Expression plasmid constructs of shRNAs

The effective shRNAs targeting Np95, Dnmt1, Dnmt3a or Dnmt3b were individually evaluated and selected from the lentiviral-based shRNA-expressing plasmid DNA library generated by the RNAi Consortium and prepared by the RNAi Core at Academia Sinica according to the results of quantitative RT-PCR (Dnmt3a or Dnmt3b) and Western blotting (Dnmt1 or Np95) analyses following transient transfection of the plasmid DNAs in ES cells for 2 days. The empty vector pLKO.1 and pLKO.1-sh plasmid expressing a shRNA (Control-sh) without targets in the mouse genome were used as controls. The RNAi Consortium Numbers (TRCNs) of the selected shRNA plasmid DNAs are the following: TRCN0000039482 and TRCN0000302343 for Np95-sh1 and Np95-sh2; TRCN0000039027 and TRCN0000225700 for Dnmt1-sh1 and Dnmt1-sh2; TRCN0000039034, TRCN0000039036 and TRCN0000039037 for Dnmt3a; TRCN0000071068, TRCN0000071070 and TRCN0000071072 for Dnmt3b.

For the purposes of proper processing of the shRNAs and generation of stable knockdown cell lines, DNA oligos encoding a control shRNA targeting the lacZ sequence (sh) and the six DNA regions encoding shRNAs targeting Dnmt3a or Dnmt3b with a high score of knockdown efficiency (<60% remaining) were chosen and individually re-cloned into the pCI.1-miR-EGFP-Puro expression vector (un-published, a derivative from pCI of Promega). The coding sequence of each of the seven shRNAs was embedded between the 5′ and 3′ flanking sequences of the murine miR-155 precursor⁶¹ within the artificial intron of the pCI.1-miR-EGFP-Puro expression vector. The CMV promoter in the plasmid directed co-expression of the shRNA(s) with a bicistronic transcript containing EGFP and an IRES-connected puromycin-resistance gene. Puromycin resistance was used for the selection of different stable ES clones and the intensity of EGFP fluorescence was used as an indicator of the expression level of the intronic shRNA(s).