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shRNA Technology Shared Resource

hand filling small pipettes

Short hairpin RNAs (shRNAs) can efficiently suppress gene expression in human cells. The shRNA Technology Shared Resource will provide investigators at the Medical University of South Carolina (MUSC) access to genome-wide human and mouse libraries that together encode a total of almost 160,000 shRNA clones against over 41,000 genes.

The resource utilizes The RNAi Consortium’s (TRC) genome-wide lentiviral mouse and human libraries, and investigators will have the option of ordering shRNA’s targeting single or multiple genes, gene family sets, and pathway-specific pooled libraries.

The library will allow access to multiple shRNAs for a single gene, which is important for validation against off-target effects. This technology holds tremendous power and is ready to help investigators at MUSC work toward breakthrough discoveries.

For more information, contact Jian Ouyang, Ph.D., at ouyangj@musc.edu.

shRNA Library Features

  • Largest and most validated shRNA collection.
  • Human library: 20,018 genes, 129,695 clones (1500-96 well plates).
  • Mouse library:21,171 genes, 118,062 clones (1374-96 well plates).
  • Hairpins comprised of a 21mer base stem and a 6 base loop designed against NCBI REFSEQ.
  • Sequence, specificity, and position scoring with the Broad Institute algorithm.
  • A minimum of three to five shRNA constructs are created for each target gene to provide varying levels of knockdown and to target different regions of mRNA transcript.
  • For any given RefSeq, there is often a shRNA clone targeting the 3'UTR for use in phenotypic rescue studies using cDNA expression constructs.

Lentiviral Vector Features

  • shRNA cloned into the pLKO vector developed by the Broad Institute.
  • Allows for stable or transient transfection.
  • Self-inactivating replication incompetent viral particles can be produced in packaging cells (HEK293T) by co-transfection with compatible packaging plasmids.
  • Stable gene silencing is selected using the puromycin selectable marker.
  • Integrates for long-term knockdown.
  • Transduces virtually any cell type (dividing or non-dividing).
  • No interferon response.
  • Lack of recombination issues.

Useful Links

Protocols