Identification and Transgenic Analysis of a Murine Promoter that Targets Cholinergic Neuron Expression

Authors

  • Jorge M. Naciff,

  • Michael M. Behbehani,

  • Hidemi Misawa,

    1. Department of Neurology, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan
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  • John R. Dedman


  • The nucleotide sequences reported in this article have been submitted to the GeneBank/EMBL Data Bank with accession nos. D12486 and AF019045 (updated).

  • Abbreviations used : ACh, acetylcholine ; CAT, chloramphenicol acetyltransferase ; CDF/LIF, cytokine differentiation factor/leukemia inhibitor factor ; ChAT, choline acetyltransferase ; CNTF, ciliary neurotrophic factor ; CSE, cholinergic-specific enhancer ; NRSLE, neuronal restrictive silencer-like element ; PBS, phosphate-buffered saline ; VAChT, vesicular acetylcholine transporter.

Address correspondence and reprint requests to Dr. J. R. Dedman at Department of Molecular and Cellular Physiology, College of Medicine, University of Cincinnati, Cincinnati, OH 45267-0576, U.S.A.

Abstract

Abstract : Choline acetyltransferase (ChAT) is a specific phenotypic marker of cholinergic neurons. Previous reports showed that different upstream regions of the ChAT gene are necessary for cell type-specific expression of reporter genes in cholinergic cell lines. The identity of the mouse ChAT promoter region controlling the establishment, maintenance, and plasticity of the cholinergic phenotype in vivo is not known. We characterized a promoter region of the mouse ChAT gene in transgenic mice, using β-galactosidase (LacZ) as a reporter gene. A 3,402-bp segment from the 5′-untranslated region of the mouse ChAT gene (from -3,356 to +46, +1 being the translation initiation site) was sufficient to direct the expression of LacZ to selected neurons of the nervous system ; however, it did not provide complete cholinergic specificity. A larger fragment (6,417 bp, from -6,371 to +46) of this region contains the requisite regulatory elements that restrict expression of the LacZ reporter gene only in cholinergic neurons of transgenic mice. This 6.4-kb DNA fragment encompasses 633 bp of the 5′-flanking region of the mouse vesicular acetylcholine transporter (VAChT), the entire open reading frame of the VAChT gene, contained within the first intron of the ChAT gene, and sequences upstream of the start coding sequences of the ChAT gene. This promoter will allow targeting of specific gene products to cholinergic neurons to evaluate the mechanisms of diseases characterized by dysfunction of cholinergic neurons and will be valuable in design strategies to correct those disorders.

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