Figure 1

shRNA cassette configuration. (A) Each single shRNA was originally expressed from a human H1 (pol III) promoter in separate vectors. Multiple cassette combinations were made by PCR amplifying each promoter-shRNA-terminator (plus ~100 bp of common flanking sequence) as a self-contained expression cassette, and sequentially inserting them into a single vector via an infinitely expandable cloning strategy. The PCR amplified shRNA expression cassette was digested with 'a' (Mlu I) and 'b' (Asi SI) restriction enzymes (REs) and was ligated to the recipient vector opened up with 'A' (Asc I) and 'B' (Pac I) REs destroying the original 'a', 'A', b', and 'B' sites in the process. The newly created vector has the 'A' and 'B' sites reconstituted via the incoming donor fragment, ready for insertion of subsequent cassettes. The series selected for this study begins with shRNA #3, followed by #8 to make combination 2.2 (shRNA #3.8). Additional shRNAs were added in order to make the combinations 3.2 (#3.8.9), 4.3 (#3.8.9.2), 5.3 (#3.8.9.2.7) and 6.3 (#3.8.9.2.7.6). (B) The average cassette length was ~300 bp long, of which 250 bp (83%) was repeated since each expression cassette was transferred into combination using generic primers.