The AAV vector pTRCGW (5.6 kb), (a kind gift from Paul Dijkhuizen, The Netherlands Institute for Brain Research, Amsterdam, The Netherlands), carrying the ampicillin resistance gene, inverted terminal repeats (ITR), human immediate early cytomegalovirus promoter (CMV), wood chuck posttranscriptional regulatory element (WPRE) and multiple restriction sites, was used to construct the rAAV-Cre vector plasmid. The 1.1 Kb fragment of Cre gene along with the nuclear localization signal (NLS) was excised from pNSE-Cre  by digesting with Hind III and NdeI restriction enzymes and blunted on both sites using Ecoli DNA polymerase I (klenow fragment). AAV-Cre was created by inserting NLS-Cre between two ITRs in pTRCGW vector that had been digested with Spe1 and Bam H1 sites, were also blunt ended by using klenow fragment.
To generate the LV-Cre-EGFP plasmid, a 2 Kb NotI/BglII fragment containing Cre-EGFP was isolated from pCMV-Cre-EGFP, blunted, and ligated into the multiple cloning site of the lentiviral transfer vector p156RRLsin-PPThCMV-GFP-pre [6, 34, 35]. This vector contains a CMV promoter along with the HIV-1-Sin 18 LTR and the HIV-1 genomic RNA packaging signal. pCMV-Cre-EGFP was created by inserting NLS-Cre derived from pJTCRE3  into the multiple cloning site of pEGFP-N3 (Clontech, CA, USA).
AAV-Cre Viral vector production
Ten culture dishes (100-mm diameter), each containing 5 × 106 293T cells, were co-transfected by calcium phosphate with 5 μg of either AAV-GFP or AAV-Cre vector plasmid and 15 μg of the packaging plasmid(PDG)  Twelve-fourteen hrs following the transfection, medium was replaced by complete Dulbecco's Modified Eagle's Medium (DMEM, Gibco) containing 10% FCS (Gibco) and 1% of a mixture of penicillin (100 lU/ml) and streptomycin (100 pg/ml), in which the cells were incubated for 48 hrs at 37°C and 5% CO2. The cells were dislodged from the culture dishes in 2 ml of PBS, pH 7.5, 1 mM MgCl2 and 10 mg/ml DNase I and freeze-thawed three times in a dry ice/100% ethanol bath and 37°C water bath. The cell debris was spun down at 4000 rpm for 20 min at 4°C. The supernatant was passed through a heparin-agarose beads column (H-6508 Sigma, Econo column, BioRad CA, USA) followed by Iodixanol purification (Nycomed Pharma AS, Oslo, Norway) and centricon concentrator (Amicon, USA) as described previously . The transgene expressing unit titre (tu/ml) was determined by co-infection of 293T cells with serial dilutions (10-2, 10-3, 10-4, 10-5 and 10-6) of the AAV-GFP or AAV-Cre viral vector stock in the presence of adenovirus (multiplicity of infection, MOI = 3). The titre of AAV-GFP was determined by counting GFP positive cells in a fluorescence microscope following 16 hrs infection. For detection of AAV-Cre, cells were washed with PBS, fixed with 4% PFA and processed for the immunohistochemical detection of Cre as described below. The titre was found to be 1 × 108 tu/ml for both AAV-GFP and AAV-Cre.
To determine if Cre was expressed in AAV-Cre infected cells in vitro, 293T cells were plated in 6 well plates (5 × 105 per well) in complete DMEM. Cells were infected with AAV-Cre (1:10,000) in the presence of adenovirus (MOI = 3). Fourteen-forty eight hrs following the infection, cells were washed with PBS and fixed with 4% PFA for 15 min, followed by washing with PBS (3 × 10 min). Cells were then incubated with antibody to Cre (1:200, Berkeley, CA, USA), diluted in 50 mM Tris-HCl, pH 7.5, 0.5 M NaCl and 0.1% Triton) overnight at 4°C. The following morning, cells were washed with PBS (3 × 5 min) and incubated with ABC solution (Avidin Biotin Complex, Vector laboratories, CA USA) for an hour at room temperature. The cells were washed with PBS (3 × 5 min) and allowed to react for 5 min in a solution of 0.05% diamino benzidine and 0.03% hydrogen peroxide in PBS. The cells were washed with PBS to terminate the reaction and subsequently observed under a bright-field microscope (Zeiss Axiovert 10).
