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psiRNA-DUO - Expression of Two shRNAs Description psiRNA-DUO generates two shRNAs from the same plasmid for the silencing of either a single target gene (with/without polymorphisms) or two different target genes. psiRNA-DUO contains special features designed to make the cloning of the shRNA inserts and selection of recombinant clones simple and rapid. The major features of the psiRNA-DUO plasmid are its two RNA Pol III cassettes and the GFP-Zeo fusion gene. 7SK RNAi Cassettes The first cassette contains the following elements: - Human 7SK promoter (see page 144) - Acc65 I / Hind III cloning sites - LacZ a-peptide expression cassette for white and blue selection The second cassette contains the following elements: - Human 7SK promoter - Bbs I / Bbs I cloning sites - GUS expression cassette for white and blue selection b-Glucuronidase (GUS, uidA) is an E. coli enzyme capable of hydrolyzing the chromogenic substrate X-Gluc to generate a blue pigment. Therefore, similarly to LacZ, the expression of GUS in permissive E. coli allows the discrimination between blue parental clones and white recombinant clones. psiRNA-DUO is provided with the E. coli GT115, a lacZ uidA mutant strain, enabling the white and blue color selection from the LacZ and GUS systems. GFP-Zeo Fusion Gene The GFP-Zeo gene is a transcriptional fusion between the Green Fluorescent Protein reporter gene and the Zeocin resistance (Zeo R ) gene. The two moieties are separated by a bacterial promoter (EC2K) located within an intron. In E. coli, Zeo R is expressed from the EC2K promoter that is spliced out in mammalian cells. A composite CMV promoter drives the expression of the GFP-Zeo fusion gene, allowing the monitoring of transfection efficiency and the selection of stable clones. Contents and Storage psiRNA-DUO is provided as a kit composed of following: - 20 μg of psiRNA-DUO-GFPzeo - 20 μg of psiRNA-LucLac-GFPzeo (control plasmid) - 1 vial of LyoComp GT115 - 2 sets of sequencing primers (2 x 10 μg each) - 2 pouches of Fast-Media ® Zeo X-Gal - 2 pouches of Fast-Media ® Zeo X-Gluc For more information on LyoComp GT115, sequencing primers and Fast-Media ® X-Gal and X-Gluc, see page 45. Products are shipped at room temperature. Store Fast-Media ® at room temperature. Store all other components at -20°C. Procedures Two Cloning Step Procedure 1- Clone first shRNA insert in first 7SK RNAi cassette opened with Acc65 I and Hind III. 2- Select white recombinant clones on X-Gal plates prepared with Fast-Media ® Zeo X-Gal. 3- Clone second shRNA insert in second 7SK RNAi cassette opened with Bbs I. 4- Select white recombinant clones on X-Gluc plates prepared with Fast-Media ® Zeo X-Gluc. One Cloning Step Procedure 1- Digest psiRNA-DUO with Acc65 I, Hind III and Bbs I. 2- Ligate both shRNA inserts with the two psiRNA-DUO fragments (Hind III/Bbs I and Bbs I/Acc65 I). 3- Select sequentially white recombinant clones on X-Gal plates and X-Gluc plates. PRODUCT QTY CAT. CODE psiRNA-DUO 1 kit ksirna4-gz3 www.invivogen.com/psirna-system 145 RNA INTERFERENCE 7
RNA INTERFERENCE 7 psiTEST System - Prevalidation of siRNAs and shRNAs The psiTEST system enables you to screen for effective siRNAs and shRNAs without using time-consuming and costly reagents that are specific to your target gene, such as antibodies. The psiTEST system can be used to evaluate RNAi on virtually any gene of any species for which the sequence is known. The silencing efficiency of a given siRNA or shRNA is evaluated by monitoring the reduction of expression of a chimeric gene consisting of a secreted alkaline phosphatase (SEAP) reporter gene transcriptionally fused to the target gene. Transfection of effective siRNAs or shRNAs triggers the RNAi process resulting in the degradation of the chimeric mRNA and therefore the absence of SEAP activity. Description The psiTEST system is based on a transcriptional fusion between a reporter gene and your target gene. The reporter gene is an optimized alkaline phosphatase gene engineered to be secreted (SEAP). The efficacy of an siRNA or shRNA to induce RNAi on your target gene is determined by measuring the expression levels of the SEAP reporter gene (see figure). Since SEAP is a secreted protein, the expression level can be evaluated from supernatants therefore allowing to perform kinetics of the silencing process. Features and Benefits The psiTEST system contains two major components, the psiTEST plasmid and QUANTI-Blue . psiTEST plasmid The psiTEST plasmid features the SEAP gene fused at the 3’ end after its Stop codon to a multiple cloning site (MCS). This MCS contains many common restriction sites compatible with many other restriction sites. psiTEST also features a GFP::zeo fusion gene: the zeo moiety