Obtain a new animal model for Huntington’s Disease

Huntington's disease (HD) is a fatal neurodegenerative disorder, which is caused by a CAG repeat expansion encoding a poly-glutamine tract in the huntingtin (htt) gene. The recapitulation of the exact disease symptoms and course is rather difficult, since the generation of HD disease (mouse) models is challenging because of the size and complexity of the htt gene locus. Using recombineering, fournovel BAC transgenes harboring full-length or truncated htt cDNA comprising 98 or 15 glutamine residues were generated, which will further research into Huntington's disease.

Therefore the notoriously difficult huntingtin cDNA was transferred into the heterologous genomic locus BDNF (brain-derived neurotrophic factor). BDNF is expressed in the cortical neurons projecting to the striatal medium spiny neurons, and was used to direct htt transgene expression to investigate the contribution of these cell types to HD.

Cloning strategy: Using three Red/ET recombination steps and a final FLP recombination huntingtin cDNA was transferred into the BDNF locus of a suitable BAC. Chimeric oligonucleotides comprising a primer binding site and a bifunctional homology arm were used in PCR-amplification of antibiotic resistance cassettes which were inserted into the substrate plasmid adjacent to the htt cDNA. Using unique restriction sites, the cDNA was excised from the source plasmid and transferred into the BAC.

The recombineering protocol enabled the generation of four mouse models of HD and is applicable to many other complex cloning exercises too.