The RNAi pathway can be activated by two means; delivery of synthetic siRNAs, which induces a transient knockdown of protein expression, or by expression of dsRNA precursor molecules that are processed by the cellular RNAi machinery into siRNAs, which results in longer lasting gene knockdown. These dsRNA precursors are often expressed as short hairpin RNA molecules from RNA polymerase-III-dependent promoters. After their transcription, shRNA molecules are processed by the RNAse-III enzyme DICER to generate 19�C21 bp long dsRNA molecules harbouring 2 nucleotide long 39 extensions, which are characteristic of siRNAs. Alternatively, the dsRNA precursors can be expressed within the context of micro-RNA molecules, expressed from RNA polymerase-II-dependent promoters. These dsRNA precursors are first processed by nuclear DROSHA, another member of the RNAse-III family, to release the pre-miRNA from the primary RNA transcript and then by DICER to generate siRNAs in the cytoplasm. All three systems are widely used for RNAi experiments that DK-AH 269 include genome-wide loss-of-function screens in selected human cell lines and the establishment of transgenic model organisms for functional gene analysis. The success of an RNAi experiment crucially depends on the choice of the DM 235 target sequence as well as the efficacy of siRNA expression, which has to be optimised for each cell line and adapted for experimental requirements. Thus, while for certain experiments in some cell lines transient transfection of synthetic siRNAs is the optimal strategy, expression of shRNAs might be more suitable in other circumstances and the best RNAi strategy has often to be determined experimentally. To overcome the limitations of transfection technologies, shRNAs are frequently expressed from viral vectors, including adeno-, retroand lentiviral vectors, which also allow the generation of stable RNAi cell lines. When analysing essential genes, however, shRNA expression in stable cell lines has to be conditional. Several different conditional RNAi systems have been developed over the past decade. The most frequently used systems are based on the expression of shRNAs from conditional RNA polymerase-III-dependent promoters. Because siRNAs can also be processed from miRNAs, a variety of cell type specific and conditional RNA polymerase-II-dependent promoter systems have been used for siRNA expression.