4 Rip-2 gene silencing attenuates allergic airway inflammation in mice 104 4.1 Results 4.1.1 In vitro characterization of Rip-2 siRNA We have screened the gene silencing effects of three Rip-2 siRNA sequences (S1 – S3) targeted at different sites of the coding region of mouse Rip-2 mRNA, in both RAW264.7 and NIH/3T3 cell lines The screening was performed by observing for Rip-2 mRNA reduction, using agarose gel electrophoresis analysis, 24 h after transfection siRNA was transfected into the cells using Lipofectamine 2000 Transfection without siRNA or with control siRNA served as controls S1, S2 and S3 markedly silenced Rip-2 mRNA expression in both cell lines 24 h after transfection by about 70%, as compared to the control siRNA (Figure 4.1A) Results from immnuoblots suggest that the same three sequences produced equivalent knockdown of Rip-2 protein expression by at least 80% in RAW264.7 cells and NIH/3T3 cells at 48 h and 72 h, respectively (Fig 4.1B) The intensity of bands from gel electrophoresis and western blots were quantified using ImageJ as described in Materials and methods Among the sequences, S2 consistently knocked down Rip-2 with the least variability and was chosen as the lead siRNA for subsequent in vivo experiments 4.1.2 Rip-2 silencing in vivo In order to verify that 30 µl of solution administered intratracheally to a mouse could reach a substantial portion of the mouse lungs, 30 µl of Evan’s blue was administered to the mice intratracheally Figure 4.2A shows that nearly all parts of the lungs were stained by Evan’s blue Therefore, in subsequent experiments, 30 µl was administered to the mice for each dose Daily intratracheal administration of nmol S2 to naïve BALB/c mice for consecutive days was able to knock-down Rip-2 protein lung level for up to 72 h after the last siRNA dose (Figure 4.2B) In OVA mouse asthma model, we observed for the first time that Rip-2 lung level was markedly elevated (Figure 4.2C) To ensure lung Rip-2 protein knock-down in asthma, we compared the gene silencing 105 Figure 4.1 A RAW264.7 NIH/3T3 Rip-2 Rip-2 β-actin β-actin B NIH/3T3 RAW 264.7 24 h 24 h 48 h 48 h 72 h 72 h β-actin β-actin Veh SiRNA - + + + + + - Con S1 S2 S3 * ** Veh SiRNA - ** + + + + + - Con S1 S2 S3 * * ** ** * *** ** * Figure 4.1 Sequence-dependent inhibition of Rip-2 mRNA and protein expression by S2 in mouse cell lines (A) Rip-2 mRNA level after transfection in RAW264.7 and NIH/3T3 cells RNA evaluation was performed 24 h after transfection β-actin was used as an internal control Agarose gel band intensities were analysed using ImageJ software and normalized to β-actin control (B) Rip-2 protein level after transfection in RAW264.7 and NIH/3T3 cells Protein evaluation was performed 24, 48 and 72 h after transfection β-actin was used as an internal control Immunoblot band intensities were analysed using ImageJ software and normalized to β-actin control * Significant difference from Con, P