Design of lentivirally expressed siRNAs.

RNA interference (RNAi) has been widely used as a tool for gene knockdown in fundamental research and for the development of new RNA-based therapeutics. The RNAi pathway is typically induced by expression of ∼22 base pair (bp) small interfering RNAs (siRNAs), which can be transfected into cells. For long-term gene silencing, short hairpin RNA (shRNA), or artificial microRNA (amiRNA) expression constructs have been developed that produce these RNAi inducers inside the cell. Currently, these types of constructs are broadly applied to knock down any gene of interest. Besides mono RNAi strategies that involve single shRNAs or amiRNAs, combinatorial RNAi approaches have been developed that allow the simultaneous expression of multiple siRNAs or amiRNAs by using polycistrons, extended shRNAs (e-shRNAs), or long hairpin RNAs (lhRNAs). Here, we provide practical information for the construction of single shRNA or amiRNA vectors, but also multi-shRNA/amiRNA constructs. Furthermore, we summarize the advantages and limitations of the most commonly used viral vectors for the expression of RNAi inducers.