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Sample records for morpholino gene knockdown

  1. Sterilization of sterlet Acipenser ruthenus by using knockdown agent, antisense morpholino oligonucleotide, against dead end gene.

    PubMed

    Linhartová, Zuzana; Saito, Taiju; Kašpar, Vojtěch; Rodina, Marek; Prášková, Eva; Hagihara, Seishi; Pšenička, Martin

    2015-10-15

    Sturgeons (chondrostean, acipenseridae) are ancient fish species, widely known for their caviar. Nowadays, most of them are critically endangered. The sterlet (Acipenser ruthenus) is a common Eurasian sturgeon species with a small body size and the fastest reproductive cycle among sturgeons. Such species can be used as a host for surrogate production; application is of value for recovery of critically endangered and huge sturgeon species with an extremely long reproductive cycle. One prerequisite for production of the donor's gametes only is to have a sterile host. Commonly used sterilization techniques in fishes such as triploidization or hybridization do not guarantee sterility in sturgeon. Alternatively, sterilization can be achieved by using a temporary germ cell exclusion-specific gene by a knockdown agent, the antisense morpholino oligonucleotide (MO). The targeted gene for the MO is the dead end gene (dnd) which is a vertebrate-specific gene encoding a RNA-binding protein which is crucial for migration and survival of primordial germ cells (PGCs). For this purpose, a dnd homologue of Russian sturgeon (Agdnd), resulting in the same sequence in the start codon region with isolated fragments of sterlet dnd (Ardnd), was used. Reverse transcription polymerase chain reaction confirmed tissue-specific expression of Ardnd only in the gonads of both sexes. Dnd-MO for depletion of PGCs together with fluorescein isothiocyanate (FITC)-biotin-dextran for PGCs labeling was injected into the vegetal region of one- to four-cell-stage sterlet embryos. In the control groups, only FITC was injected to validate the injection method and labeling of PGCs. After optimization of MO concentration together with volume injection, 250-μM MO was applied for sterilization of sturgeon embryos. Primordial germ cells were detected under a fluorescent stereomicroscope in the genital ridge of the FITC-labeled control group only, whereas no PGCs were present in the body cavities of morphants

  2. Knockdown of gene expression by antisense morpholino oligos in preimplantation mouse embryos cultured in vitro.

    PubMed

    Sato, Yuki; Sato, Shiori; Kikuchi, Takahiro; Nonaka, Asumi; Kumagai, Yuki; Sasaki, Akira; Kobayashi, Masayuki

    2016-09-15

    Knockdown of gene expression by antisense morpholino oligos (MOs) is a simple and effective method for analyzing the roles of genes in mammalian cells. Here, we demonstrate the efficient delivery of MOs by Endo-Porter (EP), a special transfection reagent for MOs, into preimplantation mouse embryos cultured in vitro. A fluorescein-labeled control MO was applied for monitoring the incorporation of MOs into developing 2-cell embryos in the presence of varying amounts of EP and bovine serum albumin. In optimized conditions, fluorescence was detected in 2-cell embryos within a 3-h incubation period. In order to analyze the validity of the optimized conditions, an antisense Oct4 MO was applied for knockdown of the synthesis of OCT4 protein in developing embryos from the 2-cell stage. In blastocysts, the antisense Oct4 MO induced a decrease in the amount in OCT4 protein to less than half. An almost complete absence of OCT4-positive cells and nearly complete disappearance of the inner cell mass in the outgrowths of blastocysts were also noted. These phenotypes corresponded with those of Oct4-deficient mouse embryos. Overall, we suggest that the delivery of MOs using EP is useful for the knockdown of gene expression in preimplantation mouse embryos cultured in vitro. PMID:27381842

  3. Characterization of zebrafish dysferlin by morpholino knockdown

    SciTech Connect

    Kawahara, Genri; Serafini, Peter R.; Myers, Jennifer A.; Alexander, Matthew S.; Kunkel, Louis M.

    2011-09-23

    Highlights: {yields} cDNAs of zebrafish dysferlin were cloned (6.3 kb). {yields} The dysferlin expression was detected in skeletal muscle, heart and eye. {yields} Injection of antisense morpholinos to dysferlin caused marked muscle disorganization. {yields} Zebrafish dysferlin expression may be involved in stabilizing muscle structures. -- Abstract: Mutations in the gene encoding dysferlin cause two distinct muscular dystrophy phenotypes: limb-girdle muscular dystrophy type 2B (LGMD-2B) and Miyoshi myopathy (MM). Dysferlin is a large transmembrane protein involved in myoblast fusion and membrane resealing. Zebrafish represent an ideal animal model to use for studying muscle disease including abnormalities of dysferlin. cDNAs of zebrafish dysferlin were cloned (6.3 kb) and the predicted amino acid sequences, showed 68% similarity to predicted amino acid sequences of mammalian dysferlin. The expression of dysferlin was mainly in skeletal muscle, heart and eye, and the expression could be detected as early as 11 h post fertilization (hpf). Three different antisense oligonucleotide morpholinos were targeted to inhibit translation of this dysferlin mRNA and the morpholino-injected fish showed marked muscle disorganization which could be detected by birefringence assay. Western blot analysis using dysferlin antibodies showed that the expression of dysferlin was reduced in each of the three morphants. Dysferlin expression was shown to be reduced at the myosepta of zebrafish muscle using immunohistochemistry, although the expression of other muscle membrane components, dystrophin, laminin, {beta}-dystroglycan were detected normally. Our data suggest that zebrafish dysferlin expression is involved in stabilizing muscle structures and its downregulation causes muscle disorganization.

  4. Development of the morpholino gene knockdown technique in Fundulus heteroclitus: a tool for studying molecular mechanisms in an established environmental model

    PubMed Central

    Matson, Cole W.; Clark, Bryan W.; Jenny, Matthew J.; Fleming, Carrie R.; Hahn, Mark E.; Di Giulio, Richard T.

    2008-01-01

    A significant challenge in environmental toxicology is that many genetic and genomic tools available in laboratory models are not developed for commonly used environmental models. The Atlantic killifish (Fundulus heteroclitus) is one of the most studied teleost environmental models, yet few genetic or genomic tools have been developed for use in this species. The advancement of genetic and evolutionary toxicology will require that many of the tools developed in laboratory models be transferred into species more applicable to environmental toxicology. Antisense morpholino oligonucleotide (MO) gene knockdown technology has been widely utilized to study development in zebrafish and has been proven to be a powerful tool in toxicological investigations through direct manipulation of molecular pathways. To expand the utility of killifish as an environmental model, MO gene knockdown technology was adapted for use in Fundulus. Morpholino microinjection methods were altered to overcome the significant differences between these two species. Morpholino efficacy and functional duration were evaluated with molecular and phenotypic methods. A cytochrome P450-1A (CYP1A) MO was used to confirm effectiveness of the methodology. For CYP1A MO-injected embryos, a 70% reduction in CYP1A activity, a 86% reduction in total CYP1A protein, a significant increase in β-naphthoflavone-induced teratogenicity, and estimates of functional duration (50% reduction in activity 10 dpf, and 86% reduction in total protein 12 dpf) conclusively demonstrated that MO technologies can be used effectively in killifish and will likely be just as informative as they have been in zebrafish. PMID:18378331

  5. Morpholinos: studying gene function in the chick

    PubMed Central

    Norris, Anneliese; Streit, Andrea

    2014-01-01

    The use of morpholinos for perturbing gene function in the chick, Gallus gallus, has led to many important discoveries in developmental biology. This technology makes use of in vivo electroporation, which allows gain and loss of function in a temporally, and spatially controlled manner. Using this method, morpholinos can be transfected into embryonic tissues from early to late developmental stages. In this article, we describe the methods currently used in our laboratory to knock down gene function using morpholinos in vivo. We also detail how morpholinos are used to provide consistency of the results, and describe two protocols to visualise the morpholino after electroporation. In addition, we provide guidance on avoiding potential pitfalls, and suggestions for troubleshooting solutions. These revised techniques provide a practical starting point for investigating gene function in the chick. PMID:24184187

  6. Spatiotemporal control of embryonic gene expression using caged morpholinos.

    PubMed

    Shestopalov, Ilya A; Chen, James K

    2011-01-01

    Embryonic development depends on spatial and temporal control of gene function, and deciphering the molecular mechanisms that underlie pattern formation requires methods for perturbing gene expression with similar precision. Emerging chemical technologies can enable such perturbations, as exemplified by the use of caged morpholino (cMO) oligonucleotides to photo-inactivate genes in zebrafish embryos with spatiotemporal control. This chapter describes general principles for cMO design and methods for cMO assembly in three steps from commercially available reagents. Experimental techniques for the microinjection and photoactivation of these reagents are described in detail, as well as the preparation and application of caged fluorescein dextran (cFD) for labeling irradiated cells. Using these protocols, cMOs can be effective tools for functional genomic studies in zebrafish and other model organisms. PMID:21924162

  7. Ruthenium-Caged Antisense Morpholinos for Regulating Gene Expression in Zebrafish Embryos

    PubMed Central

    Griepenburg, J.C.; Rapp, T.L.; Carroll, P.J.; Eberwine, J.; Dmochowski, I.J.

    2015-01-01

    Photochemical approaches afford high spatiotemporal control over molecular structure and function, for broad applications in materials and biological science. Here, we present the first example of a visible light responsive ruthenium-based photolinker, Ru(bipyridine)2(3-ethynylpyridine)2 (RuBEP), which was reacted stoichiometrically with a 25mer DNA or morpholino (MO) oligonucleotide functionalized with 3′ and 5′ terminal azides, via Cu(I)-mediated [3+2] Huisgen cycloaddition reactions. RuBEP-caged circular morpholinos (Ru-MOs) targeting two early developmental zebrafish genes, chordin and notail, were synthesized and tested in vivo. One-cell-stage zebrafish embryos microinjected with Ru-MO and incubated in the dark for 24 h developed normally, consistent with caging, whereas irradiation at 450 nm dissociated one 3-ethynylpyridine ligand (ϕ = 0.33) and uncaged the MO to achieve gene knockdown. As demonstrated, Ru photolinkers provide a versatile method for controlling structure and function of biopolymers. PMID:26023327

  8. Gene Interference with Morpholinos in a Gold Nanoparticle-Based Delivery Platform in Rat PC12 Cells.

    PubMed

    Deng, Wei; Farnham, Melissa M J; Goldys, Ewa M; Mohammed, Suja; Pilowsky, Paul M

    2015-12-01

    For the first time the efficiency of gene knockdown of the pituitary adenylate cyclase-activating polypeptide (PACAP) receptor 1 (PAC1R) is demonstrated by employing gold nanocomplexes. This gene knockdown subsequently affects the action of PACAP on neurite outgrowth in PC12 cells. These nanocomplexes comprise cholera toxin B (CTB)-gold nanoparticle conjugates loaded with double-stranded morpholinos (MOs) (photo MO and antisense MO). Nanocomplexes are introduced into cells via lipid raft-dependent endocytosis. After UV light exposure, the photolinker in the photo MO is cleaved, bisecting the photo MO and releasing the antisense MO from the conjugate. The antisense MO then binds the PAC1R mRNA and decreases gene expression. The maximal efficiency of gene knockdown is observed after 24 hours, resulting in a 65% ± 12 reduction of the protein level. This reduction in PAC1R impairs the responsiveness of cells to PACAP exposure. Following PAC1R gene knockdown, only 10% ± 8 and 11%± 9 of cells exhibit neurite outgrowth after 4-day exposure to PACAP-38 and PACAP-27, respectively. These results demonstrate an efficient PAC1R gene knockdown and noticeable difference in response to PACAP action on neural cell differentiation, adding an extra dimension to determine the involvement of PACAP and its PAC1R in the neurotropic effect to PC12 cells. PMID:26510306

  9. Rapid Screening of Gene Function by Systemic Delivery of Morpholino Oligonucleotides to Live Mouse Embryos

    PubMed Central

    McClelland, Kathryn S.; Wainwright, Elanor N.; Bowles, Josephine; Koopman, Peter

    2015-01-01

    Traditional gene targeting methods in mice are complex and time consuming, especially when conditional deletion methods are required. Here, we describe a novel technique for assessing gene function by injection of modified antisense morpholino oligonucleotides (MOs) into the heart of mid-gestation mouse embryos. After allowing MOs to circulate through the embryonic vasculature, target tissues were explanted, cultured and analysed for expression of key markers. We established proof-of-principle by partially phenocopying known gene knockout phenotypes in the fetal gonads (Stra8, Sox9) and pancreas (Sox9). We also generated a novel double knockdown of Gli1 and Gli2, revealing defects in Leydig cell differentiation in the fetal testis. Finally, we gained insight into the roles of Adamts19 and Ctrb1, genes of unknown function in sex determination and gonadal development. These studies reveal the utility of this method as a means of first-pass analysis of gene function during organogenesis before committing to detailed genetic analysis. PMID:25629157

  10. Targeting gene expression in the preimplantation mouse embryo using morpholino antisense oligonucleotides.

    PubMed

    Siddall, Laura S; Barcroft, Lisa C; Watson, Andrew J

    2002-12-01

    Morpholino antisense oligonucleotides act by blocking translation of their target gene products and are effective tools for down-regulating gene expression. The current study was conducted to define treatment conditions for the use of morpholino oligonucleotides (MOs) in mammalian preimplantation embryos, and to employ MOs to target genes and study gene function in the early embryo. For the first time, ethoxylated polyethylenimine (EPEI), Lipofectin or Lysolecithin delivery agents were employed in combination with a fluorescent control MO and an alpha-catenin specific MO, to down-regulate gene expression during murine preimplantation development. Experiments applied to both two- and eight-cell stage murine preimplantation embryos contrasted the efficacy of MO concentrations of 1, 2, 5, 10, and 20 microM and treatment delivery times of 3, 6, 24, and 48 hr. Continuous treatment of two-cell embryos with Lipofectin and 20 microM alpha-catenin MO for 48 hr resulted in a significant (P < 0.05) reduction in development to the blastocyst stage and was accompanied by a marked reduction in alpha-catenin protein. These results indicate that morpholino antisense oligonucleotides are effective tools for down-regulating gene expression during mammalian preimplantation development. PMID:12412042

  11. Delaying Gal4-Driven Gene Expression in the Zebrafish with Morpholinos and Gal80

    PubMed Central

    Faucherre, Adèle; López-Schier, Hernán

    2011-01-01

    The modular Gal4/UAS gene expression system has become an indispensable tool in modern biology. Several large-scale gene- and enhancer-trap screens in the zebrafish have generated hundreds of transgenic lines expressing Gal4 in unique patterns. However, the early embryonic expression of the Gal4 severely limits their use for studies on regeneration or behavior because UAS-driven effectors could disrupt normal organogenesis. To overcome this limitation, we explored the use of the Gal4 repressor Gal80 in transient assays and with stable transgenes to temporally control Gal4 activity. We also validated a strategy to delay Gal4-driven gene expression using a morpholino targeted to Gal4. The first approach is limited to transgenes expressing the native Gal4. The morphant approach can also be applied to transgenic lines expressing the Gal4-VP16 fusion protein. It promises to become a standard approach to delay Gal4-driven transgene expression and enhance the genetic toolkit for the zebrafish. PMID:21298067

  12. In vitro correction of a novel splicing alteration in the BTK gene by using antisense morpholino oligonucleotides.

    PubMed

    Rattanachartnarong, Natthakorn; Tongkobpetch, Siraprapa; Chatchatee, Pantipa; Daengsuwan, Tassalapa; Ittiwut, Chupong; Suphapeetiporn, Kanya; Shotelersuk, Vorasuk

    2014-10-01

    A novel sequence variant, c.240+109C>A, in the Bruton's tyrosine kinase (BTK) gene was identified in a patient with X-linked agammaglobulinemia. This alteration resulted in an incorporation of 106 nucleotides of BTK intron 3 into its mRNA. Administration of the 25-mer antisense morpholino oligonucleotide analog in the patient's cultured peripheral blood mononuclear cells was able to restore correctly spliced BTK mRNA, a potential treatment for X-linked agammaglobulinemia. PMID:24658450

  13. In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

    PubMed Central

    Thummel, Ryan; Bailey, Travis J.; Hyde, David R.

    2011-01-01

    Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident Müller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected

  14. Quantitative Microinjection of Morpholino Antisense Oligonucleotides into Mouse Oocytes to Examine Gene Function in Meiosis-I.

    PubMed

    Nakagawa, Shoma; FitzHarris, Greg

    2016-01-01

    Specific protein depletion is a powerful approach for assessing individual gene function in cellular processes, and has been extensively employed in recent years in mammalian oocyte meiosis-I. Conditional knockout mice and RNA interference (RNAi) methods such as siRNA or dsRNA microinjection are among several approaches to have been applied in this system over the past decade. RNAi by microinjection of Morpholino antisense Oligonucleotides (MO), in particular, has proven highly popular and tractable in many studies, since MOs have high specificity of interaction, low cell toxicity, and are more stable than other microinjected RNAi molecules. Here, we describe a method of MO microinjection into the mouse germinal vesicle-stage (GV) oocyte followed by a simple immunofluorescence approach for examination of gene function in meiosis-I. PMID:27557584

  15. Knockdown of col22a1 gene in zebrafish induces a muscular dystrophy by disruption of the myotendinous junction.

    PubMed

    Charvet, Benjamin; Guiraud, Alexandre; Malbouyres, Marilyne; Zwolanek, Daniela; Guillon, Emilie; Bretaud, Sandrine; Monnot, Catherine; Schulze, Jörg; Bader, Hannah L; Allard, Bruno; Koch, Manuel; Ruggiero, Florence

    2013-11-01

    The myotendinous junction (MTJ) is the major site of force transfer in skeletal muscle, and defects in its structure correlate with a subset of muscular dystrophies. Col22a1 encodes the MTJ component collagen XXII, the function of which remains unknown. Here, we have cloned and characterized the zebrafish col22a1 gene and conducted morpholino-based loss-of-function studies in developing embryos. We showed that col22a1 transcripts localize at muscle ends when the MTJ forms and that COLXXII protein integrates the junctional extracellular matrix. Knockdown of COLXXII expression resulted in muscular dystrophy-like phenotype, including swimming impairment, curvature of embryo trunk/tail, strong reduction of twitch-contraction amplitude and contraction-induced muscle fiber detachment, and provoked significant activation of the survival factor Akt. Electron microscopy and immunofluorescence studies revealed that absence of COLXXII caused a strong reduction of MTJ folds and defects in myoseptal structure. These defects resulted in reduced contractile force and susceptibility of junctional extracellular matrix to rupture when subjected to repeated mechanical stress. Co-injection of sub-phenotypic doses of morpholinos against col22a1 and genes of the major muscle linkage systems showed a synergistic gene interaction between col22a1 and itga7 (α7β1 integrin) that was not observed with dag1 (dystroglycan). Finally, pertinent to a conserved role in humans, the dystrophic phenotype was rescued by microinjection of recombinant human COLXXII. Our findings indicate that COLXXII contributes to the stabilization of myotendinous junctions and strengthens skeletal muscle attachments during contractile activity. PMID:24131632

  16. Inhibition of zebrafish fin regeneration using in vivo electroporation of morpholinos against fgfr1 and msxb.

    PubMed

    Thummel, Ryan; Bai, Shan; Sarras, Michael P; Song, Peizhen; McDermott, Jeffrey; Brewer, Jeffrey; Perry, Martin; Zhang, Xiaoming; Hyde, David R; Godwin, Alan R

    2006-02-01

    Increased interest in using zebrafish as a model organism has led to a resurgence of fin regeneration studies. This has allowed for the identification of a large number of gene families, including signaling molecules and transcription factors, which are expressed during regeneration. However, in cases where no specific inhibitor is available for the gene product of interest, determination of a functional role for these genes has been difficult. Here we demonstrate that in vivo electroporation of morpholino oligonucleotides is a feasible approach for protein knock-down during fin regeneration. Morpholino oligonucleotides against fgfr1 and msxb were utilized and knock-down of both proteins resulted in reduced fin outgrowth. Importantly, Fgfr1 knock-down phenocopied outgrowth inhibition obtained with an Fgfr1 inhibitor. Furthermore, this method provided direct evidence for a functional role for msxb in caudal fin regeneration. Finally, knock-down of Fgfr1, but not Msxb, affected the blastemal expression of msxc, suggesting this technique can be used to determine epistasis in genetic pathways affecting regeneration. Thus, this convenient reverse genetic approach allows researchers to quickly (1) assess the function of genes known to be expressed during fin regeneration, (2) screen genes for functional relevance during fin regeneration, and (3) assign genes to the molecular pathways underlying fin regeneration. PMID:16273523

  17. Knockdown of Pex11β reveals its pivotal role in regulating peroxisomal genes, numbers, and ROS levels in Xenopus laevis A6 cells.

    PubMed

    Fox, Mark A; Nieuwesteeg, Michelle A; Willson, Jessica A; Cepeda, Mario; Damjanovski, Sashko

    2014-04-01

    Peroxisomes are organelles that are ubiquitously found in all eukaryotic cells. Enzymes within their lumen are responsible for a variety of processes including the metabolism of fatty acids and eradication (neutralization) of free radicals. Peroxisomes are dynamic organelles, able to alter their numbers in response to a variety of different metabolic and cell-specific cues. Changes in peroxisome numbers can occur through division of preexisting peroxisomes or through de novo biogenesis from the ER. Proteins such as the Pex11 family of peroxins have been implicated as regulatory factors involved in peroxisome division. Division of peroxisomes involves elongation and membrane constriction followed by fission, which requires Pex11β. The regulation of peroxisome numbers in different cell types and tissues is variable and poorly understood. Here, we examine how knockdown of Pex11β affects peroxisomal genes, proteins, and peroxisome numbers in A6 kidney epithelial cells derived from Xenopus laevis. Pex11β morpholino use subsequently decreased mRNA levels of Pex1, PMP70, and PPARγ. Moreover, the Pex11β morpholino decreased PMP70 protein levels and PMP70-positive structures. Furthermore, the marker GFP-SKL revealed fewer peroxisome-like structures. These decreases resulted in increased levels of H2O2 and cellular and mitochondrial reactive oxygen species as measured by Amplex Red, DCFDA, and MitoTracker assays, respectively. PMID:24234511

  18. Deiodinase knockdown affects zebrafish eye development at the level of gene expression, morphology and function.

    PubMed

    Houbrechts, Anne M; Vergauwen, Lucia; Bagci, Enise; Van Houcke, Jolien; Heijlen, Marjolein; Kulemeka, Bernard; Hyde, David R; Knapen, Dries; Darras, Veerle M

    2016-03-15

    Retinal development in vertebrates relies extensively on thyroid hormones. Their local availability is tightly controlled by several regulators, including deiodinases (Ds). Here we used morpholino technology to explore the roles of Ds during eye development in zebrafish. Transcriptome analysis at 3 days post fertilization (dpf) revealed a pronounced effect of knockdown of both T4-activating Ds (D1D2MO) or knockdown of T3-inactivating D3 (D3bMO) on phototransduction and retinoid recycling. This was accompanied by morphological defects (studied from 1 to 7 dpf) including reduced eye size, disturbed retinal lamination and strong reduction in rods and all four cone types. Defects were more prominent and persistent in D3-deficient fish. Finally, D3-deficient zebrafish larvae had disrupted visual function at 4 dpf and were less sensitive to a light stimulus at 5 dpf. These data demonstrate the importance of TH-activating and -inactivating Ds for correct zebrafish eye development, and point to D3b as a central player. PMID:26802877

  19. Manipulating the in vivo immune response by targeted gene knockdown

    PubMed Central

    Lieberman, Judy

    2015-01-01

    Aptamers, nucleic acids selected for high affinity binding to proteins, can be used to activate or antagonize immune mediators or receptors in a location and cell-type specific manner and to enhance antigen presentation. They can also be linked to other molecules (other aptamers, siRNAs or miRNAs, proteins, toxins) to produce multifunctional compounds for targeted immune modulation in vivo. Aptamer-siRNA chimeras (AsiCs) that induce efficient cell-specific knockdown in immune cells in vitro and in vivo can be used as an immunological research tool or potentially as an immunomodulating therapeutic. PMID:26149459

  20. Manipulating the in vivo immune response by targeted gene knockdown.

    PubMed

    Lieberman, Judy

    2015-08-01

    Aptamers, nucleic acids selected for high affinity binding to proteins, can be used to activate or antagonize immune mediators or receptors in a location and cell-type specific manner and to enhance antigen presentation. They can also be linked to other molecules (other aptamers, siRNAs or miRNAs, proteins, toxins) to produce multifunctional compounds for targeted immune modulation in vivo. Aptamer-siRNA chimeras (AsiCs) that induce efficient cell-specific knockdown in immune cells in vitro and in vivo can be used as an immunological research tool or potentially as an immunomodulating therapeutic. PMID:26149459

  1. Selenoprotein X Gene Knockdown Aggravated H2O2-Induced Apoptosis in Liver LO2 Cells.

    PubMed

    Tang, Jiayong; Cao, Lei; Li, Qiang; Wang, Longqiong; Jia, Gang; Liu, Guangmang; Chen, Xiaoling; Cai, Jingyi; Shang, Haiying; Zhao, Hua

    2016-09-01

    To determine the roles of selenoprotein X gene (Selx) in protecting liver cells against oxidative damage, the influences of Selx knockdown on H2O2-induced apoptosis in human normal hepatocyte (LO2) cells were studied. pSilencer 3.1 was used to develop knockdown vector targeting the 3'-UTR of human Selx. The Selx knockdown and control cells were further exposed to H2O2, and cell viability, cell apoptosis rate, and the expression levels of mRNA and protein of apoptosis-related genes were detected. The results showed that vector targeting the 3'-UTR of Selx successfully silenced mRNA or protein expression of SelX in LO2 cells. Selx knockdown resulted in decreased cell viability, increased percentage of early apoptotic cells, decreased Bcl2A1 and Bcl-2 expression, and increased phosphorylation of P38 in LO2 cells. When Selx knockdown LO2 cells were exposed to H2O2, characteristics of H2O2-induced cell dysfunctions were further exacerbated. Taken together, our findings suggested that SelX played important roles in protecting LO2 cells against oxidative damage and reducing H2O2-induced apoptosis in liver cells. PMID:26899321

  2. RNAi mediated Tiam1 gene knockdown inhibits invasion of retinoblastoma.

    PubMed

    Subramanian, Nithya; Navaneethakrishnan, Saranya; Biswas, Jyotirmay; Kanwar, Rupinder K; Kanwar, Jagat R; Krishnakumar, Subramanian

    2013-01-01

    T lymphoma invasion and metastasis protein (Tiam1) is up-regulated in variety of cancers and its expression level is related to metastatic potential of the type of cancer. Earlier, Tiam1 was shown to be overexpressed in retinoblastoma (RB) and we hypothesized that it was involved in invasiveness of RB. This was tested by silencing Tiam1 in RB cell lines (Y79 and Weri-Rb1) using siRNA pool, targeting different regions of Tiam1 mRNA. The cDNA microarray of Tiam1 silenced cells showed gene regulations altered by Tiam1 were predominantly on the actin cytoskeleton interacting proteins, apoptotic initiators and tumorogenic potential targets. The silenced phenotype resulted in decreased growth and increased apoptosis with non-invasive characteristics. Transfection of full length and N-terminal truncated construct (C1199) clearly revealed membrane localization of Tiam1 and not in the case of C580 construct. F-actin staining showed the interaction of Tiam1 with actin in the membrane edges that leads to ruffling, and also imparts varying invasive potential to the cell. The results obtained from our study show for the first time that Tiam1 modulates the cell invasion, mediated by actin cytoskeleton remodeling in RB. PMID:23950931

  3. Gene expression profiling of selenophosphate synthetase 2 knockdown in Drosophila melanogaster.

    PubMed

    Li, Gaopeng; Liu, Liying; Li, Ping; Chen, Luonan; Song, Haiyun; Zhang, Yan

    2016-03-01

    Selenium (Se) is an important trace element for many organisms and is incorporated into selenoproteins as selenocysteine (Sec). In eukaryotes, selenophosphate synthetase SPS2 is essential for Sec biosynthesis. In recent years, genetic disruptions of both Sec biosynthesis genes and selenoprotein genes have been investigated in different animal models, which provide important clues for understanding the Se metabolism and function in these organisms. However, a systematic study on the knockdown of SPS2 has not been performed in vivo. Herein, we conducted microarray experiments to study the transcriptome of fruit flies with knockdown of SPS2 in larval and adult stages. Several hundred differentially expressed genes were identified in each stage. In spite that the expression levels of other Sec biosynthesis genes and selenoprotein genes were not significantly changed, it is possible that selenoprotein translation might be reduced without impacting the mRNA level. Functional enrichment and network-based analyses revealed that although different sets of differentially expressed genes were obtained in each stage, they were both significantly enriched in the carbohydrate metabolism and redox processes. Furthermore, protein-protein interaction (PPI)-based network clustering analysis implied that several hub genes detected in the top modules, such as Nimrod C1 and regucalcin, could be considered as key regulators that are responsible for the complex responses caused by SPS2 knockdown. Overall, our data provide new insights into the relationship between Se utilization and several fundamental cellular processes as well as diseases. PMID:26824785

  4. Mitochondria-targeted antioxidant prevents cardiac dysfunction induced by tafazzin gene knockdown in cardiac myocytes.

    PubMed

    He, Quan; Harris, Nicole; Ren, Jun; Han, Xianlin

    2014-01-01

    Tafazzin, a mitochondrial acyltransferase, plays an important role in cardiolipin side chain remodeling. Previous studies have shown that dysfunction of tafazzin reduces cardiolipin content, impairs mitochondrial function, and causes dilated cardiomyopathy in Barth syndrome. Reactive oxygen species (ROS) have been implicated in the development of cardiomyopathy and are also the obligated byproducts of mitochondria. We hypothesized that tafazzin knockdown increases ROS production from mitochondria, and a mitochondria-targeted antioxidant prevents tafazzin knockdown induced mitochondrial and cardiac dysfunction. We employed cardiac myocytes transduced with an adenovirus containing tafazzin shRNA as a model to investigate the effects of the mitochondrial antioxidant, mito-Tempo. Knocking down tafazzin decreased steady state levels of cardiolipin and increased mitochondrial ROS. Treatment of cardiac myocytes with mito-Tempo normalized tafazzin knockdown enhanced mitochondrial ROS production and cellular ATP decline. Mito-Tempo also significantly abrogated tafazzin knockdown induced cardiac hypertrophy, contractile dysfunction, and cell death. We conclude that mitochondria-targeted antioxidant prevents cardiac dysfunction induced by tafazzin gene knockdown in cardiac myocytes and suggest mito-Tempo as a potential therapeutic for Barth syndrome and other dilated cardiomyopathies resulting from mitochondrial oxidative stress. PMID:25247053

  5. Knockdown of MLO genes reduces susceptibility to powdery mildew in grapevine.

    PubMed

    Pessina, Stefano; Lenzi, Luisa; Perazzolli, Michele; Campa, Manuela; Dalla Costa, Lorenza; Urso, Simona; Valè, Giampiero; Salamini, Francesco; Velasco, Riccardo; Malnoy, Mickael

    2016-01-01

    Erysiphe necator is the causal agent of powdery mildew (PM), one of the most destructive diseases of grapevine. PM is controlled by sulfur-based and synthetic fungicides, which every year are dispersed into the environment. This is why PM-resistant varieties should become a priority for sustainable grapevine and wine production. PM resistance can be achieved in other crops by knocking out susceptibility S-genes, such as those residing at genetic loci known as MLO (Mildew Locus O). All MLO S-genes of dicots belong to the phylogenetic clade V, including grapevine genes VvMLO7, 11 and 13, which are upregulated during PM infection, and VvMLO6, which is not upregulated. Before adopting a gene-editing approach to knockout candidate S-genes, the evidence that loss of function of MLO genes can reduce PM susceptibility is necessary. This paper reports the knockdown through RNA interference of VvMLO6, 7, 11 and 13. The knockdown of VvMLO6, 11 and 13 did not decrease PM severity, whereas the knockdown of VvMLO7 in combination with VvMLO6 and VvMLO11 reduced PM severity up to 77%. The knockdown of VvMLO7 and VvMLO6 seemed to be important for PM resistance, whereas a role for VvMLO11 does not seem likely. Cell wall appositions (papillae) were present in both resistant and susceptible lines in response to PM attack. Thirteen genes involved in defense were less upregulated in infected mlo plants, highlighting the early mlo-dependent disruption of PM invasion. PMID:27390621

  6. Knockdown of MLO genes reduces susceptibility to powdery mildew in grapevine

    PubMed Central

    Pessina, Stefano; Lenzi, Luisa; Perazzolli, Michele; Campa, Manuela; Dalla Costa, Lorenza; Urso, Simona; Valè, Giampiero; Salamini, Francesco; Velasco, Riccardo; Malnoy, Mickael

    2016-01-01

    Erysiphe necator is the causal agent of powdery mildew (PM), one of the most destructive diseases of grapevine. PM is controlled by sulfur-based and synthetic fungicides, which every year are dispersed into the environment. This is why PM-resistant varieties should become a priority for sustainable grapevine and wine production. PM resistance can be achieved in other crops by knocking out susceptibility S-genes, such as those residing at genetic loci known as MLO (Mildew Locus O). All MLO S-genes of dicots belong to the phylogenetic clade V, including grapevine genes VvMLO7, 11 and 13, which are upregulated during PM infection, and VvMLO6, which is not upregulated. Before adopting a gene-editing approach to knockout candidate S-genes, the evidence that loss of function of MLO genes can reduce PM susceptibility is necessary. This paper reports the knockdown through RNA interference of VvMLO6, 7, 11 and 13. The knockdown of VvMLO6, 11 and 13 did not decrease PM severity, whereas the knockdown of VvMLO7 in combination with VvMLO6 and VvMLO11 reduced PM severity up to 77%. The knockdown of VvMLO7 and VvMLO6 seemed to be important for PM resistance, whereas a role for VvMLO11 does not seem likely. Cell wall appositions (papillae) were present in both resistant and susceptible lines in response to PM attack. Thirteen genes involved in defense were less upregulated in infected mlo plants, highlighting the early mlo-dependent disruption of PM invasion. PMID:27390621

  7. Testis-specific Fank1 gene in knockdown mice produces oligospermia via apoptosis

    PubMed Central

    Dong, Wan-Wei; Huang, Hua-Liang; Yang, Wei; Liu, Jia; Yu, Yang; Zhou, Sheng-Lai; Wang, Wei; Lv, Xiang-Chuan; Li, Zhao-Yang; Zhang, Mei-Ying; Zheng, Zhi-Hong; Yan, Wei

    2014-01-01

    Fank1 is exclusively expressed in the testis from the meiosis phase to the haploid phase of spermatogenesis. In this study, we examined the function of Fank1 by establishing a Fank1-knockdown transgenic mouse model. The apoptotic statuses of the testes of the transgenic mice were tested using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method. The FANK1 consensus DNA-binding sequence was identified using cyclic amplification of sequence target (CAST) analysis. Differentially expressed genes were examined using microarray analysis. A reduction in sperm number and an increase in apoptotic spermatocytes were observed in Fank1-knockdown mice, and the apoptotic cells were found to be primarily spermatogonia and spermatocytes. The CAST results demonstrated that the consensus DNA-binding sequence was AAAAAG, in which the percentage occurrence of each base at each position ranged from 55 to 86%. This sequence was present in the promoter regions of 10 differentially expressed genes that were examined using microarray analysis. In total, 17 genes were differentially expressed with changes in their expression levels greater than twofold. The abnormal expression of Fank1 target genes that were regulated directly or indirectly by Fank1 reduced the number of sperm in the knockdown mice. Thus, FANK1 may play a pivotal role in spermatogenesis as a transcription factor. PMID:24369145

  8. Short Hairpin RNA (shRNA): Design, Delivery, and Assessment of Gene Knockdown

    PubMed Central

    Moore, Chris B.; Guthrie, Elizabeth H.; Huang, Max Tze-Han; Taxman, Debra J.

    2013-01-01

    Shortly after the cellular mechanism of RNA interference (RNAi) was first described, scientists began using this powerful technique to study gene function. This included designing better methods for the successful delivery of small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) into mammalian cells. While the simplest method for RNAi is the cytosolic delivery of siRNA oligonucleotides, this technique is limited to cells capable of transfection and is primarily utilized during transient in vitro studies. The introduction of shRNA into mammalian cells through infection with viral vectors allows for stable integration of shRNA and long-term knockdown of the targeted gene; however, several challenges exist with the implementation of this technology. Here we describe some well-tested protocols which should increase the chances of successful design, delivery, and assessment of gene knockdown by shRNA. We provide suggestions for designing shRNA targets and controls, a protocol for sequencing through the secondary structure of the shRNA hairpin structure, and protocols for packaging and delivery of shRNA lentiviral particles. Using real-time PCR and functional assays we demonstrate the successful knockdown of ASC, an inflammatory adaptor molecule. These studies demonstrate the practicality of including two shRNAs with different efficacies of knockdown to provide an additional level of control and to verify dose dependency of functional effects. Along with the methods described here, as new techniques and algorithms are designed in the future, shRNA is likely to include further promising application and continue to be a critical component of gene discovery. PMID:20387148

  9. Inducible Knockdown of Plasmodium Gene Expression Using the glmS Ribozyme

    PubMed Central

    Prommana, Parichat; Uthaipibull, Chairat; Wongsombat, Chayaphat; Kamchonwongpaisan, Sumalee; Yuthavong, Yongyuth; Knuepfer, Ellen; Holder, Anthony A.; Shaw, Philip J.

    2013-01-01

    Conventional reverse genetic approaches for study of Plasmodium malaria parasite gene function are limited, or not applicable. Hence, new inducible systems are needed. Here we describe a method to control P. falciparum gene expression in which target genes bearing a glmS ribozyme in the 3′ untranslated region are efficiently knocked down in transgenic P. falciparum parasites in response to glucosamine inducer. Using reporter genes, we show that the glmS ribozyme cleaves reporter mRNA in vivo leading to reduction in mRNA expression following glucosamine treatment. Glucosamine-induced ribozyme activation led to efficient reduction of reporter protein, which could be rapidly reversed by removing the inducer. The glmS ribozyme was validated as a reverse-genetic tool by integration into the essential gene and antifolate drug target dihydrofolate reductase-thymidylate synthase (PfDHFR-TS). Glucosamine treatment of transgenic parasites led to rapid and efficient knockdown of PfDHFR-TS mRNA and protein. PfDHFR-TS knockdown led to a growth/arrest mutant phenotype and hypersensitivity to pyrimethamine. The glmS ribozyme may thus be a tool for study of essential genes in P. falciparum and other parasite species amenable to transfection. PMID:24023691

  10. Let-7a gene knockdown protects against cerebral ischemia/reperfusion injury

    PubMed Central

    Wang, Zhong-kun; Liu, Fang-fang; Wang, Yu; Jiang, Xin-mei; Yu, Xue-fan

    2016-01-01

    The microRNA (miRNA) let-7 was one of the first miRNAs to be discovered, and is highly conserved and widely expressed among species. let-7 expression increases in brain tissue after cerebral ischemia/reperfusion injury; however, no studies have reported let-7 effects on nerve injury after cerebral ischemia/reperfusion injury. To investigate the effects of let-7 gene knockdown on cerebral ischemia/reperfusion injury, we established a rat model of cerebral ischemia/reperfusion injury. Quantitative reverse transcription-polymerase chain reaction demonstrated that 12 hours after cerebral ischemia/reperfusion injury, let-7 expression was up-regulated, peaked at 24 hours, and was still higher than that in control rats after 72 hours. Let-7 gene knockdown in rats suppressed microglial activation and inflammatory factor release, reduced neuronal apoptosis and infarct volume in brain tissue after cerebral ischemia/reperfusion injury. Western blot assays and luciferase assays revealed that mitogen-activated protein kinase phosphatase-1 (MKP1) is a direct target of let-7. Let-7 enhanced phosphorylated p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) expression by down-regulating MKP1. These findings suggest that knockdown of let-7 inhibited the activation of p38 MAPK and JNK signaling pathways by up-regulating MKP1 expression, reduced apoptosis and the inflammatory reaction, and exerted a neuroprotective effect following cerebral ischemia/reperfusion injury. PMID:27073379

  11. Gene expression profiling of NB4 cells following knockdown of nucleostemin using DNA microarrays

    PubMed Central

    SUN, XIAOLI; JIA, YU; WEI, YUANYU; LIU, SHUAI; YUE, BAOHONG

    2016-01-01

    Nucleostemin (NS) is mainly expressed in stem and tumor cells, and is necessary for the maintenance of their self-renewal and proliferation. Originally, NS was thought to exert its effects through inhibiting p53, while recent studies have revealed that NS is also able to function independently of p53. The present study performed a gene expression profiling analysis of p53-mutant NB4 leukeima cells following knockdown of NS in order to elucidate the p53-independent NS pathway. NS expression was silenced using lentivirus-mediated RNA interference technology, and gene expression profiling of NB4 cells was performed by DNA microarray analysis. A total of 1,953 genes were identified to be differentially expressed (fold change ≥2 or ≤0.5) following knockdown of NS expression. Furthermore, reverse-transcription quantitative polymerase chain reaction analysis was used to detect the expression of certain candidate genes, and the results were in agreement with the micaroarray data. Pathway analysis indicated that aberrant genes were enhanced in endoplasmic, c-Jun N-terminal kinase and mineral absorption pathways. The present study shed light on the mechanisms of the p54-independent NS pathway in NB4 cells and provided a foundation for the discovery of promising targets for the treatment of p53-mutant leukemia. PMID:27374947

  12. Morpholinos: Antisense and Sensibility.

    PubMed

    Blum, Martin; De Robertis, Edward M; Wallingford, John B; Niehrs, Christof

    2015-10-26

    For over 15 years, antisense morpholino oligonucleotides (MOs) have allowed developmental biologists to make key discoveries regarding developmental mechanisms in numerous model organisms. Recently, serious concerns have been raised as to the specificity of MO effects, and it has been recommended to discontinue their usage, despite the long experience of the scientific community with the MO tool in thousands of studies. Reviewing the many advantages afforded by MOs, we conclude that adequately controlled MOs should continue to be accepted as generic loss-of-function approach, as otherwise progress in developmental biology will greatly suffer. PMID:26506304

  13. Response of Two Heat Shock Genes to Selection for Knockdown Heat Resistance in Drosophila Melanogaster

    PubMed Central

    McColl, G.; Hoffmann, A. A.; McKechnie, S. W.

    1996-01-01

    To identify genes involved in stress resistance and heat hardening, replicate lines of Drosophila melanogaster were selected for increased resistance to knockdown by a 39° heat stress. Two selective regimes were used, one with and one without prior hardening. Mean knockdown times were increased from ~5 min to >20 min after 18 generations. Initial realized heritabilities were as high as 10% for lines selected without hardening, and crosses between lines indicated simple additive gene effects for the selected phenotypes. To survey allelic variation and correlated selection responses in two candidate stress genes, hsr-omega and hsp68, we applied denaturing gradient gel electrophoresis to amplified DNA sequences from small regions of these genes. After eight generations of selection, allele frequencies at both loci showed correlated responses for selection following hardening, but not without hardening. The hardening process itself was associated with a hsp68 frequency change in the opposite direction to that associated with selection that followed hardening. These stress loci are closely linked on chromosome III, and the hardening selection established a disequilibrium, suggesting an epistatic effect on resistance. The data indicate that molecular variation in both hsr-omega and hsp68 contribute to natural heritable variation for hardened heat resistance. PMID:8844150

  14. Core Binding Factor-β Knockdown Alters Ovarian Gene Expression and Function in the Mouse.

    PubMed

    Wilson, Kalin; Park, Jiyeon; Curry, Thomas E; Mishra, Birendra; Gossen, Jan; Taniuchi, Ichiro; Jo, Misung

    2016-07-01

    Core binding factor (CBF) is a heterodimeric transcription factor complex composed of a DNA-binding subunit, one of three runt-related transcription factor (RUNX) factors, and a non-DNA binding subunit, CBFβ. CBFβ is critical for DNA binding and stability of the CBF transcription factor complex. In the ovary, the LH surge increases the expression of Runx1 and Runx2 in periovulatory follicles, implicating a role for CBFs in the periovulatory process. The present study investigated the functional significance of CBFs (RUNX1/CBFβ and RUNX2/CBFβ) in the ovary by examining the ovarian phenotype of granulosa cell-specific CBFβ knockdown mice; CBFβ f/f * Cyp19 cre. The mutant female mice exhibited significant reductions in fertility, with smaller litter sizes, decreased progesterone during gestation, and fewer cumulus oocyte complexes collected after an induced superovulation. RNA sequencing and transcriptome assembly revealed altered expression of more than 200 mRNA transcripts in the granulosa cells of Cbfb knockdown mice after human chorionic gonadotropin stimulation in vitro. Among the affected transcripts are known regulators of ovulation and luteinization including Sfrp4, Sgk1, Lhcgr, Prlr, Wnt4, and Edn2 as well as many genes not yet characterized in the ovary. Cbfβ knockdown mice also exhibited decreased expression of key genes within the corpora lutea and morphological changes in the ovarian structure, including the presence of large antral follicles well into the luteal phase. Overall, these data suggest a role for CBFs as significant regulators of gene expression, ovulatory processes, and luteal development in the ovary. PMID:27176614

  15. Simultaneous knockdown of six non-family genes using a single synthetic RNAi fragment in Arabidopsis thaliana

    DOE PAGESBeta

    Czarnecki, Olaf; Bryan, Anthony C.; Jawdy, Sara S.; Yang, Xiaohan; Cheng, Zong-Ming; Chen, Jin-Gui; Tuskan, Gerald A.

    2016-02-17

    Genetic engineering of plants that results in successful establishment of new biochemical or regulatory pathways requires stable introduction of one or more genes into the plant genome. It might also be necessary to down-regulate or turn off expression of endogenous genes in order to reduce activity of competing pathways. An established way to knockdown gene expression in plants is expressing a hairpin-RNAi construct, eventually leading to degradation of a specifically targeted mRNA. Knockdown of multiple genes that do not share homologous sequences is still challenging and involves either sophisticated cloning strategies to create vectors with different serial expression constructs ormore » multiple transformation events that is often restricted by a lack of available transformation markers. Synthetic RNAi fragments were assembled in yeast carrying homologous sequences to six or seven non-family genes and introduced into pAGRIKOLA. Transformation of Arabidopsis thaliana and subsequent expression analysis of targeted genes proved efficient knockdown of all target genes. In conclusion, we present a simple and cost-effective method to create constructs to simultaneously knockdown multiple non-family genes or genes that do not share sequence homology. The presented method can be applied in plant and animal synthetic biology as well as traditional plant and animal genetic engineering.« less

  16. Efficient Gene Knockdown in Mouse Oocytes through Peptide Nanoparticle-Mediated SiRNA Transfection

    PubMed Central

    Jin, Zhen; Li, Ruichao; Zhou, Chunxiang; Shi, Liya; Zhang, Xiaolan; Yang, Zhixia; Zhang, Dong

    2016-01-01

    The use of mouse oocytes as a model for studying female meiosis is very important in reproductive medicine. Gene knockdown by specific small interfering RNA (siRNA) is usually the first step in the study of the function of a target gene in mouse oocytes during in vitro maturation. Traditionally, the only way to introduce siRNA into mouse oocytes is through microinjection, which is certainly less efficient and strenuous than siRNA transfection in somatic cells. Recently, in research using somatic cells, peptide nanoparticle-mediated siRNA transfection has been gaining popularity over liposome nanoparticle-mediated methods because of its high efficiency, low toxicity, good stability, and strong serum compatibility. However, no researchers have yet tried transfecting siRNA into mouse oocytes because of the existence of the protective zona pellucida surrounding the oocyte membrane (vitelline membrane). We therefore tested whether peptide nanoparticles can introduce siRNA into mouse oocytes. In the present study, we showed for the first time that our optimized program can efficiently knock down a target gene with high specificity. Furthermore, we achieved the expected meiotic phenotypes after we knocked down a test unknown target gene TRIM75. We propose that peptide nanoparticles may be superior for preliminary functional studies of unknown genes in mouse oocytes. PMID:26974323

  17. Efficient Gene Knockdown in Mouse Oocytes through Peptide Nanoparticle-Mediated SiRNA Transfection.

    PubMed

    Jin, Zhen; Li, Ruichao; Zhou, Chunxiang; Shi, Liya; Zhang, Xiaolan; Yang, Zhixia; Zhang, Dong

    2016-01-01

    The use of mouse oocytes as a model for studying female meiosis is very important in reproductive medicine. Gene knockdown by specific small interfering RNA (siRNA) is usually the first step in the study of the function of a target gene in mouse oocytes during in vitro maturation. Traditionally, the only way to introduce siRNA into mouse oocytes is through microinjection, which is certainly less efficient and strenuous than siRNA transfection in somatic cells. Recently, in research using somatic cells, peptide nanoparticle-mediated siRNA transfection has been gaining popularity over liposome nanoparticle-mediated methods because of its high efficiency, low toxicity, good stability, and strong serum compatibility. However, no researchers have yet tried transfecting siRNA into mouse oocytes because of the existence of the protective zona pellucida surrounding the oocyte membrane (vitelline membrane). We therefore tested whether peptide nanoparticles can introduce siRNA into mouse oocytes. In the present study, we showed for the first time that our optimized program can efficiently knock down a target gene with high specificity. Furthermore, we achieved the expected meiotic phenotypes after we knocked down a test unknown target gene TRIM75. We propose that peptide nanoparticles may be superior for preliminary functional studies of unknown genes in mouse oocytes. PMID:26974323

  18. Morphological Profiles of RNAi-Induced Gene Knockdown Are Highly Reproducible but Dominated by Seed Effects

    PubMed Central

    Singh, Shantanu; Wu, Xiaoyun; Ljosa, Vebjorn; Bray, Mark-Anthony; Piccioni, Federica; Root, David E.; Doench, John G.; Boehm, Jesse S.; Carpenter, Anne E.

    2015-01-01

    RNA interference and morphological profiling—the measurement of thousands of phenotypes from individual cells by microscopy and image analysis—are a potentially powerful combination. We show that morphological profiles of RNAi-induced knockdown using the Cell Painting assay are in fact highly sensitive and reproducible. However, we find that the magnitude and prevalence of off-target effects via the RNAi seed-based mechanism make morphological profiles of RNAi reagents targeting the same gene look no more similar than reagents targeting different genes. Pairs of RNAi reagents that share the same seed sequence produce image-based profiles that are much more similar to each other than profiles from pairs designed to target the same gene, a phenomenon previously observed in small-scale gene-expression profiling experiments. Various strategies have been used to enrich on-target versus off-target effects in the context of RNAi screening where a narrow set of phenotypes are measured, mostly based on comparing multiple sequences targeting the same gene; however, new approaches will be needed to make RNAi morphological profiling (that is, comparing multi-dimensional phenotypes) viable. We have shared our raw data and computational pipelines to facilitate research. PMID:26197079

  19. First knockdown gene expression in bat (Hipposideros armiger) brain mediated by lentivirus.

    PubMed

    Chen, Qi; Zhu, Tengteng; Jones, Gareth; Zhang, Junpeng; Sun, Yi

    2013-06-01

    Lentivirus-mediated RNA interference (RNAi) is a potent experimental tool for investigating gene functions in vitro and in vivo. It has advantages that transgenic technology lacks. However, in vivo applications are difficult to apply in the central nervous system of non-model organisms due to the lack of a standard brain atlas and genetic information. Here, we report the development of an in vivo gene delivery system used in bat brain tissue for the first time, based on lentivirus (LV) vectors expressing short hairpin RNA (shRNA) targeting Hipposideros armiger forkhead box P2 (FoxP2). In vitro transfection into HEK 293T cell with the vector bearing the cassettes encoding FoxP2 shRNA verified the knockdown efficiency. Pseudovirus particles were administered via stereotactic intracerebral microinjection into the anterior cingulate cortex of H. armiger. FoxP2 is of major interest because of its role in sensorimotor coordination and probably in echolocation. Subsequent in situ hybridization validated the in vivo silencing of the target gene. This report demonstrates that LV-mediated expression of RNAi could achieve effective gene silencing in bats, a non-model organism, and will assist in elucidating the functions of bat genes. PMID:22965420

  20. Antisense precision polymer micelles require less poly(ethylenimine) for efficient gene knockdown.

    PubMed

    Fakhoury, Johans J; Edwardson, Thomas G; Conway, Justin W; Trinh, Tuan; Khan, Farhad; Barłóg, Maciej; Bazzi, Hassan S; Sleiman, Hanadi F

    2015-12-28

    Therapeutic nucleic acids are powerful molecules for shutting down protein expression. However, their cellular uptake is poor and requires transport vectors, such as cationic polymers. Of these, poly(ethylenimine) (PEI) has been shown to be an efficient vehicle for nucleic acid transport into cells. However, cytotoxicity has been a major hurdle in the development of PEI-DNA complexes as clinically viable therapeutics. We have synthesized antisense-polymer conjugates, where the polymeric block is completely monodisperse and sequence-controlled. Depending on the polymer sequence, these can self-assemble to produce micelles of very low polydispersity. The introduction of linear poly(ethylenimine) to these micelles leads to aggregation into size-defined PEI-mediated superstructures. Subsequently, both cellular uptake and gene silencing are greatly enhanced over extended periods compared to antisense alone, while at the same time cellular cytotoxicity remains very low. In contrast, gene silencing is not enhanced with antisense polymer conjugates that are not able to self-assemble into micelles. Thus, using antisense precision micelles, we are able to achieve significant transfection and knockdown with minimal cytotoxicity at much lower concentrations of linear PEI then previously reported. Consequently, a conceptual solution to the problem of antisense or siRNA delivery is to self-assemble these molecules into 'gene-like' micelles with high local charge and increased stability, thus reducing the amount of transfection agent needed for effective gene silencing. PMID:26597764

  1. Quantification of Functionalised Gold Nanoparticle-Targeted Knockdown of Gene Expression in HeLa Cells

    PubMed Central

    Jiwaji, Meesbah; Sandison, Mairi E.; Reboud, Julien; Stevenson, Ross; Daly, Rónán; Barkess, Gráinne; Faulds, Karen; Kolch, Walter; Graham, Duncan; Girolami, Mark A.; Cooper, Jonathan M.; Pitt, Andrew R.

    2014-01-01

    Introduction Gene therapy continues to grow as an important area of research, primarily because of its potential in the treatment of disease. One significant area where there is a need for better understanding is in improving the efficiency of oligonucleotide delivery to the cell and indeed, following delivery, the characterization of the effects on the cell. Methods In this report, we compare different transfection reagents as delivery vehicles for gold nanoparticles functionalized with DNA oligonucleotides, and quantify their relative transfection efficiencies. The inhibitory properties of small interfering RNA (siRNA), single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA) sequences targeted to human metallothionein hMT-IIa are also quantified in HeLa cells. Techniques used in this study include fluorescence and confocal microscopy, qPCR and Western analysis. Findings We show that the use of transfection reagents does significantly increase nanoparticle transfection efficiencies. Furthermore, siRNA, ssRNA and ssDNA sequences all have comparable inhibitory properties to ssDNA sequences immobilized onto gold nanoparticles. We also show that functionalized gold nanoparticles can co-localize with autophagosomes and illustrate other factors that can affect data collection and interpretation when performing studies with functionalized nanoparticles. Conclusions The desired outcome for biological knockdown studies is the efficient reduction of a specific target; which we demonstrate by using ssDNA inhibitory sequences targeted to human metallothionein IIa gene transcripts that result in the knockdown of both the mRNA transcript and the target protein. PMID:24926959

  2. GATA4 knockdown in MA-10 Leydig cells identifies multiple target genes in the steroidogenic pathway.

    PubMed

    Bergeron, Francis; Nadeau, Gabriel; Viger, Robert S

    2015-03-01

    GATA4 is an essential transcription factor required for the initiation of genital ridge formation, for normal testicular and ovarian differentiation at the time of sex determination, and for male and female fertility in adulthood. In spite of its crucial roles, the genes and/or gene networks that are ultimately regulated by GATA4 in gonadal tissues remain to be fully understood. This is particularly true for the steroidogenic lineages such as Leydig cells of the testis where many in vitro (promoter) studies have provided good circumstantial evidence that GATA4 is a key regulator of Leydig cell gene expression and steroidogenesis, but formal proof is still lacking. We therefore performed a microarray screening analysis of MA-10 Leydig cells in which Gata4 expression was knocked down using an siRNA strategy. Analysis identified several GATA4-regulated pathways including cholesterol synthesis, cholesterol transport, and especially steroidogenesis. A decrease in GATA4 protein was associated with decreased expression of steroidogenic genes previously suspected to be GATA4 targets such as Cyp11a1 and Star. Gata4 knockdown also led to an important decrease in other novel steroidogenic targets including Srd5a1, Gsta3, Hsd3b1, and Hsd3b6, as well as genes known to participate in cholesterol metabolism such as Scarb1, Ldlr, Soat1, Scap, and Cyp51. Consistent with the decreased expression of these genes, a reduction in GATA4 protein compromised the ability of MA-10 cells to produce steroids both basally and under hormone stimulation. These data therefore provide strong evidence that GATA4 is an essential transcription factor that sits atop of the Leydig cell steroidogenic program. PMID:25504870

  3. CRISPR/Cas9-Mediated Gene Knock-Down in Post-Mitotic Neurons

    PubMed Central

    Saulnier, Jessica L.; Sabatini, Bernardo L.

    2014-01-01

    The prokaryotic adaptive immune system CRISPR/Cas9 has recently been adapted for genome editing in eukaryotic cells. This technique allows for sequence-specific induction of double-strand breaks in genomic DNA of individual cells, effectively resulting in knock-out of targeted genes. It thus promises to be an ideal candidate for application in neuroscience where constitutive genetic modifications are frequently either lethal or ineffective due to adaptive changes of the brain. Here we use CRISPR/Cas9 to knock-out Grin1, the gene encoding the obligatory NMDA receptor subunit protein GluN1, in a sparse population of mouse pyramidal neurons. Within this genetically mosaic tissue, manipulated cells lack synaptic current mediated by NMDA-type glutamate receptors consistent with complete knock-out of the targeted gene. Our results show the first proof-of-principle demonstration of CRISPR/Cas9-mediated knock-down in neurons in vivo, where it can be a useful tool to study the function of specific proteins in neuronal circuits. PMID:25140704

  4. Antisense precision polymer micelles require less poly(ethylenimine) for efficient gene knockdown

    NASA Astrophysics Data System (ADS)

    Fakhoury, Johans J.; Edwardson, Thomas G.; Conway, Justin W.; Trinh, Tuan; Khan, Farhad; Barłóg, Maciej; Bazzi, Hassan S.; Sleiman, Hanadi F.

    2015-12-01

    Therapeutic nucleic acids are powerful molecules for shutting down protein expression. However, their cellular uptake is poor and requires transport vectors, such as cationic polymers. Of these, poly(ethylenimine) (PEI) has been shown to be an efficient vehicle for nucleic acid transport into cells. However, cytotoxicity has been a major hurdle in the development of PEI-DNA complexes as clinically viable therapeutics. We have synthesized antisense-polymer conjugates, where the polymeric block is completely monodisperse and sequence-controlled. Depending on the polymer sequence, these can self-assemble to produce micelles of very low polydispersity. The introduction of linear poly(ethylenimine) to these micelles leads to aggregation into size-defined PEI-mediated superstructures. Subsequently, both cellular uptake and gene silencing are greatly enhanced over extended periods compared to antisense alone, while at the same time cellular cytotoxicity remains very low. In contrast, gene silencing is not enhanced with antisense polymer conjugates that are not able to self-assemble into micelles. Thus, using antisense precision micelles, we are able to achieve significant transfection and knockdown with minimal cytotoxicity at much lower concentrations of linear PEI then previously reported. Consequently, a conceptual solution to the problem of antisense or siRNA delivery is to self-assemble these molecules into `gene-like' micelles with high local charge and increased stability, thus reducing the amount of transfection agent needed for effective gene silencing.Therapeutic nucleic acids are powerful molecules for shutting down protein expression. However, their cellular uptake is poor and requires transport vectors, such as cationic polymers. Of these, poly(ethylenimine) (PEI) has been shown to be an efficient vehicle for nucleic acid transport into cells. However, cytotoxicity has been a major hurdle in the development of PEI-DNA complexes as clinically viable

  5. Knockdown of ROS1 gene sensitizes breast tumor growth to doxorubicin in a syngeneic mouse model.

    PubMed

    Tiash, Snigdha; Chua, Ming Jang; Chowdhury, Ezharul Hoque

    2016-06-01

    Treatment of breast cancer, the second leading cause of female deaths worldwide, with classical drugs is often accompanied by treatment failure and relapse of disease condition. Development of chemoresistance and drug toxicity compels compromising the drug concentration below the threshold level with the consequence of therapeutic inefficacy. Moreover, amplification and over-activation of proto-oncogenes in tumor cells make the treatment more challenging. The oncogene, ROS1 which is highly expressed in diverse types of cancers including breast carcinoma, functions as a survival protein aiding cancer progression. Thus we speculated that selective silencing of ROS1 gene by carrier-mediated delivery of siRNA might sensitize the cancer cells to the classical drugs at a relatively low concentration. In this investigation we showed that intracellular delivery of c-ROS1-targeting siRNA using pH-sensitive inorganic nanoparticles of carbonate apatite sensitizes mouse breast cancer cells (4T1) to doxorubicin, but not to cisplatin or paclitaxel, with the highest enhancement in chemosensitivity obtained at 40 nM of the drug concentration. Although intravenous administrations of ROS1-loaded nanoparticles reduced growth of the tumor, a further substantial effect on growth retardation was noted when the mice were treated with the siRNA- and Dox-bound particles, thus suggesting that silencing of ROS1 gene could sensitize the mouse breast cancer cells both in vitro and in vivo to doxorubicin as a result of synergistic effect of the gene knockdown and the drug action, eventually preventing activation of the survival pathway protein, AKT1. Our findings therefore provide valuable insight into the potential cross-talk between the pathways of ROS1 and doxorubicin for future development of effective therapeutics for breast cancer. PMID:27035628

  6. Gene knockdown by ihpRNA-triggering in the ectomycorrhizal basidiomycete fungus Laccaria bicolor.

    PubMed

    Kemppainen, Minna J; Pardo, Alejandro G

    2010-01-01

    Ectomycorrhiza (ECM) is a mutualistic association between fungi and the roots of the vast majority of trees. These include numerous ecologically and economically relevant species and the participating fungal symbionts are predominantly filamentous basidiomycetes. In natural ecosystems the plant nutrient uptake from soil takes place via the extraradical mycelia of these ECM mycosimbionts as a trade for plant photosyntates. The symbiotic phase in the life cycle of ECM basidiomycetes is the dikaryotic hyphae. Therefore, studies on symbiotic relevant gene functions require the inactivation of both gene copies in these dikaryotic fungi. RNA silencing is a eukaryotic sequence homology-dependent degradation of target RNAs which is believed to have evolved as a protection mechanism against invading nucleic acids. In different eukaryotic organisms, including fungi, the RNA silencing pathway can be artificially triggered to target and degrade gene transcripts of interest, resulting in gene knock-down. Most importantly, RNA silencing can act at the cytosolic level affecting mRNAs originating from several gene copies and different nuclei thus offering an efficient means of altering gene expression in dikaryotic organisms. Therefore, the pHg/pSILBAγ silencing vector was constructed for efficient RNA silencing triggering in the model mycorrhizal fungus Laccaria bicolor. This cloning vector carries the Agaricus bisporus gpdII-promoter, two multiple cloning sites separated by a L. bicolor nitrate reductase intron and the Aspergillus nidulans trpC terminator. pSILBAγ allows an easy two-step PCR-cloning of hairpin sequences to be expressed in basidiomycetes. With one further cloning step into pHg, a pCAMBIA1300-based binary vector carrying a hygromycin resistance cassette, makes the pHg/pSILBAγ plasmid compatible with Agrobacterium-mediated transformation. The pHg/pSILBAγ-system results in predominantly single integrations of RNA silencing triggering T-DNAs in the fungal genome

  7. klf2ash317 Mutant Zebrafish Do Not Recapitulate Morpholino-Induced Vascular and Haematopoietic Phenotypes

    PubMed Central

    Novodvorsky, Peter; Watson, Oliver; Gray, Caroline; Wilkinson, Robert N.; Reeve, Scott; Smythe, Carl; Beniston, Richard; Plant, Karen; Maguire, Richard; M. K. Rothman, Alexander; Elworthy, Stone; van Eeden, Fredericus J. M.; Chico, Timothy J. A.

    2015-01-01

    Introduction and Objectives The zinc-finger transcription factor Krϋppel-like factor 2 (KLF2) transduces blood flow into molecular signals responsible for a wide range of responses within the vasculature. KLF2 maintains a healthy, quiescent endothelial phenotype. Previous studies report a range of phenotypes following morpholino antisense oligonucleotide-induced klf2a knockdown in zebrafish. Targeted genome editing is an increasingly applied method for functional assessment of candidate genes. We therefore generated a stable klf2a mutant zebrafish and characterised its cardiovascular and haematopoietic development. Methods and Results Using Transcription Activator-Like Effector Nucleases (TALEN) we generated a klf2a mutant (klf2ash317) with a 14bp deletion leading to a premature stop codon in exon 2. Western blotting confirmed loss of wild type Klf2a protein and the presence of a truncated protein in klf2ash317 mutants. Homozygous klf2ash317 mutants exhibit no defects in vascular patterning, survive to adulthood and are fertile, without displaying previously described morphant phenotypes such as high-output cardiac failure, reduced haematopoetic stem cell (HSC) development or impaired formation of the 5th accessory aortic arch. Homozygous klf2ash317 mutation did not reduce angiogenesis in zebrafish with homozygous mutations in von Hippel Lindau (vhl), a form of angiogenesis that is dependent on blood flow. We examined expression of three klf family members in wildtype and klf2ash317 zebrafish. We detected vascular expression of klf2b (but not klf4a or biklf/klf4b/klf17) in wildtypes but found no differences in expression that might account for the lack of phenotype in klf2ash317 mutants. klf2b morpholino knockdown did not affect heart rate or impair formation of the 5th accessory aortic arch in either wildtypes or klf2ash317 mutants. Conclusions The klf2ash317 mutation produces a truncated Klf2a protein but, unlike morpholino induced klf2a knockdown, does not

  8. Genetic Architecture of a Hormonal Response to Gene Knockdown in Honey Bees

    PubMed Central

    Rueppell, Olav; Huang, Zachary Y.; Wang, Ying; Fondrk, M. Kim; Page, Robert E.; Amdam, Gro V.

    2015-01-01

    Variation in endocrine signaling is proposed to underlie the evolution and regulation of social life histories, but the genetic architecture of endocrine signaling is still poorly understood. An excellent example of a hormonally influenced set of social traits is found in the honey bee (Apis mellifera): a dynamic and mutually suppressive relationship between juvenile hormone (JH) and the yolk precursor protein vitellogenin (Vg) regulates behavioral maturation and foraging of workers. Several other traits cosegregate with these behavioral phenotypes, comprising the pollen hoarding syndrome (PHS) one of the best-described animal behavioral syndromes. Genotype differences in responsiveness of JH to Vg are a potential mechanistic basis for the PHS. Here, we reduced Vg expression via RNA interference in progeny from a backcross between 2 selected lines of honey bees that differ in JH responsiveness to Vg reduction and measured JH response and ovary size, which represents another key aspect of the PHS. Genetic mapping based on restriction site-associated DNA tag sequencing identified suggestive quantitative trait loci (QTL) for ovary size and JH responsiveness. We confirmed genetic effects on both traits near many QTL that had been identified previously for their effect on various PHS traits. Thus, our results support a role for endocrine control of complex traits at a genetic level. Furthermore, this first example of a genetic map of a hormonal response to gene knockdown in a social insect helps to refine the genetic understanding of complex behaviors and the physiology that may underlie behavioral control in general. PMID:25596612

  9. Optimization of Critical Hairpin Features Allows miRNA-based Gene Knockdown Upon Single-copy Transduction

    PubMed Central

    Myburgh, Renier; Cherpin, Ophélie; Schlaepfer, Erika; Rehrauer, Hubert; Speck, Roberto F; Krause, Karl-Heinz; Salmon, Patrick

    2014-01-01

    Gene knockdown using micro RNA (miRNA)-based vector constructs is likely to become a prominent gene therapy approach. It was the aim of this study to improve the efficiency of gene knockdown through optimizing the structure of miRNA mimics. Knockdown of two target genes was analyzed: CCR5 and green fluorescent protein. We describe here a novel and optimized miRNA mimic design called mirGE comprising a lower stem length of 13 base pairs (bp), positioning of the targeting strand on the 5′ side of the miRNA, together with nucleotide mismatches in upper stem positions 1 and 12 placed on the passenger strand. Our mirGE proved superior to miR-30 in four aspects: yield of targeting strand incorporation into RNA-induced silencing complex (RISC); incorporation into RISC of correct targeting strand; precision of cleavage by Drosha; and ratio of targeting strand over passenger strand. A triple mirGE hairpin cassette targeting CCR5 was constructed. It allowed CCR5 knockdown with an efficiency of over 90% upon single-copy transduction. Importantly, single-copy expression of this construct rendered transduced target cells, including primary human macrophages, resistant to infection with a CCR5-tropic strain of HIV. Our results provide new insights for a better knockdown efficiency of constructs containing miRNA. Our results also provide the proof-of-principle that cells can be rendered HIV resistant through single-copy vector transduction, rendering this approach more compatible with clinical applications. PMID:25350582

  10. In vivo delivery of morpholino oligos by cell-penetrating peptides.

    PubMed

    Moulton, Hong M

    2013-01-01

    Morpholino oligos (Morpholinos) are widely used tools for knocking down gene expression and are currently in a clinical trial for treatment of Duchene muscular dystrophy. A Morpholino analog has been in a clinical trial as a potential anti-bioterrorism agent for inhibiting replication of deadly Marburg viral infection. The cellular uptake of Morpholinos can been greatly increased by conjugation with cell-penetrating peptides (CPP). The use of the CPP-Morpholino conjugates (PPMOs) in vivo has been broadly demonstrated in viral, bacterial, genetic and other diseases. The following aspects of PPMOs will be discussed in this paper including chemistry, stability, antisense specificity, mechanism of cellular uptake, in vivo efficacy, tissue distribution, pharmacokinetics, toxicity and the human clinical trials. PPMOs are powerful research tools for studying gene function in animals and their properties are being improved as potential human therapeutic agents. PMID:23140456

  11. A Primer for Morpholino Use in Zebrafish

    PubMed Central

    Bill, Brent R.; Petzold, Andrew M.; Clark, Karl J.; Schimmenti, Lisa A.

    2009-01-01

    Abstract Morpholino oligonucleotides are the most common anti-sense “knockdown” technique used in zebrafish (Danio rerio). This review discusses common practices for the design, preparation, and deployment of morpholinos in this vertebrate model system. Off-targeting effects of morpholinos are discussed as well as method to minimize this potentially confounding variable via co-injection of a tP53-targeting morpholino. Finally, new uses of morpholinos are summarized and contextualized with respect to the complementary, DNA-based knockout technologies recently developed for zebrafish. PMID:19374550

  12. Synthesis of Morpholino Monomers, Chlorophosphoramidate Monomers, and Solid-Phase Synthesis of Short Morpholino Oligomers.

    PubMed

    Bhadra, Jhuma; Pattanayak, Sankha; Sinha, Surajit

    2015-01-01

    Phosphorodiamidate morpholino oligomers (PMOs) are a highly capable class of synthetic antisense oligonucleotides that are used to study gene functions in in vitro and in vivo models. This unit describes the synthesis of exocyclic-amine-protected 7'-hydroxy and 7'-chlorophosphoramidate-activated morpholino monomers of A, T, G, and C, together with their incorporation into short PMO oligomers by solid-phase synthesis. Starting from ribonucleosides, the exocyclic-amine-protected 7'-hydroxy monomers are prepared following a modified Summerton protocol, which consists of a periodate cleavage/Schiff base formation/reduction cycle. The exocyclic amine protections are installed at a later stage (except G) to avoid the use of costly exocyclic-amine-protected counterparts that give control over protecting group manipulation. The 7'-hydroxy monomers with N-Trit/N-MMTr are then converted to the 7'-chlorophosphoramidate morpholino monomers in one step employing a combination of lithium bromide and DBU. These chlorophosphoramidate monomers are finally assembled by solid-support synthesis to obtain the short PMO oligomers. PMID:26380905

  13. A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila

    SciTech Connect

    Nagarkar-Jaiswal, Sonal; Lee, Pei-Tseng; Campbell, Megan E.; Chen, Kuchuan; Anguiano-Zarate, Stephanie; Cantu Gutierrez, Manuel; Busby, Theodore; Lin, Wen-Wen; He, Yuchun; Schulze, Karen L.; Booth, Benjamin W.; Evans-Holm, Martha; Venken, Koen J.T.; Levis, Robert W.; Spradling, Allan C.; Hoskins, Roger A.; Bellen, Hugo J.

    2015-03-31

    Here, we document a collection of ~7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates.

  14. A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila

    DOE PAGESBeta

    Nagarkar-Jaiswal, Sonal; Lee, Pei-Tseng; Campbell, Megan E.; Chen, Kuchuan; Anguiano-Zarate, Stephanie; Cantu Gutierrez, Manuel; Busby, Theodore; Lin, Wen-Wen; He, Yuchun; Schulze, Karen L.; et al

    2015-03-31

    Here, we document a collection of ~7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstratemore » reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates.« less

  15. Knockdown of cyclin-dependent kinase 10 (cdk10) gene impairs neural progenitor survival via modulation of raf1a gene expression.

    PubMed

    Yeh, Chi-Wei; Kao, Shoa-Hsuan; Cheng, Yi-Chuan; Hsu, Li-Sung

    2013-09-27

    In this study, we used zebrafish as an animal model to elucidate the developmental function of cdk10 in vertebrates. In situ hybridization analyses demonstrated that cdk10 is expressed throughout development with a relative enrichment in the brain in the late stages. Similar to its mammalian ortholog, cdk10 can interact with the transcription factor ETS2 and exhibit kinase activity by phosphorylating histone H1. Morpholino-based loss of cdk10 expression caused apoptosis in sox2-positive cells and decreased the expression of subsequent neuronal markers. Acetylated tubulin staining revealed a significant reduction in the number of Rohon-Beard sensory neurons in cdk10 morphants. This result is similar to that demonstrated by decreased islet2 expression in the dorsal regions. Moreover, cdk10 morphants exhibited a marked loss of huC-positive neurons in the telencephalon and throughout the spinal cord axis. The population of retinal ganglion cells was also diminished in cdk10 morphants. These phenotypes were rescued by co-injection of cdk10 mRNA. Interestingly, the knockdown of cdk10 significantly elevated raf1a mRNA expression. Meanwhile, an MEK inhibitor (U0126) recovered sox2 and ngn1 transcript levels in cdk10 morphants. Our findings provide the first functional characterization of cdk10 in vertebrate development and reveal its critical function in neurogenesis by modulation of raf1a expression. PMID:23902762

  16. Near-infrared-light-based nano-platform boosts endosomal escape and controls gene knockdown in vivo.

    PubMed

    Jayakumar, Muthu Kumara Gnanasammandhan; Bansal, Akshaya; Huang, Kai; Yao, Risheng; Li, Bing Nan; Zhang, Yong

    2014-05-27

    Current nanoparticle-based gene delivery techniques face two major limitations, namely, endosomal degradation and poor cytosolic release of the nanoparticles and nonspecificity of treatment. These limitations can be overcome with certain light-based techniques, such as photochemical internalization to enable endosomal escape of the delivered nanoparticles and light-controlled gene expression to overcome the nonspecific effects. However, these techniques require UV/visible light, which is either phototoxic and/or has low tissue penetration capabilities, thus preventing their use in deep tissues in a clinical setting. In an effort to overcome these barriers, we have successfully demonstrated a light-based gene delivery system that significantly boosts cytosolic gene delivery, with precise control over gene expression and the potential for use in nonsuperficial tissues. Core-shell fluorescent upconversion nanoparticles excited by highly penetrating near-infrared radiation and emitting simultaneously in the ultraviolet and visible ranges were synthesized and used as remote nanotransducers to simultaneously activate endosomal escape and gene knockdown. Gene knockdown using photomorpholinos was enhanced as much as 30% in vitro compared to the control without endosomal escape facilitation. A similar trend was seen in vivo in a murine melanoma model, demonstrating the enormous clinical potential of this system. PMID:24730360

  17. The effects of knockdown of rho-associated kinase 1 and zipper-interacting protein kinase on gene expression and function in cultured human arterial smooth muscle cells.

    PubMed

    Deng, Jing-Ti; Wang, Xiu-Ling; Chen, Yong-Xiang; O'Brien, Edward R; Gui, Yu; Walsh, Michael P

    2015-01-01

    Rho-associated kinase (ROCK) and zipper-interacting protein kinase (ZIPK) have been implicated in diverse physiological functions. ROCK1 phosphorylates and activates ZIPK suggesting that at least some of these physiological functions may require both enzymes. To test the hypothesis that sequential activation of ROCK1 and ZIPK is commonly involved in regulatory pathways, we utilized siRNA to knock down ROCK1 and ZIPK in cultured human arterial smooth muscle cells (SMC). Microarray analysis using a whole-transcript expression chip identified changes in gene expression induced by ROCK1 and ZIPK knockdown. ROCK1 knockdown affected the expression of 553 genes, while ZIPK knockdown affected the expression of 390 genes. A high incidence of regulation of transcription regulator genes was observed in both knockdowns. Other affected groups included transporters, kinases, peptidases, transmembrane and G protein-coupled receptors, growth factors, phosphatases and ion channels. Only 76 differentially expressed genes were common to ROCK1 and ZIPK knockdown. Ingenuity Pathway Analysis identified five pathways shared between the two knockdowns. We focused on cytokine signaling pathways since ROCK1 knockdown up-regulated 5 and down-regulated 4 cytokine genes, in contrast to ZIPK knockdown, which affected the expression of only two cytokine genes (both down-regulated). IL-6 gene expression and secretion of IL-6 protein were up-regulated by ROCK1 knockdown, whereas ZIPK knockdown reduced IL-6 mRNA expression and IL-6 protein secretion and increased ROCK1 protein expression, suggesting that ROCK1 may inhibit IL-6 secretion. IL-1β mRNA and protein levels were increased in response to ROCK1 knockdown. Differences in the effects of ROCK1 and ZIPK knockdown on cell cycle regulatory genes suggested that ROCK1 and ZIPK regulate the cell cycle by different mechanisms. ROCK1, but not ZIPK knockdown reduced the viability and inhibited proliferation of vascular SMC. We conclude that ROCK1 and

  18. The Effects of Knockdown of Rho-Associated Kinase 1 and Zipper-Interacting Protein Kinase on Gene Expression and Function in Cultured Human Arterial Smooth Muscle Cells

    PubMed Central

    Deng, Jing-Ti; Wang, Xiu-Ling; Chen, Yong-Xiang; O’Brien, Edward R.; Gui, Yu; Walsh, Michael P.

    2015-01-01

    Rho-associated kinase (ROCK) and zipper-interacting protein kinase (ZIPK) have been implicated in diverse physiological functions. ROCK1 phosphorylates and activates ZIPK suggesting that at least some of these physiological functions may require both enzymes. To test the hypothesis that sequential activation of ROCK1 and ZIPK is commonly involved in regulatory pathways, we utilized siRNA to knock down ROCK1 and ZIPK in cultured human arterial smooth muscle cells (SMC). Microarray analysis using a whole-transcript expression chip identified changes in gene expression induced by ROCK1 and ZIPK knockdown. ROCK1 knockdown affected the expression of 553 genes, while ZIPK knockdown affected the expression of 390 genes. A high incidence of regulation of transcription regulator genes was observed in both knockdowns. Other affected groups included transporters, kinases, peptidases, transmembrane and G protein-coupled receptors, growth factors, phosphatases and ion channels. Only 76 differentially expressed genes were common to ROCK1 and ZIPK knockdown. Ingenuity Pathway Analysis identified five pathways shared between the two knockdowns. We focused on cytokine signaling pathways since ROCK1 knockdown up-regulated 5 and down-regulated 4 cytokine genes, in contrast to ZIPK knockdown, which affected the expression of only two cytokine genes (both down-regulated). IL-6 gene expression and secretion of IL-6 protein were up-regulated by ROCK1 knockdown, whereas ZIPK knockdown reduced IL-6 mRNA expression and IL-6 protein secretion and increased ROCK1 protein expression, suggesting that ROCK1 may inhibit IL-6 secretion. IL-1β mRNA and protein levels were increased in response to ROCK1 knockdown. Differences in the effects of ROCK1 and ZIPK knockdown on cell cycle regulatory genes suggested that ROCK1 and ZIPK regulate the cell cycle by different mechanisms. ROCK1, but not ZIPK knockdown reduced the viability and inhibited proliferation of vascular SMC. We conclude that ROCK1 and

  19. CERKL Knockdown Causes Retinal Degeneration in Zebrafish

    PubMed Central

    Riera, Marina; Burguera, Demian; Garcia-Fernàndez, Jordi; Gonzàlez-Duarte, Roser

    2013-01-01

    The human CERKL gene is responsible for common and severe forms of retinal dystrophies. Despite intense in vitro studies at the molecular and cellular level and in vivo analyses of the retina of murine knockout models, CERKL function remains unknown. In this study, we aimed to approach the developmental and functional features of cerkl in Danio rerio within an Evo-Devo framework. We show that gene expression increases from early developmental stages until the formation of the retina in the optic cup. Unlike the high mRNA-CERKL isoform multiplicity shown in mammals, the moderate transcriptional complexity in fish facilitates phenotypic studies derived from gene silencing. Moreover, of relevance to pathogenicity, teleost CERKL shares the two main human protein isoforms. Morpholino injection has been used to generate a cerkl knockdown zebrafish model. The morphant phenotype results in abnormal eye development with lamination defects, failure to develop photoreceptor outer segments, increased apoptosis of retinal cells and small eyes. Our data support that zebrafish Cerkl does not interfere with proliferation and neural differentiation during early developmental stages but is relevant for survival and protection of the retinal tissue. Overall, we propose that this zebrafish model is a powerful tool to unveil CERKL contribution to human retinal degeneration. PMID:23671706

  20. Effective Targeted Gene Knockdown in Mammalian Cells Using the piggyBac Transposase-based Delivery System

    PubMed Central

    Owens, Jesse B; Mathews, Juanita; Davy, Philip; Stoytchev, Ilko; Moisyadi, Stefan; Allsopp, Rich

    2013-01-01

    Nonviral gene delivery systems are rapidly becoming a desirable and applicable method to overexpress genes in various types of cells. We have recently developed a piggyBac transposase-based, helper-independent and self-inactivating delivery system (pmGENIE-3) capable of high-efficiency transfection of mammalian cells including human cells. In the following study, we have assessed the potential of this delivery system to drive the expression of short hairpin RNAs to knock down genes in human cells. Two independent pmGENIE-3 vectors were developed to specifically target knockdown of an endogenous gene, telomerase reverse transcriptase (TERT), in telomerase-positive human immortalized cell lines. As compared with a transposase-deficient vector, pmGENIE-3 showed significantly improved short-term transfection efficiency (~4-fold enhancement, 48 hours posttransfection) and long-term integration efficiency (~5-fold enhancement) following antibiotic selection. We detected a significant reduction of both TERT expression and telomerase activity in both HEK293 and MCF-7 breast carcinoma cells transfected with two pmGENIE-3 construct targeting distinct regions of TERT. Importantly, this knockdown of expression was sufficient to abrogate telomerase function since telomeres were significantly shortened (3–4 Kb, P < 0.001) in both TERT-targeted cell lines following antibiotic selection of stable integrants. Together, these data show the capacity of the piggyBac nonviral delivery system to stably knockdown gene expression in mammalian cells and indicate the potential to develop novel tumor-targeting therapies. PMID:24326734

  1. Effects of TET1 knockdown on gene expression and DNA methylation in porcine induced pluripotent stem cells.

    PubMed

    Fan, Anran; Ma, Kuiying; An, Xinglan; Ding, Yu; An, Peipei; Song, Guangqi; Tang, Lina; Zhang, Sheng; Zhang, Peng; Tan, Wentao; Tang, Bo; Zhang, Xueming; Li, Ziyi

    2013-12-01

    TET1 is implicated in maintaining the pluripotency of embryonic stem cells. However, its precise effects on induced pluripotent stem cells (iPSCs), and particularly on porcine iPSCs (piPSCs), are not well defined. To investigate the role of TET1 in the pluripotency and differentiation of piPSCs, piPSCs were induced from porcine embryonic fibroblasts by overexpression of POU5F1 (OCT4), SOX2, KLF4, and MYC (C-MYC). siRNAs targeting to TET1 were used to transiently knockdown the expression of TET1 in piPSCs. Morphological abnormalities and loss of the undifferentiated state of piPSCs were observed in the piPSCs after the downregulation of TET1. The effects of TET1 knockdown on the expression of key stem cell factors and differentiation markers were analyzed to gain insights into the molecular mechanisms underlying the phenomenon. The results revealed that knockdown of TET1 resulted in the downregulated expression of pluripotency-related genes, such as LEFTY2, KLF2, and SOX2, and the upregulated expression of differentiation-related genes including PITX2, HAND1, GATA6, and LEF1. However, POU5F1, MYC, KLF4, and NANOG were actually not downregulated. Further analysis showed that the methylation levels of the promoters for POU5F1 and MYC increased significantly after TET1 downregulation, whereas there were no obvious changes in the promoters of SOX2, KLF4, and NANOG. The methylation of the whole genome increased, while hydroxymethylation slightly declined. Taken together, these results suggest that TET1 may play important roles in the self-renewal of piPSCs and the maintenance of their characteristics by regulating the expression of genes and the DNA methylation. PMID:24051058

  2. Network Analysis for the Identification of Differentially Expressed Hub Genes Using Myogenin Knock-down Muscle Satellite Cells

    PubMed Central

    Jan, Arif Tasleem; Ahmad, Sarafraz; Cho, Kyung-Hyun; Kim, Jihoe; Choi, Inho

    2015-01-01

    Muscle, a multinucleate syncytium formed by the fusion of mononuclear myoblasts, arises from quiescent progenitors (satellite cells) via activation of muscle-specific transcription factors (MyoD, Myf5, myogenin: MYOG, and MRF4). Subsequent to a decline in Pax7, induction in the expression of MYOG is a hallmark of myoblasts that have entered the differentiation phase following cell cycle withdrawal. It is evident that MYOG function cannot be compensated by any other myogenic regulatory factors (MRFs). Despite a plethora of information available regarding MYOG, the mechanism by which MYOG regulates muscle cell differentiation has not yet been identified. Using an RNA-Seq approach, analysis of MYOG knock-down muscle satellite cells (MSCs) have shown that genes associated with cell cycle and division, DNA replication, and phosphate metabolism are differentially expressed. By constructing an interaction network of differentially expressed genes (DEGs) using GeneMANIA, cadherin-associated protein (CTNNA2) was identified as the main hub gene in the network with highest node degree. Four functional clusters (modules or communities) were identified in the network and the functional enrichment analysis revealed that genes included in these clusters significantly contribute to skeletal muscle development. To confirm this finding, in vitro studies revealed increased expression of CTNNA2 in MSCs on day 12 compared to day 10. Expression of CTNNA2 was decreased in MYOG knock-down cells. However, knocking down CTNNA2, which leads to increased expression of extracellular matrix (ECM) genes (type I collagen α1 and type I collagen α2) along with myostatin (MSTN), was not found significantly affecting the expression of MYOG in C2C12 cells. We therefore propose that MYOG exerts its regulatory effects by acting upstream of CTNNA2, which in turn regulates the differentiation of C2C12 cells via interaction with ECM genes. Taken together, these findings highlight a new mechanism by which

  3. Regeneration of diabetic axons is enhanced by selective knockdown of the PTEN gene.

    PubMed

    Singh, Bhagat; Singh, Vandana; Krishnan, Anand; Koshy, Kurien; Martinez, Jose A; Cheng, Chu; Almquist, Chris; Zochodne, Douglas W

    2014-04-01

    Diabetes mellitus renders both widespread and localized irreversible damage to peripheral axons while imposing critical limitations on their ability to regenerate. A major failure of regenerative capacity thereby imposes a 'double hit' in diabetic patients who frequently develop focal neuropathies such as carpal tunnel syndrome in addition to generalized diffuse polyneuropathy. The mechanisms of diabetic neuron regenerative failure have been speculative and few approaches have offered therapeutic opportunities. In this work we identify an unexpected but major role for PTEN upregulation in diabetic peripheral neurons in attenuating axon regrowth. In chronic diabetic neuropathy models in mice, we identified significant PTEN upregulation in peripheral sensory neurons of messenger RNA and protein compared to littermate controls. In vitro, sensory neurons from these mice responded to PTEN knockdown with substantial rises in neurite outgrowth and branching. To test regenerative plasticity in a chronic diabetic model with established neuropathy, we superimposed an additional focal sciatic nerve crush injury and assessed morphological, electrophysiological and behavioural recovery. Knockdown of PTEN in dorsal root ganglia ipsilateral to the side of injury was achieved using a unique form of non-viral short interfering RNA delivery to the ipsilateral nerve injury site and paw. In comparison with scrambled sequence control short interfering RNA, PTEN short interfering RNA improved several facets of regeneration: recovery of compound muscle action potentials, reflecting numbers of reconnected motor axons to endplates, conduction velocities of both motor and sensory axons, reflecting their maturation during regrowth, numbers and calibre of regenerating myelinated axons distal to the injury site, reinnervation of the skin by unmyelinated epidermal axons and recovery of mechanical sensation. Collectively, these findings identify a novel therapeutic approach, potentially

  4. LncRNA2Target: a database for differentially expressed genes after lncRNA knockdown or overexpression

    PubMed Central

    Jiang, Qinghua; Wang, Jixuan; Wu, Xiaoliang; Ma, Rui; Zhang, Tianjiao; Jin, Shuilin; Han, Zhijie; Tan, Renjie; Peng, Jiajie; Liu, Guiyou; Li, Yu; Wang, Yadong

    2015-01-01

    Long non-coding RNAs (lncRNAs) have emerged as critical regulators of genes at epigenetic, transcriptional and post-transcriptional levels, yet what genes are regulated by a specific lncRNA remains to be characterized. To assess the effects of the lncRNA on gene expression, an increasing number of researchers profiled the genome-wide or individual gene expression level change after knocking down or overexpressing the lncRNA. Herein, we describe a curated database named LncRNA2Target, which stores lncRNA-to-target genes and is publicly accessible at http://www.lncrna2target.org. A gene was considered as a target of a lncRNA if it is differentially expressed after the lncRNA knockdown or overexpression. LncRNA2Target provides a web interface through which its users can search for the targets of a particular lncRNA or for the lncRNAs that target a particular gene. Both search types are performed either by browsing a provided catalog of lncRNA names or by inserting lncRNA/target gene IDs/names in a search box. PMID:25399422

  5. A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

    PubMed Central

    Huang, Liwei; Xiao, An; Wecker, Andrea; McBride, Daniel A.; Choi, Soo Young; Zhou, Weibin; Lipschutz, Joshua H.

    2015-01-01

    Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to “off-target” effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney. PMID:25489842

  6. Knockdown of the coenzyme Q synthesis gene Smed-dlp1 affects planarian regeneration and tissue homeostasis

    PubMed Central

    Shiobara, Yumiko; Harada, Chiaki; Shiota, Takeshi; Sakamoto, Kimitoshi; Kita, Kiyoshi; Tanaka, Saeko; Tabata, Kenta; Sekie, Kiyoteru; Yamamoto, Yorihiro; Sugiyama, Tomoyasu

    2015-01-01

    The freshwater planarian is a model organism used to study tissue regeneration that occupies an important position among multicellular organisms. Planarian genomic databases have led to the identification of genes that are required for regeneration, with implications for their roles in its underlying mechanism. Coenzyme Q (CoQ) is a fundamental lipophilic molecule that is synthesized and expressed in every cell of every organism. Furthermore, CoQ levels affect development, life span, disease and aging in nematodes and mice. Because CoQ can be ingested in food, it has been used in preventive nutrition. In this study, we investigated the role of CoQ in planarian regeneration. Planarians synthesize both CoQ9 and rhodoquinone 9 (RQ9). Knockdown of Smed-dlp1, a trans-prenyltransferase gene that encodes an enzyme that synthesizes the CoQ side chain, led to a decrease in CoQ9 and RQ9 levels. However, ATP levels did not consistently decrease in these animals. Knockdown animals exhibited tissue regression and curling. The number of mitotic cells decreased in Smed-dlp1 (RNAi) animals. These results suggested a failure in physiological cell turnover and stem cell function. Accordingly, regenerating planarians died from lysis or exhibited delayed regeneration. Interestingly, the observed phenotypes were partially rescued by ingesting food supplemented with α-tocopherol. Taken together, our results suggest that oxidative stress induced by reduced CoQ9 levels affects planarian regeneration and tissue homeostasis. PMID:26516985

  7. Knockdown of the coenzyme Q synthesis gene Smed-dlp1 affects planarian regeneration and tissue homeostasis.

    PubMed

    Shiobara, Yumiko; Harada, Chiaki; Shiota, Takeshi; Sakamoto, Kimitoshi; Kita, Kiyoshi; Tanaka, Saeko; Tabata, Kenta; Sekie, Kiyoteru; Yamamoto, Yorihiro; Sugiyama, Tomoyasu

    2015-12-01

    The freshwater planarian is a model organism used to study tissue regeneration that occupies an important position among multicellular organisms. Planarian genomic databases have led to the identification of genes that are required for regeneration, with implications for their roles in its underlying mechanism. Coenzyme Q (CoQ) is a fundamental lipophilic molecule that is synthesized and expressed in every cell of every organism. Furthermore, CoQ levels affect development, life span, disease and aging in nematodes and mice. Because CoQ can be ingested in food, it has been used in preventive nutrition. In this study, we investigated the role of CoQ in planarian regeneration. Planarians synthesize both CoQ9 and rhodoquinone 9 (RQ9). Knockdown of Smed-dlp1, a trans-prenyltransferase gene that encodes an enzyme that synthesizes the CoQ side chain, led to a decrease in CoQ9 and RQ9 levels. However, ATP levels did not consistently decrease in these animals. Knockdown animals exhibited tissue regression and curling. The number of mitotic cells decreased in Smed-dlp1 (RNAi) animals. These results suggested a failure in physiological cell turnover and stem cell function. Accordingly, regenerating planarians died from lysis or exhibited delayed regeneration. Interestingly, the observed phenotypes were partially rescued by ingesting food supplemented with α-tocopherol. Taken together, our results suggest that oxidative stress induced by reduced CoQ9 levels affects planarian regeneration and tissue homeostasis. PMID:26516985

  8. A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila

    PubMed Central

    Nagarkar-Jaiswal, Sonal; Lee, Pei-Tseng; Campbell, Megan E; Chen, Kuchuan; Anguiano-Zarate, Stephanie; Cantu Gutierrez, Manuel; Busby, Theodore; Lin, Wen-Wen; He, Yuchun; Schulze, Karen L; Booth, Benjamin W; Evans-Holm, Martha; Venken, Koen JT; Levis, Robert W; Spradling, Allan C; Hoskins, Roger A; Bellen, Hugo J

    2015-01-01

    Here, we document a collection of ∼7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates. DOI: http://dx.doi.org/10.7554/eLife.05338.001 PMID:25824290

  9. Knockdown of Midgut Genes by dsRNA-Transgenic Plant-Mediated RNA Interference in the Hemipteran Insect Nilaparvata lugens

    PubMed Central

    Zha, Wenjun; Peng, Xinxin; Chen, Rongzhi; Du, Bo; Zhu, Lili; He, Guangcun

    2011-01-01

    Background RNA interference (RNAi) is a powerful technique for functional genomics research in insects. Transgenic plants producing double-stranded RNA (dsRNA) directed against insect genes have been reported for lepidopteran and coleopteran insects, showing potential for field-level control of insect pests, but this has not been reported for other insect orders. Methodology/Principal Findings The Hemipteran insect brown planthopper (Nilaparvata lugens Stål) is a typical phloem sap feeder specific to rice (Oryza sativa L.). To analyze the potential of exploiting RNAi-mediated effects in this insect, we identified genes (Nlsid-1 and Nlaub) encoding proteins that might be involved in the RNAi pathway in N. lugens. Both genes are expressed ubiquitously in nymphs and adult insects. Three genes (the hexose transporter gene NlHT1, the carboxypeptidase gene Nlcar and the trypsin-like serine protease gene Nltry) that are highly expressed in the N. lugens midgut were isolated and used to develop dsRNA constructs for transforming rice. RNA blot analysis showed that the dsRNAs were transcribed and some of them were processed to siRNAs in the transgenic lines. When nymphs were fed on rice plants expressing dsRNA, levels of transcripts of the targeted genes in the midgut were reduced; however, lethal phenotypic effects after dsRNA feeding were not observed. Conclusions Our study shows that genes for the RNAi pathway (Nlsid-1 and Nlaub) are present in N. lugens. When insects were fed on rice plant materials expressing dsRNAs, RNA interference was triggered and the target genes transcript levels were suppressed. The gene knockdown technique described here may prove to be a valuable tool for further investigations in N. lugens. The results demonstrate the potential of dsRNA-mediated RNAi for field-level control of planthoppers, but appropriate target genes must be selected when designing the dsRNA-transgenic plants. PMID:21655219

  10. Optimizing antisense oligonucleotides using phosphorodiamidate morpholino oligomers.

    PubMed

    Popplewell, Linda J; Malerba, Alberto; Dickson, George

    2012-01-01

    Duchenne muscular dystrophy (DMD) is caused by mutations that disrupt the reading frame of the human DMD gene. Selective removal of exons flanking an out-of-frame DMD mutation can result in an in-frame mRNA transcript that may be translated into an internally deleted Becker muscular dystrophy-like functionally active dystrophin protein with therapeutic activity. Antisense oligonucleotides (AOs) can be designed to bind to complementary sequences in the targeted mRNA and modify pre-mRNA splicing to correct the reading frame of a mutated transcript. AO-induced exon skipping resulting in functional truncated dystrophin has been demonstrated in animal models of DMD both in vitro and in vivo, in DMD patient cells in vitro in culture, and in DMD muscle explants. The recent advances made in this field suggest that it is likely that AO-induced exon skipping will be the first gene therapy for DMD to reach the clinic. However, it should be noted that personalized molecular medicine may be necessary, since the various reading frame-disrupting mutations are spread across the DMD gene. The different deletions that cause DMD would require skipping of different exons, which would require the optimization and clinical trial workup of many specific AOs. This chapter describes the methodologies available for the optimization of AOs, in particular phosphorodiamidate morpholino oligomers, for the targeted skipping of specific exons on the DMD gene. PMID:22454060

  11. Novel point mutations in the German cockroach para sodium channel gene are associated with knockdown resistance (kdr) to pyrethroid insecticides.

    PubMed

    Liu, Z; Valles, S M; Dong, K

    2000-10-01

    Knockdown resistance (kdr) to pyrethroid insecticides has been attributed to point mutations in the para sodium channel gene in more than a half dozen insect pest species. In this study, we identified two novel para mutations in five highly resistant kdr-type German cockroach strains. The two mutations, from glutamic acid (E434) to lysine (K434) and from cysteine (C764) to arginine (R764), respectively, are located in the first intracellular linker connecting domains I and II. E434K is located near the beginning of the linker (closest to domain I), whereas C764R is found toward the end of the linker (closest to domain II). Two additional mutations from aspartic acid (D58) to glycine (G58), and from proline (P1880) to leucine (L1888), respectively, were found in one of the resistant strains. The four mutations coexist with the previously identified leucine to phenylalanine (L993F) kdr mutation in IIS6, and are present only in the highly resistant individuals of a given strain. These findings suggest that these mutations might be responsible for high levels of knockdown resistance toward pyrethroid insecticides in the German cockroach. PMID:10899465

  12. Knockdown of prolactin receptors in a pancreatic beta cell line: effects on DNA synthesis, apoptosis, and gene expression.

    PubMed

    Arumugam, Ramamani; Fleenor, Don; Freemark, Michael

    2014-08-01

    Prolactin (PRL) and placental lactogen stimulate beta cell replication and insulin production in vitro and in vivo. The molecular mechanisms by which lactogens promote beta cell expansion are unclear. We treated rat insulinoma cells with a PRL receptor (PRLR) siRNA to determine if PRLR signaling is required for beta cell DNA synthesis and cell survival and to identify beta cell cycle genes whose expression depends upon lactogen action. Effects of PRLR knockdown were compared with those of PRL treatment. PRLR knockdown (-80 %) reduced DNA synthesis, increased apoptosis, and inhibited expression of cyclins D2 and B2, IRS-2, Tph1, and the anti-apoptotic protein PTTG1; p21 and BCL6 mRNAs increased. Conversely, PRL treatment increased DNA synthesis, reduced apoptosis, and enhanced expression of A, B and D2 cyclins, CDK1, IRS-2, FoxM1, BCLxL, and PTTG1; BCL6 declined. PRLR signaling is required for DNA synthesis and survival of rat insulinoma cells. The effects of lactogens are mediated by down-regulation of cell cycle inhibitors (BCL6, p21) and induction of A, B, and D2 cyclins, IRS-2, Tph1, FoxM1, and the anti-apoptotic proteins BCLxL and PTTG1. PMID:24114406

  13. Knockdown of the Rhipicephalus microplus Cytochrome c Oxidase Subunit III Gene Is Associated with a Failure of Anaplasma marginale Transmission

    PubMed Central

    Bifano, Thais D.; Ueti, Massaro W.; Esteves, Eliane; Reif, Kathryn E.; Braz, Glória R. C.; Scoles, Glen A.; Bastos, Reginaldo G.; White, Stephen N.; Daffre, Sirlei

    2014-01-01

    Rhipicephalus microplus is an obligate hematophagous ectoparasite of cattle and an important biological vector of Anaplasma marginale in tropical and subtropical regions. The primary determinants for A. marginale transmission are infection of the tick gut, followed by infection of salivary glands. Transmission of A. marginale to cattle occurs via infected saliva delivered during tick feeding. Interference in colonization of either the tick gut or salivary glands can affect transmission of A. marginale to naïve animals. In this study, we used the tick embryonic cell line BME26 to identify genes that are modulated in response to A. marginale infection. Suppression-subtractive hybridization libraries (SSH) were constructed, and five up-regulated genes {glutathione S-transferase (GST), cytochrome c oxidase sub III (COXIII), dynein (DYN), synaptobrevin (SYN) and phosphatidylinositol-3,4,5-triphosphate 3-phosphatase (PHOS)} were selected as targets for functional in vivo genomic analysis. RNA interference (RNAi) was used to determine the effect of tick gene knockdown on A. marginale acquisition and transmission. Although RNAi consistently knocked down all individually examined tick genes in infected tick guts and salivary glands, only the group of ticks injected with dsCOXIII failed to transmit A. marginale to naïve calves. To our knowledge, this is the first report demonstrating that RNAi of a tick gene is associated with a failure of A. marginale transmission. PMID:24878588

  14. RNA interference knockdown of DNA methyl-transferase 3 affects gene alternative splicing in the honey bee

    PubMed Central

    Li-Byarlay, Hongmei; Li, Yang; Stroud, Hume; Feng, Suhua; Newman, Thomas C.; Kaneda, Megan; Hou, Kirk K.; Worley, Kim C.; Elsik, Christine G.; Wickline, Samuel A.; Jacobsen, Steven E.; Ma, Jian; Robinson, Gene E.

    2013-01-01

    Studies of DNA methylation from fungi, plants, and animals indicate that gene body methylation is ancient and highly conserved in eukaryotic genomes, but its role has not been clearly defined. It has been postulated that regulation of alternative splicing of transcripts was an original function of DNA methylation, but a direct experimental test of the effect of methylation on alternative slicing at the whole genome level has never been performed. To do this, we developed a unique method to administer RNA interference (RNAi) in a high-throughput and noninvasive manner and then used it to knock down the expression of DNA methyl-transferase 3 (dnmt3), which is required for de novo DNA methylation. We chose the honey bee (Apis mellifera) for this test because it has recently emerged as an important model organism for studying the effects of DNA methylation on development and social behavior, and DNA methylation in honey bees is predominantly on gene bodies. Here we show that dnmt3 RNAi decreased global genomic methylation level as expected and in addition caused widespread and diverse changes in alternative splicing in fat tissue. Four different types of splicing events were affected by dnmt3 gene knockdown, and change in two types, exon skipping and intron retention, was directly related to decreased methylation. These results demonstrate that one function of gene body DNA methylation is to regulate alternative splicing. PMID:23852726

  15. Knockdown of PRKAR1A, the Gene Responsible for Carney Complex, Interferes With Differentiation in Osteoblastic Cells

    PubMed Central

    Zhang, Mei; Manchanda, Parmeet K.; Wu, Dayong; Wang, Qianben

    2014-01-01

    PRKAR1A is the gene encoding the type 1A regulatory subunit of protein kinase A, and it is the cause of the inherited human tumor syndrome Carney complex. Data from our laboratory has demonstrated that Prkar1a loss causes tumors in multiple cell lineages, including neural crest cells and osteoblasts. We have proposed that one mechanism by which tumorigenesis occurs is through the failure of terminal differentiation. In the present study, we directly test the effects of Prkar1a reduction on osteogenic differentiation in mouse and human cells in vitro. We found that Prkar1a levels noticeably increased during osteoblastic differentiation, indicating a positive correlation between the expression of Prkar1a and osteogenic potential. To validate this hypothesis, we generated stable Prkar1a knockdown in both mouse and human cells. These cells displayed significantly suppressed bone nodule formation and decreased expression of osteoblast markers such as osteocalcin and osteopontin. These observations imply that the antiosteogenic effect of Prkar1a ablation is not species or cell line specific. Furthermore, because Runt-related transcription factor-2 (Runx2) is a key mediator of osteoblast differentiation, we reasoned that the function of this transcription factor may be inhibited by Prkar1a knockdown. Chromatin immunoprecipitation and luciferase assays demonstrated that Prkar1a ablation repressed DNA binding and function of Runx2 at its target genes. Additionally, we determined that this effect is likely due to reductions in the Runx2-cooperating transcription factors forkhead box O1 and activating transcription factor 4. Taken together, this study provides direct evidence that ablation of Prkar1a interferes with signaling pathways necessary for osteoblast differentiation. PMID:24506536

  16. Knockdown of PRKAR1A, the gene responsible for Carney complex, interferes with differentiation in osteoblastic cells.

    PubMed

    Zhang, Mei; Manchanda, Parmeet K; Wu, Dayong; Wang, Qianben; Kirschner, Lawrence S

    2014-03-01

    PRKAR1A is the gene encoding the type 1A regulatory subunit of protein kinase A, and it is the cause of the inherited human tumor syndrome Carney complex. Data from our laboratory has demonstrated that Prkar1a loss causes tumors in multiple cell lineages, including neural crest cells and osteoblasts. We have proposed that one mechanism by which tumorigenesis occurs is through the failure of terminal differentiation. In the present study, we directly test the effects of Prkar1a reduction on osteogenic differentiation in mouse and human cells in vitro. We found that Prkar1a levels noticeably increased during osteoblastic differentiation, indicating a positive correlation between the expression of Prkar1a and osteogenic potential. To validate this hypothesis, we generated stable Prkar1a knockdown in both mouse and human cells. These cells displayed significantly suppressed bone nodule formation and decreased expression of osteoblast markers such as osteocalcin and osteopontin. These observations imply that the antiosteogenic effect of Prkar1a ablation is not species or cell line specific. Furthermore, because Runt-related transcription factor-2 (Runx2) is a key mediator of osteoblast differentiation, we reasoned that the function of this transcription factor may be inhibited by Prkar1a knockdown. Chromatin immunoprecipitation and luciferase assays demonstrated that Prkar1a ablation repressed DNA binding and function of Runx2 at its target genes. Additionally, we determined that this effect is likely due to reductions in the Runx2-cooperating transcription factors forkhead box O1 and activating transcription factor 4. Taken together, this study provides direct evidence that ablation of Prkar1a interferes with signaling pathways necessary for osteoblast differentiation. PMID:24506536

  17. Knockdown of Polyphenol Oxidase Gene Expression in Potato (Solanum tuberosum L.) with Artificial MicroRNAs.

    PubMed

    Chi, Ming; Bhagwat, Basdeo; Tang, Guiliang; Xiang, Yu

    2016-01-01

    It is of great importance and interest to develop crop varieties with low polyphenol oxidase (PPO) activity for the food industry because PPO-mediated oxidative browning is a main cause of post-harvest deterioration and quality loss of fresh produce and processed foods. We recently demonstrated that potato tubers with reduced browning phenotypes can be produced by inhibition of the expression of several PPO gene isoforms using artificial microRNA (amiRNA) technology. The approach introduces a single type of 21-nucleotide RNA population to guide silencing of the PPO gene transcripts in potato tissues. Some advantages of the technology are: small RNA molecules are genetically transformed, off-target gene silencing can be avoided or minimized at the stage of amiRNA designs, and accuracy and efficiency of the processes can be detected at every step using molecular biological techniques. Here we describe the methods for transformation and regeneration of potatoes with amiRNA vectors, detection of the expression of amiRNAs, identification of the cleaved product of the target gene transcripts, and assay of the expression level of PPO gene isoforms in potatoes. PMID:26843174

  18. Knock-down of heat-shock protein 90 and isocitrate lyase gene expression reduced root-knot nematode reproduction.

    PubMed

    Lourenço-Tessutti, Isabela Tristan; Souza Junior, José Dijair Antonino; Martins-de-Sa, Diogo; Viana, Antônio Américo Barbosa; Carneiro, Regina Maria Dechechi Gomes; Togawa, Roberto Coiti; de Almeida-Engler, Janice; Batista, João Aguiar Nogueira; Silva, Maria Cristina Mattar; Fragoso, Rodrigo Rocha; Grossi-de-Sa, Maria Fatima

    2015-05-01

    Crop losses caused by nematode infections are estimated to be valued at USD 157 billion per year. Meloidogyne incognita, a root-knot nematode (RKN), is considered to be one of the most important plant pathogens due to its worldwide distribution and the austere damage it can cause to a large variety of agronomically important crops. RNA interference (RNAi), a gene silencing process, has proven to be a valuable biotechnology alternative method for RKN control. In this study, the RNAi approach was applied, using fragments of M. incognita genes that encode for two essential molecules, heat-shock protein 90 (HSP90) and isocitrate lyase (ICL). Plant-mediated RNAi of these genes led to a significant level of resistance against M. incognita in the transgenic Nicotiana tabacum plants. Bioassays of plants expressing HSP90 dsRNA demonstrated a delay in gall formation and up to 46% reduction in eggs compared with wild-type plants. A reduction in the level of HSP90 transcripts was observed in recovered eggs from plants expressing dsRNA, indicating that gene silencing persisted and was passed along to first progeny. The ICL knock-down had no clear effect on gall formation but resulted in up to 77% reduction in egg oviposition compared with wild-type plants. Our data suggest that both genes may be involved in RKN development and reproduction. Thus, in this paper, we describe essential candidate genes that could be applied to generate genetically modified crops, using the RNAi strategy to control RKN parasitism. PMID:26020830

  19. Using RNAi in C. "elegans" to Demonstrate Gene Knockdown Phenotypes in the Undergraduate Biology Lab Setting

    ERIC Educational Resources Information Center

    Roy, Nicole M.

    2013-01-01

    RNA interference (RNAi) is a powerful technology used to knock down genes in basic research and medicine. In 2006 RNAi technology using "Caenorhabditis elegans" ("C. elegans") was awarded the Nobel Prize in medicine and thus students graduating in the biological sciences should have experience with this technology. However,…

  20. Knockdown of Five Genes Encoding Uncharacterized Proteins Inhibits Entamoeba histolytica Phagocytosis of Dead Host Cells.

    PubMed

    Sateriale, Adam; Miller, Peter; Huston, Christopher D

    2016-04-01

    Entamoeba histolytica is the protozoan parasite that causes invasive amebiasis, which is endemic to many developing countries and characterized by dysentery and liver abscesses. The virulence of E. histolytica correlates with the degree of host cell engulfment, or phagocytosis, and E. histolytica phagocytosis alters amebic gene expression in a feed-forward manner that results in an increased phagocytic ability. Here, we used a streamlined RNA interference screen to silence the expression of 15 genes whose expression was upregulated in phagocytic E. histolytica trophozoites to determine whether these genes actually function in the phagocytic process. When five of these genes were silenced, amebic strains with significant decreases in the ability to phagocytose apoptotic host cells were produced. Phagocytosis of live host cells, however, was largely unchanged, and the defects were surprisingly specific for phagocytosis. Two of the five encoded proteins, which we named E. histolytica ILWEQ (EhILWEQ) and E. histolytica BAR (EhBAR), were chosen for localization via SNAP tag labeling and localized to the site of partially formed phagosomes. Therefore, both EhILWEQ and EhBAR appear to contribute to E. histolytica virulence through their function in phagocytosis, and the large proportion (5/15 [33%]) of gene-silenced strains with a reduced ability to phagocytose host cells validates the previously published microarray data set demonstrating feed-forward control of E. histolytica phagocytosis. Finally, although only limited conclusions can be drawn from studies using the virulence-deficient G3 Entamoeba strain, the relative specificity of the defects induced for phagocytosis of apoptotic cells but not healthy cells suggests that cell killing may play a rate-limiting role in the process of Entamoeba histolytica host cell engulfment. PMID:26810036

  1. Knockdown of the corazonin gene reveals its critical role in the control of gregarious characteristics in the desert locust.

    PubMed

    Sugahara, Ryohei; Saeki, Shinjiro; Jouraku, Akiya; Shiotsuki, Takahiro; Tanaka, Seiji

    2015-08-01

    The two plague locusts, Schistocerca gregaria and Locusta migratoria, exhibit density-dependent phase polyphenism. Nymphs occurring at low population densities (solitarious forms) are uniformly colored and match their body color to the background color of their habitat, whereas those occurring at high population densities (gregarious) develop black patterns. An injection of the neuropeptide, corazonin (Crz) has been shown to induce black patterns in locusts and affect the classical morphometric ratio, F/C (F, hind femur length; C, maximum head width). We herein identified and cloned the CRZ genes from S. gregaria (SgCRZ) and L. migratoria. A comparative analysis of prepro-Crz sequences among insects showed that the functional peptide was well conserved; its conservation was limited to the peptide region. Silencing of the identified SgCRZ gene in gregarious S. gregaria nymphs markedly lightened their body color and shifted the adult F/C ratio toward the value typical of solitarious forms. In addition, knockdown of the gene in solitarious nymphs strongly inhibited darkening even after a transfer to crowded conditions; however, these individuals developed black patterns after being injected with the Crz as a rescue treatment. SgCRZ was constitutively expressed in the brains of S. gregaria during nymphal development in both phases. This gene was highly expressed not only in the brain in both phases, but also in the corpora allata in the gregarious phase. This conspicuous phase-dependent difference in SgCRZ gene expression may indicate a functional role in the control of phase polyphenism in this locust. PMID:26092175

  2. Speech Sound Processing Deficits and Training-Induced Neural Plasticity in Rats with Dyslexia Gene Knockdown

    PubMed Central

    Centanni, Tracy M.; Chen, Fuyi; Booker, Anne M.; Engineer, Crystal T.; Sloan, Andrew M.; Rennaker, Robert L.; LoTurco, Joseph J.; Kilgard, Michael P.

