Highly specific and accurate selection of siRNAs for high-throughput functional assays

Santoyo, Javier; Vaquerizas, Juan M.; Dopazo, Joaquı́n

doi:10.1093/bioinformatics/bti196

Cited by 49 publications

(16 citation statements)

References 28 publications

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“…Constructs were verified by DNA sequencing. The shRNAs were designed as described (51), and sequences are as follows (sh1Rex1: 5′-ACGGATACCTAGAGTGCATCA, sh2Rex1: 5′-CACGGAGAGCTCGAAACTAAA, shRNA Gfp: 5′-AAGCGCGATCACATGGTCCTG). Plasmids used for transfections were purified on PureLink™ kits and columns (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

Expression of endogenous retroviruses is negatively regulated by the pluripotency marker Rex1/Zfp42

Guallar

Pérez-Palacios

Climent

et al. 2012

Nucleic Acids Research

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Rex1/Zfp42 is a Yy1-related zinc-finger protein whose expression is frequently used to identify pluripotent stem cells. We show that depletion of Rex1 levels notably affected self-renewal of mouse embryonic stem (ES) cells in clonal assays, in the absence of evident differences in expression of marker genes for pluripotency or differentiation. By contrast, marked differences in expression of several endogenous retroviral elements (ERVs) were evident upon Rex1 depletion. We demonstrate association of REX1 to specific elements in chromatin-immunoprecipitation assays, most strongly to muERV-L and to a lower extent to IAP and musD elements. Rex1 regulates muERV-L expression in vivo, as we show altered levels upon transient gain-and-loss of Rex1 function in pre-implantation embryos. We also find REX1 can associate with the lysine-demethylase LSD1/KDM1A, suggesting they act in concert. Similar to REX1 binding to retrotransposable elements (REs) in ES cells, we also detected binding of the REX1 related proteins YY1 and YY2 to REs, although the binding preferences of the two proteins were slightly different. Altogether, we show that Rex1 regulates ERV expression in mouse ES cells and during pre-implantation development and suggest that Rex1 and its relatives have evolved as regulators of endogenous retroviral transcription.

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Section: Methodsmentioning

confidence: 99%

Expression of endogenous retroviruses is negatively regulated by the pluripotency marker Rex1/Zfp42

Guallar

Pérez-Palacios

Climent

et al. 2012

Nucleic Acids Research

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“…To design this mini-library, small hairpins located in the open reading frame of the target genes were selected using the SIDE software. 45 To avoid off-target gene knock-downs normally associated with the presence of a perfect match into the 3Ј untranslated region of nontarget genes, 46 hairpins were selected exclusively within the open reading frame of the target genes (Table S1). Individual small hairpins (sh) were cloned by standard PCR into pENTR vector downstream of the H1 promoter generating 277 sequence-verified shRNAi pENTR clones that subsequently were subcloned into the pA179.Helix lentiviral (HIV)-based vector.…”

Section: Generation Of a Lentiviral Rna Interference Mini-library Formentioning

confidence: 99%

Lentiviral (HIV)-based RNA interference screen in human B-cell receptor regulatory networks reveals MCL1-induced oncogenic pathways

Ruiz-Vela

Aggarwal

Cueva

et al. 2008

Blood

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IntroductionReciprocal chromosomal translocations involving one of the IG loci and a proto-oncogene are hallmarks of many types of human B-cell non-Hodgkin lymphomas (B-NHL). 1 As a consequence of such translocations, several proto-oncogenes are placed under the transcriptional control of the active IG locus, causing constitutive expression of the oncogenes with subsequent B-cell transformation. 2 During B-cell tumor progression, IG chromosomal translocations are thought to be early events in the pathogenesis of these neoplasias and result in critical deregulation of multiple oncogenes such as BCL2 t(14;18)(q32;q21) in follicular lymphomas, MYC t(8;14)(q24;q32) in Burkitt lymphomas, CCND1 t(11;14)(q13;q32) in mantle cell lymphomas, MALT1 t(1;14)(p21;q32) in extranodal MALT lymphomas, or BCL6 t(3;V)(q27;V) in diffuse large cell lymphomas. 3 In addition to these chromosomal aberrations, several observations suggest that B-cell receptor (BCR) signaling supplies important tonic survival signals that might be required for the maintenance of B-cell lymphomas. 4 Considering the allelic exclusion model, chromosomal translocation events should occur at equal frequency on the expressed IGH allele and the nonexpressed IGH allele. Because translocations of proto-oncogenes into the IGH-loci are always found on the nonproductively rearranged IGH loci, with few exceptions 5 ; these findings suggest that survival of B-cell lymphoma clones is incompatible with translocations of protooncogenes into the productively rearranged IGH loci because this type of translocation would induce inability to express immunoglobulin (Ig), 2 and suggests the dependency of Ig expression for B-cell lymphoma survival. Moreover, taking into account that the somatic hypermutation machinery induces 2 types of destructive somatic mutations (nonsense mutations and duplications causing reading-frame shifts); these events would lead to the generation of B-cell lymphoma clones that lack Ig expression under nonselective conditions. 6 Because that treatment of patients with anti-idiotypic antibodies (a selective condition) did not result in the selection of Ig-negative B-cell lymphoma clones, 7 this observation also suggests the dependency of Ig expression for the survival of many B-cell lymphomas. 2 In support of this hypothesis, several molecules integrated in the BCR regulatory network appear to act as oncoproteins, such as SYK, 8 ZAP70, 9 BLK, 10 and MALT1, 11 whereas other BCR regulatory molecules surprisingly promote programmed cell death, such as LYN, 12 IP3R, 13 calpains, 14 SLP65, 15 and BTK. 16 These observations exemplify both positive and negative regulatory roles for BCR in signaling and raise the question of how the BCR signal transduction pathway can generate such distinct outcomes and what is the contribution of BCR signaling in B-cell transformation.B-cell antigen receptors specific for self-molecules are normally produced as a consequence of the random VDJ recombination. B cells that recognize a self-antigen undergo programmed cell death ser...

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“…4b). DISCUSSION Although a set of guidelines for the selection of potential siRNA duplexes has been proposed [16][17][18], there are no reliable methods for selecting the effective target regions without practical test. It is well known that different siRNA duplexes induce different levels of RNAi activity [5,12].…”

Section: Inhibitory Effects Of Aiv Replication By Stablymentioning

confidence: 99%

Screening efficient siRNAs in vitro as the candidate genes for chicken anti-avian influenza virus H5N1 breeding

et al. 2010

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The frequent disease outbreaks caused by avian influenza virus (AIV) not only affect the poultry industry but also pose a threat to human safety. To address the problem, RNA interference (RNAi) has recently been widely used as a potential antiviral approach. Transgenesis, in combination with RNAi to spe cifically inhibit AIV gene expression, has been proposed to make chickens resistant to avian influenza. For the transgenic breeding, screening the efficient siRNAs in vitro as the candidate genes is one of the most important tasks. Here, we combined an online search tool and a series of bioinformatics programs with a set of rules for designing the siRNAs targeting different mRNA regions of AIV H5N1 subtype. By this method we chose five rational siRNAs, constructed five U6 promoter driven shRNA expression plasmids contained the siRNA genes, and used these to produce stably transfected Madin Darby canine kidney (MDCK) cells. Data from virus titration, IFA, PUI stained flow cytometry, real time quantitative RT PCR and DAS ELISA analyses showed that all five stably transfected cell lines were effectively resistant to viral replication when exposed to 100 CCID 50 of AIV, and we finally chose the most effective plasmids (pSi 604i and pSi 1597i) as the candidates for making the transgenic chickens. These findings provide baseline information for breeding transgenic chickens resistant to AIV in combination with RNAi.

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Highly specific and accurate selection of siRNAs for high-throughput functional assays

Abstract: jdopazo@cnio.es.

Cited by 49 publications

References 28 publications

Expression of endogenous retroviruses is negatively regulated by the pluripotency marker Rex1/Zfp42

Expression of endogenous retroviruses is negatively regulated by the pluripotency marker Rex1/Zfp42

Lentiviral (HIV)-based RNA interference screen in human B-cell receptor regulatory networks reveals MCL1-induced oncogenic pathways

Screening efficient siRNAs in vitro as the candidate genes for chicken anti-avian influenza virus H5N1 breeding

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