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. 2006 Feb 28;2(1):154–160.

Figure 1.

Figure 1.

Assessment of ASP-RNAi with reporter alleles. (A) Schematic drawing of reporter alleles. Reporter alleles were constructed based on the Photinus and Renilla luciferase reporter genes driven by the same TK promoter, and allelic sequences of wild-type and mutant (synthetic oligonucleotides) were inserted into the 3′-UTRs of the reporter genes, i.e., the reporter alleles encode luciferase reporter genes carrying artificially inserted allele sequences of interest. Assessment of siRNA duplexes on the induction of ASP-RNAi against the Swedish APP mutant (B) and against the London APP mutants (C-E) was carried out. Synthetic siRNA duplexes against the mutants indicated were cotransfected with the mutant and wild-type reporter alleles and the β-galactosidase gene (control) into HeLa cells. The Photinus and Renilla luciferase genes carry the mutant and wild-type allelic sequences, respectively. Twenty-four hours after transfection, dual-luciferase and β-galactosidase assays were carried out. The levels of either Photinus (blue boxes) or Renilla (pink boxes) luciferase activity was normalized against the levels of β-galactosidase activity, and the ratios of mutant and wild-type luciferase activities in the presence of siRNA duplexes were normalized against the control ratio obtained in the presence of the siControl duplex (siCont). Data are averages of at least three independent determinations. Error bars represent standard deviations.