1. Volume 4, Issue 2, Pages 149-156 (August 2001)
Production and Neurotropism of Lentivirus Vectors Pseudotyped with Lyssavirus Envelope Glycoproteins 
Nathalie Desmaris, Assumpcio Bosch, Christine Salaün, Caroline Petit, Marie-Christine Prévost, Noël Tordo, Pierre Perrin, Olivier Schwartz, Hugues de Rocquigny, Jean Michel Heard 
Molecular Therapy 
Volume 4, Issue 2, Pages (August 2001)
DOI: /mthe
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

2. FIG. 1
Detection of the VSV-G or MK-G envelope expression and fusogenicity in cells producing pseudotyped HIV-GFP vectors. HeLa cells were transfected with plasmid DNA encoding the HIV-GFP vector genome, the HIV packaging components (p8.91), and either the VSV-G (pMD.G), or the MK-G envelope. Cells were fixed 36 h later and stained for VSV-G with the Cy3-conjugated monoclonal antibody P5D4, or for MK-G using a rabbit antiserum and Texas red conjugated anti-rabbit-IgG. Syncitia analyzed for GFP (in green) and envelope expression (in red) by confocal microscopy are shown. Bars, 10 μm.
Molecular Therapy 2001 4, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

3. FIG. 2
Morphology of HIV-1 vector particles pseudotyped with the VSV-G or the MK-G envelope. Viral particles contained in culture supernatants (200 ml) collected 36 h after exposure of 293T cells to plasmid DNA encoding the HIV-lacZ genome, the HIV packaging components (p8.91), and either the VSV-G (pMD.G) or the MK-G envelope were pelleted by ultracentrifugation. Pellets were resuspended in 3% glutaraldehyde, stained with uranyl acetate and osmic acid, and laid on electron microscope copper grids. Wild-type HIV-1 particles from infected HeLa cells that were similarly processed are shown as a control. Bars, 50 nm.
Molecular Therapy 2001 4, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

4. FIG. 3
Incorporation of VSV-G or MK-G envelopes into HIV-lacZ vector particles. Vector particles were collected in cell supernatants 36 h after exposure of 293T cells to plasmid DNA encoding the HIV-lacZ vector genome, the HIV packaging components (p8.91), and either the VSV-G (pMD.G) or the MK-G envelope. The envelope expression vector was omitted in mock controls. Supernatants containing equivalent amounts of p24 were pelleted by ultracentrifugation, resuspended in Laemmli sample buffer, and analyzed by western blot using the monoclonal P5D4 anti-VSV-G antibody or a rabbit serum against MK-G. Molecular mass markers are in kDa.
Molecular Therapy 2001 4, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

5. FIG. 4
Gene expression in mouse brain after intrastriatal injection of the HIV-βglu vector pseudotyped with the VSVG or the MK-G envelope. Mice deficient for βglucuronidase received a single intrastriatal stereotactic injection of the HIVβglu vector, which encodes human βglucuronidase, and were sacrificed four weeks later. Coronal cryosections were stained for βglucuronidase activity. No staining is visible in the brain of noninjected controls (mock). Injection of two doses of the VSV-G pseudotype (50 and 23 ng of p24) shows that the size and the intensity of the stained area depend on the amount of injected vector. Comparison of the signals induced by the injection of equivalent amounts of vector pseudotyped with the VSV-G or with the MK-G envelope shows a more intense and more diffuse staining with the VSV-G pseudotypes, indicating that these particles are more infectious in vivo than MK-G pseudotypes.
Molecular Therapy 2001 4, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

6. FIG. 5
Brain cell tropism and retrograde axonal transport of HIV-lacZ vectors pseudotyped with the VSV-G or the MKG envelope. Normal rats received intrastriatal stereotactic injections (8 × 104 IUs, A-D) or intranasal instillations (16 × 104 IUs, E-H), of HIV-lacZ vector pseudotyped with the VSV-G or the MK-G envelope. Animals were sacrificed six weeks later. Expression of E. coli β-galactosidase was detected in red on coronal cryosections of the striatum (A-D) or sagittal cryosections of the olfactive tubercles (E-H) by immunofluorescence. Sections were simultaneously stained in green for NeuN, a marker of neurons, or for GFAP, a marker of glial cells. Cells stained for E. coli β-galactosidase and NeuN (A, B, E, and F) and cells stained for E. coli βgalactosidase and GFAP (C, D, G, and H) are indicated (arrows). Cells stained for E. coli βgalactosidase alone are also indicated (asterisks). Bars, 10 μm.
Molecular Therapy 2001 4, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions