Fig. 5.
High-volume application of siRNA targeted against Escherichia coli lacZ sequence of C57BL/6J-TgN(MtnlacZ) transgenic mice leads to efficient down-regulation of β-gal gene expression. To induce expression of the MT-lacZ transgene C57BL/6J-TgN(MtnlacZ) mice were injected twice with Cd2+ before (sequence 1) or after (sequence 2) siRNA delivery. LacZ staining of the whole liver shows efficient down-regulation of β-gal gene expression by siRNA-mediated gene silencing. (a) Representative liver of an animal treated with sequence 2. For determination of the proportion of hepatocytes with effective uptake of siRNA, X-gal staining of isolated primary hepatocytes was performed. The proportion of X-gal-positive cells in the untreated control group was normalized to 100%. Only ≈30% of the hepatocytes show positive X-gal staining after LacZ siRNA treatment, indicating effective delivery of siRNA into 70% of the hepatocytes. (b) In three independent experiments 10 optical fields were counted for each animal. scr, scrambled. (c) Quantification of siRNA-mediated down-regulation of β-gal gene expression was also determined by measuring β-gal activity in liver cell extracts. Data shown are the mean from six animals in each group.