LV-Cre-EGFP viral vector production
For viral vector production, the LV-Cre-EGFP transfer plasmid was co-transfected together with the viral core packaging construct pCMVdeltaR8.74 and the VSV-G envelope protein vector pMD.G.2 into 293T cells as previously described. The original transfer vector and the helper plasmids were obtained from Dr. L. Naldini (Institute for Cancer Research, University of Torino, Italy). Briefly, 5 × 106 293T cells were seeded in 10 cm dishes 24 hrs prior to transfection in complete Iscove's Modified Dulbecco's Medium (IMDM) containing 10% FCS, glutamine and 1% penicillin/streptomycin in a 5% CO2 incubator, the culture medium was changed 2 hrs prior to transfection. Ten μg of transfer vector plasmid, 6.5 μg envelope plasmid, and 3.5 μg of packaging plasmid were co-transfected using the calcium phosphate method. The medium was replaced after 14–16 hrs and the conditioned medium was collected 24 hrs later, cleared by low-speed centrifugation (1000 rpm for 5 minutes) and filtered through 0.22 μm cellulose acetate filters. The supernatant was concentrated about 100 fold by ultra centrifugation (20,000 rpm for 2.5 hrs). The pellet was re-suspended in PBS and virus was aliquoted and stored at -80°C. The titre was determined by infecting 293T cells with serial dilution of the LV-Cre-EGFP virus (10-3, 10-4, 10-5 and 10-6) in complete IMDM. Cells were incubated for 16 hrs and the medium was replaced with fresh IMDM and cells were incubated for 24 hrs. The titre was determined by counting GFP positive cells under a fluorescence microscope. This was confirmed by counting positive cells after Cre-immunocytochemistry as described above. The titre of the first stock was 1.5 × 1010 tu/ml and the second stock was found to be 2 × 109.
To determine if Cre was expressed in LV-Cre-EGFP infected cells in vitro, 293T cells (5 × 105 per well) were plated in complete IMDM. The next day, cells were infected with LV-Cre-EGFP (1:10,000) in complete IMDM medium for 16 hrs, medium was replaced with fresh medium and incubated for 24–48 hrs. These experiments were replicated 3 times. For immunofluorescence labelling, cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min. followed by washing with PBS (3 × 10 min). Cells were then incubated with the Cre antibody (1:200, diluted in 50 mM Tris-HCl, pH 7.5, 0.5 M NaCl and 0.1% Triton) overnight at 4°C. Next morning cells were washed with PBS (3 × 10 min) and incubated with Cy3-conjugated goat anti-mouse (1:400, Jackson laboratories PA, USA) for an hour at room temperature. Cells were washed with PBS (3 × 10 min), mounted on slides and observed under a confocal microscope (LSM 410 Zeiss) using 488 nm and 543 nm excitations for GFP and Cy3 respectively.
AAV and LV delivery of Cre into the brain
Rosa26 reporter mice (22–30 g) were deeply anaesthetised by a fluanisone /fentanyl mixture containing Hypnorm (Janssen Pharmaceutical, UK) and Dormicum (Roche, NL) and water in a ratio of 1:1:2 (10 μl/gm I.P.). Virus was injected into the cortex and hippocampus via 30G stainless needle attached by polyethylene tubing to a 10-μl Hamilton syringe and a Harvard 22 microinjection pump. 0.8 μl of a mixture of AAV-Cre and AAV-GFP (AAV-Cre/AAV-GFP mixture, 9:1) or 0.5 μl LV-Cre-EGFP virus was injected into the cortex (anterioposterior -2, lateral + 1.3, dorsoventral -0.45) and hippocampus (anterioposterior -2, lateral + 1.3, dorsoventral -1.55)  at a rate of 0.2 μl/min. The needle remained in place for an additional 5 minutes to facilitate the controlled delivery of virus. Three days and 1, 2 and 4 weeks following the injections mice were perfused with 4% PFA. Four mice were examined for each time point. Sections of the brain were cut in the coronal plane at a thickness of 50 μm with a vibratome and processed for β-galactosidase enzyme histochemical, immunohistochemical, and double immunofluorescence studies.
β-galactosidase staining was done by incubating the sections for an hour at 37°C with X-gal-solution containing 5 mMK4Fe(CN)6, 5 mM K3Fe(CN)6, 2 mM MgCl2, 1 mg/ml 5-bromo-4-chloro-3-indolyl-D-galactoside in PBS. The X-gal reacted sections were washed with PBS (3 × 10 min), dehydrated and embedded in entellan mounting medium. Immunohistochemistry for GFP was carried out by the ABC method using diamino-benzidine as a substrate , and with rabbit anti GFP (1:100, Chemicon, CA, USA) as primary antibody and biotinylated anti-rabbit (1:200, Vector) as secondary antibody.
Colocalization of Cre and GFP was examined by using a double immunofluorescence. A mixture of biotin labelled monoclonal Cre antibody (1:1000) and polyclonal GFP antibody (1:100) was used as primary antibodies, while a mixture of streptavidin-conjugated horseradish peroxidase (1:200, Life Sciences, MA, USA) and FITC conjugated anti-rabbit (1:100, Sigma) was used as secondary antibodies. Cre signal was enhanced by incubating the sections in fluorophore labelled tyramide solution (1:200, Life Sciences) for 30 min at RT, mounted sections were analyzed by fluorescence microscope fitted with SP confocal head (Leica DMRE) using Leica confocal software.