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. 2017 Jul 27;6:159-170.
doi: 10.1016/j.omtm.2017.07.006. eCollection 2017 Sep 15.

Minimal Purkinje Cell-Specific PCP2/L7 Promoter Virally Available for Rodents and Non-human Primates

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Free PMC article

Minimal Purkinje Cell-Specific PCP2/L7 Promoter Virally Available for Rodents and Non-human Primates

Keisuke Nitta et al. Mol Ther Methods Clin Dev. .
Free PMC article

Abstract

Cell-type-specific promoters in combination with viral vectors and gene-editing technology permit efficient gene manipulation in specific cell populations. Cerebellar Purkinje cells play a pivotal role in cerebellar functions. Although the Purkinje cell-specific L7 promoter is widely used for the generation of transgenic mice, it remains unsuitable for viral vectors because of its large size (3 kb) and exceedingly weak promoter activity. Here, we found that the 0.8-kb region (named here as L7-6) upstream of the transcription initiation codon in the first exon was alone sufficient as a Purkinje cell-specific promoter, presenting a far stronger promoter activity over the original 3-kb L7 promoter with a sustained significant specificity to Purkinje cells. Intravenous injection of adeno-associated virus vectors that are highly permeable to the blood-brain barrier confirmed the Purkinje cell specificity of the L7-6 in the CNS. The features of the L7-6 were also preserved in the marmoset, a non-human primate. The high sequence homology of the L7-6 among mouse, marmoset, and human suggests the preservation of the promoter strength and Purkinje cell specificity features also in humans. These findings suggest that L7-6 will facilitate the cerebellar research targeting the pathophysiology and gene therapy of cerebellar disorders.

Keywords: L7; PCP2; Purkinje cell; adeno-associated virus; cell type-specific promoter; cerebellum; lentivirus; marmoset; non-human primate; viral vector.

