Short hairpin RNA (shRNA) mediated RNAi is a well established method for investigating cancer pathways and is a promising future therapeutic strategy. In particular, delivery of shRNAs by retroviral transduction enables RNAi knockdown in mouse models and cell lines refractory to transfection. The primary disadvantage of retroviral shRNA-based experiments is the reduced target gene knockdown frequently observed when a single copy of the shRNA vector is integrated. We have investigated the fate of shRNA precursors using a novel deep sequencing strategy we have previously developed. We find over 99 percent of precursor molecules are diverted to a non-productive biogenesis route, severely limiting the amount of mature RNA produced. In this proposed application we describe a screening strategy to optimize shRNA design and maximize target gene knockdown potential. Our proposed design will be broadly applicable to cell line and animal studies of cancer pathways.
|Effective start/end date||9/1/15 → 8/31/18|
- NIH National Cancer Institute (NCI)
Small Interfering RNA
Gene Knockdown Techniques
High-Throughput Nucleotide Sequencing