Figure 2

An infinitely expandable cloning strategy. In the example of our multiple cassette cloning strategy shown, a 7th cassette is being inserted into a vector that already has 6 cassettes integrated (A). The incoming donor fragment is a PCR amplified shRNA expression cassette (B) digested with 'a' (Mlu I) and 'b' (Asi SI) restriction enzymes (REs) which is 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. Each shRNA expression cassette included the H1 promoter, shRNA, terminator and some flanking sequence to a total length of ~ 270 - 300 bp. (C) All 10 single shRNA expression cassettes were first transferred from pSilencer type plasmids (as assembled in prior work) as complete expression cassettes into single shRNA Lentivirus transfer plasmids (setup with an infinitely expandable MCS as detailed above).