Acetylcholinesterase (AChE) gene modification in transgenic animals: functional consequences of selected exon and regulatory region deletion

Abstract

AChE is an alternatively spliced gene. Exons 2, 3 and 4 are invariantly spliced, and this sequence is responsible for catalytic function. The 3' alternatively spliced exons, 5 and 6, are responsible for AChE disposition in tissue [J. Massoulie, The origin of the molecular diversity and functional anchoring of cholinesterases. Neurosignals 11 (3) (2002) 130-143; Y. Li, S. Camp, P. Taylor, Tissue-specific expression and alternative mRNA processing of the mammalian acetylcholinesterase gene. J. Biol. Chem. 268 (8) (1993) 5790-5797]. The splice to exon 5 produces the GPI anchored form of AChE found in the hematopoietic system, whereas the splice to exon 6 produces a sequence that binds to the structural subunits PRiMA and ColQ, producing AChE expression in brain and muscle. A third alternative RNA species is present that is not spliced at the 3' end; the intron 3' of exon 4 is used as coding sequence and produces the read-through, unanchored form of AChE. In order to further understand the role of alternative splicing in the expression of the AChE gene, we have used homologous recombination in stem cells to produce gene specific deletions in mice. Alternatively and together exon 5 and exon 6 were deleted. A cassette containing the neomycin gene flanked by loxP sites was used to replace the exon(s) of interest. Tissue analysis of mice with exon 5 deleted and the neomycin cassette retained showed very low levels of AChE expression, far less than would have been anticipated. Only the read-through species of the enzyme was produced; clearly the inclusion of the selection cassette disrupted splicing of exon 4 to exon 6. The selection cassette was then deleted in exon 5, exon 6 and exons 5 + 6 deleted mice by breeding to Ella-cre transgenic mice. AChE expression in serum, brain and muscle has been analyzed. Another AChE gene targeted mouse strain involving a region in the first intron, found to be critical for AChE expression in muscle cells [S. Camp, L. Zhang, M. Marquez, B. delaTorre, P. Taylor, Knockout mice with deletions of alternatively spliced exons of Acetylcholinesterase, in: N.C. Inestrosa, E.O. Campus (Eds.), VII International Meeting on Cholinesterases, Pucon-Chile Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects. P. Universidad Catholica de Chile-FONDAP Biomedicina, 2004, pp. 43-48; R.Y.Y. Chan, C. Boudreau-Larivière, L.A. Angus, F. Mankal, B.J. Jasmin, An intronic enhancer containing an N-box motif is required for synapse- and tissue-specific expression of the acetylcholinesterase gene in skeletal muscle fibers. Proc. Natl. Acad. Sci. USA 96 (1999) 4627-4632], is also presented. The intronic region was floxed and then deleted by mating with Ella-cre transgenic mice. The deletion of this region produced a dramatic phenotype; a mouse with near normal AChE expression in brain and other CNS tissues, but no AChE expression in muscle. Phenotype and AChE tissue activities are compared with the total AChE knockout mouse [W. Xie, J.A. Chatonnet, P.J. Wilder, A. Rizzino, R.D. McComb, P. Taylor, S.H. Hinrichs, O. Lockridge, Postnatal developmental delay and supersensitivity to organophosphate in gene-targeted mice lacking acetylcholinesterase. J. Pharmacol. Exp. Ther. 293 (3) (2000) 896-902].