    2014-01-01

    In utero RNAi of the dyslexia-associated gene Kiaa0319 in rats (KIA-) degrades cortical responses to speech sounds and increases trial-by-trial variability in onset latency. We tested the hypothesis that KIA- rats would be impaired at speech sound discrimination. KIA- rats needed twice as much training in quiet conditions to perform at control levels and remained impaired at several speech tasks. Focused training using truncated speech sounds was able to normalize speech discrimination in quiet and background noise conditions. Training also normalized trial-by-trial neural variability and temporal phase locking. Cortical activity from speech trained KIA- rats was sufficient to accurately discriminate between similar consonant sounds. These results provide the first direct evidence that assumed reduced expression of the dyslexia-associated gene KIAA0319 can cause phoneme processing impairments similar to those seen in dyslexia and that intensive behavioral therapy can eliminate these impairments. PMID:24871331

  3. [Exon skipping therapy for Duchenne muscular dystrophy by using antisense Morpholino].

    PubMed

    Takeda, Shin'ichi

    2009-11-01

    Duchenne muscular dystrophy (DMD) is caused by the lack of dystrophin protein at the sarcolemma. Exon skipping by antisense oligonucleotides is a novel method to restore the reading frame of the mutated DMD gene, and rescue dystrophin production. We recently reported that systemic delivery of Morpholino antisense oligonucleotides targeting exon 6 and 8 of the canine DMD gene, efficiently recovered functional dystrophin proteins at the sarcolamma of dystrophic dogs, and improved performance of affected dogs without serious side effects (Yokota et al., Ann Neurol. 65 (6): 667-676, 2009). To optimize therapeutic antisense Morpholinos for more frequent mutations of the DMD gene, we designed antisense Morpholinos targeting exon 51 of the mouse DMD gene, and injected them separately or in combination into the muscles of mdx52 mice, in which exon 52 has been deleted by a gene targeting technique (Araki et al., 1997). We also tried systemic delivery of antisense Morpholino to skip exon 51 in mdx52 mice. It is important to verify the effectiveness and side effects of antisense Morpholino in experimental animal models such as dystrophic dogs or mdx52 mice, before clinical trials in DMD patients. PMID:20030230

  4. Core RNAi machinery and gene knockdown in the emerald ash borer (Agrilus planipennis).

    PubMed

    Zhao, Chaoyang; Alvarez Gonzales, Miguel A; Poland, Therese M; Mittapalli, Omprakash

    2015-01-01

    The RNA interference (RNAi) technology has been widely used in insect functional genomics research and provides an alternative approach for insect pest management. To understand whether the emerald ash borer (Agrilus planipennis), an invasive and destructive coleopteran insect pest of ash tree (Fraxinus spp.), possesses a strong RNAi machinery that is capable of degrading target mRNA as a response to exogenous double-stranded RNA (dsRNA) induction, we identified three RNAi pathway core component genes, Dicer-2, Argonaute-2 and R2D2, from the A. planipennis genome sequence. Characterization of these core components revealed that they contain conserved domains essential for the proteins to function in the RNAi pathway. Phylogenetic analyses showed that they are closely related to homologs derived from other coleopteran species. We also delivered the dsRNA fragment of AplaScrB-2, a β-fructofuranosidase-encoding gene horizontally acquired by A. planipennis as we reported previously, into A. planipennis adults through microinjection. Quantitative real-time PCR analysis on the dsRNA-treated beetles demonstrated a significantly decreased gene expression level of AplaScrB-2 appearing on day 2 and lasting until at least day 6. This study is the first record of RNAi applied in A. planipennis. PMID:25541004

  5. Nucleofection Mediates High-Efficiency Stable Gene Knockdown and Transgene Expression in Human Embryonic Stem Cells

    PubMed Central

    Hohenstein, Kristi A.; Pyle, April D.; Chern, Jing Yi; Lock, Leslie F.; Donovan, Peter J.

    2013-01-01

    High-efficiency genetic modification of human embryonic stem (hES) cells would enable manipulation of gene activity, routine gene targeting, and development of new human disease models and treatments. Chemical transfection, nucleofection, and electroporation of hES cells result in low transfection efficiencies. Viral transduction is efficient but has significant drawbacks. Here we describe techniques to transiently and stably express transgenes in hES cells with high efficiency using a widely available vector system. The technique combines nucleofection of single hES cells with improved methods to select hES cells at clonal density. As validation, we reduced Oct4 and Nanog expression using siRNAs and shRNA vectors in hES cells. Furthermore, we derived many hES cell clones with either stably reduced alkaline phosphatase activity or stably overexpressed green fluorescent protein. These clones retained stem cell characteristics (normal karyotype, stem cell marker expression, self-renewal, and pluripotency). These studies will accelerate efforts to interrogate gene function and define the parameters that control growth and differentiation of hES cells. PMID:18323409

  6. Knockdown of Nogo gene by short hairpin RNA interference promotes functional recovery of spinal cord injury in a rat model.

    PubMed

    Liu, Guo-Min; Luo, Yun-Gang; Li, Juan; Xu, Kun

    2016-05-01

    The specific myelin component Nogo protein is one of the major inhibitory molecules of spinal cord axonal outgrowth following spinal cord injury. The present study aimed to investigate the effects of silencing Nogo protein with shRNA interference on the promotion of functional recovery in a rat model with spinal cord hemisection. Nogo-A short hairpin RNAs (Nogo shRNAs) were constructed and transfected into rats with spinal cord hemisection by adenovirus-mediated transfection. Reverse transcription‑polymerase chain reaction and western blotting were performed to analyze the expression of Nogo-A and Growth Associated Protein 43 (GAP-43). In addition, Basso Beattie Bresnahan (BBB) scores were used to assess the functional recovery of rats following spinal cord injury. The results demonstrated that expression of the Nogo‑A gene was observed to be downregulated following transfection and GAP‑43 expression was observed to increase. The BBB scores were increased following treatment with Nogo shRNAs, indicating functional recovery of the injured nerves. Thus, Nogo-A shRNA interference can knockdown Nogo gene expression and upregulate GAP-43 to promote the functional recovery of spinal cord injury in rats. This finding may advance progress toward assisting the regeneration of injured neurons through the use of Nogo-A shRNA. PMID:27035338

  7. RNAi-mediated knockdown of the Halloween gene spookiest (CYP307B1) impedes adult eclosion in the western tarnished plant bug, Lygus hesperus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ecdysteroids play a critical role in coordinating insect growth, development, and reproduction. A suite of cytochrome P450 monooxygenases coded by what are collectively termed Halloween genes mediate ecdysteroid biosynthesis. In this study, we describe cloning and RNAi-mediated knockdown of the CYP3...

  8. Cardiac Gene Expression Knockdown Using Small Inhibitory RNA-Loaded Microbubbles and Ultrasound.

    PubMed

    Kopechek, Jonathan A; Carson, Andrew R; McTiernan, Charles F; Chen, Xucai; Klein, Edwin C; Villanueva, Flordeliza S

    2016-01-01

    RNA interference has potential therapeutic value for cardiac disease, but targeted delivery of interfering RNA is a challenge. Custom designed microbubbles, in conjunction with ultrasound, can deliver small inhibitory RNA to target tissues in vivo. The efficacy of cardiac RNA interference using a microbubble-ultrasound theranostic platform has not been demonstrated in vivo. Therefore, our objective was to test the hypothesis that custom designed microbubbles and ultrasound can mediate effective delivery of small inhibitory RNA to the heart. Microbubble and ultrasound mediated cardiac RNA interference was tested in transgenic mice displaying cardiac-restricted luciferase expression. Luciferase expression was assayed in select tissues of untreated mice (n = 14). Mice received intravenous infusion of cationic microbubbles bearing small inhibitory RNA directed against luciferase (n = 9) or control RNA (n = 8) during intermittent cardiac-directed ultrasound at mechanical index of 1.6. Simultaneous echocardiography in a separate group of mice (n = 3) confirmed microbubble destruction and replenishment during treatment. Three days post treatment, cardiac luciferase messenger RNA and protein levels were significantly lower in ultrasound-treated mice receiving microbubbles loaded with small inhibitory RNA directed against luciferase compared to mice receiving microbubbles bearing control RNA (23±7% and 33±7% of control mice, p<0.01 and p = 0.03, respectively). Passive cavitation detection focused on the heart confirmed that insonification resulted in inertial cavitation. In conclusion, small inhibitory RNA-loaded microbubbles and ultrasound directed at the heart significantly reduced the expression of a reporter gene. Ultrasound-targeted destruction of RNA-loaded microbubbles may be an effective image-guided strategy for therapeutic RNA interference in cardiac disease. PMID:27471848

  9. Cardiac Gene Expression Knockdown Using Small Inhibitory RNA-Loaded Microbubbles and Ultrasound

    PubMed Central

    McTiernan, Charles F.; Chen, Xucai; Klein, Edwin C.; Villanueva, Flordeliza S.

    2016-01-01

    RNA interference has potential therapeutic value for cardiac disease, but targeted delivery of interfering RNA is a challenge. Custom designed microbubbles, in conjunction with ultrasound, can deliver small inhibitory RNA to target tissues in vivo. The efficacy of cardiac RNA interference using a microbubble-ultrasound theranostic platform has not been demonstrated in vivo. Therefore, our objective was to test the hypothesis that custom designed microbubbles and ultrasound can mediate effective delivery of small inhibitory RNA to the heart. Microbubble and ultrasound mediated cardiac RNA interference was tested in transgenic mice displaying cardiac-restricted luciferase expression. Luciferase expression was assayed in select tissues of untreated mice (n = 14). Mice received intravenous infusion of cationic microbubbles bearing small inhibitory RNA directed against luciferase (n = 9) or control RNA (n = 8) during intermittent cardiac-directed ultrasound at mechanical index of 1.6. Simultaneous echocardiography in a separate group of mice (n = 3) confirmed microbubble destruction and replenishment during treatment. Three days post treatment, cardiac luciferase messenger RNA and protein levels were significantly lower in ultrasound-treated mice receiving microbubbles loaded with small inhibitory RNA directed against luciferase compared to mice receiving microbubbles bearing control RNA (23±7% and 33±7% of control mice, p<0.01 and p = 0.03, respectively). Passive cavitation detection focused on the heart confirmed that insonification resulted in inertial cavitation. In conclusion, small inhibitory RNA-loaded microbubbles and ultrasound directed at the heart significantly reduced the expression of a reporter gene. Ultrasound-targeted destruction of RNA-loaded microbubbles may be an effective image-guided strategy for therapeutic RNA interference in cardiac disease. PMID:27471848

  10. Knock-down of transcript abundance of a family of Kunitz proteinase inhibitor genes in white clover (Trifolium repens) reveals a redundancy and diversity of gene function.

    PubMed

    Islam, Afsana; Leung, Susanna; Burgess, Elisabeth P J; Laing, William A; Richardson, Kim A; Hofmann, Rainer W; Dijkwel, Paul P; McManus, Michael T

    2015-12-01

    The transcriptional regulation of four phylogenetically distinct members of a family of Kunitz proteinase inhibitor (KPI) genes isolated from white clover (Trifolium repens; designated Tr-KPI1, Tr-KPI2, Tr-KPI4 and Tr-KPI5) has been investigated to determine their wider functional role. The four genes displayed differential transcription during seed germination, and in different tissues of the mature plant, and transcription was also ontogenetically regulated. Heterologous over-expression of Tr-KPI1, Tr-KPI2, Tr-KPI4 and Tr-KPI5 in Nicotiana tabacum retarded larval growth of the herbivore Spodoptera litura, and an increase in the transcription of the pathogenesis-related genes PR1 and PR4 was observed in the Tr-KPI1 and Tr-KPI4 over-expressing lines. RNA interference (RNAi) knock-down lines in white clover displayed significantly altered vegetative growth phenotypes with inhibition of shoot growth and a stimulation of root growth, while knock-down of Tr-KPI1, Tr-KPI2 and Tr-KPI5 transcript abundance also retarded larval growth of S. litura. Examination of these RNAi lines revealed constitutive stress-associated phenotypes as well as altered transcription of cellular signalling genes. These results reveal a functional redundancy across members of the KPI gene family. Further, the regulation of transcription of at least one member of the family, Tr-KPI2, may occupy a central role in the maintenance of a cellular homeostasis. PMID:26377591

  11. Electroporation of cDNA/Morpholinos to targeted areas of embryonic CNS in Xenopus

    PubMed Central

    Falk, Julien; Drinjakovic, Jovana; Leung, Kin Mei; Dwivedy, Asha; Regan, Aoife G; Piper, Michael; Holt, Christine E

    2007-01-01

    Background Blastomere injection of mRNA or antisense oligonucleotides has proven effective in analyzing early gene function in Xenopus. However, functional analysis of genes involved in neuronal differentiation and axon pathfinding by this method is often hampered by earlier function of these genes during development. Therefore, fine spatio-temporal control of over-expression or knock-down approaches is required to specifically address the role of a given gene in these processes. Results We describe here an electroporation procedure that can be used with high efficiency and low toxicity for targeting DNA and antisense morpholino oligonucleotides (MOs) into spatially restricted regions of the Xenopus CNS at a critical time-window of development (22–50 hour post-fertilization) when axonal tracts are first forming. The approach relies on the design of "electroporation chambers" that enable reproducible positioning of fixed-spaced electrodes coupled with accurate DNA/MO injection. Simple adjustments can be made to the electroporation chamber to suit the shape of different aged embryos and to alter the size and location of the targeted region. This procedure can be used to electroporate separate regions of the CNS in the same embryo allowing separate manipulation of growing axons and their intermediate and final targets in the brain. Conclusion Our study demonstrates that electroporation can be used as a versatile tool to investigate molecular pathways involved in axon extension during Xenopus embryogenesis. Electroporation enables gain or loss of function studies to be performed with easy monitoring of electroporated cells. Double-targeted transfection provides a unique opportunity to monitor axon-target interaction in vivo. Finally, electroporated embryos represent a valuable source of MO-loaded or DNA transfected cells for in vitro analysis. The technique has broad applications as it can be tailored easily to other developing organ systems and to other organisms by

  12. RNAi-mediated knockdown of pituitary tumor-transforming gene-1 (PTTG1) suppresses the proliferation and invasive potential of PC3 human prostate cancer cells

    PubMed Central

    Huang, S.Q.; Liao, Q.J.; Wang, X.W.; Xin, D.Q.; Chen, S.X.; Wu, Q.J.; Ye, G.

    2012-01-01

    Pituitary tumor-transforming gene-1 (PTTG1) is a proto-oncogene that promotes tumorigenesis and metastasis in numerous cell types and is overexpressed in a variety of human tumors. We have demonstrated that PTTG1 expression was up-regulated in both human prostate cancer specimens and prostate cancer cell lines. For a more direct assessment of the function of PTTG1 in prostate tumorigenesis, RNAi-mediated knockdown was used to selectively decrease PTTG1 expression in PC3 human prostate tumor cells. After three weeks of selection, colonies stably transfected with PTTG1-targeted RNAi (the knockdown PC3 cell line) or empty vector (the control PC3 cell line) were selected and expanded to investigate the role of PTTG1 expression in PC3 cell growth and invasion. Cell proliferation rate was significantly slower (28%) in the PTTG1 knockdown line after 6 days of growth as indicated by an MTT cell viability assay (P < 0.05). Similarly, a soft agar colony formation assay revealed significantly fewer (66.7%) PTTG1 knockdown PC3 cell colonies than control colonies after three weeks of growth. In addition, PTTG1 knockdown resulted in cell cycle arrest at G1 as indicated by fluorescence-activated cell sorting. The PTTG1 knockdown PC3 cell line also exhibited significantly reduced migration through Matrigel in a transwell assay of invasive potential, and down-regulation of PTTG1 could lead to increased sensitivity of these prostate cancer cells to a commonly used anticancer drug, taxol. Thus, PTTG1 expression is crucial for PC3 cell proliferation and invasion, and could be a promising new target for prostate cancer therapy. PMID:22872288

  13. Dmp53, basket and drICE gene knockdown and polyphenol gallic acid increase life span and locomotor activity in a Drosophila Parkinson’s disease model

    PubMed Central

    Ortega-Arellano, Hector Flavio; Jimenez-Del-Rio, Marlene; Velez-Pardo, Carlos

    2013-01-01

    Understanding the mechanism(s) by which dopaminergic (DAergic) neurons are eroded in Parkinson’s disease (PD) is critical for effective therapeutic strategies. By using the binary tyrosine hydroxylase (TH)-Gal4/UAS-X RNAi Drosophila melanogaster system, we report that Dmp53, basket and drICE gene knockdown in dopaminergic neurons prolong life span (p < 0.05; log-rank test) and locomotor activity (p < 0.05; χ2 test) in D. melanogaster lines chronically exposed to (1 mM) paraquat (PQ, oxidative stress (OS) generator) compared to untreated transgenic fly lines. Likewise, knockdown flies displayed higher climbing performance than control flies. Amazingly, gallic acid (GA) significantly protected DAergic neurons, ameliorated life span, and climbing abilities in knockdown fly lines treated with PQ compared to flies treated with PQ only. Therefore, silencing specific gene(s) involved in neuronal death might constitute an excellent tool to study the response of DAergic neurons to OS stimuli. We propose that a therapy with antioxidants and selectively “switching off” death genes in DAergic neurons could provide a means for pre-clinical PD individuals to significantly ameliorate their disease condition. PMID:24385865

  14. Spatially- and temporally-controlled postnatal p53 knockdown cooperates with embryonic Schwann cell precursor Nf1 gene loss to promote malignant peripheral nerve sheath tumor formation

    PubMed Central

    Hirbe, Angela C.; Dahiya, Sonika; Friedmann-Morvinski, Dinorah; Verma, Inder M.; Clapp, D. Wade; Gutmann, David H.

    2016-01-01

    Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive sarcomas that arise sporadically or in association with the Neurofibromatosis type 1 (NF1) cancer predisposition syndrome. In individuals with NF1, MPNSTs are hypothesized to arise from Nf1-deficient Schwann cell precursor cells following the somatic acquisition of secondary cooperating genetic mutations (e.g., p53 loss). To model this sequential genetic cooperativity, we coupled somatic lentivirus-mediated p53 knockdown in the adult right sciatic nerve with embryonic Schwann cell precursor Nf1 gene inactivation in two different Nf1 conditional knockout mouse strains. Using this approach, ∼60% of mice with Periostin-Cre-mediated Nf1 gene inactivation (Periostin-Cre; Nf1flox/flox mice) developed tumors classified as low-grade MPNSTs following p53 knockdown (mean, 6 months). Similarly, ∼70% of Nf1+/− mice with GFAP-Cre-mediated Nf1 gene inactivation (GFAP-Cre; Nf1flox/null mice) developed low-grade MPNSTs following p53 knockdown (mean, 3 months). In addition, wild-type and Nf1+/− mice with GFAP-Cre-mediated Nf1 loss develop MPNSTs following somatic p53 knockout with different latencies, suggesting potential influences of Nf1+/− stromal cells in MPNST pathogenesis. Collectively, this new MPNST model system permits the analysis of somatically-acquired events as well as tumor microenvironment signals that potentially cooperate with Nf1 loss in the development and progression of this deadly malignancy. PMID:26859681

  15. Knockdown of WHIRLY1 Affects Drought Stress-Induced Leaf Senescence and Histone Modifications of the Senescence-Associated Gene HvS40.

    PubMed

    Janack, Bianka; Sosoi, Paula; Krupinska, Karin; Humbeck, Klaus

    2016-01-01

    The plastid-nucleus located protein WHIRLY1 has been described as an upstream regulator of leaf senescence, binding to the promoter of senescence-associated genes like HvS40. To investigate the impact of WHIRLY1 on drought stress-induced, premature senescence, transgenic barley plants with an RNAi-mediated knockdown of the HvWHIRLY1 gene were grown under normal and drought stress conditions. The course of leaf senescence in these lines was monitored by physiological parameters and studies on the expression of senescence- and drought stress-related genes. Drought treatment accelerated leaf senescence in WT plants, whereas WHIRLY 1 knockdown lines (RNAi-W1) showed a stay-green phenotype. Expression of both senescence-associated and drought stress-responsive genes, was delayed in the transgenic plants. Notably, expression of transcription factors of the WRKY and NAC families, which are known to function in senescence- and stress-related signaling pathways, was affected in plants with impaired accumulation of WHIRLY1, indicating that WHIRLY1 acts as an upstream regulator of drought stress-induced senescence. To reveal the epigenetic indexing of HvS40 at the onset of drought-induced senescence in WT and RNAi-W1 lines, stress-responsive loading with histone modifications of promoter and coding sequences of HvS40 was analyzed by chromatin immunoprecipitation and quantified by qRT-PCR. In the wildtype, the euchromatic mark H3K9ac of the HvS40 gene was low under control conditions and was established in response to drought treatment, indicating the action of epigenetic mechanisms in response to drought stress. However, drought stress caused no significant increase in H3K9ac in plants impaired in accumulation of WHIRLY1. The results show that WHIRLY1 knockdown sets in motion a delay in senescence that involves all aspects of gene expression, including changes in chromatin structure. PMID:27608048

  16. Microinjection of mRNA or morpholinos for reverse genetic analysis in the starlet sea anemone, Nematostella vectensis

    PubMed Central

    Layden, Michael J; Röttinger, Eric; Wolenski, Francis S; Gilmore, Thomas D; Martindale, Mark Q

    2016-01-01

    We describe a protocol for microinjection of embryos for an emerging model system, the cnidarian sea anemone, Nematostella vectensis. In addition, we provide protocols for carrying out overexpression and knockdown of gene function through microinjection of in vitro–translated mRNAs or gene-specific oligonucleotide morpholinos (Mos), respectively. our approach is simple, and it takes advantage of the natural adherence properties of the early embryo to position them in a single layer on a polystyrene dish. embryos are visualized on a dissecting microscope equipped with epifluorescence and injected with microinjection needles using a picospritzer forced-air injection system. a micromanipulator is used to guide the needle to impale individual embryos. Injection takes ∼1.5 h, and an experienced researcher can inject ∼2,000 embryos in a single session. With the availability of the published Nematostella genome, the entire protocol, including cloning and transcription of mRNAs, can be carried out in ∼1 week. PMID:23579781

  17. Reverse genetic screening reveals poor correlation between Morpholino-induced and mutant phenotypes in zebrafish

    PubMed Central

    Gupta, A.; Grosse, A. S.; van Impel, A.; Kirchmaier, B. C.; Peterson-Maduro, J.; Kourkoulis, G.; Male, I.; DeSantis, D.F.; Sheppard-Tindell, S.; Ebarasi, L.; Betsholtz, C.; Schulte-Merker, S.; Wolfe, S. A.; Lawson, N. D.

    2014-01-01

    SUMMARY The widespread availability of programmable site-specific nucleases now enables targeted gene disruption in the zebrafish. In this study, we applied site-specific nucleases to generate zebrafish lines bearing individual mutations in more than twenty genes. We found that mutations in only a small proportion of genes caused defects in embryogenesis. Moreover, mutants for ten different genes failed to recapitulate published Morpholino-induced phenotypes (morphants). The absence of phenotypes in mutant embryos was not likely due to maternal effects or failure to eliminate gene function. Consistently, a comparison of published morphant defects with the Sanger Zebrafish Mutation Project revealed that approximately eighty percent of morphant phenotypes were not observed in mutant embryos, similar to our mutant collection. Based on these results, we suggest that mutant phenotypes become the standard metric to define gene function in zebrafish, after which Morpholinos that recapitulate respective phenotypes could be reliably applied for ancillary analyses. PMID:25533206

  18. Practical Recommendations for the Use of the GeneSwitch Gal4 System to Knock-Down Genes in Drosophila melanogaster.

    PubMed

    Scialo, Filippo; Sriram, Ashwin; Stefanatos, Rhoda; Sanz, Alberto

    2016-01-01

    Drosophila melanogaster is a popular research model organism thanks to its' powerful genetic tools that allow spatial and temporal control of gene expression. The inducible GeneSwitch Gal4 system (GS) system is a modified version of the classic UAS/GAL4 system which allows inducible regulation of gene expression and eliminates background effects. It is widely acknowledged that the GS system is leaky, with low level expression of UAS transgenes in absence of the inducer RU-486 (the progesterone analog that activates the modified GAL4 protein). However, in the course of our experiments, we have observed that the extent of this leak depends on the nature of the transgene being expressed. In the absence of RU-486, when strong drivers are used to express protein coding transgenes, leaky expression is low or negligible, however expression of RNA interference (RNAi) transgenes results in complete depletion of protein levels. The majority of published studies, using the GS system and RNAi transgenes validate knock-down efficiency by comparing target gene mRNA levels between induced and non-induced groups. Here, we demonstrate that this approach is lacking and that both additional control groups and further validation is required at the protein level. Unfortunately, this experimental limitation of the GS system eliminates "the background advantage", but does offer the possibility of performing more complex experiments (e.g. studying depletion and overexpression of different proteins in the same genetic background). The limitations and new possible applications of the GS system are discussed in detail. PMID:27570965

  19. Practical Recommendations for the Use of the GeneSwitch Gal4 System to Knock-Down Genes in Drosophila melanogaster

    PubMed Central

    Scialo, Filippo; Sriram, Ashwin; Stefanatos, Rhoda; Sanz, Alberto

    2016-01-01

    Drosophila melanogaster is a popular research model organism thanks to its’ powerful genetic tools that allow spatial and temporal control of gene expression. The inducible GeneSwitch Gal4 system (GS) system is a modified version of the classic UAS/GAL4 system which allows inducible regulation of gene expression and eliminates background effects. It is widely acknowledged that the GS system is leaky, with low level expression of UAS transgenes in absence of the inducer RU-486 (the progesterone analog that activates the modified GAL4 protein). However, in the course of our experiments, we have observed that the extent of this leak depends on the nature of the transgene being expressed. In the absence of RU-486, when strong drivers are used to express protein coding transgenes, leaky expression is low or negligible, however expression of RNA interference (RNAi) transgenes results in complete depletion of protein levels. The majority of published studies, using the GS system and RNAi transgenes validate knock-down efficiency by comparing target gene mRNA levels between induced and non-induced groups. Here, we demonstrate that this approach is lacking and that both additional control groups and further validation is required at the protein level. Unfortunately, this experimental limitation of the GS system eliminates “the background advantage”, but does offer the possibility of performing more complex experiments (e.g. studying depletion and overexpression of different proteins in the same genetic background). The limitations and new possible applications of the GS system are discussed in detail. PMID:27570965

  20. Knockdown of a nutrient amino acid transporter gene LdNAT1 reduces free neutral amino acid contents and impairs Leptinotarsa decemlineata pupation

    PubMed Central

    Fu, Kai-Yun; Guo, Wen-Chao; Ahmat, Tursun; Li, Guo-Qing

    2015-01-01

    A Leptinotarsa decemlineata SLC6 NAT gene (LdNAT1) was cloned. LdNAT1 was highly expressed in the larval alimentary canal especially midgut. LdNAT1 mRNA levels were high right after the molt and low just before the molt. JH and a JH analog pyriproxyfen activated LdNAT1 expression. RNAi of an allatostatin gene LdAS-C increased JH and upregulated LdNAT1 transcription. Conversely, silencing of a JH biosynthesis gene LdJHAMT decreased JH and reduced LdNAT1 expression. Moreover, 20E and an ecdysteroid agonist halofenozide repressed LdNAT1 expression, whereas a decrease in 20E by RNAi of an ecdysteroidogenesis gene LdSHD and disruption of 20E signaling by knockdown of LdE75 and LdFTZ-F1 activated LdNAT1 expression. Thus, LdNAT1 responded to both 20E and JH. Moreover, knockdown of LdNAT1 reduced the contents of cysteine, histidine, isoleucine, leucine, methionine, phenylalanine and serine in the larval bodies and increased the contents of these amino acids in the larval feces. Furthermore, RNAi of LdNAT1 inhibited insulin/target of rapamycin pathway, lowered 20E and JH titers, reduced 20E and JH signaling, retarded larval growth and impaired pupation. These data showed that LdNAT1 was involved in the absorption of several neutral amino acids critical for larval growth and metamorphosis. PMID:26657797

  1. RNAi-mediated knockdown of the CLN3 gene inhibits proliferation and promotes apoptosis in drug-resistant ovarian cancer cells

    PubMed Central

    MAO, DONGWEI; CHE, JIANHUA; HAN, SHIYU; ZHAO, HONGHUI; ZHU, YUMEI; ZHU, HONG

    2015-01-01

    CLN3 is a recently identified anti-apoptotic gene, which has been demonstrated to be highly expressed in a diverse range of cancer cell lines, including ovarian cancer. In the present study, RNA interference, mediated by a lentivirus expressing CLN3 short hairpin RNA (shRNA) was utilized to knockdown the expression of CLN3 in the A2780 human ovarian cancer cell line, and its cisplatin-resistant and carboplatin-resistant sublines, A2780/DDP and A2780/CBP cells. It was revealed that the mRNA and protein expression levels of CLN3 were significantly reduced in the CLN3-specific shRNA-transduced cells, compared with the untransduced and control shRNA-transduced cells. In addition, specific knockdown of CLN3 in these cells inhibited cell proliferation and led to cell cycle arrest at the G0/G1 phase, with eventual apoptosis. CLN3 knockdown caused increases in the levels of Bax, FAX, cleaved-caspase 3, cleaved-caspase 8 and cleaved-RARP, but decreased the level of Bcl-2. Finally, it was observed that CLN3 depletion markedly reduced the half maximum inhibitory concentration in the A2780/DDP and A2780/CBP cells. Taken together, these data suggested that CLN3 is involved in tumorigenesis and drug resistance in ovarian cancer, and may serve as a promising therapeutic target for its treatment. PMID:26299671

  2. Knockdown of astrocyte elevated gene-1 inhibits tumor growth and modifies microRNAs expression profiles in human colorectal cancer cells

    SciTech Connect

    Huang, Sujun; Wu, Binwen; Li, Dongfeng; Zhou, Weihong; Deng, Gang; Zhang, Kaijun; Li, Youjia

    2014-02-14

    Highlights: • AEG-1 expression in CRC cell lines and down-regulation or upregulation of AEG-1 in vitro. • Knockdown of AEG-1 inhibits cell proliferation, colony formation and invasion. • Upregulation of AEG-1 enhances proliferation, invasion and colony formation. • Knockdown of AEG-1 accumulates G0/G1-phase cells and promotes apoptosis in CRC cells. • AEG-1 knockdown increases 5-FU cytotoxicity. - Abstract: Astrocyte elevated gene-1 (AEG-1), upregulated in various types of malignancies including colorectal cancer (CRC), has been reported to be associated with the carcinogenesis. MicroRNAs (miRNAs) are widely involved in the initiation and progression of cancer. However, the functional significance of AEG-1 and the relationship between AEG-1 and microRNAs in human CRC remains unclear. The aim of this study was to investigate whether AEG-1 could serve as a potential therapeutic target of human CRC and its possible mechanism. We adopted a strategy of ectopic overexpression or RNA interference to upregulate or downregulate expression of AEG-1 in CRC models. Their phenotypic changes were analyzed by Western blot, MTT and transwell matrix penetration assays. MicroRNAs expression profiles were performed using microarray analysis followed by validation using qRT-PCR. Knockdown of AEG-1 could significantly inhibit colon cancer cell proliferation, colony formation, invasion and promotes apoptosis. Conversely, upregulation of AEG-1 could significantly enhance cell proliferation, invasion and reduced apoptisis. AEG-1 directly contributes to resistance to chemotherapeutic drug. Targeted downregulation of AEG-1 might improve the expression of miR-181a-2{sup ∗}, -193b and -193a, and inversely inhibit miR-31 and -9{sup ∗}. Targeted inhibition of AEG-1 can lead to modification of key elemental characteristics, such as miRNAs, which may become a potential effective therapeutic strategy for CRC.

  3. A comprehensive transcriptomic view on the role of SMAD4 gene by RNAi-mediated knockdown in porcine follicular granulosa cells.

    PubMed

    Zhang, Lifan; Du, Xing; Wei, Shengjuan; Li, Dongfeng; Li, Qifa

    2016-07-01

    As a key mediator of the transforming growth factor-beta (TGF-β) signaling pathway, which plays a pivotal role in regulating mammalian reproductive performance, Sma- and Mad-related protein 4 (SMAD4) is closely associated with the development of ovarian follicular. However, current knowledge of the genome-wide view on the role of SMAD4 gene in mammalian follicular granulosa cells (GCs) is still largely unknown. In the present study, RNA-Seq was performed to investigate the effects of SMAD4 knockdown by RNA interference (SMAD4-siRNA) in porcine follicular GCs. A total of 1025 differentially expressed genes (DEGs), including 530 upregulated genes and 495 downregulated genes, were identified in SMAD4-siRNA treated GCs compared with that treated with NC-siRNA. Furthermore, functional enrichment analysis indicated that upregulated DEGs in SMAD4-siRNA treated cells were mainly enriched in cell-cycle related processes, interferon signaling pathway, and immune system process, while downregulated DEGs in SMAD4-siRNA treated cells were mainly involved in extracellular matrix organization/disassembly, pathogenesis, and cell adhesion. In particular, cell cycle and TGF-β signaling pathway were discovered as the canonical pathways changed under SMAD4-silencing. Taken together, our data reveals SMAD4 knockdown alters the expression of numerous genes involved in key biological processes of the development of follicular GCs and provides a novel global clue of the role of SMAD4 gene in porcine follicular GCs, thus improving our understanding of regulatory mechanisms of SMAD4 gene in follicular development. PMID:27107035

  4. AHR2 knockdown prevents PAH-mediated cardiac toxicity and XRE- and ARE-associated gene induction in zebrafish (Danio rerio)

    SciTech Connect

    Van Tiem, Lindsey A.; Di Giulio, Richard T.

    2011-08-01

    Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants often present in aquatic systems as complex mixtures. Embryonic fish are sensitive to the developmental toxicity of some PAHs, but the exact mechanisms involved in this toxicity are still unknown. This study explored the role of the aryl hydrocarbon receptor (AHR) in the oxidative stress response of zebrafish to the embryotoxicity of select PAHs. Embryos were exposed to two PAHs, benzo[k]fluoranthene (BkF; a strong AHR agonist) and fluoranthene (FL; a cytochrome P4501A (CYP1A) inhibitor), alone and in combination. CYP1A, CYP1B1, CYP1C1, and redox-responsive genes glutathione s-transferase pi 2 (GSTp2), glutathione peroxidase 1 (GPx1), the glutamate-cysteine ligase catalytic subunit (GCLc), MnSOD and CuZnSOD mRNA expression was examined. CYP1 activity was measured via an in vivo ethoxyresorufin-O-deethlyase (EROD) activity assay, and the area of the pericardium was measured as an index of cardiotoxicity. BkF or FL alone caused no deformities whereas BkF + FL resulted in extreme pericardial effusion. BkF induced CYP activity above controls and co-exposure with FL inhibited this activity. BkF induced expression of all three CYPs, GSTp2, and GCLc. BkF + FL caused greater than additive induction of the three CYPs, GSTp2, GPx1, and GCLc but had no effect on MnSOD or CuZnSOD. AHR2 knockdown protected against the cardiac deformities caused by BkF + FL and significantly inhibited the induction of the CYPs, GSTp2, GPx1, and GCLc after BkF + FL compared to non-injected controls. These results further show the protective role of AHR2 knockdown against cardiotoxic PAHs and the role of AHR2 as a mediator of redox-responsive gene induction. - Research Highlights: > Co-exposure of the PAHs BkF and FL causes cardiotoxicity in zebrafish. > BkF and FL co-exposure upregulates certain XRE- and ARE-associated genes. > AHR2 knockdown prevents the deformities caused by BkF and FL co-exposure. > AHR2

  5. Knock-down of HEXA and HEXB genes correlate with the absence of the immunostimulatory function of HSC-derived dendritic cells.

    PubMed

    Tiribuzi, Roberto; D'Angelo, Francesco; Berardi, Anna C; Martino, Sabata; Orlacchio, Aldo

    2012-01-01

    In an attempt to investigate whether the genetic defect in the HEXA and HEXB genes (which causes the absence of the lysosomal β-N-acetyl-hexosaminidase), are related to the wide inflammation in GM2 gangliosidoses (Tay-Sachs and Sandhoff disease), we have chosen the dendritic cells (DCs) as a study model. Using the RNA interference approach, we generated an in vitro model of HEXs knock-down immunogenic DCs (i-DCs) from CD34(+)-haemopoietic stem cells (CD34(+)-HSCs), thus mimicking the Tay-Sachs (HEXA-/-) and Sandhoff (HEXB-/-) cells. We showed that the absence of β-N-acetyl-hexosaminidase activity does not alter the differentiation of i-DCs from HSCs, but it is critical for the activation of CD4(+)T cells because knock-down of HEXA or HEXB gene causes a loss of function of i-DCs. Notably, the silencing of the HEXA gene had a stronger immune inhibitory effect, thereby indicating a major involvement of β-N-acetyl-hexosaminidase A isoenzyme within this mechanism. PMID:21997228

  6. Zebrafish ambra1a and ambra1b knockdown impairs skeletal muscle development.

    PubMed

    Skobo, Tatjana; Benato, Francesca; Grumati, Paolo; Meneghetti, Giacomo; Cianfanelli, Valentina; Castagnaro, Silvia; Chrisam, Martina; Di Bartolomeo, Sabrina; Bonaldo, Paolo; Cecconi, Francesco; Dalla Valle, Luisa

    2014-01-01

    The essential role of autophagy in muscle homeostasis has been clearly demonstrated by phenotype analysis of mice with muscle-specific inactivation of genes encoding autophagy-related proteins. Ambra1 is a key component of the Beclin 1 complex and, in zebrafish, it is encoded by two paralogous genes, ambra1a and ambra1b, both required for normal embryogenesis and larval development. In this study we focused on the function of Ambra1, a positive regulator of the autophagic process, during skeletal muscle development by means of morpholino (MO)-mediated knockdown and compared the phenotype of zebrafish Ambra1-depleted embryos with that of Ambra1gt/gt mouse embryos. Morphological analysis of zebrafish morphant embryos revealed that silencing of ambra1 impairs locomotor activity and muscle development, as well as myoD1 expression. Skeletal muscles in ATG-morphant embryos displayed severe histopathological changes and contained only small areas of organized myofibrils that were widely dispersed throughout the cell. Double knockdown of ambra1a and ambra1b resulted in a more severe phenotype whereas defects were much less evident in splice-morphants. The morphants phenotypes were effectively rescued by co-injection with human AMBRA1 mRNA. Together, these results indicate that ambra1a and ambra1b are required for the correct development and morphogenesis of skeletal muscle. PMID:24922546

  7. Suppression and restoration of primordial germ cell marker gene expression in channel catfish, Ictalurus punctatus, using knockdown constructs regulated by copper transport protein gene promoters: Potential for reversible transgenic sterilization.