allows to amplify the plasmid in E. coli and select stable mammalian clones while the GFP moiety enables the monitoring of transfection efficiency. psiTEST can be used to clone fragments of genes or entire genes, the optimum size being 1.5 kb, although genes up to 3.5 kb can be targeted. QUANTI-Blue QUANTI-Blue is a SEAP detection medium that turns purple/blue in the presence of phosphatase activity. Functional siRNAs or shRNAs will induce the degradation of the SEAP mRNA resulting in the reduction or absence of SEAP activity. Silencing efficiencies of the siRNAs or shRNAs can be observed visually and unlike fluorescent reporters can also be easily quantified using a microplate reader or spectrophotometer. Contents and Storage The psiTEST system is provided with several control plasmids: psiTESThp53, psiRNA-hp53 and psiRNA-Luc. psiTEST-hp53 contains a fusion between the SEAP and human p53 genes. psiRNA-hp53 produces an shRNA that functionally silences the human p53 resulting in the absence of phosphatase activity. psiRNA-Luc expresses an shRNA insert that targets luciferase GL3 and therefore will not affect the expression of the phosphatase reporter gene. The psiTEST system contains the following components: - 20 μg of psiTEST and 20 μg of psiTEST-hp53 (control plasmid) - 20 μg of psiRNA-hp53 and 20 μg of psiRNA-Luc - 2 pouches of QUANTI-Blue (100 ml each) - 4 pouches of Fast-Media ® Zeo (2 TB, 2 Agar) Products are shipped at room temperature. Store Fast-Media ® at room temperature. Store all other components at -20°C. Procedure 146 www.invivogen.com/psitest-system 1- Clone your target gene (or a fragment) into the psiTEST plasmid. 2- Cotransfect mammalian cells with recombinant psiTEST and siRNA or shRNA-producing plasmid, such as psiRNA . 3- Incubate at 37°C with 5% CO2 for 72 hours. 4- Collect supernatant and add to a QUANTI-Blue - containing well. 5- Incubate at 37°C for 30 min to 3 hours. 6- Determine the functionality of your siRNA or shRNA by observing the coloration of QUANTI-Blue visually and/or with a spectrophotometer at 620-655 nm. PRODUCT QTY CAT. CODE psiTEST 1 kit ksitest QUANTI-Blue 5 pouches rep-qb1
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INNOVATION WITHIN REACH InvivoGen
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CONTENTS AT A GLANCE 1 CELL CULTURE
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4 CYTOKINE SIGNALING TABLE of CONTE
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Mycoplasma Detection & Elimination
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PlasmoTest Contents - HEK-Blue -2
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Primocin - The Antimicrobial Shiel
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SEAP Reporter Gene System Secreted
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HEK-Blue Detection - Real-Time Det
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Blasticidin S Description Blasticid
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LyoVec New formulation LyoVec is
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Poly-Arginine-HA Tagged Reprogrammi
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LENTI-Smart - Lentiviral Vector Pr
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Lentiviral Vector Production and Ce
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Antibody generation using pFUSE-CHI
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Engineering Fc regions for altered
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pBROAD & pWHERE - Optimized Vector
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pSELECT-Tag - Expression of a GFP-,
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pVIVO - Expression of Two Genes of
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pORF Description pORF plasmids feat
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Prom A-List - Human and Rodent Pro
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PROMOTER GENE TISSUE DISTRIBUTION S
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ChemiComp E. coli ChemiComp E. coli
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Fast-Media ® Preparation 1. Empty
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RIG-I-like Receptors 54 RLR & RLR-R
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Toll-Like Receptors Toll-Like Recep
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www.invivogen.com/innate-immunity 5
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IPAF and NAIP5 IPAF (NLRC4, CLAN/CA
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C-Type Lectin Receptors C-type lect
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pUNO pUNO-HA Toll-Like Receptors (T
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GENE NAME/ALIASES DESCRIPTION CAT.
