Figure 2.

In vitro screening of designed siRNA sequences against the Mut Col1a1 exon 23. (a) Inhibition of luciferase activity in HEK 293 cells, which were modified to express either recombinant WT-LucF or Mut-LucF transcript (named WT-LucF-HEK or Mut-LucF-HEK, respectively). The results were obtained 24 h after transfection of LacZ-siRNA, LucF-siRNA or siRNAs designed against the Mut Col1a1 exon 23 (Mut siRNAs a to f). They are expressed as percentage inhibition and compared with baseline luciferase activity of untransfected LucF-HEK cells. Mut siRNA e and f showed the strongest and specific Luciferase inhibition against the Mut allele. Results are expressed as mean±SD of three independent experiments (*P<0.05 as compared with LacZ-siRNA-transfected cells). (b) The relative expression of the total Col1a1 transcript and (c) WT and Mut Col1a1 transcripts were assessed in Brtl fibroblasts transfected with 10 nM LacZ-siRNA, e-WT siRNA or siRNA e-Mut, f-Mut against the Mut Col1a1 and in untransfected cells by real-time PCR. Ratio of WT/Mut Col1a1 transcripts is also shown. Although Mut siRNA e and f significantly inhibited both WT and Mut allele, the WT/Brtl allele ratio was only significantly increased in cells transfected with e-Mut and f-Mut siRNA and not in untransfected cells. The results are expressed as mean±SD of a triplicate experiments (*P<0.05; ^#P<10⁻⁴, referred to untransfected cells).