Codon optimised NDI1 sequences, designed to encode specific amino acid changes to reduce immunogenicity profiles and a minimal polyadenylation signal, were synthesised by GeneArt, Inc. (Invitrogen, Paisley, UK) and cloned into pAAV-MCS (Agilent Technologies, CA, USA) downstream of a CMV promoter. The lead construct, pAAV-ophNdi1, contained 329 synonymous codon modifications and an I82V amino acid substitution (patent no. 10220102). An additional construct, pAAV-ophNdi1-HA, was created by cloning ophNdi1 with a C-terminal HA tag (synthesized by GeneArt®, Thermo Fischer Scientific, MA, USA) into pAAV-MCS. To enhance AAV packaging efficiency, 4.4 kb of bacteriophage lambda DNA [46 (link)] was inserted into the plasmid backbone of pAAV-ophNdi1 and the antibiotic resistance gene substituted for kanamycin. Cloning was verified by DNA sequencing. pAAV-Ndi1 and pAAV-CAG-EGFP vectors were cloned as previously described in [39 (link),47 (link)], respectively.
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