Figure 1.
Schematic representation of lentiviral constructs.
A–B. Schematic of the lentiviral constructs used in this study. Arrows represent mammalian transcriptional start sites. In all vectors, the 3′-LTR contains a deletion in the U3 region which renders the virus self-inactivating (SIN 3′-UTR) and replication incompetent. A. The MND-tdTomato lentiviral vector contains a microRNA (miR30) and chloramphenicol resistance gene (CMR) in the 3′-UTR of the tdTomato fluorescent reporter gene. B. The internal promoters are MND, modified MoMuLV LTR containing myeloproliferative sarcoma virus enhancer; MSCV, murine stem cell virus LTR; UBC, Ubiquitin C promoter; and PGK, human phosphoglyercerate kinase promoter. Ψ: packaging signal; RRE, REV response element; cPPT, central polypurine tract; EGFP, enhanced green fluorescent protein; IRES, internal ribosome entry site; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element; LTR, long terminal repeat.
Table 1.
Purkinje neuron transduction by lentiviruses and AAV.
Figure 2.
Patterns of cellular transduction by Lenti-MND-tdTomato.
A. Montage of low magnification confocal images of sagittal sections of wild type mouse cerebellum injected with MND-tdTomato (red) and immunostained for calbindin (green), a marker for Purkinje neurons. A region of tdTomato expressing cells in the Purkinje and/or molecular layer is visible near the injection site, but the vast majority of tdTomato-expressing cells are in the white matter (wm). Arrowhead indicates approximate location of injection, where some damage to brain parenchyma can be seen. A’. A higher magnification image from an adjacent slide illustrating the lack of co-localization of tdTomato-expressing processes and calbindin positive Purkinje neuron dendrites. B–E. To examine transduction patterns in more detail, MND-tdTomato was injected into L7/pcp2-GFP mouse cerebellum, and sagittal sections were examined at higher magnification. L7/pcp2-GFP mice express GFP under control of the Purkinje cell specific promoter L7/pcp2. B, GFP expression is visible in Purkinje neuron somata, dendrites, and axons (ax), which project into the white matter tract (left panel and green, right panel). tdTomato-expressing somata are located in the white matter tracts and extend short processes (center panel and red, right panel). C. Purkinje neuron axons (left panel and green, right panel) and processes expressing tdTomato (center panel and red, right panel) do not overlap. D-E. High magnification images of the Purkinje layer of L7/pcp2-GFP cerebellum injected with MND-tdTomato. D. GFP-expressing Purkinje neuron somata with characteristic highly branched dendrites (left and green, right panel). Cells expressing tdTomato are located in the Purkinje layer but somata are smaller and processes are straight and unbranched (center). tdTomato (red) expression pattern does not colocalize with GFP (green) expressing Purkinje neuron somata or dendrites (right panel). The shape and location of tdTomato expressing cells is consistent with Bergmann glia. E. Coexpression of GFP (left) and tdTomato (center) in three Purkinje neuron somata (arrowheads, and yellow, right panel). Scale bars: 100 µm (A, B), 50 µm (A’), 25 µm (D, E).
Figure 3.
Expression of reporter genes in cerebellar cells transduced with lentiviral vectors under various promoters.
Representative confocal images of sagittal cerebellar sections from mice 7–14 days following intracerebellar injection of lentiviral vectors with indicated promoters. Dotted lines demarcate the border between cerebellar cortex layers. In low magnification images (B, D, F, G, and J), the line is drawn between Purkinje and granule layers. In high magnification images (A, C, E, H, and I), two lines are drawn to separate the Purkinje layer from the molecular layer and granule layer. A. Widespread GFP expression in a cerebellar lobe injected with MND-GFP. B. Single confocal section of MND-GFP transduced cerebellum at higher magnification showing absence of GFP expression in Purkinje neuron somata (asterisks). C. Low magnification of cerebellum injected with MSCV-GFP. D. High magnification of cerebellum injected with MSCV-GFP demonstrating GFP expression in small cell bodies in the Purkinje layer with radial processes extending to the pial surface, characteristic of Bergmann glia. E. Venus expression in a cerebellar lobe injected with UBC-Venus. F, G. High magnification of UBC-Venus infected cerebellum shows venus expression in multiple small cells in the granule layer (F) and a single Purkinje neuron (G). H, I. GFP expression in cerebellar lobes of two animals injected with PGK-GFP. Several GFP-expressing Purkinje neurons are visible in H, whereas most GFP-expressing cells in I are in the white matter, with a single GFP-positive Purkinje neuron. J. High magnification view of GFP-positive Purkinje neurons from H. Abbreviations: m = molecular layer; p = Purkinje layer; g = granule layer; wm = white matter. Scale bars, 25 µm (B, D, F, G, J), 100 µm (A, C, E, H, I).
