Figure 1. Schematic representation of siRNA‐ and shRNA‐induced RNAi. Hairpin‐type vectors can be introduced in the cells by transfection or viral‐mediated transduction. Inside the cells, the hairpin is transcribed and processed by Dicer to generate siRNAs. The siRNA oligonucleotides are transfected directly into the cells.

Figure 2. Map of pLKO.1 containing an shRNA insert. The original pLKO.1‐TRC cloning vector has a 1.9 kb stuffer that is released by digestion with AgeI and EcoRI (see later).

Figure 3. Schematic representation of the complementary oligonucleotides to be designed. Red = sense sequence; green = antisense sequence.

Figure 4. Detail of the shRNA insert. The U6 promoter directs RNA Polymerase III transcription of the shRNA. The shRNA contains 21 “sense” bases that are identical to the target gene, a loop, and 21 “antisense” bases that are complementary to the “sense” bases. The shRNA is followed by a polyT termination sequence for RNA Polymerase III.

Figure 5. Lentiviral‐mediated shRNA delivery to knock‐down the expression of the transcriptional regulator YY1. (a) RT‐gPCR showing the mRNA levels of YY1; (b) Immunoblot assay to show the downregulation of YY1 protein.