Development of a lentiviral vector for an easy one-step shRNA oligonucleotide cloning procedure and potency screening of a panel of shRNAs targeting human IL-12B. (a) Schematic overview of the lentiviral vector, pCCL-PGK-Puro-H1-MCS, for an easy one-step shRNA cloning procedure. shRNA oligonucleotides with compatible overhangs can be cloned into the multiple cloning site (MCS) from which shRNA expression will be driven by the H1 promoter. (b) Schematic overview of IL-12B mRNA and the target sites for the seven designed shRNAs. (c) Potency screening of seven shRNAs targeting IL-12B mRNA using the dual luciferase assay. HEK293 cells were co-transfected with the shRNA-encoding lentiviral vector and the psiCHECK-IL12B vector encoding firefly luciferase for transfection normalization and a fusion mRNA consisting of renilla luciferase and IL-12B. Luciferase activities were measured forty-eight hours post-transfection and renilla luciferase activity was normalized to firefly luciferase activity and depicted relative to transfection with the empty lentiviral vector, pCCL-PGK-Puro-H1-MCS, not encoding an shRNA (vehicle). (d) Sequence comparison of the 3' end of the H1 promoter depicting sequence differences introduced in the H1 promoter for cloning of shRNA oligonucleotides. The TATA box is depicted in bold, and sequence differences are underlined. Furthermore, the termination signal of the H1 promoter consists of 5 thymines. (e) Confirmation of full promoter activity from the H1 promoter of pCCL-PGK-Puro-H1-MCS modified to encompass a multiple cloning site for easy one-step cloning of shRNA oligonucleotides. shRNA potency was evaluated when expressed from the two sequence contexts of the H1 promoter, namely the context described by Raoul et. al. (black bars) and the new sequence context of the pCCL-PGK-Puro-H1-MCS (white bars). Comparison of shRNA potency was performed using the dual luciferase assay as described above. All dual luciferase assay experiments were performed at least in triplicates and data are depicted as mean + SEM.
Bak et al. BMC Dermatology 2011 11:5 doi:10.1186/1471-5945-11-5