Overview

Recent advances in the field of RNA interference (RNAi) have enabled researchers to conduct in depth investigations of gene function. High throughput screens in cultured mammalian cells can now be performed using RNAi libraries such as our own Hannon-Elledge library. Our second-generation shRNA libraries consist of over 200,000 short hairpin RNA constructs targeting over 45,000 human and mouse genes modeled after primary miRNA transcripts. Each hairpin is linked to a unique 60 nucleotide identification sequence, which serves as a barcode and allows us to virtually count the number of cells that contain a specific hairpin in a cell population. Small changes in barcode copy number can be monitored through the use of microarray technology. The barcode can be amplified from a cell's genomic DNA and fluorescently labeled to produce a probe that is hybridized to a microarray. We have optimized our probe labeling methods, probe size and hybridization conditions using library plasmid DNA in a Nimblegen platform. This optimized protocol allows us to detect 78.6% of probes within 1 standard deviation above the mean background from a complex mixture of approximately 1500 hairpins. By using two color hybridizations, we can detect control subsets of hairpins known to be depleted from a sample population. I applied this RNAi barcode screening method in a screen using a complex mixture of 7500 library hairpins in a p53 isogenic HCT 116 colon cancer cells to identify genes that modify sensitivity to a common chemotherapeutic, doxorubicin. Knockdown of Chk1, a kinase that mediates the G2/M checkpoint, increases sensitivity to doxorubicin in the p53 deficient HCT116 cells but not to the p53 wt HCT116 cell line. This work illustrates the powerful use of RNAi screens to search for genes that synergize with existing therapeutics, and suggests strategies for genetically informed combination therapies.

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9781243570574

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