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Viral Vector-Based Evaluation of Regulatory Regions in the Neuron-Specific Enolase (NSE) Promoter in Mouse Cerebellum In Vivo

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Abstract

We investigated the neuron-specific enolase (NSE) promoter in terms of its promoter strength and neuronal specificity in the cerebellum in vivo. The 1.8 kb rat NSE promoter was divided into three regions, A (0.8 kb), B (0.7 kb), and C (0.3 kb), starting from the 5′ side. Then, we made various deletion constructs and assessed them by virally expressing GFP under the control of one of the deleted promoters. Removing region A reduced GFP expression to ~6% of that of the original 1.8 kb promoter. Further deletion of region B (presence of region C alone) did not influence the promoter strength, but removing region B from the original 1.8 kb promoter reduced the GFP expression to ~6% of the original level, similar to the level observed after deletion of region A. Immunohistochemistry showed robust GFP expression in Purkinje cells and modest expression in interneurons by the original promoter. Removing region A and/or region B abolished the GFP expression in Purkinje cells in most cerebellar lobules, with the expression in interneurons almost unchanged. These results suggest that region C, which is a proximal 0.3 kb sequence, contains cis-acting elements that drive transcription predominantly in interneurons. The addition of either region A or B onto region C does not alter the promoter properties; however, the addition of both regions A and B to region C drastically enhanced the promoter activity in Purkinje cells, suggesting the synergistic action of cis-acting regulatory elements in regions A and B for strong activation in Purkinje cells.

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Acknowledgments

The authors thank Asako Ohnishi and Junko Sugiyama for AAV9 vector production and maintenance of mice. This study was (partially) supported by the program for Brain Mapping by Integrated Neurotechnologies for Disease Studies (Brain/MINDS) from the Ministry of Education, Culture, and Sports Science (MEXT) and the Japan Agency for Medical Research and Development (AMED) and Gunma University Initiative for Advanced Research (GIAR) (to H. Hirai); and JSPS KAKENHI grant no. 15K18330 (to A. Konno).

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Authors and Affiliations

  1. Department of Neurophysiology & Neural Repair, Gunma University Graduate School of Medicine, Maebashi, Gunma, 371-8511, Japan

    Yoichiro Shinohara, Toshinori Ohtani, Ayumu Konno & Hirokazu Hirai

  2. Department of Ophthalmology, Gunma University Graduate School of Medicine, Maebashi, Gunma, 371-8511, Japan

    Yoichiro Shinohara

  3. Research Program for Neural Signalling, Division of Endocrinology, Metabolism and Signal Research, Gunma University Initiative for Advanced Research, Maebashi, Gunma, 371-8511, Japan

    Hirokazu Hirai

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  1. Yoichiro Shinohara
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  2. Toshinori Ohtani
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  3. Ayumu Konno
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  4. Hirokazu Hirai
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Correspondence to Hirokazu Hirai.

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Shinohara, Y., Ohtani, T., Konno, A. et al. Viral Vector-Based Evaluation of Regulatory Regions in the Neuron-Specific Enolase (NSE) Promoter in Mouse Cerebellum In Vivo. Cerebellum 16, 913–922 (2017). https://doi.org/10.1007/s12311-017-0866-5

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