Early in 2004, Paddison
et al described their strategy and vector components used to prepare version 1 of their shRNA libraries.
Further understanding of the RNAi pathway and how to exploit this pathway for enforcing
silencing in mammals permitted two strategic improvements in version 2:
- The vector was modified, which resulted in greater than 10-fold increase of shRNA transcription.
- The shRNAs were designed using improved bioinformatics derived from empirical data (Cleary et al, submitted).
Tests of these strategies on components of the proteasome revealed a large
increase in both the percentage of clones that functioned and the strength of the
phenotype that they created. The results of careful titration experiments of plasmid and
RNA transfections followed by quantification of small RNAs in the cell suggest that an
equivalent of at least 40 nM siRNA is achieved. This is well within the effective range of a good siRNA.
