B005 Characterisation of mouse embryonic fibroblast models of Huntington’s disease and their application as screening tools for therapeutic agents

<h3>Background</h3> Mouse embryonic fibroblasts (MEFs) have been isolated from the widely studied Huntington’s disease (HD) mouse models zQ175 and YAC128. zQ175 knock-in mice contain a mutated version of human HTT exon 1 integrated into mouse Htt and carry approximately 175-200 CAGs. YAC128 transgenic mice express the full-length human HTT gene with an expanded CAG repeat. Both models undergo a process of alternative processing of their huntingtin genes that lead to the production of the Htt1a/HTT1a transcripts and pathogenic HTTexon1 protein. <h3>Aims</h3> To characterise the YAC128 and zQ175 MEF lines at the RNA and protein levels and their suitability as screening tools for therapeutic agents. <h3>Methods/Techniques</h3> QuantiGene assays were implemented to analyse the expression levels of full-length and intronic huntingtin transcripts and RNAscope probes designed to visualise these transcripts at a single molecular level. Pairwise combinations of huntingtin antibodies were optimised for HTRF/AlphaLISA assays to detect huntingtin isoforms. <h3>Results/Outcome</h3> The growth rates of both MEF models were assessed in several formats and PicoGreen assays were established as a proxy for cell number. QuantiGene analysis indicated the presence of Htt1a in zQ175 MEFs and HTT1a in YAC128 MEFs and RNAscope showed their distribution in YAC128 MEFs. Levels of full-length huntingtin and HTTexon1 proteins were measured by HTRF/AlphaLISA assays. These MEFs were successfully used to validate therapeutic agents (ASO, siRNA) targeting full-length or intronic huntingtin mRNAs. <h3>Conclusions</h3> These MEF lines provide a valuable tool to evaluate potential therapeutic effects of agents aimed at lowering huntingtin levels.