Animals and surgical procedures
Mice were intracranially injected with HC-Ads expressing β-Gal under the control of the
regulatable switch TetON. Briefly, mice were anesthetized using Ketamine (75 mg/kg) and
Medetomidine (0.5 mg/kg) and placed in a stereotactic frame modified for mice. All animals
were injected into the right striatum (stereotactic coordinates: 0.5 mm anterior, 2.2 mm lateral
from bregma and 3.2 mm ventral from the brain’s surface) with 1x107 blue forming units (BFU)
of HC-Ad in 1ul saline, using a 5 ul Hamilton syringe. Each injection was performed over a
period of 3 minutes, with the needle being left in place for additional 3-5 minutes before
withdrawal. To induce expression of regulatable transgene, mice were given special diet chow
containing doxycycline (2000 ppm, NewCo Distributors, Rancho Cucamonga, CA) starting
twenty-four hours prior to intracranial HC-Ad delivery. At experimental endpoints (3 weeks and
7 weeks after brain injection) mice were anesthetized via intraperitoneal injection of an overdose
of Ketamine (50 mg/kg) and Xylazine (50 mg/kg) and perfused with oxygenated Tyrode’s
solution (0.14 M NaCl, 1.8 mM CaCl2, 2.7 mM KCl, 0.32 mM NaH2PO4, 5.6 mM glucose and
11.6 mM NaHCO3) or perfused-fixed with oxygenated Tyrode’s solution followed by 4%
paraformaldehyde in PBS by means of trans-cardial perfusion. Brain tissue was removed for
transgene expression functional assay (β-Galactosidase activity assay) or post-fixed for 48 hours
before brains were serial sectioned using an electronic VT1000S vibrating blade vibratome
(Leica, Wetzlar, Germany) to obtain 30-um free-floating sections for immunohistochemistry.
Plasmid construction
We constructed a plasmid expressing the TetON switch, comprising the rtTA2SM2 and the
tTSKid cDNAs (kindly provided by Dr. H. Bujard from ZMBH, Germany) under the control of
the CMV promoter (pCI-rtTA2SM2-IRES-tTSKid-pA, pCI-TetON, Fig. 1B) to immunize mice
against the regulatable switch. This plasmid was generated by inserting the XhoI/MluI flanked
rtTA2SM2 coding sequence (excised from p[rtTA2SM2-IRES-tTSKid-pA]12 into the XhoI/MluI
site of pCI (pCI-rtTA2SM2) and then inserting a MluI/SalI flanked IRES-tTSKid-pA coding
sequence into the Mlu/SalI site of pCI-rtTA2SM2. A plasmid encoding the reporter gene βGalactosidase under the control of the CMV promoter (pCI-β-Gal, Fig. 1B) was constructed to
immunize mice against the transgene. This plasmid was developed by ligating a SalI flanked βGalactosidase gene from pAL120-LacZ44 into the SalI site of pCI Vector (Promega, Madison,
WI) (Fig. 1 B). The engineering of the β-Gal and regulatable TetON switch components was
previously performed and described by us in detail elsewhere12.
Production, scale up and purification of HC-Ad vectors
HC-Ad vectors expressing β-Galactosidase under the control of the regulatable mCMV- TetON
(HC-Ad-mTetON-β-Gal) switch were scaled up from vector seed stocks, purified, and titrated as
described by us and others previously12,
30, 31, 43
. Briefly, 293-N3S cells expressing Cre
recombinase (116) growing in suspension were infected with 100 viral particles per cell and coinfected with a helper virus with its packaging domain flanked by loxP sites. Two days later
cells were harvested and virus was purified by three cesium chloride gradients and titrated for
viral particles, PFU, and blue forming units (BFU).
Production of anti-TetON antibody
We generated a rabbit polyclonal antibody (anti-TetON) specific for the transactivator sequence,
i.e., rtTA2S-M2. An antigenic peptide was synthesized (New England Peptide, Gadner, MA) and
then conjugated to KLH using a disulphide linkage between the C-terminal cytosine (C) and an
internal KLH residue (New England Peptides, Gardner, MA). This was then used to raise
antibodies against TetON in New Zealand white rabbits. In brief, New Zealand white rabbits
were immunized three times at 14 days apart with the purified peptide. After the third
immunization, the titer of the antibody was verified by ELISA (1:64,000) and whole serum was
recovered for characterization by immunohistochemistry in cells transfected with pCI-TetON
and Western Blot in COS-7 cells infected with an adenovirus encoding the TetON switch (not
shown) or with a control vector without transgene (Ad-0). Cells were harvested in RIPA lysis
buffer 48 hours later and 20µg total protein was loaded in a 12% SDS PAGE gel with 5% for
stacking and run at 130 volts for 90 mins. The gels were then transferred onto nitrocellulose
membranes (Bio-Rad, Hercules, CA) and probed with the novel rabbit anti-TetON (1:100) for 4
hours. A Horseradish Peroxidase (HRP) conjugated sheep anti-rabbit (Bio-Rad, Hercules, CA)
was used as secondary antibody at a dilution of 1:2000 and incubated for 2 hours. The blots
were then developed with the ECL Western Blotting Analysis Kit (Amersham Biosciences,
Piscataway, NJ).
