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The University of Southampton
Institute for Life Sciences

Research project: Biology of DNA triplex formation in C. elegans and Drosophila

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Defining the function of genes requires specific and potent methods for regulating their expression both temporally and in a tissue specific manner. This can be achieved by a number of different methods, including those which generate genetic mutants and those which employ molecules which interfere with gene expression e.g. morpholinos or double-stranded RNA. Of the methods that have been developed, most notable is RNAi.

The speed and enthusiasm with which this technique has been adopted by biologists seeking to define gene function is testament to the urgent need for new tools to manipulate gene expression.

Here we propose to develop another approach for this in intact animals. This targets DNA and therefore has several theoretical advantages over techniques such as antisense and RNAi, which target RNA. It utilizes triplex-forming oligonucleotides (TFOs) which bind in the major groove of duplex DNA, forming specific hydrogen bond contacts with exposed groups on the base pairs, thereby generating DNA base triplets. Unlike RNAi, it does not require complex cellular machinery and should be similar for all cell types, from prokaryote to eukaryote, from invertebrate to mammals. Furthermore, there are fewer sites to target (only two for a diploid cell); it is not subject to up-regulation of the target gene; and finally and most importantly, covalent attachment of the third strand can lead to irreversible gene inactivation or targeted mutagenesis, which could provide a universal means for gene targeting in the germ line of any organism.

This project is a collaboration between chemists who have developed TFOs that are suitable for use in physiological conditions, and a group of biologists who seek to exploit them for targeting specific genes in the model genetic animals C. elegans and Drosophila. Essentially, the work will involve experiments aimed at phenocopying well characterised genetic mutants by administering TFOs to wild-type animals. C. elegans and Drosophila offer the opportunity to test a number of different methods for delivering TFOs to cells in the intact animals and to provide an accurate read-out of the functional consequences. Thus we will provide information on the efficiency, penetrance and heritability of TFO-mediated regulation of gene expression. Furthermore, we will make use of transcriptome profiling in TFO treated animals to rigorously test for the specifity of TFO effects. The outcome of the study will be a much-needed definitive biological analysis of the actions of TFOs in intact animals. This will underpin future developments which aim to use these molecules either in the clinic, or as tools to define gene function.

Related research groups

Biomedical Sciences
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