FIG. 2.

A: Target sequence of the luciferase mRNA. The sequence targeted by the clone 952-derived shRNA is underlined, and the sequence targeted by the clone 954-derived shRNA is overlined. B and C: Sequences and secondary structure of shRNAs encoded by clone 952 and clone 954, respectively. D: For the construction of clone 952 shRNA expression vectors, complementary ODNs are synthesized and annealed. The sequence complementary to the forward ODN contains 4-nucleotide overhangs to the EcoRI and ApaI restriction sites at 5′- and 3′-end, respectively, to direct subcloning into the cohesive ends of the expression vector driven by the U6 promoter shown in Figure 1A.³⁷ The T5 terminator sequence for RNA polymerase III is introduced in the sequence of the ODNs. E, left: Percent inhibition in luciferase activity at 2 and 4 days caused by cotransfection at zero time of the U87 cells with clone 790, a pCEP4-derived luciferase expression plasmid,⁴⁸ and shRNA clones 952 or 954 plasmid DNA, respectively. There is no inhibition of luciferase expression in the cells transfected with clone 954 at 4 days. E, right: Percent inhibition in luciferase activity at 2 and 4 days caused by exposure of the U87 cells to PIL encapsulated clone 952 and 954, respectively. Cells were exposed to lipofectamine and clone 790 plasmid DNA for 4 h, washed, and the HIRmAb-PILs carrying either clone 952 or clone 954 were added, and the cells were incubated for 2 or 4 days before measurement of luciferase activity. All data are mean ± SEM (n = 3 dishes per point). Reproduced with permission from Zhang et al. In vivo knockdown of gene expression in brain cancer with intravenous RNAi in adult rats. J Gene Med 5:1039–1045. Copyright © 2003, John Wiley & Sons, Ltd. All rights reserved.⁴⁴