Assessment of antigen specific immunity by IFN-γ ELISPOT assay
The number of IFN--producing T cells was assessed using the enzyme-linked immunospot
(ELISPOT) kit assay (R&D Systems Inc., Minneapolis, MN) according to the manufacturer's
instructions. Briefly, splenocytes (1 x 106 cells/well) were cultured in Millipore MultiScreen
plates (coated with anti-IFN- antibody) for 24 hrs in X-Vivo media (Cambrex, Baltimore, MD)
containing either the tetracycline transactivator (tTA2) pure protein (1 g/ml, kindly provided by
Dr. Philippe Moullier, INSERM ERM, Nantes, France)18 or the -Galactosidase pure protein (5
g/ml) (Sigma Aldrich, St-Louis). Twenty four hours later, the ELISPOT wells were washed
and incubated overnight at 4°C with biotinylated anti-IFN- detection antibody (R&D Systems
Inc., Minneapolis, MN). Reactions were visualized using streptavidin-alkaline phosphatase, 5bromo-4-chromo-3-indolylphosphatase p-toluidine salt, and nitro blue tetrazolium chloride as
substrate. The number of spots per 106 splenocytes, which represents the number of IFN-producing cells, were counted with the KS ELISPOT automated image analysis system (Zeiss,
Jena, Germany).
Immunohistochemistry
Transgene expression in coronal brain section or in cryostat sections from muscle was
determined using rabbit polyclonal anti-β-Galactosidase (1:1,000) or anti-TetON antibodies
(1:300) generated in our laboratory10,
45
. To detect infiltration of macrophages and T cells,
floating brain sections were pretreated with 10 mM citrate buffer (pH: 6) for 20 minutes at 65oC
and then incubated with rat anti-mouse F4/80 antibody (1:500, Serotec, Raleigh, NC) and
polyclonal rabbit anti-human CD3 (1:500, DakoCytomation, Glostrup, Denmark), respectively.
Dopaminergic neurons were stained using rabbit anti-TH (1:5000, Calbiochem, Darmstadt,
Germany) and myelinized fibers using mouse anti-MBP (1:1000, Chemicon, Temecula, CA).
Antibodies were diluted in TBS containing 1% horse serum, 0.5% Triton X-100, and 0.1%
sodium azide, followed by biotin-conjugated secondary antibodies (1:800, DAKO, Glostrup,
Denmark). Secondary antibody binding was revealed using Vectastain ABC Elite kit (Vector
laboratories, Burlingame, CA) followed by diaminobenzidone (DAB) and glucose oxidase.
Sections were mounted on gelatin-coated glass slides and dehydrated in graded ethanol series
solutions and xylene before being mounted.
β-Galactosidase enzymatic activity assay
Three and seven weeks after intracranial administration of HC-Ads, mice were perfused and a
block of brain tissue around the injection site was dissected, homogenized, and subjected to
multiple freeze thaw cycles. Cell debris were removed by centrifugation and the supernatant,
containing protein extracts in PBS with a cocktail of protease inhibitors cocktail EDTA-Free
(Pierce, Rockford, IL), was stored at -70ºC until use. Regulatable expression of β-Galactosidase
from HC-Ad vector was tested in brain tissues by measuring -Galactosidase activity. Galactosidase assay, protein quantification assay, and enzymatic activity rate were performed
and measured as described earlier12. β-Galactosidase activity data were normalized by protein
content and incubation time.
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Gerdes, CA, Castro, MG and Lowenstein, PR (2000). Strong promoters are the
key to highly efficient, noninflammatory and noncytotoxic adenoviral-mediated
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Smith-Arica, JR, Morelli, AE, Larregina, AT, Smi, J, Lowenstein, PR and Castro,
MG (2000). Cell-type-specific and regulatable transgenesis in the adult brain:
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expression. Mol Ther 2: 579–587.