    PubMed

    Su, Baofeng; Shang, Mei; Grewe, Peter M; Patil, Jawahar G; Peatman, Eric; Perera, Dayan A; Cheng, Qi; Li, Chao; Weng, Chia-Chen; Li, Ping; Liu, Zhanjiang; Dunham, Rex A

    2015-12-01

    Complementary DNA overexpression and short hairpin RNA interference approaches were evaluated for decreasing expression of primordial germ cell (PGC) marker genes and thereby sterilizing channel catfish, Ictalurus punctatus, by delivering knockdown constructs driven by a constitutive promoter from yeast and a copper transport protein gene into fish embryos by electroporation. Two PGC marker genes, nanos and dead end, were the target knockdown genes, and their expressions, along with that of an off-target gene, vasa, were evaluated temporally using real-time polymerase chain reaction. Copper sulfate was evaluated as a repressor compound. Some of the constructs knocked down PGC marker gene expression, and some of the constructs were partially repressed by application of 0.1-ppm copper sulfate. When the rate of sexual maturity was compared for three-year-old broodfish that had been exposed to the sterilizing constructs during embryologic development and controls that had not been exposed, several treatments had reduced sexual maturity for the exposed fish. Of two promoter systems evaluated, the one which had been designed to be less sensitive to copper generally was more effective at achieving sterilization and more responsive to repression. Knockdown constructs based on 3' nanos short hairpin RNA interference appeared to result in the best repression and restoration of normal sexual maturity. We conclude that these copper-based systems exhibited good potential for repressible transgenic sterilization. Optimization of this system could allow environmentally safe application of transgenic technology and might be applicable to other applications for aquatic organisms. PMID:26341409

  8. Expression of phosphoribosyl pyrophosphate synthetase genes in U87 glioma cells with ERN1 knockdown: effect of hypoxia and endoplasmic reticulum stress.

    PubMed

    Minchenko, O H; Garmash, I A; Kovalevska, O V; Tsymbal, D O; Minchenko, D O

    2014-01-01

    Activation of pentose phosphate pathway is an important factor of enhanced cell proliferation and tumor growth. Phosphoribosyl pyrophosphate synthetase (PRPS) is a key enzyme of this pathway and plays a central role in the synthesis of purines and pyrimidines. Hypoxia as well as ERN1 (from endoplasmic reticulum to nuclei-1) mediated endoplasmic reticulum stress response-signalling pathway is linked to the proliferation because the blockade of ERN1 suppresses tumor growth, including glioma. We studied the expression of different PRPS genes in glioma cells with ERN1 knockdown under hypoxic condition. It was shown that hypoxia decreases the expression of PRPS1 and PRPS2 genes in both types of glioma cells, being more pronounced in cells without ERN1 function, but PRPSAP1 and PRPSAP2 gene expressions are suppressed by hypoxia only in glioma cells with blockade of ERN1. Moreover, the blockade of endoribonuclease activity of ERN1 does not affect the expression of PRPS1 and PRPS2 as well as PPRS-associated protein genes in U87 glioma cells. At the same time, the induction of endoplasmic reticulum stress by tunicamycin in glioma cells with suppressed activity of ERN1 endoribonuclease decreases the expression level of PRPS1 and PRPS2 genes only. Results of this investigation clearly demonstrated that the expression of different genes encoding subunits of PRPS enzyme is affected by hypoxia in U87 glioma cells, but the effect of hypoxia is modified by suppression of endoplasmic reticulum stress signaling enzyme ERN1. PMID:25816608

  9. Gucy2f zebrafish knockdown – a model for Gucy2d-related leber congenital amaurosis

    PubMed Central

    Stiebel-Kalish, Hadas; Reich, Ehud; Rainy, Nir; Vatine, Gad; Nisgav, Yael; Tovar, Anna; Gothilf, Yoav; Bach, Michael

    2012-01-01

    Mutations in retinal-specific guanylate cyclase (Gucy2d) are associated with Leber congenital amaurosis-1 (LCA1). Zebrafish offer unique advantages relative to rodents, including their excellent color vision, precocious retinal development, robust visual testing strategies, low cost, relatively easy transgenesis and shortened experimental times. In this study we will demonstrate the feasibility of using gene-targeting in the zebrafish as a model for the photoreceptor-specific GUCY2D-related LCA1, by reporting the visual phenotype and retinal histology resulting from Gucy2f knockdown. Gucy2f zebrafish LCA-orthologous cDNA was identified and isolated by PCR amplification. Its expression pattern was determined by whole-mount in-situ hybridization and its function was studied by gene knockdown using two different morpholino-modified oligos (MO), one that blocks translation of Gucy2f and one that blocks splicing of Gucy2f. Visual function was assessed with an optomotor assay on 6-days-post-fertilization larvae, and by analyzing changes in retinal histology. Gucy2f knockdown resulted in significantly lower vision as measured by the optomotor response compared with uninjected and control MO-injected zebrafish larvae. Histological changes in the Gucy2f-knockdown larvae included loss and shortening of cone and rod outer segments. A zebrafish model of Gucy2f-related LCA1 displays early visual dysfunction and photoreceptor layer dystrophy. This study serves as proof of concept for the use of zebrafish as a simple, inexpensive model with excellent vision on which further study of LCA-related genes is possible. PMID:22378290

  10. siRNA Knockdown of Ribosomal Protein Gene RPL19 Abrogates the Aggressive Phenotype of Human Prostate Cancer

    PubMed Central

    Bee, Alix; Brewer, Daniel; Beesley, Carol; Dodson, Andrew; Forootan, Shiva; Dickinson, Timothy; Gerard, Patricia; Lane, Brian; Yao, Sheng; Cooper, Colin S.; Djamgoz, Mustafa B. A.; Gosden, Christine M.; Ke, Youqiang; Foster, Christopher S.

    2011-01-01

    We provide novel functional data that posttranscriptional silencing of gene RPL19 using RNAi not only abrogates the malignant phenotype of PC-3M prostate cancer cells but is selective with respect to transcription and translation of other genes. Reducing RPL19 transcription modulates a subset of genes, evidenced by gene expression array analysis and Western blotting, but does not compromise cell proliferation or apoptosis in-vitro. However, growth of xenografted tumors containing the knocked-down RPL19 in-vivo is significantly reduced. Analysis of the modulated genes reveals induction of the non-malignant phenotype principally to involve perturbation of networks of transcription factors and cellular adhesion genes. The data provide evidence that extra-ribosomal regulatory functions of RPL19, beyond protein synthesis, are critical regulators of cellular phenotype. Targeting key members of affected networks identified by gene expression analysis raises the possibility of therapeutically stabilizing a benign phenotype generated by modulating the expression of an individual gene and thereafter constraining a malignant phenotype while leaving non-malignant tissues unaffected. PMID:21799931

  11. Zebrafish Cardiotoxicity: The Effects of CYP1A Inhibition and AHR2 Knockdown Following Exposure to Weak Aryl Hydrocarbon Receptor Agonists

    PubMed Central

    Clark, Bryan William; Van Tiem Garner, Lindsey; Di Giulio, Richard Thomas

    2014-01-01

    The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates many of the toxic effects of dioxin-like compounds (DLCs) and some polycyclic aromatic hydrocarbons (PAHs). Strong AHR agonists, such as certain polychlorinated biphenyls and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), cause severe cardiac teratogenesis in fish embryos. Moderately strong AHR agonists, such as benzo[a]pyrene and β-naphthoflavone, have been shown to cause similar cardiotoxic effects when coupled with a cytochrome P450 1A (CYP1A) inhibitor, such as fluoranthene (FL). We sought to determine if weak AHR agonists, when combined with a CYP1A inhibitor (FL) or CYP1A morpholino gene knockdown, are capable of causing cardiac deformities similar to moderately strong AHR agonists (Wassenberg and Di Giulio 2004; Wassenberg and Di Giulio 2004; Billiard, Timme-Laragy et al. 2006; Van Tiem and Di Giulio 2011). The weak AHR agonists included the following: carbaryl, phenanthrene, 2-methylindole, 3-methylindole, indigo, and indirubin. The results showed a complex pattern of cardiotoxic response to weak agonist inhibitor exposure and morpholino-knockdown. Danio rerio (zebrafish) embryos were first exposed to weak AHR agonists at equimolar concentrations. The agonists were assessed for their relative potency as inducers of CYP1 enzyme activity, measured by the ethoxyresorufin-o-deethylase (EROD) assay, and cardiac deformities. Carbaryl, 2-methylindole, and 3-methylindole induced the highest CYP1A activity in zebrafish. Experiments were then conducted to determine the individual cardiotoxicity of each compound. Next, zebrafish were co-exposed to each agonist (at concentrations below those determined to be cardiotoxic) and FL in combination to assess if CYP1A inhibition could induce cardiac deformities. Carbaryl, 2-methylindole, 3-methylindole, and phenanthrene significantly increased pericardial edema relative to controls when combined with FL. To further evaluate the

  12. Knockdown of phosphoethanolamine transmethylation enzymes decreases viability of Haemonchus contortus.

    PubMed

    Witola, William H; Cooks-Fagbodun, Sheritta; Ordonez, Adriana Reyes; Matthews, Kwame; Abugri, Daniel A; McHugh, Mark

    2016-06-15

    The phosphobase methylation pathway, in which phosphoethanolamine N-methyltransferases (PMTs) successively catalyze the methylation of phosphoethanolamine to phosphocholine, is essential in the free-living nematode Caenorhabditis elegans. Two PMT-encoding genes (HcPMT1 and HcPMT2) cloned from Haemonchus contortus have been shown, by in vitro assays, to possess enzymatic characteristics similar to those of C. elegans PMTs, but their physiological significance in H. contortus is yet to be elucidated. Therefore, in this study, we endeavored to determine the importance of HcPMT1 and HcPMT2 in the survival of H. contortus by adapting the use of phosphorodiamidate morpholino oligomers (PPMO) antisense approach to block the translation of HcPMT1 and HcPMT2 in the worms. We found that PPMOs targeting HcPMT1 and HcPMT2 down-regulated the expression of HcPMT1 and HcPMT2 proteins in adult H. contortus. Analysis of the effect of HcPMT1 and HcPMT2 knockdown showed that it significantly decreased worm motility and viability, thus validating HcPMT1 and HcPMT2 as essential enzymes for survival of H. contortus. Studies of gene function in H. contortus have been constrained by limited forward and reverse genetic technologies for use in H. contortus. Thus, our success in adaptation of use of PPMO antisense approach in H. contortus provides an important reverse genetic technological advance for studying this parasitic nematode of veterinary significance. PMID:27198768

  13. AHR2 knockdown prevents PAH-mediated cardiac toxicity and XRE- and ARE-associated gene induction in zebrafish (Danio rerio)

    PubMed Central

    Van Tiem, Lindsey A.; Di Giulio, Richard T.

    2011-01-01

    Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants often present in aquatic systems as complex mixtures. Embryonic fish are sensitive to the developmental toxicity of some PAHs, but the exact mechanisms involved in this toxicity are still unknown. This study explored the role of the aryl hydrocarbon receptor (AHR) in the oxidative stress response of zebrafish to the embryotoxicity of select PAHs. Embryos were exposed to two PAHs, benzo[k]fluoranthene (BkF; a strong AHR agonist) and fluoranthene (FL; a cytochrome P4501A (CYP1A) inhibitor), alone and in combination. CYP1A, CYP1B1, CYP1C1, and redox-responsive genes glutathione s-transferase pi 2 (GSTp2), glutathione peroxidase 1 (GPx1), the glutamate-cysteine ligase catalytic subunit (GCLc), MnSOD and CuZnSOD mRNA expression was examined. CYP1 activity was measured via an in vivo ethoxyresorufin-O-deethlyase (EROD) activity assay, and the area of the pericardium was measured as an index of cardiotoxicity. BkF or FL alone caused no deformities whereas BkF + FL resulted in extreme pericardial effusion. BkF induced CYP activity above controls and co-exposure with FL inhibited this activity. BkF induced expression of all three CYPs, GSTp2, and GCLc. BkF + FL caused greater than additive induction of the three CYPs, GSTp2, GPx1, and GCLc but had no effect on MnSOD or CuZnSOD. AHR2 knockdown protected against the cardiac deformities caused by BkF + FL and significantly inhibited the induction of the CYPs, GSTp2, GPx1 and GCLc after BkF + FL compared to non-injected controls. These results further show the protective role of AHR2 knockdown against cardiotoxic PAHs and the role of AHR2 as a mediator of redox-responsive gene induction. PMID:21600235

  14. Network analysis of genes involved in the enhancement of hyperthermia sensitivity by the knockdown of BAG3 in human oral squamous cell carcinoma cells.

    PubMed

    Yunoki, Tatsuya; Tabuchi, Yoshiaki; Hayashi, Atsushi; Kondo, Takashi

    2016-07-01

    BCL2-associated athanogene 3 (BAG3), a co-chaperone of the heat shock 70 kDa protein (HSPA) family of proteins, is a cytoprotective protein that acts against various stresses, including heat stress. The aim of the present study was to identify gene networks involved in the enhancement of hyperthermia (HT) sensitivity by the knockdown (KD) of BAG3 in human oral squamous cell carcinoma (OSCC) cells. Although a marked elevation in the protein expression of BAG3 was detected in human the OSCC HSC-3 cells exposed to HT at 44˚C for 90 min, its expression was almost completely suppressed in the cells transfected with small interfering RNA against BAG3 (siBAG) under normal and HT conditions. The silencing of BAG3 also enhanced the cell death that was increased in the HSC-3 cells by exposure to HT. Global gene expression analysis revealed many genes that were differentially expressed by >2-fold in the cells exposed to HT and transfected with siBAG. Moreover, Ingenuity® pathways analysis demonstrated two unique gene networks, designated as Pro-cell death and Anti-cell death, which were obtained from upregulated genes and were mainly associated with the biological functions of induction and the prevention of cell death, respectively. Of note, the expression levels of genes in the Pro-cell death and Anti-cell death gene networks were significantly elevated and reduced in the HT + BAG3-KD group compared to those in the HT control group, respectively. These results provide further insight into the molecular mechanisms involved in the enhancement of HT sensitivity by the silencing of BAG3 in human OSCC cells. PMID:27245201

  15. Inhibition of iridovirus protein synthesis and virus replication by antisense morpholino oligonucleotides targeted to the major capsid protein, the 18 kDa immediate-early protein, and a viral homolog of RNA polymerase II

    SciTech Connect

    Sample, Robert; Bryan, Locke; Long, Scott; Majji, Sai; Hoskins, Glenn; Sinning, Allan; Olivier, Jake; Chinchar, V. Gregory . E-mail: vchinchar@microbio.umsmed.edu

    2007-02-20

    Frog virus 3 (FV3) is a large DNA virus that encodes {approx} 100 proteins. Although the general features of FV3 replication are known, the specific roles that most viral proteins play in the virus life cycle have not yet been elucidated. To address the question of viral gene function, antisense morpholino oligonucleotides (asMOs) were used to transiently knock-down expression of specific viral genes and thus infer their role in virus replication. We designed asMOs directed against the major capsid protein (MCP), an 18 kDa immediate-early protein (18K) that was thought to be a viral regulatory protein, and the viral homologue of the largest subunit of RNA polymerase II (vPol-II{alpha}). All three asMOs successfully inhibited translation of the targeted protein, and two of the three asMOs resulted in marked phenotypic changes. Knock-down of the MCP resulted in a marked reduction in viral titer without a corresponding drop in the synthesis of other late viral proteins. Transmission electron microscopy (TEM) showed that in cells treated with the anti-MCP MO assembly sites were devoid of viral particles and contained numerous aberrant structures. In contrast, inhibition of 18K synthesis did not block virion formation, suggesting that the 18K protein was not essential for replication of FV3 in fathead minnow (FHM) cells. Finally, consistent with the view that late viral gene expression is catalyzed by a virus-encoded or virus-modified Pol-II-like protein, knock-down of vPol-II{alpha} triggered a global decline in late gene expression and virus yields without affecting the synthesis of early viral genes. Collectively, these results demonstrate the utility of using asMOs to elucidate the function of FV3 proteins.

  16. Activation of endocrine-related gene expression in placental choriocarcinoma cell lines following DNA methylation knock-down.

    PubMed

    Hogg, K; Robinson, W P; Beristain, A G

    2014-07-01

    Increasingly, placental DNA methylation is assessed as a factor in pregnancy-related complications, yet the transcriptional impact of such findings is not always clear. Using a proliferative in vitro placental model, the effect of DNA methylation loss on gene activation was evaluated at a number of genes selected for being differentially methylated in pre-eclampsia-associated placentae in vivo. We aimed to determine whether reduced DNA methylation at specific loci was associated with transcriptional changes at the corresponding gene, thus providing mechanistic underpinnings for previous clinical findings and to assess the degree of transcriptional response amongst our candidate genes. BeWo and JEG3 choriocarcinoma cells were exposed to 1 μM 5-Aza-2'-deoxycytidine (5-Aza-CdR) or vehicle control for 48 h, and re-plated and cultured for a further 72 h in normal media before cells were harvested for RNA and DNA. Bisulphite pyrosequencing confirmed that DNA methylation was reduced by ∼30-50% points at the selected loci studied in both cell lines. Gene activation, measured by qRT-PCR, was highly variable and transcript specific, indicating differential sensitivity to DNA methylation. Most notably, loss of DNA methylation at the leptin (LEP) promoter corresponded to a 200-fold and 40-fold increase in LEP expression in BeWo and JEG3 cells, respectively (P < 0.01). Transcripts of steroidogenic pathway enzymes CYP11A1 and HSD3B1 were up-regulated ∼40-fold in response to 5-Aza-CdR exposure in BeWo cells (P < 0.01). Other transcripts, including aromatase (CYP19), HSD11B2, inhibin (INHBA) and glucocorticoid receptor (NR3C1) were more moderately, although significantly, affected by loss of associated DNA methylation. These data present a mixed effect of DNA methylation changes at selected loci supporting cautionary interpretation of DNA methylation results in the absence of functional data. PMID:24623739

  17. Postnatal knockdown of dok-7 gene expression in mice causes structural defects in neuromuscular synapses and myasthenic pathology.

    PubMed

    Eguchi, Takahiro; Tezuka, Tohru; Miyoshi, Sadanori; Yamanashi, Yuji

    2016-06-01

    The neuromuscular junction (NMJ) is a synapse between a motor neuron and skeletal muscle and is required for muscle contraction. The formation and maintenance of NMJs are governed by the muscle-specific receptor tyrosine kinase MuSK. We previously showed that the muscle cytoplasmic protein Dok-7 is an essential activator of MuSK. Indeed, mice lacking either Dok-7 or MuSK form no NMJs, and defects in the human DOK7 gene underlie a congenital myasthenic syndrome (an NMJ disorder). However, it remains unproven whether Dok-7 is required for the postnatal maintenance of NMJs. In this study, we generated recombinant adeno-associated virus (AAV) vectors encoding short hairpin RNAs targeting the mouse dok-7 gene (AAV-shD7). Systemic administration of AAV-shD7 into 2-week-old mice down-regulated dok-7 expression in muscle and induced myasthenic symptoms including reduction in body weight and motor function. Moreover, AAV-shD7 treatment suppressed MuSK-dependent gene expression of NMJ components and reduced the size of NMJs. These results demonstrate that correct, physiological levels of dok-7 expression are required for the postnatal maintenance of NMJs. PMID:27091576

  18. Knockdown of the dyslexia-associated gene Kiaa0319 impairs temporal responses to speech stimuli in rat primary auditory cortex.

    PubMed

    Centanni, T M; Booker, A B; Sloan, A M; Chen, F; Maher, B J; Carraway, R S; Khodaparast, N; Rennaker, R; LoTurco, J J; Kilgard, M P

    2014-07-01

    One in 15 school age children have dyslexia, which is characterized by phoneme-processing problems and difficulty learning to read. Dyslexia is associated with mutations in the gene KIAA0319. It is not known whether reduced expression of KIAA0319 can degrade the brain's ability to process phonemes. In the current study, we used RNA interference (RNAi) to reduce expression of Kiaa0319 (the rat homolog of the human gene KIAA0319) and evaluate the effect in a rat model of phoneme discrimination. Speech discrimination thresholds in normal rats are nearly identical to human thresholds. We recorded multiunit neural responses to isolated speech sounds in primary auditory cortex (A1) of rats that received in utero RNAi of Kiaa0319. Reduced expression of Kiaa0319 increased the trial-by-trial variability of speech responses and reduced the neural discrimination ability of speech sounds. Intracellular recordings from affected neurons revealed that reduced expression of Kiaa0319 increased neural excitability and input resistance. These results provide the first evidence that decreased expression of the dyslexia-associated gene Kiaa0319 can alter cortical responses and impair phoneme processing in auditory cortex. PMID:23395846

  19. Two knockdown models of the autism genes SYNGAP1 and SHANK3 in zebrafish produce similar behavioral phenotypes associated with embryonic disruptions of brain morphogenesis.

    PubMed

    Kozol, Robert A; Cukier, Holly N; Zou, Bing; Mayo, Vera; De Rubeis, Silvia; Cai, Guiqing; Griswold, Anthony J; Whitehead, Patrice L; Haines, Jonathan L; Gilbert, John R; Cuccaro, Michael L; Martin, Eden R; Baker, James D; Buxbaum, Joseph D; Pericak-Vance, Margaret A; Dallman, Julia E

    2015-07-15

    Despite significant progress in the genetics of autism spectrum disorder (ASD), how genetic mutations translate to the behavioral changes characteristic of ASD remains largely unknown. ASD affects 1-2% of children and adults, and is characterized by deficits in verbal and non-verbal communication, and social interactions, as well as the presence of repetitive behaviors and/or stereotyped interests. ASD is clinically and etiologically heterogeneous, with a strong genetic component. Here, we present functional data from syngap1 and shank3 zebrafish loss-of-function models of ASD. SYNGAP1, a synaptic Ras GTPase activating protein, and SHANK3, a synaptic scaffolding protein, were chosen because of mounting evidence that haploinsufficiency in these genes is highly penetrant for ASD and intellectual disability (ID). Orthologs of both SYNGAP1 and SHANK3 are duplicated in the zebrafish genome and we find that all four transcripts (syngap1a, syngap1b, shank3a and shank3b) are expressed at the earliest stages of nervous system development with pronounced expression in the larval brain. Consistent with early expression of these genes, knockdown of syngap1b or shank3a cause common embryonic phenotypes including delayed mid- and hindbrain development, disruptions in motor behaviors that manifest as unproductive swim attempts, and spontaneous, seizure-like behaviors. Our findings indicate that both syngap1b and shank3a play novel roles in morphogenesis resulting in common brain and behavioral phenotypes. PMID:25882707

  20. Two knockdown models of the autism genes SYNGAP1 and SHANK3 in zebrafish produce similar behavioral phenotypes associated with embryonic disruptions of brain morphogenesis

    PubMed Central

    Kozol, Robert A.; Cukier, Holly N.; Zou, Bing; Mayo, Vera; De Rubeis, Silvia; Cai, Guiqing; Griswold, Anthony J.; Whitehead, Patrice L.; Haines, Jonathan L.; Gilbert, John R.; Cuccaro, Michael L.; Martin, Eden R.; Baker, James D.; Buxbaum, Joseph D.; Pericak-Vance, Margaret A.; Dallman, Julia E.

    2015-01-01

    Despite significant progress in the genetics of autism spectrum disorder (ASD), how genetic mutations translate to the behavioral changes characteristic of ASD remains largely unknown. ASD affects 1–2% of children and adults, and is characterized by deficits in verbal and non-verbal communication, and social interactions, as well as the presence of repetitive behaviors and/or stereotyped interests. ASD is clinically and etiologically heterogeneous, with a strong genetic component. Here, we present functional data from syngap1 and shank3 zebrafish loss-of-function models of ASD. SYNGAP1, a synaptic Ras GTPase activating protein, and SHANK3, a synaptic scaffolding protein, were chosen because of mounting evidence that haploinsufficiency in these genes is highly penetrant for ASD and intellectual disability (ID). Orthologs of both SYNGAP1 and SHANK3 are duplicated in the zebrafish genome and we find that all four transcripts (syngap1a, syngap1b, shank3a and shank3b) are expressed at the earliest stages of nervous system development with pronounced expression in the larval brain. Consistent with early expression of these genes, knockdown of syngap1b or shank3a cause common embryonic phenotypes including delayed mid- and hindbrain development, disruptions in motor behaviors that manifest as unproductive swim attempts, and spontaneous, seizure-like behaviors. Our findings indicate that both syngap1b and shank3a play novel roles in morphogenesis resulting in common brain and behavioral phenotypes. PMID:25882707

  1. Gene Knockdown by EpCAM Aptamer-siRNA Chimeras Suppresses Epithelial Breast Cancers and Their Tumor-Initiating Cells.

    PubMed

    Gilboa-Geffen, Adi; Hamar, Peter; Le, Minh T N; Wheeler, Lee Adam; Trifonova, Radiana; Petrocca, Fabio; Wittrup, Anders; Lieberman, Judy

    2015-10-01

    Effective therapeutic strategies for in vivo siRNA delivery to knockdown genes in cells outside the liver are needed to harness RNA interference for treating cancer. EpCAM is a tumor-associated antigen highly expressed on common epithelial cancers and their tumor-initiating cells (TIC, also known as cancer stem cells). Here, we show that aptamer-siRNA chimeras (AsiC, an EpCAM aptamer linked to an siRNA sense strand and annealed to the siRNA antisense strand) are selectively taken up and knock down gene expression in EpCAM(+) cancer cells in vitro and in human cancer biopsy tissues. PLK1 EpCAM-AsiCs inhibit colony and mammosphere formation (in vitro TIC assays) and tumor initiation by EpCAM(+) luminal and basal-A triple-negative breast cancer (TNBC) cell lines, but not EpCAM(-) mesenchymal basal-B TNBCs, in nude mice. Subcutaneously administered EpCAM-AsiCs concentrate in EpCAM(+) Her2(+) and TNBC tumors and suppress their growth. Thus, EpCAM-AsiCs provide an attractive approach for treating epithelial cancer. PMID:26264278

  2. RNA interference-mediated knockdown of CD49e (α5 integrin chain) in human thymic epithelial cells modulates the expression of multiple genes and decreases thymocyte adhesion

    PubMed Central

    2010-01-01

    Background The thymus is a central lymphoid organ, in which bone marrow-derived T cell precursors undergo a complex process of maturation. Developing thymocytes interact with thymic microenvironment in a defined spatial order. A component of thymic microenvironment, the thymic epithelial cells, is crucial for the maturation of T-lymphocytes through cell-cell contact, cell matrix interactions and secretory of cytokines/chemokines. There is evidence that extracellular matrix molecules play a fundamental role in guiding differentiating thymocytes in both cortical and medullary regions of the thymic lobules. The interaction between the integrin α5β1 (CD49e/CD29; VLA-5) and fibronectin is relevant for thymocyte adhesion and migration within the thymic tissue. Our previous results have shown that adhesion of thymocytes to cultured TEC line is enhanced in the presence of fibronectin, and can be blocked with anti-VLA-5 antibody. Results Herein, we studied the role of CD49e expressed by the human thymic epithelium. For this purpose we knocked down the CD49e by means of RNA interference. This procedure resulted in the modulation of more than 100 genes, some of them coding for other proteins also involved in adhesion of thymocytes; others related to signaling pathways triggered after integrin activation, or even involved in the control of F-actin stress fiber formation. Functionally, we demonstrated that disruption of VLA-5 in human TEC by CD49e-siRNA-induced gene knockdown decreased the ability of TEC to promote thymocyte adhesion. Such a decrease comprised all CD4/CD8-defined thymocyte subsets. Conclusion Conceptually, our findings unravel the complexity of gene regulation, as regards key genes involved in the heterocellular cell adhesion between developing thymocytes and the major component of the thymic microenvironment, an interaction that is a mandatory event for proper intrathymic T cell differentiation. PMID:21210968

  3. RNA silencing in the model mycorrhizal fungus Laccaria bicolor: gene knock-down of nitrate reductase results in inhibition of symbiosis with Populus.

    PubMed

    Kemppainen, Minna; Duplessis, Sébastien; Martin, Francis; Pardo, Alejandro G

    2009-07-01

    profile of the fungal partner is not proper and mutual benefit from the symbiotic structure cannot be assured. Our results are the first report showing that the alteration of expression of a fungal gene impairs mycorrhization. Moreover, this work is the first demonstration of RNA silencing in mycorrhizal fungi and clearly shows that gene knock-down is a powerful tool for further functional genomic studies in mycorrhizal research. PMID:19397683

  4. Morpholino-functionalized nanochannel array for label-free single nucleotide polymorphisms detection.

    PubMed

    Gao, Hong-Li; Wang, Min; Wu, Zeng-Qiang; Wang, Chen; Wang, Kang; Xia, Xing-Hua

    2015-04-01

    The sensitive identification of single nucleotide polymorphisms becomes increasingly important for disease diagnosis, prevention, and practical applicability of pharmacogenomics. Herein, we propose a simple, highly selective, label-free single nucleotide polymorphisms (SNPs) sensing device by electrochemically monitoring the diffusion flux of ferricyanide probe across probe DNA/morpholino duplex functionalized nanochannels of porous anodic alumina. When perfectly matched or mismatched target DNA flows through the nanochannels modified with probe DNA/morpholino duplex, it competes for the probe DNA from morpholino, resulting in a change of the surface charges. Thus, the diffusion flux of negatively charged electroactive probe ferricyanide is modulated since it is sensitive to the surface charge due to the electrostatic interactions in electric double layer-merged nanochannels. Monitoring of the change in diffusion flux of probe enables us to detect not only a single base or two base mismatched sequence but also the specific location of the mismatched base. As is demonstrated, SNPs in the PML/RARα fusion gene, known as a biomarker of acute promyelocytic leukemia (APL), have been successfully detected. PMID:25734499

  5. Vivo-Morpholinos: a non-peptide transporter delivers Morpholinos into a wide array of mouse tissues.

    PubMed

    Morcos, Paul A; Li, Yongfu; Jiang, Shan

    2008-12-01

    We have developed a new transporter structure that provides effective delivery of Morpholino antisense oligomers into a wide variety of tissues in living mice. This transporter comprises a dendritic structure assembled around a triazine core which serves to position eight guanidinium head groups in a conformation effective to penetrate cell membranes. This transporter structure is conjugated to a Morpholino oligomer to form a delivery-enabled product referred to as a Vivo-Morpholino. Vivo-Morpholinos are shown to effectively enter and function within cultured cells in the presence of 100% serum using a rigorous positive test system based on correction of a defined splicing error in a pre-messenger RNA. In addition, Vivo-Morpholinos are demonstrated to enter into a wide variety of tissues in a similar positive test system in transgenic mice, as evidenced by correction of the targeted splicing error in all tissues assessed, including near-complete splice correction in the small intestine, colon, stomach, liver kidney, and a number of muscles. Finally, Vivo-Morpholinos, which target the exon-skipping of exon 23 harboring a premature termination codon in the mdx mouse model, effectively restore the reading frame of dystrophin and restore expression of a functional dystrophin protein. PMID:19238792

  6. Impaired cellular immune response to injected bacteria after knockdown of ferritin genes in the hard tick Haemaphysalis longicornis.

    PubMed

    Galay, Remil Linggatong; Takechi, Rie; Umemiya-Shirafuji, Rika; Talactac, Melbourne Rio; Maeda, Hiroki; Kusakisako, Kodai; Mochizuki, Masami; Fujisaki, Kozo; Tanaka, Tetsuya

    2016-06-01

    Iron is an indispensable element for most microorganisms, including many pathogenic bacteria. Iron-withholding is a known component of the innate immunity, particularly of vertebrate hosts. Ticks are vectors of multiple pathogens and reports have shown that they naturally harbor several bacterial species. Thus, tick innate immunity must be crucial in limiting bacterial population to tolerable level that will not cause adverse effects. We have previously characterized two types of the iron-binding protein ferritin (HlFER) in the hard tick Haemaphysalis longicornis, known to be a vector of some protozoan parasites and rickettsiae, and showed their antioxidant function and importance in blood feeding and reproduction. Here we examined the possible role of HlFERs in tick immunity against bacterial infection. After silencing Hlfer genes, adult ticks were injected with live enhanced green fluorescence protein-expressing Escherichia coli, and then monitored for survival rate. Hemolymph that included hemocytes was collected for microscopic examination to observe cellular immune response, and for E. coli culture to determine bacterial viability after injection in the ticks. The expression of some antimicrobial peptides in whole ticks was also analyzed by RT-PCR. Hlfer-silenced ticks had a significantly lower survival rate than control ticks after E. coli injection. Greater number of bacteria inside and outside the hemocytes and higher bacterial colony counts after culture with hemolymph were also observed in Hlfer-silenced ticks. However, no difference on the expression of antimicrobial peptides was observed. These results suggest that ferritin molecules might be important in the cellular immune response of ticks to some bacteria. PMID:26792075

  7. Lentiviral-Mediated RNAi Knockdown of Cbfa1 Gene Inhibits Endochondral Ossification of Antler Stem Cells in Micromass Culture

    PubMed Central

    Sun, Hongmei; Yang, Fuhe; Chu, Wenhui; Zhao, Haiping; McMahon, Chris; Li, Chunyi

    2012-01-01

    Articular cartilage (AC) lacks ability to repair defects due to its avascular nature as healing process relies on cells being brought in by blood vessels. Multiple approaches have been taken to facilitate cartilage repair in clinics, to date there is no effective treatment available that can restores the AC lesion to a normally functioning level over extended periods. In this regard, antler cartilage is unique in being richly vascularised and hence can effectively repair and regenerate. Interestingly, antler stem cells, from which the vascularised cartilage is derived, can form avascular cartilage when taken away from their original niche, suggesting that the vascular or avascular state of antler cartilage is controlled by extrinsic factors. Understanding the mechanisms underlying this phenotype switch may help us to devise a way to trigger the effective intrinsic repair of AC. However, adoption of antler cartilage model for AC repair requires the demonstration that the cartilage specific signalling pathways also prevail in antler chondrogenesis. To achieve this, in the present study we silenced expression of Cbfa1, a key factor regulatingendochondral ossification, using RNAi, and showed that expression of the downstream genes type I collagen and osteocalcin were suppressed which, in turn, inhibited endochondral ossification process taking place in the antler stem cell-formed nodules. Therefore, we provided further evidence at molecular level that antler could be developed as novel model for the study of AC repair. The eventual identification of the extrinsic factors dictating the phenotype switch between the vascular and avascular state of antler cartilage will open up a new avenue for the cure of osteoarthritis. PMID:23056636

  8. Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

    PubMed Central

    Konantz, Judith; Antos, Christopher L.

    2014-01-01

    The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to employ reverse genetics have not yet been adequately developed to assess gene function in regeneration or tissue homeostasis during larval stages after zebrafish embryogenesis, and several tissues within the zebrafish larva are difficult to target. Intraventricular injections of gene-specific morpholinos offer an alternative method for the current inability to genomically target zebrafish genes in a temporally controlled manner at these stages. This method allows for complete dispersion and subsequent incorporation of the morpholino into various tissues throughout the body, including structures that were formerly impossible to reach such as those in the larval caudal fin, a structure often used to noninvasively research tissue regeneration. Several genes activated during larval finfold regeneration are also present in regenerating adult vertebrate tissues, so the larva is a useful model to understand regeneration in adults. This morpholino dispersion method allows for the quick and easy identification of genes required for the regeneration of larval tissues as well as other physiological phenomena regulating tissue homeostasis after embryogenesis. Therefore, this delivery method provides a currently needed strategy for temporal control to the evaluation of gene function after embryogenesis.  PMID:24961304

  9. THE EFFECTS OF EMBRYONIC KNOCKDOWN OF THE CANDIDATE DYSLEXIA SUSCEPTIBILITY GENE HOMOLOGUE DYX1C1 ON THE DISTRIBUTION OF GABAERGIC NEURONS IN THE CEREBRAL CORTEX

    PubMed Central

    Currier, Timothy A.; Etchegaray, Mikel A.; Haight, Joshua L.; Galaburda, Albert M.; Rosen, Glenn D.

    2010-01-01

    Developmental dyslexia is a language-based learning disability, and a number of candidate dyslexia susceptibility genes have been identified, including DYX1C1, KIAA0319, and DCDC2. Knockdown of function by embryonic transfection of small hairpin RNA (shRNA) of rat homologues of these genes dramatically disrupts neuronal migration to the cerebral cortex by both cell autonomous and non-cell autonomous effects. Here we sought to investigate the extent of non-cell autonomous effects following in utero disruption of the candidate dyslexia susceptibility gene homolog Dyx1c1 by assessing the effects of this disruption on GABAergic neurons. We transfected the ventricular zone of embryonic day (E) 15.5 rat pups with either Dyx1c1 shRNA, DYX1C1 expression construct, both Dyx1c1 shRNA and DYX1C1 expression construct, or a scrambled version of Dyx1c1 shRNA, and sacrificed them at postnatal day 21. The mothers of these rats were injected with BrdU at either E13.5, E15.5, or E17.5. Neurons transfected with Dyx1c1 shRNA were bi-modally distributed in the cerebral cortex with one population in heterotopic locations at the white matter border and another migrating beyond their expected location in the cerebral cortex. In contrast, there was no disruption of migration following transfection with the DYX1C1 expression construct. We found untransfected GABAergic neurons (parvalbumin, calretinin, and neuropeptide Y) in the heterotopic collections of neurons in Dyx1c1 shRNA treated animals, supporting the hypothesis of non-cell autonomous effects. In contrast, we found no evidence that the position of the GABAergic neurons that made it to the cerebral cortex was disrupted by the embryonic transfection with any of the constructs. Taken together, these results support the notion that neurons within heterotopias caused by transfection with Dyx1c1 shRNA result from both cell autonomous and non-cell autonomous effects, but there is no evidence to support non-cell autonomous disruption of

  10. Knockdown of the juvenile hormone receptor gene inhibits soldier-specific morphogenesis in the damp-wood termite Zootermopsis nevadensis (Isoptera: Archotermopsidae).

    PubMed

    Masuoka, Yudai; Yaguchi, Hajime; Suzuki, Ryutaro; Maekawa, Kiyoto

    2015-09-01

    The Methoprene-tolerant (Met) protein has been established as a juvenile hormone (JH) receptor. Knockdown of the Met gene caused precocious metamorphosis and suppression of ovarian development. However, the function of Met in caste development of social insects is unclear. In termites, JH acts as a central factor for caste development, especially for soldier differentiation, which involves two molts from workers via a presoldier stage. Increased JH titer in workers is needed for the presoldier molt, and the high JH titer is maintained throughout the presoldier period. Although presoldiers have the fundamental morphological features of soldiers, the nature of the cuticle is completely different from that of soldiers. We expected that JH signals via Met are involved in soldier-specific morphogenesis of the head and mandibles during soldier differentiation, especially in the presoldier period, in natural conditions. To test this hypothesis, we focused on soldier differentiation in an incipient colony of the damp-wood termite Zootermopsis nevadensis. Met homolog (ZnMet) expression in heads increased just after the presoldier molt. This high expression was reduced by ZnMet double stranded (dsRNA) injection before the presoldier molt. Although this treatment did not cause any morphological changes in presoldiers, it caused strong effects on soldiers, their mandibles being significantly shorter and head capsules smaller than those of control soldiers. Injection of ZnMet dsRNA throughout the presoldier stage did not affect the formation of soldier morphology, including cuticle formation. These results suggested that the rapid increase in ZnMet expression and subsequent activation of JH signaling just after the presoldier molt are needed for the formation of soldier-specific weapons. Therefore, besides its established role in insect metamorphosis, the JH receptor signaling also underlies soldier development in termites. PMID:26188329

  11. Modeling and gene knockdown to assess the contribution of nonsense-mediated decay, premature termination, and selenocysteine insertion to the selenoprotein hierarchy

    PubMed Central

    Meplan, Catherine; Huguenin, Grazielle V.B.; Hesketh, John E.; Shanley, Daryl P.

