A single-step mixing cloning method for assembly of lentiviral short hairpin RNA expression vectors for gene silencing

Lentiviral expression vectors encoding short hairpin RNA (shRNA) are widely used for RNAi-based gene silencing in mammalian cells. However, current methods for the construction of shRNA expression vectors require multiple steps, which are expensive, time-consuming, and error-prone. Here, we developed a single-step mixing cloning method for the generation of lentiviral shRNA expression vectors. With this method, a pair of short oligonucleotides (∼50nt) is required and a lentiviral shRNA vector can be constructed with only one step. This method has been used to construct 30 lentiviral shRNA expression vectors successfully.