pARIS-htt an Adaptable, RNAi Insensitive & Synthetic construct encoding human huntingtin. A) Schematic representation of pARIS-htt. The entire coding sequence is divided into 8 different fragments, each fragment being flanked by unique restriction sites every 1-1.5 kbp and cloned independently into a modified pUC19 backbone. A multi-cassette full-length htt plasmid (pARIS-htt) was generated by assembly of these 8 individual fragments. pARIS-htt construct was tagged with 6×His followed by a mCherry on the amino terminus. The carboxy-terminal part contains HA and tetracysteine (TC) tags. The synthetic construct is fully compatible with the Gateway technology thanks to the introduction of flanking attL sites. (B) pARIS-htt triggers the expression of full-length htt in HEK cells. Cells mock transfected and transfected with pARIS-mCherry-httQ23 or pARIS-mCherry-httQ100 were analyzed by western blot using antibodies raised against different regions of htt: the amino-terminal part (htt-4C8), the carboxy-terminal part (htt-2C1) or the pathogenic polyQ stretch (1C2). The exact epitopes for these antibodies are illustrated in (A). Expression of the different constructs was detected using a high affinity anti-HA antibody. (C) Confocal images of Cos7 mock transfected cells and cells transiently transfected with pARIS-mCherry-httQ23 or pARIS-mCherry-httQ100 constructs. Expression of pARIS-htt is detected as a cytosolic mCherry fluorescent signal which codistributes with htt-4C8 antibody staining. Scale bar 10 μm.
Pardo et al. Molecular Brain 2010 3:17 doi:10.1186/1756-6606-3-17