5160 Nucleic Acids Research, 2007, Vol. 35, No. 15 Figure 4. ShRNAs reduce the activity of microRNA constructs while microRNAs are not eﬀective competitors. (a) A target site for the Mir-B and Mir-CG is inserted in the 30 UTR of the Renilla luciferase gene, which is used as reporter construct in this experiment (RLT). Co-transfection of an irrelevant shRNA (shSI) nearly abolished the activity of the microRNAs (compare Mir-B and Mir-CG with Mir-B+shSI, Mir-CG+shSI) while an irrelevant microRNA (Mir-Irr) had no eﬀect on Mir-B and Mir-CG (Mir-B+Mir-Irr and Mir-CG+Mir-Irr). The Renilla luciferase units were normalized against the ﬁreﬂy luciferase which was co-transfected with the experimental samples and used as an internal transfection control. (b) An irrelevant shRNA (shSI) strongly reduces the eﬃcacy of Luc (Luc versus Luc+shSI), but an irrelevant microRNA (Mir-B or Mir-CG) did not have a signiﬁcant eﬀect on the activity of the speciﬁc shRNA (compare Luc versus Luc+Mir-B and Luc+Mir-CG). The units of the ﬁreﬂy reporter target were normalized against the Renilla luciferase which was used as internal transfection control. (c) Left; northern analysis of total RNA from 293 cells transfected with shSII or mirCG. A 4-fold excess of mirCG over shSII was used for the transfection. The probe is complementary to the mature SII which is the sequence expressed from both constructs. A probe complementary to the endogenous U6snRNA (U6) was used as control. Right; shSII or mirCG were used as competitors against the shLuc construct. Five hundred nanogram or 5 ng of mirCG and shSII respectively were used to compete against 5 ng of shLuc. (d) Synthetic si and shRNAs, but not expressed microRNAs, can compete with the endogenous microRNA pathway. A stable clonal cell line with an integrated EGFP gene that includes the target site for the endogenous mir21 at its 30 end (HCT116-GFPmiR21) was mock transfected (Lip-2000) or transfected with: (i) a 20O-Me-oligonucleotide designed to bind and block mir21 activity (Mir-21–20O-Me), (ii) an irrelevant 20O-Me-oligonucleotide to test for non-speciﬁc reactivation of EGFP (IRR-20O-Me), (iii) a microRNA construct expressing the SII guide sequence (Mir-CG), (iv) an shRNA construct expressing the SII guide sequence (shSII) and (v) a synthetic siRNA (siH1). EGFP reactivation indicates interference with the activity of the endogenous mir21. the anti HIV inhibitory activity of shSII alone (shSII) or in combination with two other shRNAs (shSII+2). CEM cells were stably transduced with a single shRNA or with three diﬀerent shRNAs each independently expressed from a separate U6 promoter and then infected with HIV IIIB. The results from these experiments show that shSII results in potent inhibition of HIV replication whereas when combined with two additional shRNAs provide protection for the ﬁrst seven days, but viral replication breaks through after one week (Figure 5a). The loss of inhibitory activity by the triple construct was not due to loss of expression, since the levels of siRNAs were comparable for the single shRNA and triple constructs (Figure 5b). The most plausible explanation for the viral breakthrough in the triple construct expressing cells is competition among the shRNAs/siRNAs for export and incorporation into RISC, allowing the virus to escape RNAi surveillance and replicate.