Extended Abstracts / Chemico-Biological Interactions 157–158 (2005) 353–434 373 to the house keeping gene cyclophillin, is around 200 fold. We were unable to quantify the splicing occurring at the E1-E2 junction, but we used absolute quantifica- tion to measure the abundance of the precursor of the mature message containing intron 1 or intron 2. Serial dilutions of a plasmid containing the whole AChE gene were used for absolute quantification. Our results show that both precursor species, containing either intron 1 or intron 2, increase with differentiation although to a lower extent (around 10-fold) compared to the spliced message. We do not observe a significant difference in the levels of two precursors, suggesting that a pre-mRNA species containing intron 1 is not preferentially retained upon myoblast differentiation. We generated KO mice in which the 255bp critical region has been deleted [5]. We flanked the regulatory region with loxP sites so that it could be removed by breeding with mice carrying the Cre recombinase trans- gene. The AChE activity levels of each construct were tested by transfecting C2C12 cells. The intron 1 deletion KO mice show a trembling phenotype and small size compared to their wild type litter mates. AChE spliced message increases with myogenic dif- ferentiation as does unspliced precursor. The increase in the precursor forms might be explained by a possible transcriptional up-regulation of the gene during myo- genesis that has been also postulated by other groups [3] although the 200-fold increase observed in the levels of the spliced message indicate that post-transcriptional regulation is also occurring. The absence of AChE activ- ity in skeletal muscle tissue of KO mice in which the 255 bp intronic region has been deleted provides in vivo evidence of the critical involvement of the 5 ′ intronic region in the regulation of the AChE expression during myogenesis. References [1] T.L. Rachinsky, S. Camp, Y. Li, T.J. Ekstrom, M. Newton, P. Taylor, Molecular cloning of mouse acetylcholinesterase: tissue distri- bution of alternatively spliced mRNA species, Neuron 5 (1990) 317–327. [2] A. Mutero, S. Camp, P. Taylor, Promoter elements of the mouse acetylcholinesterase gene, J. Biol. Chem. 270 (4) (1995) 1866–1872. [3] L.M. Angus, R.Y. Chan, B.J. Jasmin, Role of intronic E- and N-box motifs in the transcriptional induction of the acetylcholinesterase gene during myogenic differentiation, J. Biol. Chem. 276 (20) (2001) 17603–17609. [4] M.E. Fuentes, P. Taylor, Control of acetylcholinesterase gene expression during myogenesis, Neuron 10 (4) (1993) 679– 687. [5] S. Camp, L Zhang, M. Marquez, B. de La Torre, J.M. Long, G. Bucht, P. Taylor, Acetylcholinesterase (AChE) gene modification in transgenic animals: functional consequences of selected exon and regulatory region deletion, VIII IMC Proceedings. doi:10.1016/j.cbi.2005.10.058 (14) Acetylcholinesterase genes and insecticide resistance in aphids S.L. Dong a,b , M.C. Andrews a , F. Li b , G.D. Moores a , Z.J. Han b , M.S. Williamson a,∗ a Rothamsted Research, Harpenden, Herts, AL5 2JQ, UK b Department of Entomology, Nanjing Agricultural Uni- versity, Nanjing 210095, PR China 1. Introduction The peach-potato aphid (Myzus persicae) and the cot- ton aphid (Aphis gossypii) are important agricultural pests in many parts of the world, causing direct feeding damage and transmission of virus diseases on a range of crops. The widespread use of organophosphorus (OP) and carbamate insecticides for their control has led to the selection of resistant populations and biochemical stud- ies of resistant strains have shown that this often results from alterations in the main synaptic AChE that renders it insensitive to these compounds. We have recently iden- tified two mutations within the active site of the enzyme that correlate with resistance (A302S and S431F) and are now expressing the aphid AChE genes using the baculovirus system in order to: (1) confirm the func- tional properties of these mutations and (2) to generate a source of the enzyme for crystallography and structural studies. 1.1. Cloning of AChE genes and identification of mutations It now seems that aphids, like most insects, have two genes that encode AChE-like proteins, termed ace-1 and ace-2. The nomenclature for these genes is highly con- fused because Drosophila has only a single ace gene, but the orthologue of this gene in other insects does not appear to be the major synaptic AChE. Hence, whilst some authors continue to refer to this gene as ace-1 because it was the first to be cloned, others have renamed ∗ Corresponding author. Tel.: +44 1582 763133; fax: +44 1582 762595. E-mail address: martin.williamson@bbsrc.ac.uk (M.S. Williamson).