Figures

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Figure 1
Figure 1
Schematic Representation of the Former 8-kb L7 Promoter and Its Association with the Four Splice Forms Four splice variants of L7 expressed in the cerebellum and/or eye were renamed chronologically as forms A–D. Boxes show the exons, in which the filled portions correspond to the coding region denoted with the start (ATG) and terminal (TGA) codons.
Figure 2
Figure 2
Deletion of the L7 Distal Structural Gene Significantly Upregulates the Promoter Strength and Maintains the Specificity to Purkinje Cells (A) Schema showing the original L7 promoter used for Purkinje cell-specific transgenic mouse (L7-1) and the deletion constructs (L7-2 to L7-5). The GFP gene was inserted at the BamHI site in exon 4 (L7-1 and L7-2) or after the original transcription initiation codon (ATG) present in exon 2 (L7-3 to L7-5). The size (base pair [bp]) of each construct without the GFP sequence is shown at the right side of each scheme. The L7 fragments were subcloned to the lentiviral plasmid as depicted. (B) Representative immunocytochemical images of the lentiviral GFP expression in cerebellar neurons under the control of L7-1 (upper) or L7-4 (lower). The neuronal culture was infected with lentiviral vectors at the day of plating and immunolabeled with GFP and calbindin D-28K at 14 days post-infection. Arrowheads indicate Purkinje cells. Scale bars, 100 μm. (C) Graph showing the GFP intensity relative to that induced by the L7-1 promoter. The GFP intensity in each promoter was calculated from 40 images randomly acquired from the culture. F(6,273) = 180.10; p < 0.001 by ANOVA. Error bars indicate SEM. (D) Graph showing the specificity of Purkinje cell transduction. Percent ratios of GFP-positive Purkinje cells to total GFP-positive cells are presented. F(6,33) = 4589.00; p < 0.001 by ANOVA. Error bars indicate SEM. (E) Graph showing the transduction efficiency of Purkinje cells. The ratios of GFP-positive Purkinje cells to total Purkinje cells are presented. F(6,33) = 55.50; p < 0.001 by ANOVA. Error bars indicate SEM. Data were obtained from eight (CMV, L7-3, and L7-4) and four (MSCV, L7-1, L7-2, and L7-5) independent cultures. **p < 0.01 by Bonferroni correction for post hoc test, as compared with the original L7-1 promoter.
Figure 3
Figure 3
L7-4 Works as a Purkinje Cell-Specific Promoter in the Mouse Cerebellum In Vivo (A) Schema depicting the lentiviral vectors expressing GFP by the L7-1 or L7-4 promoter and the viral injection to the mouse cerebellum. (B–E) Representative images of native GFP fluorescence on sagittal cerebellar sections at 1 week after viral injection. (B–E) The sections expressing GFP by the L7-1 (B and C) or L7-4 (D and E) promoter are presented. (F) Graph showing the relative GFP intensity of cerebellar sections lentivirally expressing GFP by the L7-4 promoter as compared with those induced by the original L7-1 promoter (n = 4 mice; t(6) = −6.55; ***p = 0.001 by Student’s t test). Error bars indicate SEM. (G) Schema describing the method of lentivirus-based generation of a transgenic mouse expressing GFP by the L7-4 promoter. (H–J) Bright-field (H) and native GFP fluorescent (I and J) images of a sagittal section of the whole brain (H and I) and the enlarged GFP image of the cerebellar cortex (J). Scale bars, 50 μm (A–D and J) and 2 mm (H and I).
Figure 4
Figure 4
The 0.8-kb Genome Region Upstream of the Transcription Initiation Codon in Exon 1B, L7-6, Shows a Similar Promoter Strength as L7-4 and Much Less Leaky Activity in Cortical Cells Other Than Purkinje Cells (A) Schema depicting the L7-4 promoter and the 5′ side deletion constructs. Whereas the L7-4 promoter uses the transcription initiation codon in exon 2 as the form A and form D, the L7-6 and L7-9 promoters use different transcription initiation codons in exon 1B (L7-6) and exon 1C (L7-9) as the form B and form C, respectively. The L7-7 and L7-8 promoters are present or absent from exon 1A in the form A, respectively. The size (base pairs [bp]) of each construct without the GFP sequence is shown at the right side of each scheme. (B–F) Representative native GFP fluorescent images on sagittal sections of the cerebellum virally expressing GFP by the deleted promoters as depicted. Robust GFP expression was observed by L7-4 (B) and L7-6 (C), whereas the GFP expression was significantly decreased by deletion of the 3′ side (D–F). (G) Summary graph showing the GFP intensity relative to that by the L7-4 promoter. F(4,39) = 19.96; p < 0.001, ANOVA; **p < 0.01; ††p < 0.01 by Bonferroni correction for post hoc test, as compared with L7-4 or L7-6 promoter, respectively. Error bars indicate SEM. (H and I) Enlarged GFP fluorescent images of the cerebellar cortex virally expressing GFP by the L7-4 (H) or L7-6 (I) promoter. Arrowheads indicate GFP-labeled non-Purkinje cells. (J) Graph showing the number of GFP-positive non-Purkinje cells in the molecular layer. t(10) = 4.86; ***p = 0.001 by Student’s t test. Error bars indicate SEM. Statistical data were obtained using 11 (L7-4), 12 (L7-6), 8 (L7-7), 8 (L7-8), and 5 (L7-9) mice. Scale bars, 200 μm (B–F) and 50 μm (H and I).
Figure 5
Figure 5
Regions Critical for Promoter Strength and Purkinje Cell Specificity in the L7-6 (A) Schema depicting the deletion constructs of the L7-6. L7-10 lacks approximately 380-bp deletion upstream of the L7-6. L7-11 is almost identical to L7-6 but lacks the RORα binding sequence. (B–D) Representative native GFP fluorescent images on sagittal sections of the cerebellum virally expressing GFP by the promoters as depicted. The bright GFP fluorescence was observed in the slice transduced by the L7-6 (B), which was significantly decreased in the slice transduced by L7-10 (C) and L7-11 (D). (E) Summary graph showing the GFP intensity relative to that by the L7-6 promoter. F(2,11) = 24.78; p < 0.001 by ANOVA. Error bars indicate SEM. (F–H) Enlarged images of the cerebellar cortex virally expressing GFP by the L7-6 (F) or L7-10 (G and H) promoter. Sections were double-labeled with GFP and GAD67. Arrowheads indicate examples of non-Purkinje cells double-labeled for GFP and GAD67 in the granule cell layer. (I) Graph showing the number of cells double-labeled for GFP and GAD67 in the granule cell layer. F(2,11) = 25.72; p < 0.001 by ANOVA. Error bars indicate SEM. Data were obtained using four (L7-6), five (L7-10), and five (L7-11) mice. *p < 0.05; **p < 0.01 by Bonferroni correction for post hoc test. Scale bars, 500 μm (B–D) and 20 μm (F–H).
Figure 6
Figure 6
Validation of the Cellular Specificity of the L7-6 by Intravenous Administration of AAV PHP.B Vectors That Are Highly Permeable to the Blood-Brain Barrier (A–D) Bright-field (A and C) and native GFP fluorescent (B and D) images of the whole brain and the sagittal section. The mouse received intravenous injection of 100 μL of AAV PHP.B (3.0 × 1013 viral genomes/mL) and was sacrificed 3 weeks post-injection. (E) Magnification of the GFP fluorescent image at the cerebellar cortex. Scale bars, 2 mm (AD) and 50 μm (E).
Figure 7
Figure 7
The L7-6 Promoter Behaves Identically as a Purkinje Cell-Specific Promoter in Marmoset Brain (A–C) Native GFP fluorescent image of the sagittal section of the marmoset cerebellum 5 weeks after the viral injection. A 1.9-year-old marmoset received cerebellar injection of 100 μL of AAV9 vectors expressing GFP by the mouse L7-6 promoter (1.0 × 1013 viral genomes/mL). Low-magnified images of GFP fluorescence alone (A) and GFP fluorescence superimposed on the bright field (B) and the enlarged GFP fluorescent image (C) were presented. (D–F) Immunohistochemical analysis of the sagittal section of the cerebellum expressing GFP under the control of the L7-6 promoter. The section was double-labeled with GFP and parvalbumin. Fluorescent images of GFP (D) and parvalbumin (E) and the merged image (F) are shown. Scale bars, 2 mm (A and B), 400 μm (C), and 50 μm (D–F).
Figure 8
Figure 8
Sequences for Validated RORα and Putative Transcription Factor Binding Sites in L7-6 Promoter Are Highly Conserved among Mouse, Marmoset, and Human Boxed regions are proposed transcription factor binding sites of the L7-6 promoter. Numbers above the sequence are relative positions when the transcription initiation codon begins from +1. Notably, the retinoic-acid-receptor-related orphan receptor α (RORα) binding sites completely match between mouse and human, suggesting that the mouse L7-6 promoter effectively works in the human brain as proven in the marmoset brain.

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