Figure 4.
MND-tdTomato expression following injection with pulled glass pipette.
Confocal images of sagittal cerebellar slices from wild type mice injected with MND-tdTomato lentivirus. Injections were performed using pulled glass pipettes and a picospritzer (see methods) to determine if injection technique affected cellular transduction pattern. Dotted lines in A and B represent the border between the Purkinje layer and granule layer. In C, dotted lines are drawn to separate the Purkinje layer from the molecular layer and granule layer. A. Widespread tdTomato expression in cells in the granule layer and processes in the molecular layer. B. A few small tdTomato expressing cells are visible in the Purkinje layer, but the majority of the tdTomato expressing cell bodies are located in the white matter (wm). C. High magnification view of tdTomato expressing cells with somata in the Purkinje layer show that their processes are relatively straight and unbranched, characteristic of Bergmann glia. m, molecular layer; p, Purkinje layer; g, granule layer. Scale bars: 100 µm (A, B); 25 µm (C).
Figure 5.
AAV1 transduction of Purkinje neurons and localization of viral delivered FGF14B-GFP.
A. Schematic representation of AAV transfer plasmid constructs for AAV-CAG-GFP and AAV-CAG-FGF14B-GFP. CAG, chicken β-actin promoter with CMV enhancer; in, SV40 intron; pA, polyadylation site; ITR, inverted terminal repeat. B. Confocal image from a sagittal cerebellar section injected with AAV1-CAG-GFP showing predominant GFP expression in Purkinje neuron somata and dendrites. Some GFP expressing cell bodies are visible in the granule layer. C, D. Immunostaining for FGF14 in an Fgf14−/− mouse with intracerebellar injection AAV1-CAG-FGF14B-GFP. C. Low magnification montage of FGF14-specific immunostaining in an Fgf14−/− mouse injected with AAV-CAG-FGF14B-GFP. FGF14 expression is evident in an entire lobe. D. Immunostaining for FGF14 in an Fgf14−/− mouse injected with AAV-CAG-FGF14B-GFP shows no FGF14 expression in areas distal to the injection (top) whereas areas near the injection show rescue of FGF14 expression in Purkinje neuron soma and AIS (middle). For comparison, normal FGF14 expression in a wild type cerebellum is shown at the bottom. Asterisks mark the location of the Purkinje neuron soma and arrows mark the approximate start and end of the AIS. Arrowheads mark the AIS of FGF14-expressing stellate and basket cells in the molecular layer. Scale bars: 200 µm (C); 25 µm (B, left panels in D); 5 µm (right panels in D).
Figure 6.
AAV1 delivery of FGF14B-IRES-tdTomato and FGF14B-P2A-GFP results in efficient FGF14 expression but failure of reporter gene fluorescence.
