1. Volume 2, Issue 5, Pages 446-457 (November 2000)
In Vivo Transduction of Cerebellar Purkinje Cells Using Adeno-Associated Virus Vectors 
William F. Kaemmerer, Rukmini G. Reddy, Christopher A. Warlick, Seth D. Hartung, R. Scott McIvor, Walter C. Low 
Molecular Therapy 
Volume 2, Issue 5, Pages (November 2000)
DOI: /mthe
Copyright © 2000 American Society for Gene Therapy Terms and Conditions

2. FIG. 1
Diagrams of the AAV vector constructs used. See Materials and Methods for a description of the reporter genes and their promoters. PA, polyadenylation signal; neo, neomycin resistance coding sequence under control of the thymidine kinase (TK) promoter; arrows, transcription start sites.
Molecular Therapy 2000 2, DOI: ( /mthe )
Copyright © 2000 American Society for Gene Therapy Terms and Conditions

3. FIG. 2
Diagram of a sagittal view of a mouse cerebellum illustrating some injection sites used (dark circles). Depending on syringe depth, injections were to the deep cerebellar nuclei (DCN) or the cerebellar cortex. PCL, Purkinje cell layer.
Molecular Therapy 2000 2, DOI: ( /mthe )
Copyright © 2000 American Society for Gene Therapy Terms and Conditions

4. FIG. 3
β-Galactosidase gene transfer and expression in mouse cerebellum. (a) Sagittal section of the cerebellum of a mouse injected with β-gal protein. Brightfield image showing β-gal activity by X-gal staining merged with image under fluorescent illumination for Cy2 showing calbindin immunoreactivity of Purkinje cells; arrows indicate the Purkinje cell layer; no Purkinje cell uptake of β-gal protein was evident; scale bar = 300 µm. (b) Sagittal section of the cerebellum of a mouse injected with 3 × 108 pfu Ad.RSV-βgal per site. In addition to β-gal activity in cell bodies throughout the white matter (white arrow), intensive β-gal activity in ependymal cells lining the fourth ventricle was visible (black arrow, bottom); scale bar = 300 µm. (c) Coronal section of the cerebellum of a mouse injected with 3 × 107 pfu Ad.RSV-βgal per site; β-gal activity was visible in cells in the white matter and in a sulcus. Much of the β-gal activity appearing in the Purkinje cell layer (white arrow) was not colocalized with the interior of Purkinje cells as visualized by calbindin (inset, white arrow); scale bar = 300 µm. (d) Mouse injected with 3 × 108 pfu Ad.RSV-βgal per site, sagittal section; 4× objective, scale bar = 300 µm. Inset: β-gal activity colocalized with the interior of Purkinje cell soma could occasionally be detected (white arrow); 20× objective. (e) Brightfield image of the cerebellum of a mouse injected with 1.7 × 104 fu vTR-CMVβ and Ad5 per site, showing β-gal activity in cell soma in the Purkinje cell layer (black arrows) and dendritic arbors projecting into the molecular layer. Some cells in the DCN are also transduced (asterisk); very little β-gal activity was detectable in the white matter; sagittal section, scale bar = 500 µm. (f) High magnification view of calbindin and X-gal stained section from same mouse as (e), showing detailed morphology of Purkinje cell soma and dendrites (arrows) containing β-gal activity transduced by AAV; inset: brightfield view of same Purkinje cell dendrites; scale bar = 50 µm.
Molecular Therapy 2000 2, DOI: ( /mthe )
Copyright © 2000 American Society for Gene Therapy Terms and Conditions

5. FIG. 4
Distribution of cells with β-gal activity in the cerebellar hemisphere of a mouse injected with vTR-CMVβ plus Ad5. Although the majority of Purkinje cells with β-gal activity were near the sagittal planes containing the injection sites (1250 and 2250 µm from medial), Purkinje cells with β-gal activity were detected throughout the medial–lateral extent of the hemisphere. In the hemisphere as a whole, the majority of cells with β-gal activity were Purkinje cells.
Molecular Therapy 2000 2, DOI: ( /mthe )
Copyright © 2000 American Society for Gene Therapy Terms and Conditions

