EXPRESSION ARREST shrna mir GENOME- WIDE LIBRARIES

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2 C GUGAAG EXPRESSION ARREST shrna mir GENOME- WIDE LIBRARIES MicroRNA-adapted shrna (shrna mir ) for increased, specific and consistent knockdown. MicroRNA PROCESSING PATHWAY UTILIZED FOR shrna mir Developed in collaboration with Greg Hannon (CSHL) and Steve Elledge (Harvard) the shrna mir libraries provide unique solutions for your diverse RNAi needs. Features include: Unique microrna-30 based design greatly increases knockdown specificity and efficiency Pre-cloned into viral vectors Transient, stable and in vivo options for RNAi Transfection or infection options for delivery Guaranteed knockdown Why Expression Arrest shrna mir? UNIQUE microrna-adapted DESIGN Endogenous mir-30 primary transcript mir-30 context Open biosystems mir-30 hairpin design Drosha mir-30 sequence cleavage site mir-30 loop LTR Pol II/III shrna mir mir5 mir3 Bact R Mamm R sinltr Drosha Dicer..U GAGC GXNN NNNNNNNNNNNNNNNNNNN UA----- NNNNNNNNNNNNNNNNNN NN NNUG CCUA.. AGAUGUA CAC Antisense sequence of shrna shrna mir design details 1,2,3 Replaced mature microrna sequence in primary mir-30 with genespecific shrna duplexes Added Drosha cleavage site to the hairpin construct Increased Drosha/Dicer processing = More sirnas = Greater knockdown Advantages of shrna mir design Human microrna-30 (mir-30) loop and context sequences adds endogenous processing by Drosha Dicer recognition and specificity is increased by Drosha processing Dicer processing promotes active loading of the RISC complex Destabilizing 5 end of antisense strand results in strand specific incorporation into RISC shrnamir produces more effective knockdown

3 EXPRESSION ARREST shrna mir SHOW 12-FOLD GREATER KNOCKDOWN (A) (B) MW shrna mir shrna 22mer 5S rrna (A) shrna mir constructs produce greater and more consistent knockdown of target genes when compared against conventionally designed shrna to the same genes. Multiple shrnas against various proteasomal genes were tested in a functional assay and the data averaged 3. (B) Northern blotting was used to detect the mature sirna produced after transfection of Hek293 cells with shrna or shrna mir. Transfection was normalized using a co-delivered dsred expression plasmid. A 12-fold increase in the sirna produced after shrna mir processing was detected relative to non-mir shrna processing 3. Evolution of RNAi technology Comparison of silencing triggers sirna, the earliest synthetic RNAi trigger produces rapid, transient-only knockdown. shrna overcomes several limitations inherent in sirna, including the ability to create stable knockdowns in vitro, transduce hard to transfect cell lines, and create animal models. shrna mir does all of the above and produces increased, more specific knockdown relative to sirna or shrna. shrnamir is superior to sirna and conventional shrna

4 GUARANTEED KNOCKDOWN WITH EXPRESSION ARREST shrna mir OPEN BIOSYSTEMS EXPESSION ARREST All Expression Arrest shrna mir constructs are guaranteed* to silence your target gene at least 70% when used along with shrna mir controls and Arrest-In transfection reagent for shrna delivery. These validated and optimized reagents come pre-packaged in RNAintro shrna transfection kits and provide a controlled solution for gene silencing experiments. RNAintro shrna kits include: shrna mir constructs of your choice pick from human and mouse genomes Arrest-In Transfection Reagent for shrna delivery Positive control Luciferase or egfp shrna Negative control Non-silencing shrna construct Transfection efficiency control ß gal reporter shrna mir TO KINESIN RELATED MOTOR PROTEIN eg5 LEADS TO DISRUPTION OF NORMAL CELL DIVI- SION eg5 antibody Tubulin antibody Triple overlay cell 1 cell 2 The kinesin related motor protein EG5 is known to be involved in centrosome separation. HEK293 cells were transfected with eg5 shrna (v2hs_48561) and 48 hrs later stained for tubulin (anti-tubulin, green), DNA (DAPI, blue) and EG5 (anti-eg5, red). Targeting of the EG5 gene by shrna mir results in the formation of half spindles, and cells transfected with EG5 shrna mir are arrested in mitosis and show monoastral microtubular arrays (see cell 1 remains in prometaphase). By contrast, control cells (cell 2) show normal bipolar spindles and microtubule networks in mitosis. As a negative control, HEK293 cells were transfected with a non-silencing shrna. STABLE KNOCKDOWN OF ß-SECRETASE (BACE) WITH shrna mir Anti BACE Anti Actin do not have new image Five shrna mir constructs directed against the human BACE gene were transfected into SH-SY5Y neuroblastoma cells. Stable transfected cells were selected with puromycin and assayed for BACE protein five weeks later. BACE protein expression was reduced between 85-99% by all five shrna mir constructs tested. Actin was used as a loading control. Data courtesy of Dr Aleister Saunders and Preeti Khandelwal at Drexel University * When used with RNAintro protocols and normalized for transfection efficiency. References 1. Paddison, P et al (2004) Nature Methods 1:2, Cleary, M et al (2004) Nature Methods 1:3, Silva, JM et al (2005) Nature Genetics, in Press Save time with guaranteed to work shrnamir

5 shrna mir LIBRARY DELIVERABLES AND FORMATS OPEN BIOSYSTEMS EXPESSION ARREST The Expression Arrest human and mouse shrna mir libraries are available as individual constructs, rearrays of gene families and individually arrayed whole genome libraries. Glycerol stocks and plasmid DNA formats of the shrna mir libraries are available. Individual constructs are available as glycerol stocks while gene families and complete libraries are also available as transfection ready plasmid DNA. Querying for shrna 1. Enter your gene symbol(s) or GenBank accession number(s) 2. Results prefaced by a yellow or green icon are exact matches to your search criteria 3. Click the View link for additional information including hairpin sequences 4. Order directly online. Constructs are shipped in 48 hours! Make Open Biosystems your source for the latest RNAi technology

6 OPEN BIOSYSTEMS EXPESSION ARREST Go to to place an order or call: to speak with a representative. Your expert partner for Genomic, RNAi, and Proteomic resources. GENOMICS E X P R E S S RNAi R E P R E S S PROTEOMICS D E T E C T Full-length cdnas Human ORFs SNAP-tag protein labeling kits MicroRNA-adapted shrna libraries RNAintro shrna transfection kits Drosophila and C. elegans RNAi libraries Custom antibodies Custom peptides Catalog antibodies *SNAP-tag is a trademark of Covalys Biosciences AG LIT