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 partnered with the TRC to make the shRNA libraries available to researchers worldwide. The libraries, targeting human and mouse annotated genes, contain shRNA constructs designed to include a hairpin of 21 base pair sense and antisense stem, and a 6 basepair loop.
Each hairpin sequence was cloned into the lentiviral vector (pLKO.1) and sequence verified. Multiple constructs (4 to 5) were created per gene to ensure adequate coverage of the 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. 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.
Rules-based shRNA design for efficient gene knockdown
Already cloned into lentiviral vectors
Amenable to in vitro and in vivo applications, such as the creation of stable knockdowns
Lentiviral vector enables transduction of primary and non-dividing cell lines
Broad coverage: 4 to 5 constructs per gene
Further information is available at http://www.broad.mit.edu/genome_bio/trc/rnai.html
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).
The TRC lentiviral shRNA libraries are provided in 96-well microtiter plates containing frozen stock cultures of E. coli
in 2XLB broth with 8% glycerol and carbenicillin (100 µg/mL). We check all cultures for growth prior to shipment. Individual shRNA constructs are shipped as glycerol stock cultures on wet ice.
TRC pLKO.1 lentiviral vector
Gene silencing with TRC lentiviral shRNA
- J. Moffat et al., A Lentiviral RNAi Library for Human and Mouse Genes Applied to an Arrayed Viral High-Content Screen. Cell.124, 1283-1298 (2006).
- S. A. Stewart et al., Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA. 9, 493-501 (2003).
- R. Zufferey et al., Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat. Biotechnol.15, 871-885 (1997).
- R. Zufferey et al., Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol. 72, 9873-9880 (1998).
- 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 (2008).
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