Cell line and viral vectors
U251 human astrocytoma cell line was a gift from Victor Levin, University of Texas. To express transgenes, we used replication-deficient, type 5 adenovirus (AdV5, deleted for E1/E3) based on the Adeno-XTM Expression System (BD Biosciences Clontech, Carlsbad, CA). Molecular clones of human H and L were kindly provided by Paolo Arosio, University of Brescia, Italy. The cDNAs were inserted into appropriate restriction sites in pShuttle-2 and subcloned into pAd-X plasmid. The viruses were titered on HEK293 cells. The titers were as follows: LacZ AdV 3.7×1010 pfu/mL, L*H AdV 2×1010 pfu/mL, H AdV 1.4×1010 pfu/mL, L AdV 4.5×1010 pfu/mL. The EGFP AdV titer was 1×1011 pfu/mL and purchased from the University of Pittsburgh Vector Core Facility.
Primary antibodies used for staining of molecular markers were mouse anti-FLAG (F3165, Sigma-Aldrich, St. Louis, MO), rabbit anti-mouse glial fibrillary associated protein (GFAP) (G9269, Sigma), rabbit anti-mouse neurofilament (N4142, Sigma), rabbit polyclonal to Ki67 (ab15580, Abcam, Cambridge, MA), goat polyclonal to doublecortin (C-18 epitope, sc-8066, Santa Cruz Biotechnology, CA), and rabbit polyclonal to polysialylated neural cell adhesion molecule (PSA-NCAM, AB5032, Millipore, Billerica, MA). Secondary antibodies were donkey anti-rabbit Alexa Fluor 488 (A-21206, Molecular Probes, Carlsbad, CA), rabbit anti-goat Alexa Fluor 488 FAB (A-21222, Molecular Probes), goat anti-mouse Alexa Fluor 594 FAB (A-11020, Molecular Probes), and donkey anti-mouse Alexa Fluor 594 (A-21203, Molecular Probes). Primary antibodies used for the western blot analysis were mouse monoclonal for β-actin (sc-47778, Santa Cruz), mouse monoclonal for iron regulatory protein-2 (IRP2, sc-33682, Santa Cruz), mouse monoclonal to transferrin receptor-1 (TfR1, T8199-41, US Biological, Swampscott, MA), rabbit polyclonal to divalent metal transporter-1 (DMT1, NRAMP24-A, Alpha Diagnostic, San Antonio, TX), rabbit polyclonal to DMT1+IRE (NRAMP22-A, Alpha), where IRE is iron regulatory element, and goat polyclonal to the metalloreductase STEAP3 (sc-20531, Santa Cruz). Secondary antibodies were goat anti-rabbit horseradish peroxidase (HRP) conjugate (1858415, Pierce, Rockford, IL) and goat anti-mouse HRP conjugate (1858413, Pierce).
U251 cells were grown on glass slides and transduced with reporter transgenes. At 48 h post-transduction, cells were fixed using 4% paraformaldehyde (PFA). The cells were washed with phosphate buffer saline (PBS) and 0.2% Tween 20 (Bio-Rad, Hercules, CA) and then probed using antigen-specific antibodies followed by the secondary reagents as described above.
Western blot and enzyme-linked immunosorbent assay (ELISA)
U251 cells expressing the different ferritins and control reporter (LacZ) were incubated for 48 h post-transduction in 95% Dulbecco's Modified Eagle Medium, 5% fetal bovine serum and 1 mg/mL holotransferrin (#T0665, Sigma). Total protein content was measured with a bicinchoninic acid (BCA) assay kit (Pierce). Equivalent amounts of clarified samples were resolved on 4–20% polyacrylamide gradient gels (Pierce). We used chemiluminescence to expose the immunoreactive bands on film (1651454, Eastman Kodak, Rochester, NY). The films were scanned at 600 dpi and 16 bit grayscale using a flat bed scanner (Hewlett-Packard 8200), and the lanes were quantified using ImageJ software (http://rsbweb.nih.gov
). All protein levels were expressed as a percent of LacZ expression. For enzyme-linked immunosorbent assay (ELISA) analysis, cells were assayed with the Quantikine TfR kit (R&D Systems, Minneapolis, MN).
All animal experiments were approved by the Carnegie Mellon Institutional Animal Care and Use Committee (IACUC). Adult female C57BL mice (Harlan, Indianapolis, IN), 5–7 weeks old, were anesthetized using an intraperitoneal cocktail of ketamine and xylazine and placed in a head stereotactic device. Animals were injected with 5 μL of L*H AdV in the right striatum (anterior, lateral and ventral coordinates from Bregma 1.0, 2.2, 3.0 mm, n=7) and SVZ area (−1.0,−1.0, 2.2 mm, n=20). The contralateral control side was injected with the same viral load using LacZ AdV or GFP AdV. Animals were monitored until recovered and housed with food and water ad libitum. Mice injected in the striatum were imaged at day 5, and mice injected in the SVZ were imaged at 10 days post-injection. After imaging, animals were perfused transcardially with PBS and then with 4% PFA, and the brains flash frozen in optimal cutting temperature (OCT) compound (EMS, Hatfield, PA) and stored at −80 °C.
All MRI was performed at 11.7 Tesla using a Bruker microimaging system (Bruker, Billerica, MA). T2*-weighted images were acquired using a 3D gradient-echo (GRE) sequence. The mice were anesthetized, intubated, placed on a mechanical ventilator, and maintained on 0.75% isoflurane in 70% O2 and 30% N2O inhalation gas during the imaging sessions. For the in vivo imaging we used a 2.5 cm diameter, laboratory-built surface coil, 3D GRE with TE/TR=7/50 ms, 20 flip angle, 4 averages, 200×200×200 image points, field of view=1.2×1.2×1.2 cm and 60 μm isotropic resolution. The total in vivo scan time was 4.4 hours. For the ex vivo imaging we used a 20 mm diameter volume coil, 3D GRE with TE/TR=8/100 ms, 20° flip angle, 9 averages, 256×256×256 image points, field of view=1.28×1.28×1.28 cm and 50 μm isotropic resolution. The total ex vivo scan time was 16.5 hours.
Immunohistochemistry and histology
Frozen brain tissue was cryosectioned in 20 μm thick slices and incubated for one hour at room temperature with primary antibodies in a humidified chamber, washed and incubated for 45 min with secondary antibodies. Nuclei were counterstained with Hoechst 3342 (Calbiochem, La Jolla, CA). The control was an incubation of slices with secondary antibodies without the presence of primary. For the Perls' iron stain the slices were immersed in water with 2% potassium ferrocyanide and 2% concentrated hydrochloric acid for 30 min. Cell nuclei were counterstained with 0.5% nuclear fast red (H-3403, Vector Labs, Burlingame, CA). The X-gal stain for detection of β-galactosidase activity was performed using 5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside (B4252, Sigma).
Glass cover slips were mounted after the final wash, and the slides with tissue slices were imaged using a Carl Zeiss LSM 510 Meta UV DuoScan inverted confocal laser-scanning microscope. RGB channels were collected sequentially with a matrix resolution of 1024×1024 and 4 averages.
The significance of the western blot quantification was calculated using Origin 7.5 software (Northampton, MA). We used one-way analysis of variance (ANOVA) to look at differences of the group means, followed by Tukey's method for pair-wise differences with a confidence interval of 0.95. We also used a paired student t-test with a confidence interval of 0.95.