1. Volume 5, Issue 5, Pages 528-537 (May 2002)
Targeted Transduction Patterns in the Mouse Brain by Lentivirus Vectors Pseudotyped with VSV, Ebola, Mokola, LCMV, or MuLV Envelope Proteins 
Deborah J. Watson, Gary P. Kobinger, Marco A. Passini, James M. Wilson, John H. Wolfe 
Molecular Therapy 
Volume 5, Issue 5, Pages (May 2002)
DOI: /mthe
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

2. FIG. 1
Selective transduction of striatum and external capsule by Mokola-, VSV-G-, MuLV-, Ebola-, and LCMV-pseudotyped vectors. (D), (F), (H), and (I) are counterstained with nuclear fast red. (A) Coronal hemisection of a mouse brain at bregma mm indicating level of injection. Larger outside box corresponds to (C–G); lower inside box corresponds to region shown in (H); upper inside box corresponds to (I). (B) Low-power photomicrograph showing robust β-galactosidase expression (blue) in the external capsule white-matter tract and striatum from Mokola-pseudotyped vector at 7.8 × 107 TU/ml. (C) Transduction of striatum and white matter by VSV-G-pseudotyped vector at 3.5 × 107 TU/ml. (D) Fewer cells in the striatum and white matter were transduced by a VSV-G-pseudotyped vector at a higher titer (1.2 × 109 TU/ml) but lacking the cPPT and WPRE. (E) Transduction of striatum and white matter by MuLV-pseudotyped vector. (F) Lack of β-galactosidase expression in striatum and white matter after transduction by Ebola-Z–pseudotyped lentivirus. (G) Minimal transduction of striatum and white matter by LCMV-pseudotyped vector. (H) High-power micrograph of β-galactosidase expression in striatum from VSV-G-pseudotyped vector. (I) Expression in white matter (external capsule) from Mokola-pseudotyped vector. Arrows indicate the dorsal and ventral boundaries of the white-matter tract. Bars: (A) 1 mm; (B and E) 500 µm; (C, D, F, and G) 200 µm; (H and I) 50 µm. lv, Lateral ventricle; str, striatum.
Molecular Therapy 2002 5, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

3. FIG. 2
Selective transduction of oligodendrocyte precursors and mature oligodendrocytes by VSV-G and Mokola pseudotypes. (A–D) Oligodendrocyte precursors. (E–H) Mature differentiated oligodendrocytes. Untransduced cultures (A and E) showed no background staining. Both the VSV-G (B) and Mokola (C) pseudotypes transduced oligodendrocyte precursor cells, but the Ebola pseudotype (D) did not. After differentiation, β-galactosidase expression was maintained in cultures that were transduced with VSV-G (F) or Mokola (G) vectors. Bar in (G) applies to (A–H): 20 µm. (I–L) Toxicity of VSV-G-pseudotyped vectors on cultured oligodendrocyte precursors. Inset panels are fluorescence photomicrographs of DAPI-stained nuclei. (I) Untransduced culture. (J) The VSV-G pseudotype was toxic at 8.0 × 106 TU/ml, but not at 8.0 × 105 TU/ml (K). (L) The Mokola-pseudotyped vector was not toxic at 4.8 × 106 TU/ml. Bars in (I) and inset apply to (I–L): 50 µm.
Molecular Therapy 2002 5, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

4. FIG. 3
Selective transduction of hippocampal and thalamic cells by Mokola-, MuLV-, LCMV-, Ebola-, and VSV-G-pseudotyped vectors. (A) Coronal hemisection of a mouse brain at bregma –2.12 mm indicating level of injection. Upper inside box corresponds to (B) and (E); lower inside box corresponds to region shown in (I–L); larger outside box corresponds to (H). (B) β-Galactosidase expression in the upper blade of the dentate granule neuron layer. (C) Limited transduction of the granule and subgranular layers by the LCMV pseudotype. (D) A rare transduced granule neuron with a process extending apically through the granule cell layer from the dentate gyrus after transduction with the VSV-G pseudotype. (E) Expression at the hilar border of the dentate gyrus from Mokola vector. (F) Enzyme histochemistry showing β-glucuronidase activity (red histochemical staining) at the hilar border of the dentate gyrus after transduction with a VSV-G-pseudotyped lentiviral vector encoding the human β-glucuronidase promoter and cDNA. Staining represents both transduced cells expressing enzyme and neighboring cells that have endocytosed secreted enzyme. (G) In situ hybridization with an antisense riboprobe recognizing β-glucuronidase, showing localization of only the transduced cells at the hilar border of the dentate granule cell layer. (H) Lack of expression in hippocampus and thalamus after transduction by Ebola-Z–pseudotyped lentivirus. (I–L) Transduction of thalamus. (I) MuLV-pseudotyped vector. (J) VSV-G-pseudotyped vector. (K) Mokola-pseudotyped vector. (L) LCMV-pseudotyped vector. Bars: (A) 1 mm; (B, J, L) 100 µm; (C) 20 µm; (E, I, K) 50 µm; (D) 10 µm; (F, G, H) 200 µm.
Molecular Therapy 2002 5, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

5. FIG. 4
Transduction of primary cultured CNS cells. (A–E) Cocultures of rat hippocampal neurons and astrocytes stained for β-galactosidase activity. (A) Untransduced culture. (B) VSV-G pseudotype. (C) Mokola pseudotype. (D) Ebola pseudotype. (E) A VSV-G-pseudotyped vector encoding eGFP transduced astrocytes in the culture; note ramified morphology of green cells similar to (B) and lack of colocalization with neuronal nuclear (NeuN) staining in red. (F) Culture of purified astrocytes transduced with a VSV-G-pseudotyped vector encoding eGFP. (G) β-Galactosidase expression in a Purkinje neuron in a culture of cerebellar cells transduced with a VSV-G-pseudotyped vector. (H) Bipolar granule neurons in a cerebellar culture after transduction with a VSV-G-pseudotyped vector. Bar in (H) represents 20 µm in (A–D) and (F–H), 40 µm in (E).
Molecular Therapy 2002 5, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

6. FIG. 5
Stability of pseudotyped lentiviral vectors. Stability of unconcentrated viral supernatant after 3 hours or 18 hours at 4°C (A) or three freeze–thaw cycles at –80°C and 37°C (B). Stability of concentrated lentiviral vectors after 3 or 18 hours at 4°C (C) or four freeze–thaw cycles at –80°C and 37°C (D). Values are shown as percentage of original titer from a representative experiment. Data for the VSV-G pseudotype are shown in the filled bars, the Ebola pseudotype in the striped bars, the Mokola pseudotype in the shaded bars, the LCMV pseudotype in the open bars, and the MuLV pseudotype in the hatched bars. Error bars represent standard deviations. Starting titers were (in TU/ml on 293T cells), for unconcentrated vectors, 2.4 × 107 (VSV-G), 6 × 104 (Ebola), 9 × 105 (Mokola), 1.51 × 106 (MuLV, 4°C), 1.2 × 106 (MuLV, –80°C), 1.3 × 106 (LCMV, –80°C), and 2.1 × 106 (LCMV, 4°C). For concentrated vectors, starting titers were 4.2 × 1010 (VSV-G), 2.9 × 107 (Ebola), 1.3 × 109 (Mokola), 1.87 × 108 (MuLV), and 2.9 × 109 (LCMV).
Molecular Therapy 2002 5, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions