shRNA Technology Shared Resource
Short hairpin RNA’s (shRNAs) can efficiently suppress gene expression in human cells. The shRNA Technology Shared Resource will provide investigators at 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 as well as 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.
Scientific Director
Shared Resource Location
James E. Clyburn Research Center
Bioengineering Building - 422
Bioengineering Building - 422
shRNA Library Features
- Largest and most validated shRNA collection.
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Human Library: 20,018 genes, 129,695 clones (1500-96 well plates).
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Mouse Library: 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 & position scoring with the Broad Institute algorithm.
- A minimum of 3-5 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.
Useful Links
Protocols
- Culture Amplification & Vector Purification
- Lentivirus Production (PDF)
- Lentivirus Transduction (PDF)
- Vector Maps
- Available Control Vectors (PDF)
Lentiviral Vector Features
- shRNA cloned into the pLKO vector developed by the Broad Institute.
- Allows for both 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.
About Hollings Cancer Center
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