The Functional Genomics Shared Resource now provides access to the lentiviral human short hairpin RNA (shRNA) library generated by The RNAi Consortium (TRC) of the Broad Institute, purchased through Sigma-Aldrich as their Mission shRNA collection. This collection contains 82,017 unique clones targeting 16,019 genes and is maintained as 958 bacterial glycerol plates, in 96 well format. The lentiviral vector (pLKO.1) expresses the shRNA from a U6 promoter and confers puromycin resistance. Typical viral titers generated with these vectors range from 5 x 106 to 5 x 107 TU/ml. Duke researchers may receive individual clones for $40, which is the charge based on a cost-recovery model.
Why lentiviral shRNA?
Delivery of shRNA through lentiviral transduction permits highly efficient, stable expression of RNAi molecules for long-term gene suppression in most cell types.
-Lentiviral transduction is effective in almost all cell types, including difficult to transfect, primary or post-mitotic cells, expanding the range of possible target cells.
-Drug-resistance marker allows selection of target cells that have been successfully transduced, i.e. 100% of your cells will carry shRNA.
-Stable gene suppression permits investigation of long-term phenotypes, over weeks or months, such as differentiation or in vivo tumor formation.
-Integration of shRNA expression cassettes into the genomes of target cells allows pooling of shRNA-transduced cells into a single sample, followed by a selection strategy that isolates your cells of interest, from which the shRNA sequences can then be recovered by PCR from genomic DNA. This forms the basis of pooled shRNA screening.
NOTE: All lentiviral work must be registered with the Duke Institutional Biosafety Committee (IBC) and SOP must be approved before you can begin.