Figure 3.

Use of pRosa26-DEST for Cre-regulated RNAi. Target sequences can be cloned as double-stranded oligonucleotides into a Gateway-compatible miRNA vector. This vector can directly be used to test different target sequences or the miRNA fragment can be transferred to pRosa26-DEST via a combined BP/LR reaction. The resulting vector is ready for targeting as described in Figure 1. Expression of the miRNA is blocked by the lox-STOP-lox cassette. Cre-mediated removal of this cassette links the miRNA to exon 1 of the endogenous Rosa26 locus to transcribe a chimeric transcript consisting of Rosa26 exon 1 and the miRNA sequence. The miRNA will be processed out of this primary transcript by Drosha, Dicer and the RISC complex and lead to knock-down of the target gene like other endogenous miRNAs.