    2016-01-01

    The expression of selenoproteins, a specific group of proteins that incorporates selenocysteine, is hierarchically regulated by the availability of Se, with some, but not all selenoprotein mRNA transcripts decreasing in abundance with decreasing Se. Selenocysteine insertion into the peptide chain occurs during translation following recoding of an internal UGA stop codon. There is increasing evidence that this UGA recoding competes with premature translation termination, which is followed by nonsense-mediated decay (NMD) of the transcript. In this study, we tested the hypothesis that the susceptibility of different selenoprotein mRNAs to premature termination during translation and differential sensitivity of selenoprotein transcripts to NMD are major factors in the selenoprotein hierarchy. Selenoprotein transcript abundance was measured in Caco-2 cells using real-time PCR under different Se conditions and the data obtained fitted to mathematical models of selenoprotein translation. A calibrated model that included a combination of differential sensitivity of selenoprotein transcripts to NMD and different frequency of non-NMD related premature translation termination was able to fit all the measurements. The model predictions were tested using SiRNA to knock down expression of the crucial NMD factor UPF1 (up-frameshift protein 1) and selenoprotein mRNA expression. The calibrated model was able to predict the effect of UPF1 knockdown on gene expression for all tested selenoproteins, except SPS2 (selenophosphate synthetase), which itself is essential for selenoprotein synthesis. These results indicate an important role for NMD in the hierarchical regulation of selenoprotein mRNAs, with the exception of SPS2 whose expression is likely regulated by a different mechanism. PMID:27208313

  12. Modeling and gene knockdown to assess the contribution of nonsense-mediated decay, premature termination, and selenocysteine insertion to the selenoprotein hierarchy.

    PubMed

    Zupanic, Anze; Meplan, Catherine; Huguenin, Grazielle V B; Hesketh, John E; Shanley, Daryl P

    2016-07-01

    The expression of selenoproteins, a specific group of proteins that incorporates selenocysteine, is hierarchically regulated by the availability of Se, with some, but not all selenoprotein mRNA transcripts decreasing in abundance with decreasing Se. Selenocysteine insertion into the peptide chain occurs during translation following recoding of an internal UGA stop codon. There is increasing evidence that this UGA recoding competes with premature translation termination, which is followed by nonsense-mediated decay (NMD) of the transcript. In this study, we tested the hypothesis that the susceptibility of different selenoprotein mRNAs to premature termination during translation and differential sensitivity of selenoprotein transcripts to NMD are major factors in the selenoprotein hierarchy. Selenoprotein transcript abundance was measured in Caco-2 cells using real-time PCR under different Se conditions and the data obtained fitted to mathematical models of selenoprotein translation. A calibrated model that included a combination of differential sensitivity of selenoprotein transcripts to NMD and different frequency of non-NMD related premature translation termination was able to fit all the measurements. The model predictions were tested using SiRNA to knock down expression of the crucial NMD factor UPF1 (up-frameshift protein 1) and selenoprotein mRNA expression. The calibrated model was able to predict the effect of UPF1 knockdown on gene expression for all tested selenoproteins, except SPS2 (selenophosphate synthetase), which itself is essential for selenoprotein synthesis. These results indicate an important role for NMD in the hierarchical regulation of selenoprotein mRNAs, with the exception of SPS2 whose expression is likely regulated by a different mechanism. PMID:27208313

  13. Zebrafish Models for Ectopic Mineralization Disorders: Practical Issues from Morpholino Design to Post-Injection Observations

    PubMed Central

    Hosen, Mohammad Jakir; Vanakker, Olivier M.; Willaert, Andy; Huysseune, Ann; Coucke, Paul; De Paepe, Anne

    2013-01-01

    Zebrafish (ZF, Danio rerio) has emerged as an important and popular model species to study different human diseases. Key regulators of skeletal development and calcium metabolism are highly conserved between mammals and ZF. The corresponding orthologs share significant sequence similarities and an overlap in expression patterns when compared to mammals, making ZF a potential model for the study of mineralization-related disorders and soft tissue mineralization. To characterize the function of early mineralization-related genes in ZF, these genes can be knocked down by injecting morpholinos into early stage embryos. Validation of the morpholino needs to be performed and the concern of aspecific effects can be addressed by applying one or more independent techniques to knock down the gene of interest. Post-injection assessment of early mineralization defects can be done using general light microscopy, calcein staining, Alizarin red staining, Alizarin red-Alcian blue double staining, and by the use of transgenic lines. Examination of general molecular defects can be done by performing protein and gene expression analysis, and more specific processes can be explored by investigating ectopic mineralization-related mechanisms such as apoptosis and mitochondrial dysfunction. In this paper, we will discuss all details about the aforementioned techniques; shared knowledge will be very useful for the future investigation of ZF models for ectopic mineralization disorders and to understand the underlying pathways involved in soft tissue calcification. PMID:23760765

  14. Phosphorodiamidate morpholino oligomers suppress mutant huntingtin expression and attenuate neurotoxicity.

    PubMed

    Sun, Xin; Marque, Leonard O; Cordner, Zachary; Pruitt, Jennifer L; Bhat, Manik; Li, Pan P; Kannan, Geetha; Ladenheim, Ellen E; Moran, Timothy H; Margolis, Russell L; Rudnicki, Dobrila D

    2014-12-01

    Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin (HTT) gene. Disease pathogenesis derives, at least in part, from the long polyglutamine tract encoded by mutant HTT. Therefore, considerable effort has been dedicated to the development of therapeutic strategies that significantly reduce the expression of the mutant HTT protein. Antisense oligonucleotides (ASOs) targeted to the CAG repeat region of HTT transcripts have been of particular interest due to their potential capacity to discriminate between normal and mutant HTT transcripts. Here, we focus on phosphorodiamidate morpholino oligomers (PMOs), ASOs that are especially stable, highly soluble and non-toxic. We designed three PMOs to selectively target expanded CAG repeat tracts (CTG22, CTG25 and CTG28), and two PMOs to selectively target sequences flanking the HTT CAG repeat (HTTex1a and HTTex1b). In HD patient-derived fibroblasts with expanded alleles containing 44, 77 or 109 CAG repeats, HTTex1a and HTTex1b were effective in suppressing the expression of mutant and non-mutant transcripts. CTGn PMOs also suppressed HTT expression, with the extent of suppression and the specificity for mutant transcripts dependent on the length of the targeted CAG repeat and on the CTG repeat length and concentration of the PMO. PMO CTG25 reduced HTT-induced cytotoxicity in vitro and suppressed mutant HTT expression in vivo in the N171-82Q transgenic mouse model. Finally, CTG28 reduced mutant HTT expression and improved the phenotype of Hdh(Q7/Q150) knock-in HD mice. These data demonstrate the potential of PMOs as an approach to suppressing the expression of mutant HTT. PMID:25035419

  15. Lentivirus-Mediated Knockdown of Astrocyte Elevated Gene-1 Inhibits Growth and Induces Apoptosis through MAPK Pathways in Human Retinoblastoma Cells

    PubMed Central

    Chang, Ying; Li, Bin; Xu, Xiaolin; Shen, Ling; Bai, Haixia; Gao, Fei; Zhang, Zhibao; Jonas, Jost B.

    2016-01-01

    Purpose To explore expression and function of astrocyte elevated gene-1 (AEG-1) in human retinoblastoma (RB). Methods The expression of AEG-1 in histological sections of human RBs and in RB cell lines was examined using immunohistochemical staining and RT-PCR and Western blotting respectively. We knocked down AEG-1 gene levels by AEG-1-siRNA lentivirus transfection of human RB cell lines SO-RB50 and Y79, and using an MTT assay, we assessed the role of AEG-1 on RB cell proliferation. The biological significance of lentivirus transfection induced AEG-1 down-regulation was examined by assessing the apoptosis rate in the transfected RB cells by Annexin V-APC staining and flow cytometry. We additionally measured the expression of Bcl-2, Bax, cleaved-caspase-3 and caspase-3, and the phosphorylation and non-phosphorylation alternation of MAPKs. Results AEG-1 expression was detected to be strongly positive in the histological slides of 35 out of 54 (65%) patients with RB. AEG-1 expression increased significantly (P<0.05) with tumor stage. In the RB cell lines SO-RB50, Y79 and WERI-RB1 as compared with retinal pigment epithelium cells, expression of AEG-1 mRNA and AEG-1 protein was significantly higher. In AEG-1-siRNA lentivirus transfected cell cultures as compared with negative control lentivirus transfected cell cultures, levels of AEG-1 mRNA and of AEG-1 protein (P<0.05) and cell growth rates (P<0.01) were significantly lower, and apoptosis rate (P<0.001), Bax/Bcl-2 ratio and cleaved-caspase-3 protein level were significantly increased. The P-ERK/ERK ratio was significantly decreased in the AEG-1-siRNA lentivirus transfected cell lines. Conclusions Expression of AEG-1 was associated with RB, in histological slides of patients and in cell culture experiments. Lentivirus transfection induced knockdown of AEG-1 had a tumor suppressive effect, potentially by tumor cell apoptosis induction through inhibition of ERK. PMID:26894431

  16. The morpholino molecular beacon for specific RNA visualization in vivo.

    PubMed

    Chen, Jianbin; Wu, Jikui; Hong, Yunhan

    2016-02-21

    A non-invasive fluorescent probe, morpholino molecular beacon (MO-MB), was designed for RNA visualization in vivo. Featuring negligible toxicity, stability, and high target specificity in living embryos, MO-MB is superior to conventional probes and has the potential for specific RNA visualization in basic biological and clinical research. PMID:26810703

  17. Differential Responses to Wnt and PCP Disruption Predict Expression and Developmental Function of Conserved and Novel Genes in a Cnidarian

    PubMed Central

    Lapébie, Pascal; Ruggiero, Antonella; Barreau, Carine; Chevalier, Sandra; Chang, Patrick; Dru, Philippe; Houliston, Evelyn; Momose, Tsuyoshi

    2014-01-01

    We have used Digital Gene Expression analysis to identify, without bilaterian bias, regulators of cnidarian embryonic patterning. Transcriptome comparison between un-manipulated Clytia early gastrula embryos and ones in which the key polarity regulator Wnt3 was inhibited using morpholino antisense oligonucleotides (Wnt3-MO) identified a set of significantly over and under-expressed transcripts. These code for candidate Wnt signaling modulators, orthologs of other transcription factors, secreted and transmembrane proteins known as developmental regulators in bilaterian models or previously uncharacterized, and also many cnidarian-restricted proteins. Comparisons between embryos injected with morpholinos targeting Wnt3 and its receptor Fz1 defined four transcript classes showing remarkable correlation with spatiotemporal expression profiles. Class 1 and 3 transcripts tended to show sustained expression at “oral” and “aboral” poles respectively of the developing planula larva, class 2 transcripts in cells ingressing into the endodermal region during gastrulation, while class 4 gene expression was repressed at the early gastrula stage. The preferential effect of Fz1-MO on expression of class 2 and 4 transcripts can be attributed to Planar Cell Polarity (PCP) disruption, since it was closely matched by morpholino knockdown of the specific PCP protein Strabismus. We conclude that endoderm and post gastrula-specific gene expression is particularly sensitive to PCP disruption while Wnt-/β-catenin signaling dominates gene regulation along the oral-aboral axis. Phenotype analysis using morpholinos targeting a subset of transcripts indicated developmental roles consistent with expression profiles for both conserved and cnidarian-restricted genes. Overall our unbiased screen allowed systematic identification of regionally expressed genes and provided functional support for a shared eumetazoan developmental regulatory gene set with both predicted and previously

  18. Knockdown of a JmjC domain-containing gene JMJ524 confers altered gibberellin responses by transcriptional regulation of GRAS protein lacking the DELLA domain genes in tomato

    PubMed Central

    Li, Jinhua; Yu, Chuying; Wu, Hua; Luo, Zhidan; Ouyang, Bo; Cui, Long; Zhang, Junhong; Ye, Zhibiao

    2015-01-01

    Plants integrate responses to independent hormonal and environmental signals to survive adversity. In particular, the phytohormone gibberellin (GA) regulates a variety of developmental processes and stress responses. In this study, the Jumonji-C (JmjC) domain-containing gene JMJ524 was characterized in tomato. JMJ524 responded to circadian rhythms and was upregulated by GA treatment. Knockdown of JMJ524 by RNAi caused a GA-insensitive dwarf phenotype with shrunken leaves and shortened internodes. However, in these transgenic plants, higher levels of endogenous GAs were detected. A genome-wide gene expression analysis by RNA-seq indicated that the expression levels of two DELLA-like genes, SlGLD1 (‘GRAS protein Lacking the DELLA domain’) and SlGLD2, were increased in JMJ524-RNAi transgenic plants. Nevertheless, only the overexpression of SlGLD1 in tomato resulted in a GA-insensitive dwarf phenotype, suggesting that SlGLD1 acts as a repressor of GA signalling. This study proposes that JMJ524 is required for stem elongation by altering GA responses, at least partially by regulating SlGLD1. PMID:25680796

  19. Knockdown of Carboxypeptidase A6 in Zebrafish Larvae Reduces Response to Seizure-Inducing Drugs and Causes Changes in the Level of mRNAs Encoding Signaling Molecules

    PubMed Central

    Lopes, Mark William; Sapio, Matthew R.; Leal, Rodrigo B.; Fricker, Lloyd D.

    2016-01-01

    Carboxypeptidase A6 (CPA6) is an extracellular matrix metallocarboxypeptidase that modulates peptide and protein function by removal of hydrophobic C-terminal amino acids. Mutations in the human CPA6 gene that reduce enzymatic activity in the extracellular matrix are associated with febrile seizures, temporal lobe epilepsy, and juvenile myoclonic epilepsy. The characterization of these human mutations suggests a dominant mode of inheritance by haploinsufficiency through loss of function mutations, however the total number of humans with pathologic mutations in CPA6 identified to date remains small. To better understand the relationship between CPA6 and seizures we investigated the effects of morpholino knockdown of cpa6 mRNA in zebrafish (Danio rerio) larvae. Knockdown of cpa6 mRNA resulted in resistance to the effect of seizure-inducing drugs pentylenetetrazole and pilocarpine on swimming behaviors. Knockdown of cpa6 mRNA also reduced the levels of mRNAs encoding neuropeptide precursors (bdnf, npy, chga, pcsk1nl, tac1, nts, edn1), a neuropeptide processing enzyme (cpe), transcription factor (c-fos), and molecules implicated in glutamatergic signaling (grin1a and slc1a2b). Treatment of zebrafish embryos with 60 mM pilocarpine for 1 hour led to reductions in levels of many of the same mRNAs when measured 1 day after pilocarpine exposure, except for c-fos which was elevated 1 day after pilocarpine treatment. Pilocarpine treatment, like cpa6 knockdown, led to a reduced sensitivity to pentylenetetrazole when tested 1 day after pilocarpine treatment. Taken together, these results add to mounting evidence that peptidergic systems participate in the biological effects of seizure-inducing drugs, and are the first in vivo demonstration of the molecular and behavioral consequences of cpa6 insufficiency. PMID:27050163

  20. Knockdown of Carboxypeptidase A6 in Zebrafish Larvae Reduces Response to Seizure-Inducing Drugs and Causes Changes in the Level of mRNAs Encoding Signaling Molecules.

    PubMed

    Lopes, Mark William; Sapio, Matthew R; Leal, Rodrigo B; Fricker, Lloyd D

    2016-01-01

    Carboxypeptidase A6 (CPA6) is an extracellular matrix metallocarboxypeptidase that modulates peptide and protein function by removal of hydrophobic C-terminal amino acids. Mutations in the human CPA6 gene that reduce enzymatic activity in the extracellular matrix are associated with febrile seizures, temporal lobe epilepsy, and juvenile myoclonic epilepsy. The characterization of these human mutations suggests a dominant mode of inheritance by haploinsufficiency through loss of function mutations, however the total number of humans with pathologic mutations in CPA6 identified to date remains small. To better understand the relationship between CPA6 and seizures we investigated the effects of morpholino knockdown of cpa6 mRNA in zebrafish (Danio rerio) larvae. Knockdown of cpa6 mRNA resulted in resistance to the effect of seizure-inducing drugs pentylenetetrazole and pilocarpine on swimming behaviors. Knockdown of cpa6 mRNA also reduced the levels of mRNAs encoding neuropeptide precursors (bdnf, npy, chga, pcsk1nl, tac1, nts, edn1), a neuropeptide processing enzyme (cpe), transcription factor (c-fos), and molecules implicated in glutamatergic signaling (grin1a and slc1a2b). Treatment of zebrafish embryos with 60 mM pilocarpine for 1 hour led to reductions in levels of many of the same mRNAs when measured 1 day after pilocarpine exposure, except for c-fos which was elevated 1 day after pilocarpine treatment. Pilocarpine treatment, like cpa6 knockdown, led to a reduced sensitivity to pentylenetetrazole when tested 1 day after pilocarpine treatment. Taken together, these results add to mounting evidence that peptidergic systems participate in the biological effects of seizure-inducing drugs, and are the first in vivo demonstration of the molecular and behavioral consequences of cpa6 insufficiency. PMID:27050163

  1. Analysis of 14-3-3 Family Member Function in Xenopus Embryos by Microinjection of Antisense Morpholino Oligos

    NASA Astrophysics Data System (ADS)

    Lau, Jeffrey M. C.; Muslin, Anthony J.

    The 14-3-3 intracellular phosphoserine/threonine-binding proteins are adapter molecules that regulate signal transduction, cell cycle, nutrient sensing, apoptotic, and cytoskeletal pathways. There are seven 14-3-3 family members, encoded by separate genes, in vertebrate organisms. To evaluate the role of individual 14-3-3 proteins in vertebrate embryonic development, we utilized an antisense morpholino oligo microinjection technique in Xenopus laevis embryos. By use of this method, we showed that embryos lacking specific 14-3-3 proteins displayed unique phenotypic abnormalities. Specifically, embryos lacking 14-3-3 τ exhibited gastrulation and axial patterning defects, but embryos lacking 14-3-3 γ exhibited eye defects without other abnormalities, and embryos lacking 14-3-3 ζ appeared completely normal. These and other results demonstrate the power and specificity of the morpholino antisense oligo microinjection technique.

  2. Targeted Inhibition of miRNA Maturation with Morpholinos Reveals a Role for miR-375 in Pancreatic Islet Development

    PubMed Central

    Kloosterman, Wigard P; Lagendijk, Anne K; Ketting, René F; Moulton, Jon D; Plasterk, Ronald H. A

    2007-01-01

    Several vertebrate microRNAs (miRNAs) have been implicated in cellular processes such as muscle differentiation, synapse function, and insulin secretion. In addition, analysis of Dicer null mutants has shown that miRNAs play a role in tissue morphogenesis. Nonetheless, only a few loss-of-function phenotypes for individual miRNAs have been described to date. Here, we introduce a quick and versatile method to interfere with miRNA function during zebrafish embryonic development. Morpholino oligonucleotides targeting the mature miRNA or the miRNA precursor specifically and temporally knock down miRNAs. Morpholinos can block processing of the primary miRNA (pri-miRNA) or the pre-miRNA, and they can inhibit the activity of the mature miRNA. We used this strategy to knock down 13 miRNAs conserved between zebrafish and mammals. For most miRNAs, this does not result in visible defects, but knockdown of miR-375 causes defects in the morphology of the pancreatic islet. Although the islet is still intact at 24 hours postfertilization, in later stages the islet cells become scattered. This phenotype can be recapitulated by independent control morpholinos targeting other sequences in the miR-375 precursor, excluding off-target effects as cause of the phenotype. The aberrant formation of the endocrine pancreas, caused by miR-375 knockdown, is one of the first loss-of-function phenotypes for an individual miRNA in vertebrate development. The miRNA knockdown strategy presented here will be widely used to unravel miRNA function in zebrafish. PMID:17676975

  3. DNAH6 and Its Interactions with PCD Genes in Heterotaxy and Primary Ciliary Dyskinesia.

    PubMed

    Li, You; Yagi, Hisato; Onuoha, Ezenwa Obi; Damerla, Rama Rao; Francis, Richard; Furutani, Yoshiyuki; Tariq, Muhammad; King, Stephen M; Hendricks, Gregory; Cui, Cheng; Saydmohammed, Manush; Lee, Dong Min; Zahid, Maliha; Sami, Iman; Leatherbury, Linda; Pazour, Gregory J; Ware, Stephanie M; Nakanishi, Toshio; Goldmuntz, Elizabeth; Tsang, Michael; Lo, Cecilia W

    2016-02-01

    Heterotaxy, a birth defect involving left-right patterning defects, and primary ciliary dyskinesia (PCD), a sinopulmonary disease with dyskinetic/immotile cilia in the airway are seemingly disparate diseases. However, they have an overlapping genetic etiology involving mutations in cilia genes, a reflection of the common requirement for motile cilia in left-right patterning and airway clearance. While PCD is a monogenic recessive disorder, heterotaxy has a more complex, largely non-monogenic etiology. In this study, we show mutations in the novel dynein gene DNAH6 can cause heterotaxy and ciliary dysfunction similar to PCD. We provide the first evidence that trans-heterozygous interactions between DNAH6 and other PCD genes potentially can cause heterotaxy. DNAH6 was initially identified as a candidate heterotaxy/PCD gene by filtering exome-sequencing data from 25 heterotaxy patients stratified by whether they have airway motile cilia defects. dnah6 morpholino knockdown in zebrafish disrupted motile cilia in Kupffer's vesicle required for left-right patterning and caused heterotaxy with abnormal cardiac/gut looping. Similarly DNAH6 shRNA knockdown disrupted motile cilia in human and mouse respiratory epithelia. Notably a heterotaxy patient harboring heterozygous DNAH6 mutation was identified to also carry a rare heterozygous PCD-causing DNAI1 mutation, suggesting a DNAH6/DNAI1 trans-heterozygous interaction. Furthermore, sequencing of 149 additional heterotaxy patients showed 5 of 6 patients with heterozygous DNAH6 mutations also had heterozygous mutations in DNAH5 or other PCD genes. We functionally assayed for DNAH6/DNAH5 and DNAH6/DNAI1 trans-heterozygous interactions using subthreshold double-morpholino knockdown in zebrafish and showed this caused heterotaxy. Similarly, subthreshold siRNA knockdown of Dnah6 in heterozygous Dnah5 or Dnai1 mutant mouse respiratory epithelia disrupted motile cilia function. Together, these findings support an oligogenic disease

  4. DNAH6 and Its Interactions with PCD Genes in Heterotaxy and Primary Ciliary Dyskinesia

    PubMed Central

    Onuoha, Ezenwa Obi; Damerla, Rama Rao; Francis, Richard; Furutani, Yoshiyuki; Tariq, Muhammad; King, Stephen M.; Hendricks, Gregory; Cui, Cheng; Saydmohammed, Manush; Lee, Dong Min; Zahid, Maliha; Sami, Iman; Leatherbury, Linda; Pazour, Gregory J.; Ware, Stephanie M.; Nakanishi, Toshio; Goldmuntz, Elizabeth; Tsang, Michael; Lo, Cecilia W.

    2016-01-01

    Heterotaxy, a birth defect involving left-right patterning defects, and primary ciliary dyskinesia (PCD), a sinopulmonary disease with dyskinetic/immotile cilia in the airway are seemingly disparate diseases. However, they have an overlapping genetic etiology involving mutations in cilia genes, a reflection of the common requirement for motile cilia in left-right patterning and airway clearance. While PCD is a monogenic recessive disorder, heterotaxy has a more complex, largely non-monogenic etiology. In this study, we show mutations in the novel dynein gene DNAH6 can cause heterotaxy and ciliary dysfunction similar to PCD. We provide the first evidence that trans-heterozygous interactions between DNAH6 and other PCD genes potentially can cause heterotaxy. DNAH6 was initially identified as a candidate heterotaxy/PCD gene by filtering exome-sequencing data from 25 heterotaxy patients stratified by whether they have airway motile cilia defects. dnah6 morpholino knockdown in zebrafish disrupted motile cilia in Kupffer’s vesicle required for left-right patterning and caused heterotaxy with abnormal cardiac/gut looping. Similarly DNAH6 shRNA knockdown disrupted motile cilia in human and mouse respiratory epithelia. Notably a heterotaxy patient harboring heterozygous DNAH6 mutation was identified to also carry a rare heterozygous PCD-causing DNAI1 mutation, suggesting a DNAH6/DNAI1 trans-heterozygous interaction. Furthermore, sequencing of 149 additional heterotaxy patients showed 5 of 6 patients with heterozygous DNAH6 mutations also had heterozygous mutations in DNAH5 or other PCD genes. We functionally assayed for DNAH6/DNAH5 and DNAH6/DNAI1 trans-heterozygous interactions using subthreshold double-morpholino knockdown in zebrafish and showed this caused heterotaxy. Similarly, subthreshold siRNA knockdown of Dnah6 in heterozygous Dnah5 or Dnai1 mutant mouse respiratory epithelia disrupted motile cilia function. Together, these findings support an oligogenic disease

  5. The Serine-Threonine Protein Kinase PAK4 Is Dispensable in Zebrafish: Identification of a Morpholino-Generated Pseudophenotype

    PubMed Central

    Law, Sheran H. W.; Sargent, Thomas D.

    2014-01-01

    TALEN-based inactivation of the zebrafish pak4 gene resulted in embryos and adult fish that appear normal and fertile. This is in contrast to our previously published studies which were based on the use of antisense morpholino oligonucleotides (MOs). We have excluded potential explanations such as gene duplication, alternate splicing, cryptic initiation of translation, and translation-independent RNA function. Our conclusion is that pak4 is dispensable in zebrafish, and that even when corroborated by robust controls, such as RNA rescue, MOs may elicit misleading pseudophenotypes that do not correspond to results obtained by genetic mutations, and should thus be used with caution. PMID:24945275

  6. p53-Mediated Biliary Defects Caused by Knockdown of cirh1a, the Zebrafish Homolog of the Gene Responsible for North American Indian Childhood Cirrhosis

    PubMed Central

    Wilkins, Benjamin J.; Lorent, Kristin; Matthews, Randolph P.; Pack, Michael

    2013-01-01

    North American Indian Childhood Cirrhosis (NAIC) is a rare, autosomal recessive, progressive cholestatic disease of infancy affecting the Cree-Ojibway first Nations of Quebec. All NAIC patients are homozygous for a missense mutation (R565W) in CIRH1A, the human homolog of the yeast nucleolar protein Utp4. Utp4 is part of the t-Utp subcomplex of the small subunit (SSU) processome, a ribonucleoprotein complex required for ribosomal RNA processing and small subunit assembly. NAIC has thus been proposed to be a primary ribosomal disorder (ribosomopathy); however, investigation of the pathophysiologic mechanism of this disease has been hindered by lack of an animal model. Here, using a morpholino oligonucleotide (MO)-based loss-of-function strategy, we have generated a model of NAIC in the zebrafish, Danio rerio. Zebrafish Cirhin shows substantial homology to the human homolog, and cirh1a mRNA is expressed in developing hepatocytes and biliary epithelial cells. Injection of two independent MOs directed against cirh1a at the one-cell stage causes defects in canalicular and biliary morphology in 5 dpf larvae. In addition, 5 dpf Cirhin-deficient larvae have dose-dependent defects in hepatobiliary function, as assayed by the metabolism of an ingested fluorescent lipid reporter. Previous yeast and in vitro studies have shown that defects in ribosome biogenesis cause stabilization and nuclear accumulation of p53, which in turn causes p53-mediated cell cycle arrest and/or apoptosis. Thus, the nucleolus appears to function as a cellular stress sensor in some cell types. In accordance with this hypothesis, transcriptional targets of p53 are upregulated in Cirhin-deficient zebrafish embryos, and defects in biliary function seen in Cirhin-deficient larvae are completely abrogated by mutation of tp53. Our data provide the first in vivo evidence of a role for Cirhin in biliary development, and support the hypothesis that congenital defects affecting ribosome biogenesis can activate

  7. Long-Term Efficacy of Systemic Multiexon Skipping Targeting Dystrophin Exons 45–55 With a Cocktail of Vivo-Morpholinos in Mdx52 Mice

    PubMed Central

    Echigoya, Yusuke; Aoki, Yoshitsugu; Miskew, Bailey; Panesar, Dharminder; Touznik, Aleksander; Nagata, Tetsuya; Tanihata, Jun; Nakamura, Akinori; Nagaraju, Kanneboyina; Yokota, Toshifumi

    2015-01-01

    Antisense-mediated exon skipping, which can restore the reading frame, is a most promising therapeutic approach for Duchenne muscular dystrophy. Remaining challenges include the limited applicability to patients and unclear function of truncated dystrophin proteins. Multiexon skipping targeting exons 45–55 at the mutation hotspot of the dystrophin gene could overcome both of these challenges. Previously, we described the feasibility of exons 45–55 skipping with a cocktail of Vivo-Morpholinos in vivo; however, the long-term efficacy and safety of Vivo-Morpholinos remains to be determined. In this study, we examined the efficacy and toxicity of exons 45–55 skipping by intravenous injections of 6 mg/kg 10-Vivo-Morpholino cocktail (0.6 mg/kg each vPMO) every 2 weeks for 18 weeks to dystrophic exon-52 knockout (mdx52) mice. Systemic skipping of the entire exons 45–55 region was induced, and the Western blot analysis exhibited the restoration of 5–27% of normal levels of dystrophin protein in skeletal muscles, accompanied by improvements in histopathology and muscle strength. No obvious immune response and renal and hepatic toxicity were detected at the end-point of the treatment. We demonstrate our new regimen with the 10-Vivo-Morpholino cocktail is effective and safe for long-term repeated systemic administration in the dystrophic mouse model. PMID:25647512

  8. Long-term efficacy of systemic multiexon skipping targeting dystrophin exons 45-55 with a cocktail of vivo-morpholinos in mdx52 mice.

    PubMed

    Echigoya, Yusuke; Aoki, Yoshitsugu; Miskew, Bailey; Panesar, Dharminder; Touznik, Aleksander; Nagata, Tetsuya; Tanihata, Jun; Nakamura, Akinori; Nagaraju, Kanneboyina; Yokota, Toshifumi

    2015-01-01

    Antisense-mediated exon skipping, which can restore the reading frame, is a most promising therapeutic approach for Duchenne muscular dystrophy. Remaining challenges include the limited applicability to patients and unclear function of truncated dystrophin proteins. Multiexon skipping targeting exons 45-55 at the mutation hotspot of the dystrophin gene could overcome both of these challenges. Previously, we described the feasibility of exons 45-55 skipping with a cocktail of Vivo-Morpholinos in vivo; however, the long-term efficacy and safety of Vivo-Morpholinos remains to be determined. In this study, we examined the efficacy and toxicity of exons 45-55 skipping by intravenous injections of 6 mg/kg 10-Vivo-Morpholino cocktail (0.6 mg/kg each vPMO) every 2 weeks for 18 weeks to dystrophic exon-52 knockout (mdx52) mice. Systemic skipping of the entire exons 45-55 region was induced, and the Western blot analysis exhibited the restoration of 5-27% of normal levels of dystrophin protein in skeletal muscles, accompanied by improvements in histopathology and muscle strength. No obvious immune response and renal and hepatic toxicity were detected at the end-point of the treatment. We demonstrate our new regimen with the 10-Vivo-Morpholino cocktail is effective and safe for long-term repeated systemic administration in the dystrophic mouse model. PMID:25647512

  9. Novel Genes Participating in the Formation of Prismatic and Nacreous Layers in the Pearl Oyster as Revealed by Their Tissue Distribution and RNA Interference Knockdown

    PubMed Central

    Koyama, Hiroki; Mizutani, Saeri; Ota, Ayaka; Osakabe, Yuki; Nagai, Kiyohito; Maeyama, Kaoru; Okamoto, Kikuhiko; Kanoh, Satoshi; Asakawa, Shuichi; Watabe, Shugo

    2014-01-01

    In our previous publication, we identified novel gene candidates involved in shell formation by EST analyses of the nacreous and prismatic layer-forming tissues in the pearl oyster Pinctada fucata. In the present study, 14 of those genes, including two known genes, were selected and further examined for their involvement in shell formation using the RNA interference. Molecular characterization based on the deduced amino acid sequences showed that seven of the novel genes encode secretory proteins. The tissue distribution of the transcripts of the genes, as analyzed by RT-PCR and in situ hybridization, was mostly consistent with those obtained by the EST analysis reported previously. Shells in the pearl oysters injected with dsRNAs targeting genes 000027, 000058, 000081, 000096, 000113 (nacrein), 000118, 000133 and 000411 (MSI60), which showed expression specific to the nacreous layer forming tissues, showed abnormal surface appearance in this layer. Individuals injected with dsRNAs targeting genes 000027, 000113 and 000133 also exhibited abnormal prismatic layers. Individuals injected with dsRNAs targeting genes 000031, 000066, 000098, 000145, 000194 and 000200, which showed expression specific to prismatic layer forming tissues, displayed an abnormal surface appearance in both the nacreous and prismatic layers. Taken together, the results suggest that the genes involved in prismatic layer formation might also be involved in the formation of the nacreous layers. PMID:24454739

  10. Morpholino Treatment Improves Muscle Function and Pathology of Pitx1 Transgenic Mice

    PubMed Central

    Pandey, Sachchida Nand; Lee, Yi-Chien; Yokota, Toshifumi; Chen, Yi-Wen

    2014-01-01

    Paired-like homeodomain transcription factor 1 (PITX1) was proposed to be part of the disease mechanisms of facioscapulohumeral muscular dystrophy (FSHD). We generated a tet-repressible muscle-specific Pitx1 transgenic mouse model which develops phenotypes of muscular dystrophy after the PITX1 expression is induced. In this study, we attempted to block the translation of PITX1 protein using morpholinos. Three groups of the transgenic mice received intravenous injections of phosphorodiamidate morpholino oligomers (PMO) (100 mg/kg), octaguanidinium dendrimer-conjugated morpholino (vivo-morpholino) (10 mg/kg), or phosphate-buffered saline (PBS) after the PITX1 expression was induced. Immunoblotting data showed that PITX1 expression in the triceps and quadriceps was significantly reduced 70% and 63% by the vivo-morpholino treatment, respectively. Muscle pathology of the mice treated with the vivo-morpholino was improved by showing 44% fewer angular-shaped atrophic myofibers. Muscle function determined by grip strength was significantly improved by the vivo-morpholino treatment. The study showed that systemic delivery of the vivo-morpholino reduced the PITX1 expression and improved the muscle phenotypes. Aberrant expression of DUX4 from the last unit of the D4Z4 array has been proposed to be the cause of FSHD. The findings of this study suggest that the same principle may be applied to suppress the aberrantly expressed DUX4 in FSHD. PMID:24232919

  11. 40 CFR 721.10417 - Biphenyl alkyl morpholino ketone (generic) (P-11-338).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... (generic) (P-11-338). 721.10417 Section 721.10417 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.10417 Biphenyl alkyl morpholino ketone (generic) (P-11-338). (a... generically as biphenyl alkyl morpholino ketone (PMN P-11-338) is subject to reporting under this section...

  12. 40 CFR 721.10417 - Biphenyl alkyl morpholino ketone (generic) (P-11-338).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... (generic) (P-11-338). 721.10417 Section 721.10417 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.10417 Biphenyl alkyl morpholino ketone (generic) (P-11-338). (a... generically as biphenyl alkyl morpholino ketone (PMN P-11-338) is subject to reporting under this section...

  13. 40 CFR 721.10417 - Biphenyl alkyl morpholino ketone (generic) (P-11-338).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... (generic) (P-11-338). 721.10417 Section 721.10417 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.10417 Biphenyl alkyl morpholino ketone (generic) (P-11-338). (a... generically as biphenyl alkyl morpholino ketone (PMN P-11-338) is subject to reporting under this section...

  14. Morpholino treatment improves muscle function and pathology of Pitx1 transgenic mice.

    PubMed

    Pandey, Sachchida Nand; Lee, Yi-Chien; Yokota, Toshifumi; Chen, Yi-Wen

    2014-02-01

    Paired-like homeodomain transcription factor 1 (PITX1) was proposed to be part of the disease mechanisms of facioscapulohumeral muscular dystrophy (FSHD). We generated a tet-repressible muscle-specific Pitx1 transgenic mouse model which develops phenotypes of muscular dystrophy after the PITX1 expression is induced. In this study, we attempted to block the translation of PITX1 protein using morpholinos. Three groups of the transgenic mice received intravenous injections of phosphorodiamidate morpholino oligomers (PMO) (100 mg/kg), octaguanidinium dendrimer-conjugated morpholino (vivo-morpholino) (10 mg/kg), or phosphate-buffered saline (PBS) after the PITX1 expression was induced. Immunoblotting data showed that PITX1 expression in the triceps and quadriceps was significantly reduced 70% and 63% by the vivo-morpholino treatment, respectively. Muscle pathology of the mice treated with the vivo-morpholino was improved by showing 44% fewer angular-shaped atrophic myofibers. Muscle function determined by grip strength was significantly improved by the vivo-morpholino treatment. The study showed that systemic delivery of the vivo-morpholino reduced the PITX1 expression and improved the muscle phenotypes. Aberrant expression of DUX4 from the last unit of the D4Z4 array has been proposed to be the cause of FSHD. The findings of this study suggest that the same principle may be applied to suppress the aberrantly expressed DUX4 in FSHD. PMID:24232919

  15. Impaired Cytogenetic Damage Repair and Cell Cycle Regulation in Response to Ionizing Radiation in Human Fibroblast Cells with Individual Knock-down of 25 Genes

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Rohde, Larry; Emami, Kamal; Hammond, Dianne; Casey, Rachael; Mehta, Satish; Jeevarajan, Antony; Pierson, Duane; Wu, Honglu

    2008-01-01

    Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have demonstrated that genes with upregulated expression induced by IR may play important roles in DNA damage sensing, cell cycle checkpoint and chromosomal repair, the relationship between the regulation of gene expression by IR and its impact on cytogenetic responses to ionizing radiation has not been systematically studied. In our present study, the expression of 25 genes selected based on their transcriptional changes in response to IR or from their known DNA repair roles were individually knocked down by siRNA transfection in human fibroblast cells. Chromosome aberrations (CA) and micronuclei (MN) formation were measured as the cytogenetic endpoints. Our results showed that the yield of MN and/or CA formation were significantly increased by suppressed expression of 5 genes that included Ku70 in the DSB repair pathway; XPA in the NER pathway; RPA1 in the MMR pathway; RAD17 and RBBP8 in cell cycle control. Knocked-down expression of 4 genes including MRE11A, RAD51 in the DSB pathway, and SESN1 and SUMO1 showed significant inhibition of cell cycle progression, possibly because of severe impairment of DNA damage repair. Furthermore, loss of XPA, p21 and MLH1 expression resulted in both enhanced cell cycle progression and significantly higher yield of cytogenetic damage, indicating the involvement of these gene products in both cell cycle control and DNA damage repair. Of these 11 genes that affected the cytogenetic response, 9 were up-regulated in the cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulating the biological consequences after IR. Failure to express these IR-responsive genes, such as by gene mutation, could seriously change the outcome of the post IR scenario and lead to carcinogenesis.

