The analysis of dose response curve comes in useful for the assembly of multi-siRNAs expressing cassettes

The construction of vectors that would allow the simultaneous expression of multiple siRNAs targeted against different genes is hampered by the competition between siRNAs. In this work, the simultaneous knock-down of four genes involved in smooth muscle cells activation, migration and proliferation was considered. We used the knock- down of EGFP reporter assay to evaluate the dose response curves of the four shRNA expressing plasmids. We found that each siRNA reached the highest acitivity (as evaluated from the plateau phase) with different kinetics (as evaluated from the KD). Due the specificity of KD, the mono-specific plasmids were tested against their targets by the addiction of saturating amounts of each of the other shRNA-expressing plasmid. In this way, stronger from weaker shRNAs were distinguished and KD seemed to account for it. Moreover, when stronger shRNAs were assembled, the resulting plasmid was able to simultaneously transcribe active shRNAs genes. These results indicate that expression cassettes for different siRNAs having similar KD can be efficiently and rapidly assembled into multi-specific multi-siRNA plasmids. A practical correlate of these observations is that, in order to obtain effective multi-gene knock down, only siRNAs with similar inhibitory kinetics need to be delivered to the cells.