The aim of this grant is to develop insecticide resistance genes a selectable markers suitable for the transformation of both cultured mosquito cell lines and mosquitos themselves. The availability of these markers is critical to the success of the transformation of mosquitos with genes conferring refractoriness to parasites. Due to the present paucity of a valuable genes and suitable promoters for use as selectable markers this work will be conducted in a number of different stages. Firstly, functional promoters will be identified by their ability to drive reporter constructs in mosquito cell cultures. Secondly, suitable Aedes resistance genes will be cloned and culture. Fourthly, we will attempt to transform mosquitoes by the insertion of resistance-associated mutations into the characterized genes via homologous recombination. Finally, we will construct selectable marker mini-genes from these resistance genes for use alongside transformation vectors as they become available. This work will focus on three potential selectable markers. The dihydrofolate reductase dhfr gene from Aedes albopictus which confers resistance to methotrexate, insensitive acetylcholinesterase or Ace from Aedes aegypti which confers resistance to organophosphorus and carbamate insecticides and the cyclodiene resistance gene or Rdl from Aedes aegypti, an insecticide insensitive gamma-aminobutyric acid (GABA) gated chloride ion channel.
The specific aims of the proposal in relation to these three genes are thus: 1) To test available promoters for expression in Aedes cell cultures. Promoters will be tested both for their ability to drive reporter genes and to express acetylcholinesterase from Ace and GABA gated chloride ion channels from Rdl. 2) To determine the genomic organization of the genes. This has already been performed for Rdl. 3) To introduce and test the effect of known resistance associated mutations in Rdl and Ace. 4) To transform mosquitos by the insertion of these resistance associated mutations via homologous recombination. Finally, in order to complement work on transformation using vector mediated systems we will construct mini-genes from Ace and Rdl as selectable markers capable of carrying inserted genes. This program project is designed to identify the genes controlling parasite refractoriness (projects 1 and 2) and then to provide delivery systems for their expression in, or transformation into, mosquitoes themselves (projects 3 and 4). Our proposal (number 3) is therefore designed specifically to develop transformation technologies capable of finally inserting genes conferring refractoriness into mosquitoes via a series of experiments leading to the construction of selectable marker genes.
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