A. Schematic representation of CAG-FGF14B-IRES-tdTomato AAV transfer construct. CAG, chicken β-actin promoter with CMV enhancer; in, SV40 intron; IRES, internal ribosome entry site; pA, polyadenylation site; ITR, inverted terminal repeat. B. CAG-FGF14B-IRES-tdTomato transfected into CHL1610 cells produces a diffuse cytoplasmic tdTomato expression pattern. C. Confocal image from an Fgf14−/− mouse injected with CAG-FGF14B-IRES-tdTomato and immunostained for FGF14. Viral delivered FGF14 is properly localized at the Purkinje neuron AIS but tdTomato expression is not visible. D. Schematic representation of CAG-FGF14B-P2A-GFP AAV transfer construct. The arrow represents approximate location where ribosomal skipping should occur to generate two independent polypeptides. E. GFP expression in CHL1610 cells transfected with CAG-FGF14B-GFP (top) or CAG-FGF14B-P2A-GFP (bottom). FGF14B-GFP fusion protein is expressed in punctate foci surrounding the nucleus whereas FGF14B-P2A-GFP is expressed as a diffuse cytoplasmic protein, suggesting cleavage of GFP from FGF14B. F. Western blot analysis of P2A cleavage efficiency in CHL cells. CHL1610 cells were transfected with either CAG-FGF14B-GFP or CAG-FGF14B-P2A-GFP and processed for western blot 24 h after transfection. Immunoblotting for both FGF14 and GFP revealed a ∼50kDa band in CAG-FGF14B-GFP transfected cells, which is consistent with the expected size of the fusion protein. Immunoblotting for FGF14 and GFP in CAG-FGF14B-P2A-GFP transfected cells revealed ∼25kDa bands for FGF14 and GFP and no detectable ∼50kDa band, indicating efficient cleavage of the P2A peptide. G. Confocal image of Fgf14−/− cerebellum injected with CAG-FGF14B-P2A-GFP and immunostained for FGF14 (red) and AnkyrinG (AnkG, blue). No GFP fluorescence is visible but viral delivered FGF14 is properly expressed at the AIS of Purkinje neurons where it colocalizes with AnkyrinG. H. Confocal image of Fgf14−/− cerebellum injected with CAG-FGF14B-P2A-GFP and immunostained for FGF14 (red) and GFP (blue). Immunostaining reveals that GFP is expressed and colocalizes with FGF14 in the Purkinje neuron AIS, suggesting that P2A cleavage did not occur in vivo. Scale bars: 20 µm (B, E); 10 µm (C, G, H).
Table 2.
AIS expression of virally transduced FGF14 protein in Fgf14−/− Purkinje neurons.
Figure 7.
AAV1 delivery of a dual promoter construct reveals that the CAG promoter produces better Purkinje neuron expression than the PGK promoter.
A. Schematic representation of the dual promoter AAV transfer construct which contains the CAG promoter followed by FGF14A and the PGK promoter followed by GFP. CAG, chicken β-actin promoter containing the CMV enhancer; in, SV40 intron; PGK, human phosphoglycerate kinase promoter; pA, polyadenylation site; ITR, inverted terminal repeat. B. Confocal images of sagittal sections from an Fgf14−/− mouse injected with the AAV-CAG-dual promoter virus and immunostained for FGF14 (red) and AnkyrinG (blue). Viral delivered FGF14 is properly localized at the Purkinje neuron AIS where it colocalizes with AnkyrinG. A lower level of FGF14 expression is visible on the soma membrane. Viral delivered GFP is expressed in small cells in the Purkinje layer that extend radial processes into the molecular layer (a pattern that is consistent with Bergmann glia) but is clearly absent from Purkinje neurons. Scale bars: 25 µm (B, top); 5 µm (B, bottom).
Figure 8.
Co-injection of two AAV1-CAG viruses results in optimal Purkinje neuron expression of two different genes.
A. Schematic representation of the two AAV transfer constructs used for co-injection. AAV1-CAG-GFP and AAV1-CAG-FGF14B-IRES-tdTomato viruses were mixed together at a ratio of 1∶5 prior to injecting into Fgf14−/− cerebellum. CAG, chicken β-actin promoter with CMV enhancer; in, SV40 intron; IRES, internal ribosome entry site; pA, polyadenylation site; ITR, inverted terminal repeat. B. Confocal images of sagittal sections from an Fgf14−/− cerebellum co-injected with AAV1-CAG-GFP and AAV1-CAG-FGF14B-IRES-tdTomato and immunostained for FGF14 (red). GFP expression is readily visible in Purkinje neuron somata and dendrites, and FGF14 is properly localized to the AIS. While some Purkinje neurons express FGF14 but not GFP, all GFP expressing Purkinje neurons express FGF14. Scale bars: 25 µm (B, top); 5 µm (B, bottom).