Our previous studies in yeast have identified genes that when genetically inhibited (deleted) ameliorate toxicity of a mutant huntingtin (htt) fragment, including the promising therapeutic target kynurenine 3-monooxygenase (KMO). It is likely that a similar rescue would be seen if these targets were inhibited pharmacologically, making them candidate therapeutic targets for Huntington's disease (HD). Therefore, the aims of this project are to identify further such gene targets in yeast and validate them in mammalian cell culture models. The yeast gene knock-out collection is being systematically screened to ensure all strains are tested, as previous approaches used random pooled strategies which may have missed candidate suppressors. Promising candidates with one to one mammalian orthologues are then selected for further analysis. Real-time qPCR is employed to ascertain the level of expression of the candidates in PC12 HD model cells to ensure the suppressors are in fact expressed in these cells and to quantitate levels of knock down by RNAi. Amelioration of a variety of HD relevant phenotypes (ie, caspase activation, cellular toxicity) after RNAi knock down of the candidate suppressors is being assessed. Thus far the yeast screen has identified multiple new deletion suppressors involved in several biological processes, including the ubiquitin pathway, response to oxidative stress, transcription and pyruvate metabolism. Stable knock down lines of the genes of interest have been generated in PC12 HD model cells and are now being analysed.
- Biological modifiers
- genetic screens
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