Fig 1. Schematic representations of the major steps from pSUPER to concatenable and regulatable multi-shRNA-expression vectors.

(A) pSUPER. A sequence encoding a specific shRNA would be introduced using the Bgl II and Hin dIII sites. Middle: pSUPER-Sac I (= pSUS). A sequence encoding a specific shRNA would be introduced using the Bgl II and the newly generated Sac I site (in red). Upstream of the H1 promoter the formerly present Sac I site has been destroyed, and the original Bst XI site has been modified in such a way that the 6 optional core nucleotides represent a 3-prime-corrupted Sac I motif (GAGCTG) which is cleaved by Bst XI to generate Sac I compatible ends. Bottom: pSUSTER2 (= pS2). Two tet-operator (TetO) sequences [9, 10] within the H1 promoter yield the shRNA expression cassette doxycyclin-inducible in cells that constitutively express the tet repressor protein. (B) Versatile concatenability of the pS2-system. Top: Schematic representation of a pS2-series vector for expression of a single short hairpin RNA targeting the MAP kinase ERK2. Bottom: Schematic representation of a pS2-series vector containing 4 different inducible shRNA expression cassettes targeting MEK2, MEK1, ERK2 and ERK1. Head-to-tail subcloning of a Bst XI/Sac I-cut pS2 shRNA expression cassette into another pS2 vector linearized with Bst XI regenerates the 5'-Bst XI site but does not re-create any internal sites. Additional cassettes can thus be added ad libitum. For stable transfections the whole multi-shRNA expression cassette can be subcloned via Bst XI/Sac I-digest into any suitable vector that contains a unique Sac I compatible cloning site. MCS: multiple cloning site (of pBluescript KS(+), the parental plasmid of pSUPER).