TRC Lentiviral shRNA Libraries

Simple hairpin shRNAs in the pLKO.1 lentiviral vector designed by The RNAi Consortium (TRC)
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The RNAi Consortium, or TRC, is a public-private effort based at the Broad whose mission is to create a shRNA library to enable the scientific community to use RNAi to determine the function of human and mouse genes.
  
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The RNAi consortium (TRC) is a collaborative effort based at the Broad Institute of MIT and Harvard, and includes six MIT and Harvard associated research institutions and five international life sciences organizations. We have partnered with the TRC to make shRNA libraries available to researchers worldwide and has compiled shRNA libraries for applications, such as screening.

The pLKO.1 reagents can be packaged into virus particles for use in transduction experiments. Specifically, the replication-incompetent viral particles can be efficiently produced using lentiviral packaging plasmids co-transfected in 293T packaging cells. The Trans-Lentiviral Packaging System, which offers maximum biosafety and high titers, is recommended.

Highlights

  • Libraries available for applications, such as high throughput screening
  • Cloned into lentiviral vectors
  • Amenable to in vitro and in vivo applications, such as the creation of knockdown cell lines
  • Lentiviral vector enables transduction of primary and non-dividing cells
  • Further information is available at http://www.broad.mit.edu/genome_bio/trc/rnai.html

Human TRC gene families and pathways

Gene Ontology (GO) annotated collections targeting all genes in a particular gene family or biological pathway have been created from the human TRC lentiviral shRNA library. These arrayed sets contain all shRNA from the human TRC lentiviral shRNA library that target genes falling under specific GO terms. These collections will greatly simplify pathway analysis, target identification, and validation.

Available Gene family and pathway sets

Gene Family # Genes # Constructs
Angiogenesis 117 606
Apoptosis 763 4037
Cell Cycle 768 4229
GPCRs 549 2732
Kinases 781 4518
Phosphatases 164 817
Transcription Factors 852 4330
Ubiquitins 145 737
Ubiquitin-Ligases 350 1769

GO annotations are applied to genes by curators within the Gene Ontology Consortium and do not originate from us. Consult the Gene Ontology website for details.

TRC shRNA library details

shRNA design and cloning:

The TRC shRNA constructs were designed using an siRNA rules based algorithm consisting of sequence, specificity, and position scoring for optimal hairpins. The hairpin consists of a 21-base stem and a 6-base loop. The hairpins were cloned into the pLKO1 vector and sequence verified.

Multiple (4 to 5) constructs were created for each target gene. The pLKO1 lentiviral vector enables efficient transduction of primary and non-dividing cells, such as neuronal cells, making it easy to perform RNAi studies in these hard to transfect cell lines. Stable selection is also possible using the puromycin selectable marker.

Viral packaging:

Replication-incompetent viral particles can be efficiently produced using  lentiviral packaging plasmids co-transfected in 293T packaging cells. The Trans-Lentiviral packaging system, which offers maximum biosafety and high titers, is recommended.

Controls

Available controls include the pLKO.1 empty vector as a negative control and an eGFP shRNA as a positive control.

Citing the library in Scientific Publications

In scientific publications, the libraries should be referred to as TRC-Hs1.0 (Human) and TRC-Mm1.0 (Mouse). Individual clones are uniquely identified by their TRC ID number (eg. TRCN0000014783).

  
HazardousNo
Shelf Life12 Months
Shipping ConditionDry Ice
Storage Condition-80 C
The TRC lentiviral shRNA libraries are provided in 96-well microtiter plates containing frozen stock cultures of E. coli in 2X LB broth with 8% glycerol and carbenicillin (100 µg/mL). Individual shRNA constructs are shipped as glycerol stock cultures on wet ice. We check all cultures for growth prior to shipment.
The RNAi Consortium (TRC) pLKO.1 lentiviral vector

The RNAi Consortium (TRC) pLKO.1 lentiviral vector

The RNAi Consortium (TRC) pLKO.1 lentiviral vector

The RNAi Consortium (TRC) pLKO.1 lentiviral vector


Gene silencing with TRC lentiviral shRNA

Gene silencing with TRC lentiviral shRNA

Gene silencing with TRC lentiviral shRNA


References

  1. J. Moffat et al., A Lentiviral RNAi Library for Human and Mouse Genes Applied to an Arrayed Viral High-Content Screen. Cell. 124(6), 1283-1298 (24 March 2006).
  2. S. A. Stewart et al., Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA. 9(4), 493-501 (April 2003).
  3. R. Zufferey et al., Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat. Biotechnol. 15(9), 871-885 (September 1997).
  4. R. Zufferey et al., Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J. Virol. 72(12), 9873-9880 (December 1998).
  5. T. Yamamoto, Y. Tsunetsugu-Yokota, Prospects for the therapeutic application of lentivirus-based gene therapy to HIV-1 infection. Curr. Gene Ther. 8(1), 1-8 (February 2008).

Citations