Figure 1

The CellSelectRNAi approach. (a) CellSelectRNAi principle. HL60 reporter cells are transduced with a library of candidate shRNAs targeting gene of interest. Each cell is interrogated by flow cytometry; cells with reduced reporter levels are sorted for subsequent PCR and shRNA sequence identification by 454 deep sequencing. Sequences are plotted by frequency representation in the sorted population. (b) Schematic of the CellSelectRNAi system. The RNAi reporter comprises a destabilized GFP (dsGFP), with the untranslated target gene cloned downstream of a translational stop codon (*). In the scenario on the left, a RISC complex is loaded with an effective shRNA that will bind to the target mRNA sequence in the reporter mRNA and initiate mRNA cleavage and prevention of dsGFP reporter translation. In the scenario on the right, the RISC complex is loaded with an ineffective shRNA that is unable to bind the target mRNA. The efficiency of each shRNA is proportional to the amount of dsGFP in the cell, and can be measured by flow cytometry. (c) RNAi reporter correlates with endogeneous gene expression. Flow cytometric analysis showing the level of RNAi reporter and endogeneous Axl cell surface protein in each epi-allelic MDA-MB-231 cell line demonstrates a linear relationship between RNAi reporter intensity and Axl protein stained with Axl antibody. Each point on the graph represents the geometric mean and the graph is representative of four individual experiments. The error bars represent 95% confidence-limit on the mean.