for over-expression of human pri-miRNA transcripts
The miR-express™ lentiviral microRNA (miRNA) library is a collection of individual miRNA precursors cloned into a lentiviral vector allowing efficient over-expression of human primary microRNA (pri-miRNA). This collection was created in collaboration with Dr. Muneesh Tewari at the Fred Hutchinson Cancer Research Center. The goal of this project was to clone all known human pri-miRNA transcripts with their natural flanking sequences in lentiviral vectors.
Human miRNAs were selected from the Sanger miRbase database version 11.0 (http://microrna.sanger.ac.uk/sequences) and the genome coordinates were used as a reference to design primers to PCR amplify the pri-miRNA sequences with 5’ and 3’ flank sequences. The primers for amplification of all human primary miRNAs were designed to retain as much flanking sequence as possible for efficient Drosha processing and endogenous regulation. On average 200 bases of flanking sequence was retained on both sides of the precursor miRNA. The pri-miRNA inserts were then cloned into the lentiviral vector pLemiR™, under the control of a CMV promoter.
Highlights of the miR-express collection include:
- miRNA transcripts are in their native sequence context to ensure endogenous RNAi processing into mature miRNAs
- miRNAs are expressed in target cells through transfection or transduction
- TurboRFP allows tracking of miRNA expression
- Puromycin selectable marker
- Lentivirus-based system for expression in a wide range of cell lines including primary and non-dividing cells
- Transient, stable and in vivo studies are possible
Additional technical details
Transduction or transfection starter kits are available.
miR-Express starter kits combine the versatility of lentiviral delivery, the simplicity of validated controls, and optimized transfection reagents - all in one convenient package.
Both kits include:
- Two miR-express constructs of your choice
- Arrest-In™ transfection reagent for optimized DNA delivery
- Validated non-silencing control
- Empty vector control
Transduction kits also include Trans-Lentiviral™ packaging mix sufficient for 10 packaging events.
Useful links:
miRbase at the Sanger Institute
Please
note that the pLemiR vector is not compatible with third generation
packaging systems such as ViraPower™ from Invitrogen. We recommend the Trans-Lentiviral™ Packaging System for use with our vectors.
Finding your microRNA of interest
- Enter microRNA ID, accession, symbol(s), or catalog numbers into the gene search. Results will be returned in a tabbed format. Click on shRNA under the shRNAs/RNAi tab.
- Click on the oligo ID link for additional cloning and sequence information. Complete insert sequence is displayed. Precursor miRNA sequence can be obtained from the datafile in the purchase grid or from miRbase.
- Order directly online
Individual miR-express microRNA constructs are shipped as glycerol stock cultures on wet ice. Open Biosystems checks all cultures for growth prior to shipment. Store at -80ºC.
miRBase: microRNA sequences, targets and gene nomenclature.
Griffiths-Jones S, Grocock RJ, van Dongen S, Bateman A, Enright AJ.
NAR, 2006, 34, Database Issue, D140-D144
The microRNA Registry.
Griffiths-Jones S.
NAR, 2004, 32, Database Issue, D109-D111
A uniform system for microRNA annotation.
Ambros V, Bartel B, Bartel DP, Burge CB, Carrington JC, Chen X, Dreyfuss G, Eddy SR, Griffiths-Jones S, Marshall M, Matzke M, Ruvkun G, Tuschl T.
RNA, 2003, 9(3), 277-279
Stegmeier F et al (2005) "A lentiviral microRNA-based system for single-copy polymerase II-regulated RNA interference in mammalian cells" PNAS Vol 102:37; 13212-13217
Yamamoto T. and Y. Tsunetsugu-Yokota (2008) "Prospects for the therapeutic application of lentivirus-based gene therapy to HIV-1 infection" Curr Gene Ther. Vol 8:1; 1-8.