  16. Role of zebrafish cytochrome P450 CYP1C genes in the reduced mesencephalic vein blood flow caused by activation of AHR2

    SciTech Connect

    Kubota, Akira; Stegeman, John J.; Woodin, Bruce R.; Iwanaga, Toshihiko; Harano, Ryo; Peterson, Richard E.; Hiraga, Takeo; Teraoka, Hiroki

    2011-06-15

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes various signs of toxicity in early life stages of vertebrates through activation of the aryl hydrocarbon receptor (AHR). We previously reported a sensitive and useful endpoint of TCDD developmental toxicity in zebrafish, namely a decrease in blood flow in the dorsal midbrain, but downstream genes involved in the effect are not known. The present study addressed the role of zebrafish cytochrome P450 1C (CYP1C) genes in association with a decrease in mesencephalic vein (MsV) blood flow. The CYP1C subfamily was recently discovered in fish and includes the paralogues CYP1C1 and CYP1C2, both of which are induced via AHR2 in zebrafish embryos. We used morpholino antisense oligonucleotides (MO or morpholino) to block initiation of translation of the target genes. TCDD-induced mRNA expression of CYP1Cs and a decrease in MsV blood flow were both blocked by gene knockdown of AHR2. Gene knockdown of CYP1C1 by two different morpholinos and CYP1C2 by two different morpholinos, but not by their 5 nucleotide-mismatch controls, was effective in blocking reduced MsV blood flow caused by TCDD. The same CYP1C-MOs prevented reduction of blood flow in the MsV caused by {beta}-naphthoflavone (BNF), representing another class of AHR agonists. Whole-mount in situ hybridization revealed that mRNA expression of CYP1C1 and CYP1C2 was induced by TCDD most strongly in branchiogenic primordia and pectoral fin buds. In situ hybridization using head transverse sections showed that TCDD increased the expression of both CYP1Cs in endothelial cells of blood vessels, including the MsV. These results indicate a potential role of CYP1C1 and CYP1C2 in the local circulation failure induced by AHR2 activation in the dorsal midbrain of the zebrafish embryo. - Research Highlights: > We examine the roles of zebrafish CYP1C1 and CYP1C2 in TCDD developmental toxicity. > TCDD induces mRNA expression of both CYP1Cs in the mesencephalic vein. > Knockdown of each

  17. Knockdown of RNA Interference Pathway Genes in Western Corn Rootworms (Diabrotica virgifera virgifera Le Conte) Demonstrates a Possible Mechanism of Resistance to Lethal dsRNA

    PubMed Central

    Vélez, Ana María; Khajuria, Chitvan; Wang, Haichuan; Narva, Kenneth E.; Siegfried, Blair D.

    2016-01-01

    RNA interference (RNAi) is being developed as a potential tool for insect pest management. Increased understanding of the RNAi pathway in target insect pests will provide information to use this technology effectively and to inform decisions related to resistant management strategies for RNAi based traits. Dicer 2 (Dcr2), an endonuclease responsible for formation of small interfering RNA’s and Argonaute 2 (Ago2), an essential catalytic component of the RNA-induced silencing complex (RISC) have both been associated with the RNAi pathway in a number of different insect species including the western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae). We identified both genes from a transcriptome library generated from different tissues and developmental stages of the western corn rootworm, an important target pest for transgenic plants expressing dsRNA targeting essential genes. The expression of these genes was suppressed by more than 90% after injecting gene specific dsRNA into adult rootworms. The injected beetles were then fed vATPase A dsRNA which has previously been demonstrated to cause mortality in western corn rootworm adults. The suppression of both RNAi pathway genes resulted in reduced mortality after subsequent exposure to lethal concentrations of vATPase A dsRNA as well as increased vATPase A expression relative to control treatments. Injections with dsRNA for a non-lethal target sequence (Laccase 2) did not affect mortality or expression caused by vATPase A dsRNA indicating that the results observed with Argo and Dicer dsRNA were not caused by simple competition among different dsRNA’s. These results confirm that both genes play an important role in the RNAi pathway for western corn rootworms and indicate that selection pressures that potentially affect the expression of these genes may provide a basis for future studies to understand potential mechanisms of resistance. PMID:27310918

  18. Knockdown of a putative insulin-like peptide gene LdILP2 in Leptinotarsa decemlineata by RNA interference impairs pupation and adult emergence.

    PubMed

    Fu, Kai-Yun; Zhu, Tao-Tao; Guo, Wen-Chao; Ahmat, Tursun; Li, Guo-Qing

    2016-05-01

    Five insulin-like peptide LdILP genes were identified in Leptinotarsa decemlineata. All of them contained three exons and two introns, with three genes tandemly arrayed and well separated from the other two. Phylogenetic analysis revealed that the three LdILPs from three tandemly-arrayed genes grouped with TcILP1, whereas the other two resembled with TcILP2 and TcILP4 from Tribolium castaneum. Thus, the five LdILP genes were provisionally named LdILP1a, LdILP1b, LdILP1c, LdILP2 and LdILP4. LdILP2 was widely expressed in several tissues such as the brain-corpora cardiaca-corpora allata (BR-CC-CA) complex, gut and fat body. In contrast, LdILP1a and LdILP1b were only transcribed in BR-CC-CA, LdILP4 was in ovaries, and LdILP1c was in both BR-CC-CA and ovaries. Ingestion of double-stranded RNAs (dsRNAs) targeting LdILP2 (dsLdILP2-1 and dsLdILP2-2) specifically knocked down LdILP2 and upregulated the transcription of both LdInR and Ld4EBP, indicating insulin/insulin-like growth factor signaling pathway (IIS) was inhibited. Approximately 50% of the LdILP2 RNAi larvae did not normally pupate and about 50% of the LdILP2 RNAi pupae did not emerge. Moreover, silencing LdILP2 reduced the expression of a juvenile hormone (JH) biosynthesis gene, lowered JH titer and disturbed JH signaling. Finally, knocking down LdILP2 inhibited an ecdysteroidogenesis gene, decreased 20-hydroxyecdysone (20E) titer, and repressed the expression of two 20E-response genes LdHR3 and LdFTZ-F1. Thus, the IIS pathway is involved in larval-pupal metamorphosis by modification of both JH and 20E signaling in L. decemlineata. PMID:26812356

  19. Targeting protein translation, RNA splicing, and degradation by morpholino-based conjugates in Plasmodium falciparum

    PubMed Central

    Garg, Aprajita; Wesolowski, Donna; Alonso, Dulce; Deitsch, Kirk W.; Ben Mamoun, Choukri; Altman, Sidney

    2015-01-01

    Identification and genetic validation of new targets from available genome sequences are critical steps toward the development of new potent and selective antimalarials. However, no methods are currently available for large-scale functional analysis of the Plasmodium falciparum genome. Here we present evidence for successful use of morpholino oligomers (MO) to mediate degradation of target mRNAs or to inhibit RNA splicing or translation of several genes of P. falciparum involved in chloroquine transport, apicoplast biogenesis, and phospholipid biosynthesis. Consistent with their role in the parasite life cycle, down-regulation of these essential genes resulted in inhibition of parasite development. We show that a MO conjugate that targets the chloroquine-resistant transporter PfCRT is effective against chloroquine-sensitive and -resistant parasites, causes enlarged digestive vacuoles, and renders chloroquine-resistant strains more sensitive to chloroquine. Similarly, we show that a MO conjugate that targets the PfDXR involved in apicoplast biogenesis inhibits parasite growth and that this defect can be rescued by addition of isopentenyl pyrophosphate. MO-based gene regulation is a viable alternative approach to functional analysis of the P. falciparum genome. PMID:26351679

  20. Targeting protein translation, RNA splicing, and degradation by morpholino-based conjugates in Plasmodium falciparum.

    PubMed

    Garg, Aprajita; Wesolowski, Donna; Alonso, Dulce; Deitsch, Kirk W; Ben Mamoun, Choukri; Altman, Sidney

    2015-09-22

    Identification and genetic validation of new targets from available genome sequences are critical steps toward the development of new potent and selective antimalarials. However, no methods are currently available for large-scale functional analysis of the Plasmodium falciparum genome. Here we present evidence for successful use of morpholino oligomers (MO) to mediate degradation of target mRNAs or to inhibit RNA splicing or translation of several genes of P. falciparum involved in chloroquine transport, apicoplast biogenesis, and phospholipid biosynthesis. Consistent with their role in the parasite life cycle, down-regulation of these essential genes resulted in inhibition of parasite development. We show that a MO conjugate that targets the chloroquine-resistant transporter PfCRT is effective against chloroquine-sensitive and -resistant parasites, causes enlarged digestive vacuoles, and renders chloroquine-resistant strains more sensitive to chloroquine. Similarly, we show that a MO conjugate that targets the PfDXR involved in apicoplast biogenesis inhibits parasite growth and that this defect can be rescued by addition of isopentenyl pyrophosphate. MO-based gene regulation is a viable alternative approach to functional analysis of the P. falciparum genome. PMID:26351679

  1. Establishment of Functional Genomics Pipeline in Epiblast-Like Tissue by Combining Transcriptomic Analysis and Gene Knockdown/Knockin/Knockout, Using RNA Interference and CRISPR/Cas9.

    PubMed

    Takata, Nozomu; Sakakura, Eriko; Kasukawa, Takeya; Sakuma, Tetsushi; Yamamoto, Takashi; Sasai, Yoshiki

    2016-06-01

    The epiblast (foremost embryonic ectoderm) generates all three germ layers and therefore has crucial roles in the formation of all mammalian body cells. However, regulation of epiblast gene expression is poorly understood because of the difficulty of manipulating epiblast tissues in vivo. In the present study, using the self-organizing properties of embryonic stem cell (ESC), we generated and characterized epiblast-like tissue in three-dimensional culture. We identified significant genome-wide gene expression changes in this epiblast-like tissue by transcriptomic analysis. In addition, we identified the particular significance of the Erk/Mapk and integrin-linked kinase pathways, and genes related to ectoderm/epithelial formation, using the bioinformatics resources IPA and DAVID. Here, we focused on Fgf5, which ranked in the top 10 among the discovered genes. To develop a functional analysis of Fgf5, we created an efficient method combining CRISPR/Cas9-mediated genome engineering and RNA interference (RNAi). Notably, we show one-step generation of various Fgf5 reporter lines including heterozygous and homozygous knockins (the GET method). For time- and dose-dependent depletion of fgf5 over the course of development, we generated an ESC line harboring Tol2 transposon-mediated integration of an inducible short hairpin RNA interference system (pdiRNAi). Our findings raised the possibility that Fgf/Erk signaling and apicobasal epithelial integrity are important factors in epiblast development. In addition, our methods provide a framework for a broad array of applications in the areas of mammalian genetics and molecular biology to understand development and to improve future therapeutics. PMID:26839115

  2. RNAi knockdown of acetyl-CoA carboxylase gene eliminates jinggangmycin-enhanced reproduction and population growth in the brown planthopper, Nilaparvata lugens

    PubMed Central

    Zhang, Yi-Xin; Ge, Lin-Quan; Jiang, Yi-Ping; Lu, Xiu-Li; Li, Xin; Stanley, David; Song, Qi-Sheng; Wu, Jin-Cai

    2015-01-01

    A major challenge in ecology lies in understanding the coexistence of intraguild species, well documented at the organismal level, but not at the molecular level. This study focused on the effects of the antibiotic, jinggangmycin (JGM), a fungicide widely used in Asian rice agroecosystems, on reproduction of insects within the planthopper guild, including the brown planthopper (BPH) Nilaparvata lugens and the white-backed planthopper (WBPH) Sogatella furcifera, both serious resurgence rice pests. JGM exposure significantly increased BPH fecundity and population growth, but suppressed both parameters in laboratory and field WBPH populations. We used digital gene expression and transcriptomic analyses to identify a panel of differentially expressed genes, including a set of up-regulated genes in JGM-treated BPH, which were down-regulated in JGM-treated WBPH. RNAi silencing of Acetyl Co-A carboxylase (ACC), highly expressed in JGM-treated BPH, reduced ACC expression (by > 60%) and eliminated JGM-induced fecundity increases in BPH. These findings support our hypothesis that differences in ACC expression separates intraguild species at the molecular level. PMID:26482193

  3. Knockdown of the ionotropic γ-aminobutyric acid receptor (GABAR) RDL gene decreases fipronil susceptibility of the small brown planthopper, Laodelphax striatellus (Hemiptera: Delphacidae).

    PubMed

    Wei, Qi; Wu, Shun-Fan; Niu, Chun-Dong; Yu, Hua-Yang; Dong, Yao-Xue; Gao, Cong-Fen

    2015-04-01

    Insect γ-aminobutyric acid receptors (GABARs) are important molecular targets of cyclodiene and phenylpyrazole insecticides. Previously GABARs encoding rdl (resistant to dieldrin) genes responsible for dieldrin and fipronil resistance were identified in various economically important insect pests. In this study, we cloned the open reading frame cDNA sequence of rdl gene from fipronil-susceptible and fipronil-resistant strains of Laodelphax striatellus (Lsrdl). Sequence analysis confirmed the presence of a previously identified resistance-conferring mutation. Different alternative splicing variants of Lsrdl were noted. Injection of dsLsrdl reduced the mRNA abundance of Lsrdl by 27-82%, and greatly decreased fipronil-induced mortality of individuals from both susceptible and resistant strains. These data indicate that Lsrdl encodes a functional RDL subunit that mediates susceptibility to fipronil. Additionally, temporal and spatial expression analysis showed that Lsrdl was expressed at higher levels in eggs, fifth-instar nymphs, and female adults than in third-instar and fourth-instar nymphs. Lsrdl was predominantly expressed in the heads of 2-day-old female adults. All these results provide useful background knowledge for better understanding of fipronil resistance related ionotropic GABA receptor rdl gene expressed variants and potential functional differences in insects. PMID:25808850

  4. Potent Intracellular Knock-Down of Influenza A Virus M2 Gene Transcript by DNAzymes Considerably Reduces Viral Replication in Host Cells.

    PubMed

    Kumar, Binod; Rajput, Roopali; Pati, Dibya Ranjan; Khanna, Madhu

    2015-09-01

    Influenza A virus has been known to be an important respiratory pathogen and cause of several epidemics and devastating pandemics leading to loss of life and resources across the globe. The M2 ion channel protein is highly conserved and essentially required during the trafficking, assembly, and budding processes of virus, thus an attractive target for designing antiviral drugs. We designed several 10-23 DNAzymes (Dz) targeting different regions of the M2 gene and analyzed their ability to specifically cleave the target RNA in both cell-free system as well as in cell culture using transient transfections. Dz114, among several others, directed against the predicted single-stranded bulge regions showed 70% inhibition of M2 gene expression validated by PCR and Western blot analysis. The activity was dependent on Mg(2+) (10-50 mM) in a dose-dependent manner. The mutant-Dz against M2 gene showed no down-regulation thereby illustrating high level of specificity of designed Dz114 towards the target RNA. Our findings suggest that Dz may be used as potential inhibitor of viral RNA replication and can be explored further for development of an effective therapeutic agent against influenza infection. These catalytic nucleic acid molecules may further be investigated as an alternative to the traditional influenza vaccines that require annual formulation. PMID:26021603

  5. Impairment of Preimplantation Porcine Embryo Development by Histone Demethylase KDM5B Knockdown Through Disturbance of Bivalent H3K4me3-H3K27me3 Modifications1

    PubMed Central

    Huang, Jiaojiao; Zhang, Hongyong; Wang, Xianlong; Dobbs, Kyle B.; Yao, Jing; Qin, Guosong; Whitworth, Kristin; Walters, Eric M.; Prather, Randall S.; Zhao, Jianguo

    2015-01-01

    ABSTRACT KDM5B (JARID1B/PLU1) is a H3K4me2/3 histone demethylase that is implicated in cancer development and proliferation and is also indispensable for embryonic stem cell self-renewal, cell fate, and murine embryonic development. However, little is known about the role of KDM5B during preimplantation embryo development. Here we show that KDM5B is critical to porcine preimplantation development. KDM5B was found to be expressed in a stage-specific manner, consistent with demethylation of H3K4me3, with the highest expression being observed from the 4-cell to the blastocyst stages. Knockdown of KDM5B by morpholino antisense oligonucleotides injection impaired porcine embryo development to the blastocyst stage. The impairment of embryo development might be caused by increased expression of H3K4me3 at the 4-cell and blastocyst stages, which disturbs the balance of bivalent H3K4me3-H3K27me3 modifications at the blastocyst stage. Decreased abundance of H3K27me3 at blastocyst stage activates multiple members of homeobox genes (HOX), which need to be silenced for faithful embryo development. Additionally, the histone demethylase KDM6A was found to be upregulated by knockdown of KDM5B, which indicated it was responsible for the decreased abundance of H3K27me3 at the blastocyst stage. The transcriptional levels of Ten-Eleven Translocation gene family members (TET1, TET2, and TET3) are found to be increased by knockdown of KDM5B, which indicates cross talk between histone modifications and DNA methylation. The studies above indicate that KDM5B is required for porcine embryo development through regulating the balance of bivalent H3K4me3-H3K27me3 modifications. PMID:25609834

  6. ANG II receptor subtype 1a gene knockdown in the subfornical organ prevents increased drinking behavior in bile duct-ligated rats

    PubMed Central

    Walch, Joseph D.; Nedungadi, T. Prashant

    2014-01-01

    Bile duct ligation (BDL) causes congestive liver failure that initiates hemodynamic changes, resulting in dilutional hyponatremia due to increased water intake and vasopressin release. This project tested the hypothesis that angiotensin signaling at the subfornical organ (SFO) augments drinking behavior in BDL rats. A genetically modified adeno-associated virus containing short hairpin RNA (shRNA) for ANG II receptor subtype 1a (AT1aR) gene was microinjected into the SFO of rats to knock down expression. Two weeks later, BDL or sham surgery was performed. Rats were housed in metabolic chambers for measurement of fluid and food intake and urine output. The rats were euthanized 28 days after BDL surgery for analysis. A group of rats was perfused for immunohistochemistry, and a second group was used for laser-capture microdissection for analysis of SFO AT1aR gene expression. BDL rats showed increased water intake that was attenuated in rats that received SFO microinjection of AT1aR shRNA. Among BDL rats treated with scrambled (control) and AT1aR shRNA, we observed an increased number of vasopressin-positive cells in the supraoptic nucleus that colocalized with ΔFosB staining, suggesting increased vasopressin release in both groups. These results indicate that angiotensin signaling through the SFO contributes to increased water intake, but not dilutional hyponatremia, during congestive liver failure. PMID:25009217

  7. Knockdown of Litopenaeus vannamei HtrA2, an up-regulated gene in response to WSSV infection, leading to delayed shrimp mortality.

    PubMed

    Peepim, Termsri; Phiwsaiya, Kornsunee; Charoensapsri, Walaiporn; Khunrae, Pongsak; Senapin, Saengchan; Rattanarojpong, Triwit

    2016-02-10

    HtrA2 is an apoptosis-activating gene that enhances the apoptotic process by preventing the formation of the IAP-caspase complex, thereby freeing caspase to trigger the apoptosis pathway. In this study, we presented the full-length cDNA sequence of HtrA2 from Litopenaeus vannamei (LvHtrA2). The full-length LvHtrA2 was 1335 bp, encoding 444 amino acids. This deduced amino acid sequence contained five conserved domains: a mitochondrial targeting signal (MTS), a transmembrane (TM) domain, an IAP-binding motif (IBM), a trimerization motif, a serine protease domain, and a PDZ domain normally found in the HtrA2 proteins of other organisms. A phylogenetic analysis revealed that LvHtrA2 clustered with the HtrA2 from other invertebrates and was closely related to Penaeus monodon HtrA2 (PmHtrA2). RT-PCR with RNA extracts from L. vannamei revealed that LvHtrA2 expression was found in several tissues, including the lymphoid organs, the haemocytes, the hepatopancreas, the gill, and the stomach, with different expression levels. When determining the role of LvHtrA2 in WSSV infection, it was found that LvHtrA2 transcription was early up-regulated in the WSSV-infected shrimp at 8h post-infection (p.i.) and expression still remained high at 48 h p.i.. It also demonstrated that dsRNA specific to LvHtrA2 reduced the cumulative mortality in the WSSV-infected shrimp compared with the control group. Additionally, depletion of the LvHtrA2 transcripts reduced expression levels for caspase-3 (Cap-3) gene in shrimp. This result could suggest that LvHtrA2 may involved in apoptosis mediated mortality rather than providing immune protection during WSSV infection. PMID:26712477

  8. Simultaneous knockdown of uPA and MMP9 can reduce breast cancer progression by increasing cell-cell adhesion and modulating EMT genes

    PubMed Central

    Moirangthem, Anuradha; Bondhopadhyay, Banashree; Mukherjee, Mala; Bandyopadhyay, Arghya; Mukherjee, Narendranath; Konar, Karabi; Bhattacharya, Shubham; Basu, Anupam

    2016-01-01

    In cancer progression, proteolytic enzymes like serine proteases and metalloproteinases degrade the basement membrane enabling the tumor cells to invade the adjacent tissues. Thus, invasion and metastasis are augmented by these enzymes. Simultaneous silencing of uPA and MMP9 in breast cancer cells decreased the wound healing, migratory, invasive and adhesive capacity of the cells. After simultaneous down regulation, cells were seen to be arrested in the cell cycle. There was a remarkable increase in the expression of cell to cell adhesion molecule E–cadherin, and decrease in Vimentin and Snail expression. In addition, there was a significant decrease in the expression of the stem cell marker Oct-4. In the breast tumor samples it has been observed that, tumors, expressing higher level of uPA and MMP9, express less amount of E–cadherin. It has also been observed that few tumors also show, Vimentin positive in the ductal epithelial area. Thus, our model can help for checking the aggressive tumor invasion by blocking of uPA and MMP9. Our present observations also give the concept of the presence of aggressive epithelial cells with mesenchymal nature in the tumor micro-environment, altering the expression of EMT genes. PMID:26906973

  9. Simultaneous knockdown of uPA and MMP9 can reduce breast cancer progression by increasing cell-cell adhesion and modulating EMT genes.

    PubMed

    Moirangthem, Anuradha; Bondhopadhyay, Banashree; Mukherjee, Mala; Bandyopadhyay, Arghya; Mukherjee, Narendranath; Konar, Karabi; Bhattacharya, Shubham; Basu, Anupam

    2016-01-01

    In cancer progression, proteolytic enzymes like serine proteases and metalloproteinases degrade the basement membrane enabling the tumor cells to invade the adjacent tissues. Thus, invasion and metastasis are augmented by these enzymes. Simultaneous silencing of uPA and MMP9 in breast cancer cells decreased the wound healing, migratory, invasive and adhesive capacity of the cells. After simultaneous down regulation, cells were seen to be arrested in the cell cycle. There was a remarkable increase in the expression of cell to cell adhesion molecule E-cadherin, and decrease in Vimentin and Snail expression. In addition, there was a significant decrease in the expression of the stem cell marker Oct-4. In the breast tumor samples it has been observed that, tumors, expressing higher level of uPA and MMP9, express less amount of E-cadherin. It has also been observed that few tumors also show, Vimentin positive in the ductal epithelial area. Thus, our model can help for checking the aggressive tumor invasion by blocking of uPA and MMP9. Our present observations also give the concept of the presence of aggressive epithelial cells with mesenchymal nature in the tumor micro-environment, altering the expression of EMT genes. PMID:26906973

  10. Knock-Down of Both eIF4E1 and eIF4E2 Genes Confers Broad-Spectrum Resistance against Potyviruses in Tomato

    PubMed Central

    Mazier, Marianne; Flamain, Fabrice; Nicolaï, Maryse; Sarnette, Verane; Caranta, Carole

    2011-01-01

    Background The eukaryotic translation initiation factor eIF4E plays a key role in plant-potyvirus interactions. eIF4E belongs to a small multigenic family and three genes, eIF4E1, eIF4E2 and eIF(iso)4E, have been identified in tomato. It has been demonstrated that eIF4E-mediated natural recessive resistances against potyviruses result from non-synonymous mutations in an eIF4E protein, which impair its direct interaction with the potyviral protein VPg. In tomato, the role of eIF4E proteins in potyvirus resistance is still unclear because natural or induced mutations in eIF4E1 confer only a narrow resistance spectrum against potyviruses. This contrasts with the broad spectrum resistance identified in the natural diversity of tomato. These results suggest that more than one eIF4E protein form is involved in the observed broad spectrum resistance. Methodology/Principal Findings To gain insight into the respective contribution of each eIF4E protein in tomato-potyvirus interactions, two tomato lines silenced for both eIF4E1 and eIF4E2 (RNAi-4E) and two lines silenced for eIF(iso)4E (RNAi-iso4E) were obtained and characterized. RNAi-4E lines are slightly impaired in their growth and fertility, whereas no obvious growth defects were observed in RNAi-iso4E lines. The F1 hybrid between RNAi-4E and RNAi-iso4E lines presented a pronounced semi-dwarf phenotype. Interestingly, the RNAi-4E lines silenced for both eIF4E1 and eIF4E2 showed broad spectrum resistance to potyviruses while the RNAi-iso4E lines were fully susceptible to potyviruses. Yeast two-hybrid interaction assays between the three eIF4E proteins and a set of viral VPgs identified two types of VPgs: those that interacted only with eIF4E1 and those that interacted with either eIF4E1 or with eIF4E2. Conclusion/Significance These experiments provide evidence for the involvement of both eIF4E1 and eIF4E2 in broad spectrum resistance of tomato against potyviruses and suggest a role for eIF4E2 in tomato

  11. Early Embryonic Gene Expression Profiling of Zebrafish Prion Protein (Prp2) Morphants

    PubMed Central

    Nourizadeh-Lillabadi, Rasoul; Seilø Torgersen, Jacob; Vestrheim, Olav; König, Melanie; Aleström, Peter; Syed, Mohasina

    2010-01-01

    Background The Prion protein (PRNP/Prp) plays a crucial role in transmissible spongiform encephalopathies (TSEs) like Creutzfeldt-Jakob disease (CJD), scrapie and mad cow disease. Notwithstanding the importance in human and animal disease, fundamental aspects of PRNP/Prp function and transmission remains unaccounted for. Methodology/Principal Findings The zebrafish (Danio rerio) genome contains three Prp encoding genes assigned prp1, prp2 and prp3. Currently, the second paralogue is believed to be the most similar to the mammalian PRNP gene in structure and function. Functional studies of the PRNP gene ortholog was addressed by prp2 morpholino (MO) knockdown experiments. Investigation of Prp2 depleted embryos revealed high mortality and apoptosis at 24 hours post fertilization (hpf) as well as impaired brain and neuronal development. In order to elucidate the underlying mechanisms, a genome-wide transcriptome analysis was carried out in viable 24 hpf morphants. The resulting changes in gene expression profiles revealed 249 differently expressed genes linked to biological processes like cell death, neurogenesis and embryonic development. Conclusions/Significance The current study contributes to the understanding of basic Prp functions and demonstrates that the zebrafish is an excellent model to address the role of Prp in vertebrates. The gene knockdown of prp2 indicates an essential biological function for the zebrafish ortholog with a morphant phenotype that suggests a neurodegenerative action and gene expression effects which are apoptosis related and effects gene networks controlling neurogenesis and embryo development. PMID:21042590

  12. The Popeye domain containing 2 (popdc2) gene in zebrafish is required for heart and skeletal muscle development

    PubMed Central

    Kirchmaier, Bettina C.; Poon, Kar Lai; Schwerte, Thorsten; Huisken, Jan; Winkler, Christoph; Jungblut, Benno; Stainier, Didier Y.; Brand, Thomas

    2013-01-01

    The Popeye domain containing (Popdc) genes encode a family of transmembrane proteins with an evolutionary conserved Popeye domain. These genes are abundantly expressed in striated muscle tissue, however their function is not well understood. In this study we have investigated the role of the popdc2 gene in zebrafish. Popdc2 transcripts were detected in the embryonic myocardium and transiently in the craniofacial and tail musculature. Morpholino oligonucleotide-mediated knockdown of popdc2 resulted in aberrant development of skeletal muscle and heart. Muscle segments in the trunk were irregularly shaped and craniofacial muscles were severely reduced or even missing. In the heart, pericardial edema was prevalent in the morphants and heart chambers were elongated and looping was abnormal. These pathologies in muscle and heart were alleviated after reducing the morpholino concentration. However the heart still was abnormal displaying cardiac arrhythmia at later stages of development. Optical recordings of cardiac contractility revealed irregular ventricular contractions with a 2:1, or 3:1 atrial/ventricular conduction ratio, which caused a significant reduction in heart frequency. Recordings of calcium transients with high spatiotemporal resolution using a transgenic calcium indicator line (Tg(cmlc2:gCaMP)s878) and SPIM microscopy confirmed the presence of a severe arrhythmia phenotype. Our results identify popdc2 as a gene important for striated muscle differentiation and cardiac morphogenesis. In addition it is required for the development of the cardiac conduction system. PMID:22290329

  13. Kinetic Mechanisms in Morpholino-DNA Surface Hybridization

    PubMed Central

    Liu, Yatao; Irving, Damion; Qiao, Wanqiong; Ge, Dongbiao

    2011-01-01

    Morpholinos (MOs) are DNA analogues whose uncharged nature can bring fundamental advantages to surface hybridization technologies such as DNA microarrays, by using MOs as the immobilized, or “probe”, species. Advancement of MO-based diagnostics, however, is challenged by limited understanding of the surface organization of MO molecules and of how this organization impacts hybridization kinetics and thermodynamics. The present study focuses on hybridization kinetics between monolayers of MO probes and DNA targets as a function of the instantaneous extent of hybridization (i.e. duplex coverage), total probe coverage, and ionic strength. Intriguingly, these experiments reveal distinct kinetic stages, none of which are consistent with Langmuir kinetics. The initial stage, in which duplex coverage remains relatively sparse, indicates confluence of two effects: blockage of target access to unhybridized probes by previously formed duplexes, and deactivation of the solid support due to consumption of probe molecules. This interpretation is consistent with a surface organization in which unhybridized MO probes localize near the solid support, underneath a layer of MO-DNA duplexes. As duplex coverage builds, provided saturation is not reached first, the initial stage can transition to an unusual regime characterized by near independence of hybridization rate on duplex coverage, followed by a prolonged approach to equilibrium. The possible origins of these more complex latter behaviors are discussed. Comparison with published data for DNA and peptide nucleic acid (PNA) probes is carried out to look for universal trends in kinetics. This comparison reveals qualitative similarities when comparable surface organization of probes is expected. In addition, MO monolayers are found capable of a broad range of reactivities that span reported values for PNA and DNA probes. PMID:21699181

  14. Molecular Mechanisms in Morpholino-DNA Surface Hybridization

    PubMed Central

    Gong, Ping; Wang, Kang; Liu, Yatao; Shepard, Kenneth

    2010-01-01

    Synthetic nucleic acid mimics provide opportunity for redesigning the specificity and affinity of hybridization with natural DNA or RNA. Such redesign is of great interest for diagnostic applications where it can enhance the desired signal against a background of competing interactions. This report compares hybridization of DNA analyte strands with morpholinos (MOs), which are uncharged nucleic acid mimics, to the corresponding DNA-DNA case in solution and on surfaces. In solution, MO-DNA hybridization is found to be independent of counterion concentration, in contrast to DNA-DNA hybridization. On surfaces, when immobilized MO or DNA “probe” strands hybridize with complementary DNA “targets” from solution, both the MO-DNA and DNA-DNA processes depend on ionic strength but exhibit qualitatively different behaviors. At lower ionic strengths, MO-DNA surface hybridization exhibits hallmarks of kinetic limitations when separation between hybridized probe sites becomes comparable to target dimensions, whereas extents of DNA-DNA surface hybridization are instead consistent with limits imposed by buildup of surface (Donnan) potential. The two processes also fundamentally differ at high ionic strength, under conditions when electrostatic effects are weak. Here, variations in probe coverage have a much diminished impact on MO-DNA than on DNA-DNA hybridization for similarly crowded surface conditions. These various observations agree with a structural model of MO monolayers in which MO-DNA duplexes segregate to the buffer interface while unhybridized probes localize near the solid support. A general perspective is presented on using uncharged DNA analogues, which also include compounds such as peptide nucleic acids (PNA), in surface hybridization applications. PMID:20572663

  15. Kinetic mechanisms in morpholino-DNA surface hybridization.

    PubMed

    Liu, Yatao; Irving, Damion; Qiao, Wanqiong; Ge, Dongbiao; Levicky, Rastislav

    2011-08-01

    Morpholinos (MOs) are DNA analogues whose uncharged nature can bring fundamental advantages to surface hybridization technologies such as DNA microarrays, by using MOs as the immobilized, or "probe", species. Advancement of MO-based diagnostics, however, is challenged by limited understanding of the surface organization of MO molecules and of how this organization impacts hybridization kinetics and thermodynamics. The present study focuses on hybridization kinetics between monolayers of MO probes and DNA targets as a function of the instantaneous extent of hybridization (i.e., duplex coverage), total probe coverage, and ionic strength. Intriguingly, these experiments reveal distinct kinetic stages, none of which are consistent with Langmuir kinetics. The initial stage, in which duplex coverage remains relatively sparse, indicates confluence of two effects: blockage of target access to unhybridized probes by previously formed duplexes and deactivation of the solid support due to consumption of probe molecules. This interpretation is consistent with a surface organization in which unhybridized MO probes localize near the solid support, underneath a layer of MO-DNA duplexes. As duplex coverage builds, provided saturation is not reached first, the initial stage can transition to an unusual regime characterized by near independence of hybridization rate on duplex coverage, followed by a prolonged approach to equilibrium. The possible origins of these more complex latter behaviors are discussed. Comparison with published data for DNA and peptide nucleic acid (PNA) probes is carried out to look for universal trends in kinetics. This comparison reveals qualitative similarities when comparable surface organization of probes is expected. In addition, MO monolayers are found capable of a broad range of reactivities that span reported values for PNA and DNA probes. PMID:21699181

  16. PCTAIRE1-knockdown sensitizes cancer cells to TNF family cytokines.

    PubMed

    Yanagi, Teruki; Shi, Ranxin; Aza-Blanc, Pedro; Reed, John C; Matsuzawa, Shu-ichi

    2015-01-01

    While PCTAIRE1/PCTK1/Cdk16 is overexpressed in malignant cells and is crucial in tumorigenesis, its function in apoptosis remains unclear. Here we investigated the role of PCTAIRE1 in apoptosis, especially in the extrinsic cell death pathway. Gene-knockdown of PCTAIRE1 sensitized prostate cancer PPC1 and Du145 cells, and breast cancer MDA-MB-468 cells to TNF-family cytokines, including TNF-related apoptosis-inducing ligand (TRAIL). Meanwhile, PCTAIRE1-knockdown did not sensitize non-malignant cells, including diploid fibroblasts IMR-90 and the immortalized prostate epithelial cell line 267B1. PCTAIRE1-knockdown did not up-regulate death receptor expression on the cell surface or affect caspase-8, FADD and FLIP expression levels. PCTAIRE1-knockdown did promote caspase-8 cleavage and RIPK1 degradation, while RIPK1 mRNA knockdown sensitized PPC1 cells to TNF-family cytokines. Furthermore, the kinase inhibitor SNS-032, which inhibits PCTAIRE1 kinase activity, sensitized PPC1 cells to TRAIL-induced apoptosis. Together these results suggest that PCTAIRE1 contributes to the resistance of cancer cell lines to apoptosis induced by TNF-family cytokines, which implies that PCTAIRE1 inhibitors could have synergistic effects with TNF-family cytokines for cytodestruction of cancer cells. PMID:25790448

  17. PCTAIRE1-Knockdown Sensitizes Cancer Cells to TNF Family Cytokines

    PubMed Central

    Yanagi, Teruki; Shi, Ranxin; Aza-Blanc, Pedro; Reed, John C.; Matsuzawa, Shu-ichi

    2015-01-01

    While PCTAIRE1/PCTK1/Cdk16 is overexpressed in malignant cells and is crucial in tumorigenesis, its function in apoptosis remains unclear. Here we investigated the role of PCTAIRE1 in apoptosis, especially in the extrinsic cell death pathway. Gene-knockdown of PCTAIRE1 sensitized prostate cancer PPC1 and Du145 cells, and breast cancer MDA-MB-468 cells to TNF-family cytokines, including TNF-related apoptosis-inducing ligand (TRAIL). Meanwhile, PCTAIRE1-knockdown did not sensitize non-malignant cells, including diploid fibroblasts IMR-90 and the immortalized prostate epithelial cell line 267B1. PCTAIRE1-knockdown did not up-regulate death receptor expression on the cell surface or affect caspase-8, FADD and FLIP expression levels. PCTAIRE1-knockdown did promote caspase-8 cleavage and RIPK1 degradation, while RIPK1 mRNA knockdown sensitized PPC1 cells to TNF-family cytokines. Furthermore, the kinase inhibitor SNS-032, which inhibits PCTAIRE1 kinase activity, sensitized PPC1 cells to TRAIL-induced apoptosis. Together these results suggest that PCTAIRE1 contributes to the resistance of cancer cell lines to apoptosis induced by TNF-family cytokines, which implies that PCTAIRE1 inhibitors could have synergistic effects with TNF-family cytokines for cytodestruction of cancer cells. PMID:25790448

  18. Bioinformatic and functional optimization of antisense phosphorodiamidate morpholino oligomers (PMOs) for therapeutic modulation of RNA splicing in muscle.

