we used Living Image Analysis software (Xenogen Biosciences Perkin Elmer, Waltham, MA, USA). With this software, a box was drawn around the area of signal in the region of the head to define a region of interest (ROI). The area of the ROI for each mouse within the same image was equal. The average radiance within the ROI was measured and compared.
Ex vivo assessment of luciferase activity
Brains were harvested from mice as described above and stored at −80 °C. To prepare lysates, the brains were weighed and then homogenized in PBS (10 volumes relative to mass) as described above to produce a 10 % homogenate. The PBS contained protease inhibitor [1 μL of SIGMA protease inhibitor cocktail (catalog number: P8340-5ML; Sigma-Aldrich, St. Louis, MO, USA) for every 99 μL of PBS 1×]. The mass of the PBS 1× buffer with protease inhibitor was approximated volumetrically by assuming a density of 1 mg/μL at room temperature. To detect luciferase activity, 10 and 20 μL of each sample were dispensed to separate wells in an opaque 96 well plate and 40 and 30 μL of PBS 1× were added to the wells, respectively. Fifty microliters of luciferase substrate solution from the Promega Bright-Glo Luciferase Assay System (catalog number: E2610, Madison, WI, USA) were then added to each well and luminescence values were immediately collected by a plate reader (Biotek Synergy HT Microplate Reader, Winooski, VT, USA).