An optimized design for single copy short hairpin RNAi

Research project

Description

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.
StatusActive
Effective start/end date9/1/158/31/18

Funding

  • NIH National Cancer Institute (NCI)

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RNA Interference
Small Interfering RNA
Gene Knockdown Techniques
Cell Line
Neoplasms
High-Throughput Nucleotide Sequencing
RNA Precursors
Transfection
RNA
Therapeutics