P150CAF-1 depletion by RNAi.
The RNAi plasmid vector that we used in this study contains the mouse H1 promoter and a puromycin selection gene (SB and MG, unpublished data). We characterized in detail the properties of this new vector using a GFP target gene and FACS: Extinction of target genes in ES cells is highly efficient in 70% of the cells, of intermediate efficiency in 15%, and inefficient in the remaining 15% of cells analysis. The sequence of p150CAF-1 siRNA (short interfering RNA) duplex [16] was cloned into this plasmid. The control vector expressed a siRNA that targets GFP RNA degradation. ES cells were transfected by electroporation with the RNA plasmid vector, seeded onto gelatin-coated slides, and cultured for 24 h in the absence of selection. Puromycin (2 μg/ml) was added to the culture medium and cells were cultured for an additional 48-h period. Using the p150CAF-1 RNAi vector, IF microscopy quantification reveals a complete CAF-1 depletion in most cells (Figure S1B). Western blot analysis (Figure S1A) reveals residual p150CAF-1 expression, as expected from the 15% of cells that do not inhibit the target gene.
MEFs (passage 3, 90% confluence) were transfected during 4 h with the RNAi plasmid vector using Lipofectamine 2000 (Invitrogene, Carlsbad, California, United States) according to manufacturer's conditions. Cells were trypsinized, plated at 1/3 dilution, and cultured for 48 h in the presence of puromycin.