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226 Cheng and ChangFig. 1. Structures of shRNA and siRNA-expression systems. (A) Construct of RNAPol III-controlled promoter-based shRNA-expression vector. In this system, a singleRNA Pol III promoter drives the expression of a shRNA transcript in which the senseand antisense strands of the shRNA are linked by a 9-nt loop sequence. The connectionof five consecutive Ts (T 5) at the 3′-end of the cassette provides not only a transcriptiontermination signal for RNA Pol III but also a 2-nt uridine overhang at the 3′-terminusof shRNA transcript. (B) Construct of two tandem array RNA Pol III-controlledpromoters-based siRNA-expression vector. In this system, two tandem array RNA PolIII promoter-based expression cassettes are used to drive separately the expression ofthe sense and antisense RNA transcripts. Both the sense and antisense RNAs thenanneal to form a ds-siRNA. (C) Construct of two convergent array RNA Pol IIIcontrolledpromoters-based siRNA-expression vector. In this system, two convergentarray RNA Pol III promoters-based expression unit is used to drive simultaneously thetranscription of the sense and antisense strands. Both the complementary sense andantisense strands then anneal to form a ds-siRNA.animals. In this chapter, the discussion is focused on these improved DNAvector-based shRNA-expression systems used in this laboratory. The protocolsdescribed in this chapter provide a comprehensive procedure for constructingthe simple and efficient shRNA-expression systems for specificgene silencing in mammalian cells.