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pDUO - Gene Associations Features a
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pZERO-TLR & pDeNy - Dominant Negati
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293/TLR & 293/NOD Clones 293/TLR Cl
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Response of HEK-Blue hTLR2 cells t
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Ramos-Blue Cells - NF-kB/AP-1 Repo
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MEF Cells C3H/TLR4mut & C3H/WT MEFs
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Antibodies for Detection ANTIBODY D
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- NF-kB binding site: Nuclear facto
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PRODUCT ORIGIN/DESCRIPTION TLR2 Ago
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PRODUCT ORIGIN/DESCRIPTION TLR9 Ago
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PRODUCT ORIGIN/DESCRIPTION Dectin-1
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LPS-PG (Porphyromonas gingivalis) R
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• FLA-ST ultrapure was purified b
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1. Zhao H. et al., 2004. Contributi
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1. Hornung V. et al., 2006. 5'-Trip
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TLR/NOD Ligand Screening InvivoGen
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Signal Transduction Inhibitors Invi
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Page 100 and 101: Nigericin - NLRP3 Inflammasome Indu
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Page 104 and 105: 4 CYTOKINE SIGNALING Cytokine Genes
Page 106 and 107: Blue Cytokine Reporter Cells Blue
Page 108 and 109: HEK-Blue IFN-a/b Cells - Human Typ
Page 110 and 111: HEK-Blue IL-6 Cells - IL6 Reporter
Page 112 and 113: HEK-Blue IL-1b Cells - IL-1b Repor
Page 114 and 115: HEK-Blue IL-33/IL-1b Cells - IL-33
Page 116 and 117: HEK-Blue CD40L Cells - CD40L Repor
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Page 120 and 121: Antibody generation using pFUSE-CHI
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Page 124 and 125: IgA Detection and Quantification In
Page 126 and 127: IgG Antibodies InvivoGen offers a s
Page 128 and 129: Mediators? 1. Leroux-Roels G., 2010
Page 130 and 131: Poly(I:C) Synthetic double-stranded
Page 132 and 133: pVAC - Induction of Neutralizing An
Page 134 and 135: CpG-Free DNA CpGs and the Immune Re
Page 136 and 137: pCpGfree-basic & pCpGfree-promoter
Page 138 and 139: pCpGfree-siRNA & pCpGfree-siRNADUO
Page 140 and 141: Custom-Made CpG-Free Genes InvivoGe
Page 143 and 144: RNA INTERFERENCE 7 psiRNA System I
Page 145: RNA INTERFERENCE 7 psiRNA-h7SK - 7S
Page 149 and 150: RNA INTERFERENCE 7 Ready-Made psiRN
Page 151 and 152: RNA INTERFERENCE 7 Custom-Made psiR
Page 153 and 154: INHIBITORS 8 Inhibitors PRODUCT DES
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Page 157 and 158: INHIBITORS 8 Triptolide - NF-kB Inh
Page 159 and 160: INHIBITORS 8 Heat Shock Protein Inh
Page 161 and 162: 9 CUSTOM SERVICES TLR/NOD Ligand Sc
Page 163 and 164: CUSTOM SERVICES 9 Custom Cloning :
Page 165 and 166: CUSTOM SERVICES 9 Prices 164 TERMS
Page 167 and 168: 166 PRODUCT (QUANTITY) CAT. CODE PA
Page 173 and 174: A 17-AAG 17-AAGH2 17-AEP-GA 2-Amino
Page 175 and 176: - ODN 4084-F - ODN INH-1 - ODN INH-
Page 177 and 178: ARGENTINA Genbiotech S.R.L. Tel: +5
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