6. FIG. 5
PCR assay for lacZ sequences. PCR products were generated using primers that amplify a 316-base-pair segment. Lanes: 1, Ad.RSV-βgal injected mouse; 2, vTR-CMVβ plus Ad5-injected mouse; 3 positive control [TgN(MTnlacZ)204Bri mouse]; 4, negative control (wild-type mouse); 5, 100-bp ladder; 6, HindIII-digested lambda phage marker. A product of the expected size was detected in mice injected with Ad.RSV-βgal, vTR-CMVβ plus Ad5, and the positive control.
Molecular Therapy 2000 2, DOI: ( /mthe )
Copyright © 2000 American Society for Gene Therapy Terms and Conditions

7. FIG. 6
eGFP expression in vTR-UF5 injected mice. (a) Sagittal section from a mouse injected with vTR-UF5 alone (5 × 106 fu in 2 µl per site) showing calbindin–Cy3 immunofluorescence (red) and eGFP expression. No eGFP expression was evident in Purkinje cells or in the white matter. Scale bar = 100 µm. Image is representative of results obtained from six mice. (b) Higher magnification image of the boxed portion in (a), showing the eGFP signal from the region of the deep cerebellar nuclei. Cellular morphology indicative of neurons within the nuclei is apparent. Scale bar = 20 µm. (c–f) Fluorescence microscopy of sagittal cerebellar sections from two mice injected with 7.5 × 106 fu each of vTR-UF5 and Ad5 in 4 µl per site. (c) Sagittal section showing eGFP-positive Purkinje cells surrounding the primary fissure; scale bar = 50 µm. (d) Section from a second mouse, with numerous eGFP-positive Purkinje cells rostral to the primary fissure in a section 1.5 mm from lateral, scale = 50 µm. (e) Same section as (d), showing eGFP-positive cells in the region of the DCN (asterisk), and eGFP signal visible in axons coursing through the white matter to the deep nuclei (arrow); scale bar = 100 µm. (f) Same mouse as (d), 3.2 mm from lateral, showing eGFP-positive Purkinje cells near dorsal surface; scale = 50 µm. This section is 0.6 mm from the plane of the nearest needle track; the inset diagram shows the location of these cells (box) in the sagittal plane relative to the needle track and DCN injection site.
Molecular Therapy 2000 2, DOI: ( /mthe )
Copyright © 2000 American Society for Gene Therapy Terms and Conditions

8. FIG. 7
Sagittal sections of mice injected with vTR-UF11 with or without Ad5, to cortical or DCN sites; all scale bars = 100 µm. (a) Mouse injected with vTR-UF11 and Ad5 to DCN sites. Arrow shows angle of needle track approaching the DCN (asterisk); note transduction of Purkinje cells distal from needle path, and numerous eGFP-positive cells in white matter and granular layer as well as Purkinje cell layer. (b) Mouse injected with vTR-UF11 alone to DCN sites; (c) Mouse injected with vTR-UF11 and Ad5 to cortical sites; (d) Mouse injected with vTR-UF11 alone to cortical sites. Coinjection with Ad5 (a, c) yields less specificity of transduction to Purkinje cells than does vTR-UF11 alone (b, d).
Molecular Therapy 2000 2, DOI: ( /mthe )
Copyright © 2000 American Society for Gene Therapy Terms and Conditions

9. FIG. 8
Counts of eGFP-positive Purkinje cells and other cell types in mice 7 days after injection of vTR-UF11 with or without Ad5, to cortical or DCN sites. (a) Coinjection of Ad5 has no effect on Purkinje cell transduction by vTR-UF11, but cortical injection yields a greater number of transduced Purkinje cells than DCN injection (**P ≤ 0.001). (b) In the absence of Ad5 coinjection, a greater percentage of the cells transduced by vTR-UF11 are Purkinje cells (*P ≤ 0.025). (c–f) medial–lateral distribution of eGFP-positive Purkinje cells and other cells, averaged across mice; (c) vTR-UF11 plus Ad5 to DCN, (d) vTR-UF11 plus Ad5 to cortex, (e) vTR-UF11 alone to DCN, (f) vTR-UF11 alone to cortex.
Molecular Therapy 2000 2, DOI: ( /mthe )
Copyright © 2000 American Society for Gene Therapy Terms and Conditions