TRIPZ Lentiviral Inducible shRNAmir

Now part of Thermo Fisher ScientificTRIPZ Lentiviral Inducible shRNAmirSpecific and regulatable RNAiInduction of shRNAmir (tracked byTurboRFP) is tightly regulatedTRIPZ lentiviral shRNAmir, developed in collaborationwith Greg Hannon (CSHL) and Steve Elledge (Harvard),combines the advantages of shRNAmir design, Tet-On®inducibility and powerful lentiviral delivery to producean RNAi trigger that allows tightly regulatable RNAi inmost cell types.Advantages include:• Tet-On® or Tet-Off® configurations possible forregulation• shRNAmir design for increased specificity andknockdown• TurboRFP to mark inducible shRNAmir expression• Lentiviral delivery expands regulatable RNAi optionsin vitro and in vivoHEK293T cells were transduced at an MOI of 0.3, puromycin selected (2ug/ml)for 96 hours after which 500ng/ml of doxycycline (Dox) was added. TurboRFPexpression is assessed at 72 hours (B). Post-Dox samples (C, D) were split,washed with PBS and photographed at 24 hours and 72 hours.TRIPZ Vector DetailsThe pTRIPZ vector has been engineered by OpenBiosystems to be a Tet-On® vector enabling regulatedinduction with:• A modified tetracycline response element (TRE) forreduced basal expression and tighter binding to thetransactivator• A reverse transactivator (rtTA3) modified to includethree mutations that increase its sensitivity todoxycycline by 25 fold over the initial rtTA withoutincreasing background activity (Das, et al. 2004)• A Tet-Off® configuration is also possible using Cre/LoxP technology since the rtTA3 site is flanked byLoxP sites or classical restriction digest with BamH1restriction sitesVector ElementTRErtTA3UBC promotercPPTWRETurboRFPPuro r5’LTRSIN-LTRZEO rUtilityTet-inducible promoterReverse tet-transactivatorDrives rtTA3 expression of rtTA3 and IRES-puroCentral Polypurine tract -helps translocation intothe nucleus of non-dividing cellsEnhances the stability and translation oftranscriptsMarker to track inducible shRNAmir expressionMammalian selectable markerWildtype 5’ long terminal repeat3’ Self-inactivating long terminal repeatBacterial selectable marker

OPEN BIOSYSTEMS TRIPZ Lentiviral Inducible shRNAmirKnockdown induced by DoxycyclineBasal expression as indicated byGAPDH knockdownHigh levels of knockdown achieved after induction with doxycycline:Knockdown of indicated target genes in HEK293T cells compared to a non-silencingcontrol as determined by QPCR. Each bar represents an experimental duplicatewhile QPCR was performed in triplicate using 18SrRNA as an internal reference.HEK293T cells were transduced at an MOI=0.3, puromycin selected (5µg/ml), andinduced with doxycycline (1µg/ml). Dashed line indicates 70% knockdown.Basal expression of shRNA is not dependent on MOI: In the absence ofdoxycycline, GAPDH knockdown was measured in HEK293T cells using QPCR ascompared to a non-silencing control at increasing MOI’s. Each bar represents 4experimental replicates/3 QPCR replicates. A consistent ~20% knockdown wasobserved across a three-fold increase in MOI.Finding your TRIPZ shRNAmirof interestCatalogRHS4696DescriptionTRIPZ lentiviral inducible shRNAmir individual clone1.Enter gene symbol(s), GenBank accession number(s), cloneIDs, unigene clusters or catalog numbers into the gene searchor into the shRNA search ( ) in the purchase grid. Resultswill be returned in a tabbed format. Click on shRNAs under theshRNAs/RNAi tab.RHS4750RHS4741TRIPZ lentiviral inducible empty vectorRNAintro TRIPZ inducible lentiviral shRNAmirtransfection kit2.3.Target gene sets will be shown for all targets that have greaterthan two shRNAmir/ target. Click on the oligo ID link foradditional information including hairpin sequences.Order directly online. Constructs are shipped in 48 hours.RHS4743RHS4744Non-silencing-TRIPZ lentiviral inducibleshRNAmir controlGAPDH-TRIPZ lentiviral inducible shRNAmirpositive controlRHS4697TRIPZ lentiviral inducible shRNAmir libraryGo to www.openbiosystems.com to place an order or call: 1-888-412-2225 to speak with a representative.Your expert partner for Genomic, RNAi, and Proteomic resources.GENOMICS EXPRESS RNAi REPRESS PROTEOMICS DETECTFull-length cDNAsHuman ORFsPluripotency Tool KitMicroRNA-adapted shRNA librariesRNAintro shRNA transfection kitsDrosophila and C. elegans RNAi librariesCustom antibodiesCustom peptidesCatalog antibodiesLIT5022Now part of Thermo Fisher Scientific