Adenoviral mediated knock-down in primary cell types. (A) Knock-down activity was measured for CHUK (IKKα, conserved helix-loop-helix ubiquitous kinase), IKBKB and GNAS at 3 and 6 d postinfection of primary keratinocytes (NHEK) at m.o.i. 500 using adenoviral siRNA expression constructs with a modified capsid. Analyses were performed by real-time RT-PCR as described. (B) Adenoviral knock-down activity was measured 6 d after infecting primary synoviocytes at m.o.i. 7500 for IKBKB and MMP2 (matrix metalloproteinase) using two constructs for each target. Analyses were performed as described. (C) Knock-down activity of siRNA-expressing adenoviruses (m.o.i. 1000) containing 13 different target sequences was analyzed in primary HUVEC cells (human umbilical vein endothelial cells). Relative mRNA expression levels were determined by real-time PCR analyses, normalized to internal GAPD values, and plotted relative to samples infected with Ad-siRNA-GL2 control viruses. Samples were obtained from HUVEC cells at two time points postinfection (3 or 6 d). For all eight mRNA targets, gene-specific real-time analysis was performed: PSEN1 (presenilin 1); CTSK (cathepsin K); ADAM10 (a disintegrin and metalloproteinase domain 10); CHUK; M6PR; BACE (β-site APP-cleaving enzyme); IKBKB; and GNAS.
