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toside (X-Gal) staining. Titers were calculated by counting the number of foci of blue cells per well and dividing it by the dilution factor. Expression of luciferase was assayed by washing the cultures twice with TBS (50 mM Tris·HCl, pH 7.8/130 mM NaCl/10 mM KCl/5 mM MgCl2), extracting them with 0.5% Nonidet P-40 in TBS containing 1 mM dithiotreithol, and measuring luminescence in a luminometer. Concentrated vector stocks were prepared by ultracentrifugation of conditioned medium at 50,000×g for 90 mm, resuspension of the pellets in a small volume (half percent of the starting volume of medium) of TBS containing 10 mM MgCl2, pooling, and incubation with or without 0.1 mM of each of the four deoxynucleotides (dNTPs), 3 mM spermine, and 0.3 mM spermidine for 2 hr at 37°C. After dilution in TBS, the vector particles were concentrated by a second ultracentrifugation, and the final pellet was resuspended in a very small volume (half thousandth of the starting volume of medium) of sterile saline containing 4 μg of polybrene per ml. Resuspension of the second pellet required prolonged incubation and pipetting. Stocks were stored frozen at −80°C and titered before and after freezing. MLV-based β-gal vector was similarly produced by the transient transfection into 293T cells of a plasmid driving the MLV gag and pol genes from the hCMV promoter (the vector pLNL-CMVLacZ, which carries the same β-gal expression cassette as the HIV-based vector) and the pMD.G plasmid. Before injection, all batches of vector were tested for the absence of replication-competent virus by infecting HeLa cells at high multiplicity of infection (HeLa P4 cells, which express CD4 and contain an integrated LacZ gene driven by the HIV long terminal repeat), obtained from the American Type Culture Collection stock (23), and HeLa cells previously transduced with a lentiviral vector carrying a different reporter gene and selected for its expression. The transduced cells were passaged two to three times, and the conditioned medium was tested for transfer of the markers to virgin 208F fibroblasts or for β-gal-inducing activity on P4 cells.

In Vivo Delivery of Vectors and Immunostaining of Sections from the Injected Brains. All animal procedures were performed according to an institution-approved protocol and under strict biological containment. Adult female Fischer 344 rats were anesthetized (ketamine, 44 mg/kg; acepromazine, 0.75 mg/kg; and xylazine, 4 mg/kg, in 0.9% NaCl i.p.) and positioned in a stereotactic head frame. After midline incision of the skin, holes were drilled in the appropriate locations in the skull with a dental bur, and a 5-μl Hamilton syringe with needle was used to slowly inject 2 μl of vector suspension in sterile saline into the striatum (AP +0.2, ML±3.5, DV −4.5) and hippocampus (AP −3.5, ML 3.0, DV −4.0) bilaterally. Holes were then filled with bone wax, the incision was sutured, and the animals were returned to their cages. For assessing transduction, the rats were deeply anesthetized and perfused with 4% cold paraformaldehyde and 0.2% glutaraldehyde intracardially. The brains were removed, post-fixed at 24 hr, saturated in 30% sucrose, and sectioned on a freezing microtome (50-μm serial sections). For light microscopy, sections were incubated with rabbit anti-β-gal antibodies (1:1000; Cortex Pharmaceuticals, Irvine, CA) and stained using avidinbiotin-peroxidase (Vectastain ABC Elite; Vector Laboratories) and diaminobenzidine. For immunofluorescence labeling, mouse monoclonal anti-NeuN and guinea pig anti-GFAP antibodies, secondary antibodies coupled to the fluorescent markers CY5, dichlorotriazinyl amino fluorescein, and Texas Red were also used, and the mounted sections were analyzed by confocal scanning laser microscopy (Bio-Rad model MRTC600). Fluorescent signals were collected, digitally color enhanced, and superimposed.

FIG. 1. Schematic of the generation of lentiviral vector. The relevant portions of the three plasmids cotransfected into 293T cells are depicted at the top; their contribution to the vector particles harvested in the transfectant conditioned medium are depicted at the bottom. pCMVΔR8.2, the packaging construct, provides all vector proteins but the envelope. The viral genes are expressed from the hCMV promoter and the insulin polyadenylylation site. Proviral 5′ leader and Ψ sequences have been deleted together with a large portion of the env gene. A translation stop codon was inserted upstream of the remaining env segment. The major 5′ splice donor site (SD) has been conserved. pHR′, the transducing vector, provides the vector genome. The viral long terminal repeats (LTRs) and the Ψ sequence are indicated. The gag gene is truncated after 350 bp and is out of frame (×), and it follows the Rev responsive element (RRE) and a splice acceptor site (SA). The position of a hCMV-driven expression cassette for the β-gal cDNA (LacZ) is shown. pMD.G encodes the heterologous VSV envelope that pseudotypes the vector. The hCMV promoter drives the VSV.G reading frame, which has β-globin sequences upstream (exons 2 and 3, intervening sequence 2) and downstream [poly(A) site].



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