    PubMed

    Popplewell, Linda J; Graham, Ian R; Malerba, Alberto; Dickson, George

    2011-01-01

    Duchenne muscular dystrophy (DMD) is caused by mutations that disrupt the reading frame of the human DMD gene. Selective removal of exons flanking an out-of-frame DMD mutation can result in an in-frame mRNA transcript that may be translated into an internally deleted, Becker muscular dystrophy (BMD)-like, but functionally active dystrophin protein with therapeutic activity. Antisense oligonucleotides (AOs) can be designed to bind to complementary sequences in the targeted mRNA and modify pre-mRNA splicing to correct the reading frame of a mutated transcript so that gene expression is restored. AO-induced exon skipping producing functional truncated dystrophin exon has been demonstrated in animal models of DMD both in vitro and in vivo, and in DMD patient cells in vitro in culture, and in DMD muscle explants. More recently, AO-mediated exon skipping has been confirmed in DMD patients in Phase I clinical trials. However, it should be noted that personalized molecular medicine may be necessary, since the various reading frame-disrupting mutations are spread across the DMD gene. The different deletions that cause DMD would require skipping of different exons, which would require the optimization and clinical trial workup of many specific AOs. This chapter describes the methodologies available for the optimization of AOs, and in particular phosphorodiamidate morpholino oligomers (PMOs), for the targeted skipping of specific exons on the DMD gene. PMID:21194027

  19. Aryl morpholino triazenes inhibit cytochrome P450 1A1 and 1B1.

    PubMed

    Lee, Daniel; Perez, Pedro; Jackson, William; Chin, Taylor; Galbreath, Michael; Fronczek, Frank R; Isovitsch, Ralph; Iimoto, Devin S

    2016-07-15

    Many cytochrome P450 1A1 and 1B1 (CYP1A1 and CYP1B1) inhibitors, such as resveratrol, have planar, hydrophobic, aromatic rings in their structure and exhibit anti-cancer activity. Aryl morpholino triazenes have similar structural features and in addition contain a triazene unit consisting of three consecutive, conjugated nitrogen atoms. Several aryl morpholino triazenes, including 4-[(E)-2-(3,4,5-trimethoxyphenyl)diazenyl]-morpholine (2), were prepared from a reaction involving morpholine and a diazonium ion produced from different aniline derivatives, such as 3,4,5-trimethoxyaniline. The aryl morpholino triazenes were then screened at 100μM for their ability to inhibit CYP1A1 and CYP1B1 using ethoxyresorufin as the substrate. Triazenes that inhibited the enzymes to less than 80% of the uninhibited enzyme activity were assayed to determine their IC50 value. Compound 2 was the only triazene to inhibit both CYP1A1 and CYP1B1 to the same degree as resveratrol with IC50 values of 10μM and 18μM, respectively. Compounds 3 and 6 selectively inhibited CYP1B1 over CYP1A1 with IC values of 2μM and 7μM, respectively. Thus, aryl morpholino triazenes are a new class of compounds that can inhibit CYP1A1 and CYP1B1 and potentially prevent cancer. PMID:27265259

  20. Extensive and prolonged restoration of dystrophin expression with vivo-morpholino-mediated multiple exon skipping in dystrophic dogs.

    PubMed

    Yokota, Toshifumi; Nakamura, Akinori; Nagata, Tetsuya; Saito, Takashi; Kobayashi, Masanori; Aoki, Yoshitsugu; Echigoya, Yusuke; Partridge, Terence; Hoffman, Eric P; Takeda, Shin'ichi

    2012-10-01

    Duchenne muscular dystrophy (DMD) is a severe and the most prevalent form of muscular dystrophy, characterized by rapid progression of muscle degeneration. Antisense-mediated exon skipping is currently one of the most promising therapeutic options for DMD. However, unmodified antisense oligos such as morpholinos require frequent (weekly or bi-weekly) injections. Recently, new generation morpholinos such as vivo-morpholinos are reported to lead to extensive and prolonged dystrophin expression in the dystrophic mdx mouse, an animal model of DMD. The vivo-morpholino contains a cell-penetrating moiety, octa-guanidine dendrimer. Here, we sought to test the efficacy of multiple exon skipping of exons 6-8 with vivo-morpholinos in the canine X-linked muscular dystrophy, which harbors a splice site mutation at the boundary of intron 6 and exon 7. We designed and optimized novel antisense cocktail sequences and combinations for exon 8 skipping and demonstrated effective exon skipping in dystrophic dogs in vivo. Intramuscular injections with newly designed cocktail oligos led to high levels of dystrophin expression, with some samples similar to wild-type levels. This is the first report of successful rescue of dystrophin expression with morpholino conjugates in dystrophic dogs. Our results show the potential of phosphorodiamidate morpholino oligomer conjugates as therapeutic agents for DMD. PMID:22888777

  1. Knockdown of ttc26 disrupts ciliogenesis of the photoreceptor cells and the pronephros in zebrafish.

    PubMed

    Zhang, Qi; Liu, Qin; Austin, Chrissy; Drummond, Iain; Pierce, Eric A

    2012-08-01

    In our effort to understand genetic disorders of the photoreceptor cells of the retina, we have focused on intraflagellar transport in photoreceptor sensory cilia. From previous mouse proteomic data we identified a cilia protein Ttc26, orthologue of dyf-13 in Caenorhabditis elegans, as a target. We localized Ttc26 to the transition zone of photoreceptor and to the transition zone of cilia in cultured murine inner medullary collecting duct 3 (mIMCD3) renal cells. Knockdown of Ttc26 in mIMCD3 cells produced shortened and defective primary cilia, as revealed by immunofluorescence and scanning electron microscopy. To study Ttc26 function in sensory cilia in vivo, we utilized a zebrafish vertebrate model system. Morpholino knockdown of ttc26 in zebrafish embryos caused ciliary defects in the pronephric kidney at 27 h postfertilization and distension/dilation of pronephros at 5 d postfertilization (dpf). In the eyes, the outer segments of photoreceptor cells appeared shortened or absent, whereas cellular lamination appeared normal in retinas at 5 dpf. This suggests that loss of ttc26 function prevents normal ciliogenesis and differentiation in the photoreceptor cells, and that ttc26 is required for normal development and differentiation in retina and pronephros. Our studies support the importance of Ttc26 function in ciliogenesis and suggest that screening for TTC26 mutations in human ciliopathies is justified. PMID:22718903

  2. Open and closed evolutionary paths for drastic morphological changes, involving serial gene duplication, sub-functionalization, and selection

    PubMed Central

    Abe, Gembu; Lee, Shu-Hua; Li, Ing-Jia; Chang, Chun-Ju; Tamura, Koji; Ota, Kinya G.

    2016-01-01

    Twin-tail goldfish strains are examples of drastic morphological alterations that emerged through domestication. Although this mutation is known to be caused by deficiency of one of two duplicated chordin genes, it is unknown why equivalent mutations have not been observed in other domesticated fish species. Here, we compared the chordin gene morphant phenotypes of single-tail goldfish and common carp (close relatives, both of which underwent chordin gene duplication and domestication). Morpholino-induced knockdown depleted chordin gene expression in both species; however, while knockdown reproduced twin-tail morphology in single-tail goldfish, it had no effect on common carp morphology. This difference can be explained by the observation that expression patterns of the duplicated chordin genes overlap completely in common carp, but are sub-functionalized in goldfish. Our finding implies that goldfish drastic morphological changes might be enhanced by the subsequent occurrence of three different types of evolutionary event (duplication, sub-functionalization, and selection) in a certain order. PMID:27220684

  3. Open and closed evolutionary paths for drastic morphological changes, involving serial gene duplication, sub-functionalization, and selection.

    PubMed

    Abe, Gembu; Lee, Shu-Hua; Li, Ing-Jia; Chang, Chun-Ju; Tamura, Koji; Ota, Kinya G

    2016-01-01

    Twin-tail goldfish strains are examples of drastic morphological alterations that emerged through domestication. Although this mutation is known to be caused by deficiency of one of two duplicated chordin genes, it is unknown why equivalent mutations have not been observed in other domesticated fish species. Here, we compared the chordin gene morphant phenotypes of single-tail goldfish and common carp (close relatives, both of which underwent chordin gene duplication and domestication). Morpholino-induced knockdown depleted chordin gene expression in both species; however, while knockdown reproduced twin-tail morphology in single-tail goldfish, it had no effect on common carp morphology. This difference can be explained by the observation that expression patterns of the duplicated chordin genes overlap completely in common carp, but are sub-functionalized in goldfish. Our finding implies that goldfish drastic morphological changes might be enhanced by the subsequent occurrence of three different types of evolutionary event (duplication, sub-functionalization, and selection) in a certain order. PMID:27220684

  4. Manipulation of Developing Juvenile Structures in Purple Sea Urchins (Strongylocentrotus purpuratus) by Morpholino Injection into Late Stage Larvae

    PubMed Central

    2014-01-01

    Sea urchins have been used as experimental organisms for developmental biology for over a century. Yet, as is the case for many other marine invertebrates, understanding the development of the juveniles and adults has lagged far behind that of their embryos and larvae. The reasons for this are, in large part, due to the difficulty of experimentally manipulating juvenile development. Here we develop and validate a technique for injecting compounds into juvenile rudiments of the purple sea urchin, Strongylocentrotus purpuratus. We first document the distribution of rhodaminated dextran injected into different compartments of the juvenile rudiment of sea urchin larvae. Then, to test the potential of this technique to manipulate development, we injected Vivo-Morpholinos (vMOs) designed to knock down p58b and p16, two proteins involved in the elongation of S. purpuratus larval skeleton. Rudiments injected with these vMOs showed a delay in the growth of some juvenile skeletal elements relative to controls. These data provide the first evidence that vMOs, which are designed to cross cell membranes, can be used to transiently manipulate gene function in later developmental stages in sea urchins. We therefore propose that injection of vMOs into juvenile rudiments, as shown here, is a viable approach to testing hypotheses about gene function during development, including metamorphosis. PMID:25436992

  5. Achieving targeted and quantifiable alteration of mRNA splicing with Morpholino oligos

    SciTech Connect

    Morcos, Paul A. . E-mail: pmorcos@gene-tools.com

    2007-06-29

    This work represents the first guide for using steric-block antisense oligos as tools for effective and targeted modification of RNA splicing. Comparison of several steric-block oligo types shows the properties of Morpholinos provide significant advantages over other potential splice-blocking oligos. The procedures and complications of designing effective splice-blocking Morpholino oligos are described. The design process requires complete pre-mRNA sequence for defining suitable targets, which usually generate specific predictable messengers. To validate the targeting procedure, the level and nature of transcript alteration is characterized by RT-PCR analysis of splice modification in a {beta}-globin splice model system. An oligo-walking study reveals that while U1 and U2 small nuclear RiboNucleoProtein (snRNP) binding sites are the most effective targets for blocking splicing, inclusion of these sites is not required to achieve effective splice modifications. The most effective targeting strategy employs simultaneously blocking snRNP binding sites and splice-junctions. The work presented here continues to be the basis for most of the successful Morpholino oligos designed for the worldwide research community to block RNA splicing.

  6. Bubble liposomes and ultrasound exposure improve localized morpholino oligomer delivery into the skeletal muscles of dystrophic mdx mice.

    PubMed

    Negishi, Yoichi; Ishii, Yuko; Shiono, Hitomi; Akiyama, Saki; Sekine, Shoko; Kojima, Takuo; Mayama, Sayaka; Kikuchi, Taiki; Hamano, Nobuhito; Endo-Takahashi, Yoko; Suzuki, Ryo; Maruyama, Kazuo; Aramaki, Yukihiko

    2014-03-01

    Duchenne muscular dystrophy (DMD) is a genetic disorder that is caused by mutations in the DMD gene that lead to an absence of functional protein. The mdx dystrophic mouse contains a nonsense mutation in exon 23 of the dystrophin gene; a phosphorodiamidate morpholino oligomer (PMO) designed to skip this mutated exon in the mRNA induces dystrophin expression. However, an efficient PMO delivery method is needed to improve treatment strategies for DMD. We previously developed polyethylene glycol (PEG)-modified liposomes (Bubble liposomes) that entrap ultrasound contrast gas and demonstrated that the combination of Bubble liposomes with ultrasound exposure is an effective gene delivery tool in vitro and in vivo. In this study, to evaluate the ability of Bubble liposomes as a PMO delivery tool, we tested the potency of the Bubble liposomes combined with ultrasound exposure to boost the delivery of PMO and increase the skipping of the mutated exon in the mdx mouse. The results indicated that the combination of Bubble liposomes and ultrasound exposure increased the uptake of the PMO targeting a nonsense mutation in exon 23 of the dystrophin gene and consequently increased the PMO-mediated exon-skipping efficiency compared with PMO injection alone, leading to significantly enhanced dystrophin expression. This increased efficiency indicated the potential of the combination of Bubble liposomes with ultrasound exposure to enhance PMO delivery for treating DMD. Thus, this ultrasound-mediated Bubble liposome technique may provide an effective, noninvasive, nonviral method for PMO therapy for DMD muscle as well as for other muscular dystrophies. PMID:24433046

  7. Delayed Time-to-Treatment of an Antisense Morpholino Oligomer Is Effective against Lethal Marburg Virus Infection in Cynomolgus Macaques.

    PubMed

    Warren, Travis K; Whitehouse, Chris A; Wells, Jay; Welch, Lisa; Charleston, Jay S; Heald, Alison; Nichols, Donald K; Mattix, Marc E; Palacios, Gustavo; Kugleman, Jeffrey R; Iversen, Patrick L; Bavari, Sina

    2016-02-01

    Marburg virus (MARV) is an Ebola-like virus in the family Filovirdae that causes sporadic outbreaks of severe hemorrhagic fever with a case fatality rate as high as 90%. AVI-7288, a positively charged antisense phosphorodiamidate morpholino oligomer (PMOplus) targeting the viral nucleoprotein gene, was evaluated as a potential therapeutic intervention for MARV infection following delayed treatment of 1, 24, 48, and 96 h post-infection (PI) in a nonhuman primate lethal challenge model. A total of 30 cynomolgus macaques were divided into 5 groups of 6 and infected with 1,830 plaque forming units of MARV subcutaneously. AVI-7288 was administered by bolus infusion daily for 14 days at 15 mg/kg body weight. Survival was the primary endpoint of the study. While none (0 of 6) of the saline group survived, 83-100% of infected monkeys survived when treatment was initiated 1, 24, 48, or 96 h post-infection (PI). The antisense treatment also reduced serum viremia and inflammatory cytokines in all treatment groups compared to vehicle controls. The antibody immune response to virus was preserved and tissue viral antigen was cleared in AVI-7288 treated animals. These data show that AVI-7288 protects NHPs against an otherwise lethal MARV infection when treatment is initiated up to 96 h PI. PMID:26901785

  8. Delayed Time-to-Treatment of an Antisense Morpholino Oligomer Is Effective against Lethal Marburg Virus Infection in Cynomolgus Macaques

    PubMed Central

    Warren, Travis K.; Whitehouse, Chris A.; Wells, Jay; Welch, Lisa; Charleston, Jay S.; Heald, Alison; Nichols, Donald K.; Mattix, Marc E.; Palacios, Gustavo; Kugleman, Jeffrey R.; Iversen, Patrick L.; Bavari, Sina

    2016-01-01

    Marburg virus (MARV) is an Ebola-like virus in the family Filovirdae that causes sporadic outbreaks of severe hemorrhagic fever with a case fatality rate as high as 90%. AVI-7288, a positively charged antisense phosphorodiamidate morpholino oligomer (PMOplus) targeting the viral nucleoprotein gene, was evaluated as a potential therapeutic intervention for MARV infection following delayed treatment of 1, 24, 48, and 96 h post-infection (PI) in a nonhuman primate lethal challenge model. A total of 30 cynomolgus macaques were divided into 5 groups of 6 and infected with 1,830 plaque forming units of MARV subcutaneously. AVI-7288 was administered by bolus infusion daily for 14 days at 15 mg/kg body weight. Survival was the primary endpoint of the study. While none (0 of 6) of the saline group survived, 83–100% of infected monkeys survived when treatment was initiated 1, 24, 48, or 96 h post-infection (PI). The antisense treatment also reduced serum viremia and inflammatory cytokines in all treatment groups compared to vehicle controls. The antibody immune response to virus was preserved and tissue viral antigen was cleared in AVI-7288 treated animals. These data show that AVI-7288 protects NHPs against an otherwise lethal MARV infection when treatment is initiated up to 96 h PI. PMID:26901785

  9. Cationic polyelectrolyte-mediated delivery of antisense morpholino oligonucleotides for exon-skipping in vitro and in mdx mice

    PubMed Central

    Wang, Mingxing; Wu, Bo; Tucker, Jason D; Lu, Peijuan; Lu, Qilong

    2015-01-01

    In this study, we investigated a series of cationic polyelectrolytes (PEs) with different size and composition for their potential to improve delivery of an antisense phosphorodiamidate morpholino oligomer (PMO) both in vitro and in vivo. The results showed that the poly(diallyldimethylammonium chloride) (PDDAC) polymer series, especially PE-3 and PE-4, improves the delivery efficiency of PMO, comparable with Endoporter-mediated PMO delivery in vitro. The enhanced PMO delivery and targeting to dystrophin exon 23 was further observed in mdx mice, up to fourfold with the PE-4, compared with PMO alone. The cytotoxicity of the PEs was lower than that of Endoporter and polyethylenimine 25,000 Da in vitro, and was not clearly detected in muscle in vivo under the tested concentrations. Together, these results demonstrate that optimization of PE molecular size, composition, and distribution of cationic charge are key factors to achieve enhanced PMO exon-skipping efficiency. The increased efficiency and lower toxicity show this PDDAC series to be capable gene/antisense oligonucleotide delivery-enhancing agents for treating muscular dystrophy and other diseases. PMID:26366082

  10. Cationic polyelectrolyte-mediated delivery of antisense morpholino oligonucleotides for exon-skipping in vitro and in mdx mice.

    PubMed

    Wang, Mingxing; Wu, Bo; Tucker, Jason D; Lu, Peijuan; Lu, Qilong

    2015-01-01

    In this study, we investigated a series of cationic polyelectrolytes (PEs) with different size and composition for their potential to improve delivery of an antisense phosphorodiamidate morpholino oligomer (PMO) both in vitro and in vivo. The results showed that the poly(diallyldimethylammonium chloride) (PDDAC) polymer series, especially PE-3 and PE-4, improves the delivery efficiency of PMO, comparable with Endoporter-mediated PMO delivery in vitro. The enhanced PMO delivery and targeting to dystrophin exon 23 was further observed in mdx mice, up to fourfold with the PE-4, compared with PMO alone. The cytotoxicity of the PEs was lower than that of Endoporter and polyethylenimine 25,000 Da in vitro, and was not clearly detected in muscle in vivo under the tested concentrations. Together, these results demonstrate that optimization of PE molecular size, composition, and distribution of cationic charge are key factors to achieve enhanced PMO exon-skipping efficiency. The increased efficiency and lower toxicity show this PDDAC series to be capable gene/antisense oligonucleotide delivery-enhancing agents for treating muscular dystrophy and other diseases. PMID:26366082

  11. The microcephaly gene aspm is involved in brain development in zebrafish

    SciTech Connect

    Kim, Hyun-Taek; Lee, Mi-Sun; Choi, Jung-Hwa; Jung, Ju-Yeon; Ahn, Dae-Gwon; Yeo, Sang-Yeob; Choi, Dong-Kug; Kim, Cheol-Hee

    2011-06-17

    Highlights: {yields} We identified a zebrafish aspm/mcph5 gene that is expressed in proliferating cells in the CNS during early development. {yields} Embryos injected with the aspm MO consistently showed a reduced head and eye size but were otherwise grossly normal, closely mimicking the known phenotypes of human microcephaly patients. {yields} Knock-down of aspm causes cell cycle arrest and apoptotic cell death during early development. -- Abstract: MCPH is a neurodevelopmental disorder characterized by a global reduction in cerebral cortical volume. Homozygous mutation of the MCPH5 gene, also known as ASPM, is the most common cause of the MCPH phenotype. To elucidate the roles of ASPM during embryonic development, the zebrafish aspm was identified, which is specifically expressed in proliferating cells in the CNS. Morpholino-mediated knock-down of aspm resulted in a significant reduction in head size. Furthermore, aspm-deficient embryos exhibited a mitotic arrest during early development. These findings suggest that the reduction in brain size in MCPH might be caused by lack of aspm function in the mitotic cell cycle and demonstrate that the zebrafish can provide a model system for congenital diseases of the human nervous system.

  12. Sleeping Beauty-mediated knockdown of sheep myostatin by RNA interference.

    PubMed

    Hu, Shengwei; Ni, Wei; Sai, Wujiafu; Zhang, Hui; Cao, Xudong; Qiao, Jun; Sheng, Jinliang; Guo, Fei; Chen, Chuangfu

    2011-10-01

    Myostatin is a negative regulator of skeletal muscle growth. Myostatin dysfunction therefore offers a strategy for promoting animal muscle growth in livestock production. Knockdown of myostatin was achieved by combining RNA interference and the Sleeping Beauty (SB) transposon system in sheep cells. Four targeting sites of sheep myostatin were designed and measured for myostatin silencing in sheep fetal fibroblasts by real-time PCR. The sh3 construct induced significant decrease of myostatin gene expression by 90% (P<0.05). Myostatin silencing induced by SB-mediated sh3 was further tested in stably transfected cells. SB transposition increased the integration frequency of genes into sheep genomes and mediated a more efficient myostatin knockdown than random integration of sh3. We suggest that SB-mediated shRNA provides a novel potential tool for gene knockdown in the donor cells of animal cloning. PMID:21698446

  13. The role of the Pax1/9 gene in the early development of amphioxus pharyngeal gill slits.

    PubMed

    Liu, Xin; Li, Guang; Liu, Xian; Wang, Yi-Quan

    2015-01-01

    The pharynx is a major characteristic of chordates. Compared with vertebrates, amphioxus has an advantage for the study of pharynx development, as embryos lack neural crest, and the pharynx is mainly derived from endoderm cells. The Pax1/9 subfamily genes have essential roles in vertebrate pharyngeal patterning, but it is not known if the Pax1/9 gene has similar functions in amphioxus pharynx development. To answer this question, we examined the Pax1/9 gene expression pattern in amphioxus embryos at different developmental stages, and observed morphological changes following Pax1/9 knockdown. RT-qPCR analysis indicated that Pax1/9 expression was initiated during early neurula stage and rapidly peaked during mid-neurula stage. Furthermore, in situ hybridization analysis showed that Pax1/9 transcripts were localized exclusively in the most endodermal region of the developing pharynx in early neurula stage embryos; however, Pax1/9 expression was strikingly down-regulated in the region where gill slits would form from the fusion of endoderm and ectoderm in subsequent developmental stages and was maintained in the border regions between adjacent gill slits. Knockdown of Pax1/9 function using both morpholino and siRNA approaches led to embryonic defects in the first three gill slits, and fusion of the first two gill slits. Moreover, the expression levels of the pharyngeal marker genes Six1/2 and Tbx1/10 were reduced in Pax1/9 knockdown embryos. From these observations, we concluded that the Pax1/9 gene has an important role in the initial differentiation of amphioxus pharyngeal endoderm and in the formation of gill slits, most likely via modulation of Six1/2 and Tbx1/10 expression. PMID:25504927

  14. Deiodinase Knockdown during Early Zebrafish Development Affects Growth, Development, Energy Metabolism, Motility and Phototransduction

    PubMed Central

    Bagci, Enise; Heijlen, Marjolein; Vergauwen, Lucia; Hagenaars, An; Houbrechts, Anne M.; Esguerra, Camila V.; Blust, Ronny; Darras, Veerle M.; Knapen, Dries

    2015-01-01

    Thyroid hormone (TH) balance is essential for vertebrate development. Deiodinase type 1 (D1) and type 2 (D2) increase and deiodinase type 3 (D3) decreases local intracellular levels of T3, the most important active TH. The role of deiodinase-mediated TH effects in early vertebrate development is only partially understood. Therefore, we investigated the role of deiodinases during early development of zebrafish until 96 hours post fertilization at the level of the transcriptome (microarray), biochemistry, morphology and physiology using morpholino (MO) knockdown. Knockdown of D1+D2 (D1D2MO) and knockdown of D3 (D3MO) both resulted in transcriptional regulation of energy metabolism and (muscle) development in abdomen and tail, together with reduced growth, impaired swim bladder inflation, reduced protein content and reduced motility. The reduced growth and impaired swim bladder inflation in D1D2MO could be due to lower levels of T3 which is known to drive growth and development. The pronounced upregulation of a large number of transcripts coding for key proteins in ATP-producing pathways in D1D2MO could reflect a compensatory response to a decreased metabolic rate, also typically linked to hypothyroidism. Compared to D1D2MO, the effects were more pronounced or more frequent in D3MO, in which hyperthyroidism is expected. More specifically, increased heart rate, delayed hatching and increased carbohydrate content were observed only in D3MO. An increase of the metabolic rate, a decrease of the metabolic efficiency and a stimulation of gluconeogenesis using amino acids as substrates may have been involved in the observed reduced protein content, growth and motility in D3MO larvae. Furthermore, expression of transcripts involved in purine metabolism coupled to vision was decreased in both knockdown conditions, suggesting that both may impair vision. This study provides new insights, not only into the role of deiodinases, but also into the importance of a correct TH balance

  15. Peptide nanoparticle delivery of charge-neutral splice-switching morpholino oligonucleotides.

    PubMed

    Järver, Peter; Zaghloul, Eman M; Arzumanov, Andrey A; Saleh, Amer F; McClorey, Graham; Hammond, Suzan M; Hällbrink, Mattias; Langel, Ülo; Smith, C I Edvard; Wood, Matthew J A; Gait, Michael J; El Andaloussi, Samir

    2015-04-01

    Oligonucleotide analogs have provided novel therapeutics targeting various disorders. However, their poor cellular uptake remains a major obstacle for their clinical development. Negatively charged oligonucleotides, such as 2'-O-Methyl RNA and locked nucleic acids have in recent years been delivered successfully into cells through complex formation with cationic polymers, peptides, liposomes, or similar nanoparticle delivery systems. However, due to the lack of electrostatic interactions, this promising delivery method has been unsuccessful to date using charge-neutral oligonucleotide analogs. We show here that lipid-functionalized cell-penetrating peptides can be efficiently exploited for cellular transfection of the charge-neutral oligonucleotide analog phosphorodiamidate morpholino. The lipopeptides form complexes with splice-switching phosphorodiamidate morpholino oligonucleotide and can be delivered into clinically relevant cell lines that are otherwise difficult to transfect while retaining biological activity. To our knowledge, this is the first study to show delivery through complex formation of biologically active charge-neutral oligonucleotides by cationic peptides. PMID:25594433

  16. Knockdown of ApoL1 in Zebrafish Larvae Affects the Glomerular Filtration Barrier and the Expression of Nephrin

    PubMed Central

    Kotb, Ahmed M.; Simon, Ole; Blumenthal, Antje; Vogelgesang, Silke; Dombrowski, Frank; Amann, Kerstin; Zimmermann, Uwe; Endlich, Karlhans; Endlich, Nicole

    2016-01-01

    APOL1, a secreted high-density lipoprotein, is expressed in different human tissues. Genetic variants of APOL1 are described to be associated with the development of end stage renal diseases in African Americans. In human kidney, APOL1 is mainly expressed in podocytes that are responsible for proper blood filtration. Since mice do not express ApoL1, the zebrafish is an ideal model to study the role of ApoL1. Injection of morpholinos against zApoL1 into zebrafish eggs and larvae, respectively, induces severe edema indicating a leakage of the filtration barrier. This was demonstrated in zApoL1 knockdown larvae by intravascular injection of fluorescently-labeled 10- and 500-kDa dextrans and by clearance of the vitamin D-binding protein from the circulation. Immunohistochemistry and RT-PCR revealed the reduction of nephrin, a podocyte-specific protein essential for blood filtration. Coinjection of human nephrin mRNA rescued the zApoL1 knockdown induced phenotype. Reduced APOL1 and nephrin levels were also found in biopsies of patients suffering from end stage renal diseases. Our results demonstrate that zApoL1 is essential for proper blood filtration in the zebrafish glomerulus and that zApoL1 affects the expression of nephrin. PMID:27138898

  17. Knockdown of ApoL1 in Zebrafish Larvae Affects the Glomerular Filtration Barrier and the Expression of Nephrin.

    PubMed

    Kotb, Ahmed M; Simon, Ole; Blumenthal, Antje; Vogelgesang, Silke; Dombrowski, Frank; Amann, Kerstin; Zimmermann, Uwe; Endlich, Karlhans; Endlich, Nicole

    2016-01-01

    APOL1, a secreted high-density lipoprotein, is expressed in different human tissues. Genetic variants of APOL1 are described to be associated with the development of end stage renal diseases in African Americans. In human kidney, APOL1 is mainly expressed in podocytes that are responsible for proper blood filtration. Since mice do not express ApoL1, the zebrafish is an ideal model to study the role of ApoL1. Injection of morpholinos against zApoL1 into zebrafish eggs and larvae, respectively, induces severe edema indicating a leakage of the filtration barrier. This was demonstrated in zApoL1 knockdown larvae by intravascular injection of fluorescently-labeled 10- and 500-kDa dextrans and by clearance of the vitamin D-binding protein from the circulation. Immunohistochemistry and RT-PCR revealed the reduction of nephrin, a podocyte-specific protein essential for blood filtration. Coinjection of human nephrin mRNA rescued the zApoL1 knockdown induced phenotype. Reduced APOL1 and nephrin levels were also found in biopsies of patients suffering from end stage renal diseases. Our results demonstrate that zApoL1 is essential for proper blood filtration in the zebrafish glomerulus and that zApoL1 affects the expression of nephrin. PMID:27138898

  18. Dual Myostatin and Dystrophin Exon Skipping by Morpholino Nucleic Acid Oligomers Conjugated to a Cell-penetrating Peptide Is a Promising Therapeutic Strategy for the Treatment of Duchenne Muscular Dystrophy

    PubMed Central

    Malerba, Alberto; Kang, Jagjeet K; McClorey, Graham; Saleh, Amer F; Popplewell, Linda; Gait, Michael J; Wood, Matthew JA; Dickson, George

    2012-01-01

    The knockdown of myostatin, a negative regulator of skeletal muscle mass may have important implications in disease conditions accompanied by muscle mass loss like cancer, HIV/AIDS, sarcopenia, muscle atrophy, and Duchenne muscular dystrophy (DMD). In DMD patients, where major muscle loss has occurred due to a lack of dystrophin, the therapeutic restoration of dystrophin expression alone in older patients may not be sufficient to restore the functionality of the muscles. We recently demonstrated that phosphorodiamidate morpholino oligomers (PMOs) can be used to re-direct myostatin splicing and promote the expression of an out-of-frame transcript so reducing the amount of the synthesized myostatin protein. Furthermore, the systemic administration of the same PMO conjugated to an octaguanidine moiety (Vivo-PMO) led to a significant increase in the mass of soleus muscle of treated mice. Here, we have further optimized the use of Vivo-PMO in normal mice and also tested the efficacy of the same PMO conjugated to an arginine-rich cell-penetrating peptide (B-PMO). Similar experiments conducted in mdx dystrophic mice showed that B-PMO targeting myostatin is able to significantly increase the tibialis anterior (TA) muscle weight and when coadministered with a B-PMO targeting the dystrophin exon 23, it does not have a detrimental interaction. This study confirms that myostatin knockdown by exon skipping is a potential therapeutic strategy to counteract muscle wasting conditions and dual myostatin and dystrophin skipping has potential as a therapy for DMD. PMID:23250360

  19. Biodegradable charged polyester-based vectors (BCPVs) as an efficient non-viral transfection nanoagent for gene knockdown of the BCR-ABL hybrid oncogene in a human chronic myeloid leukemia cell line

    NASA Astrophysics Data System (ADS)

    Yang, Chengbin; Panwar, Nishtha; Wang, Yucheng; Zhang, Butian; Liu, Maixian; Toh, Huiting; Yoon, Ho Sup; Tjin, Swee Chuan; Chong, Peter Han Joo; Law, Wing-Cheung; Chen, Chih-Kuang; Yong, Ken-Tye

    2016-04-01

    First-line therapy of chronic myelogenous leukemia (CML) has always involved the use of BCR-ABL tyrosine-kinase inhibitors which is associated with an abnormal chromosome called Philadelphia chromosome. Although the overall survival rate has been improved by the current therapeutic regime, the presence of resistance has resulted in limited efficacy. In this study, an RNA interference (RNAi)-based therapeutic regime is proposed with the aim to knockdown the BCR-ABL hybrid oncogene using small interfering RNA (siRNA). The siRNA transfection rates have usually been limited due to the declining contact probability among polyplexes and the non-adherent nature of leukemic cells. Our work aims at addressing this limitation by using a biodegradable charged polyester-based vector (BCPV) as a nanocarrier for the delivery of BCR-ABL-specific siRNA to the suspension culture of a K562 CML cell line. BCR-ABL siRNAs were encapsulated in the BCPVs by electrostatic force. Cell internalization was facilitated by the BCPV and assessed by confocal microscopy and flow cytometry. The regulation of the BCR-ABL level in K562 cells as a result of RNAi was analyzed by real-time polymerase chain reaction (RT-PCR). We observed that BCPV was able to form stable nanoplexes with siRNA molecules, even in the presence of fetal bovine serum (FBS), and successfully assisted in vitro siRNA transfection in the non-adherent K562 cells. As a consequence of downregulation of BCR-ABL, BCPV-siRNA nanoplexes inhibited cell proliferation and promoted cell apoptosis. All results were compared with a commercial transfection reagent, Lipofectamine2000™, which served as a positive control. More importantly, this class of non-viral vector exhibits biodegradable features and negligible cytotoxicity, thus providing a versatile platform to deliver siRNA to non-adherent leukemia cells with high transfection efficiency by effectively overcoming extra- and intra-cellular barriers. Due to the excellent in vitro

  20. Biodegradable charged polyester-based vectors (BCPVs) as an efficient non-viral transfection nanoagent for gene knockdown of the BCR-ABL hybrid oncogene in a human chronic myeloid leukemia cell line.

    PubMed

    Yang, Chengbin; Panwar, Nishtha; Wang, Yucheng; Zhang, Butian; Liu, Maixian; Toh, Huiting; Yoon, Ho Sup; Tjin, Swee Chuan; Chong, Peter Han Joo; Law, Wing-Cheung; Chen, Chih-Kuang; Yong, Ken-Tye

    2016-04-28

    First-line therapy of chronic myelogenous leukemia (CML) has always involved the use of BCR-ABL tyrosine-kinase inhibitors which is associated with an abnormal chromosome called Philadelphia chromosome. Although the overall survival rate has been improved by the current therapeutic regime, the presence of resistance has resulted in limited efficacy. In this study, an RNA interference (RNAi)-based therapeutic regime is proposed with the aim to knockdown the BCR-ABL hybrid oncogene using small interfering RNA (siRNA). The siRNA transfection rates have usually been limited due to the declining contact probability among polyplexes and the non-adherent nature of leukemic cells. Our work aims at addressing this limitation by using a biodegradable charged polyester-based vector (BCPV) as a nanocarrier for the delivery of BCR-ABL-specific siRNA to the suspension culture of a K562 CML cell line. BCR-ABL siRNAs were encapsulated in the BCPVs by electrostatic force. Cell internalization was facilitated by the BCPV and assessed by confocal microscopy and flow cytometry. The regulation of the BCR-ABL level in K562 cells as a result of RNAi was analyzed by real-time polymerase chain reaction (RT-PCR). We observed that BCPV was able to form stable nanoplexes with siRNA molecules, even in the presence of fetal bovine serum (FBS), and successfully assisted in vitro siRNA transfection in the non-adherent K562 cells. As a consequence of downregulation of BCR-ABL, BCPV-siRNA nanoplexes inhibited cell proliferation and promoted cell apoptosis. All results were compared with a commercial transfection reagent, Lipofectamine2000™, which served as a positive control. More importantly, this class of non-viral vector exhibits biodegradable features and negligible cytotoxicity, thus providing a versatile platform to deliver siRNA to non-adherent leukemia cells with high transfection efficiency by effectively overcoming extra- and intra-cellular barriers. Due to the excellent in vitro

  1. The use of urinary and kidney SILAM proteomics to monitor kidney response to high dose morpholino oligonucleotides in the mdx mouse

    PubMed Central

    Zhang, Aiping; Uaesoontrachoon, Kitipong; Shaughnessy, Conner; Das, Jharna R.; Rayavarapu, Sree; Brown, Kristy J; Ray, Patricio E.; Nagaraju, Kanneboyina; van den Anker, John N.; Hoffman, Eric P; Hathout, Yetrib

    2015-01-01

    Phosphorodiamidate morpholino oligonucleotides (PMO) are used as a promising exon-skipping gene therapy for Duchenne Muscular Dystrophy (DMD). One potential complication of high dose PMO therapy is its transient accumulation in the kidneys. Therefore new urinary biomarkers are needed to monitor this treatment. Here, we carried out a pilot proteomic profiling study using stable isotope labeling in mammals (SILAM) strategy to identify new biomarkers to monitor the effect of PMO on the kidneys of the dystrophin deficient mouse model for DMD (mdx-23). We first assessed the baseline renal status of the mdx-23 mouse compared to the wild type (C57BL10) mouse, and then followed the renal outcome of mdx-23 mouse treated with a single high dose intravenous PMO injection (800 mg/kg). Surprisingly, untreated mdx-23 mice showed evidence of renal injury at baseline, which was manifested by albuminuria, increased urine output, and changes in established urinary biomarker of acute kidney injury (AKI). The PMO treatment induced further transient renal injury, which peaked at 7 days, and returned to almost the baseline status at 30 days post-treatment. In the kidney, the SILAM approach followed by western blot validation identified changes in Meprin A subunit alpha at day 2, then returned to normal levels at day 7 and 30 after PMO injection. In the urine, SILAM approach identified an increase in Clusterin and γ-glutamyl transpeptidase 1 as potential candidates to monitor the transient renal accumulation of PMO. These results, which were confirmed by Western blots or ELISA, demonstrate the value of the SILAM approach to identify new candidate biomarkers of renal injury in mdx-23 mice treated with high dose PMO. Chemical compounds studied in this article: Phosphorodiamidate morpholino (PubChem CID: 22140692); isoflurane (PubChem CID: 3763); formic acid (PubChem CID: 284); acetonitrile (PubChem CID: 6342); acetone (PubChem CID: 180); methanol (PubChem CID: 887) PMID:26213685

  2. Effective Dystrophin Restoration by a Novel Muscle-Homing Peptide–Morpholino Conjugate in Dystrophin-Deficient mdx Mice

    PubMed Central

    Gao, Xianjun; Zhao, Jingwen; Han, Gang; Zhang, Yajie; Dong, Xue; Cao, Limin; Wang, Qingsong; Moulton, Hong M; Yin, HaiFang

    2014-01-01

    Antisense oligonucleotide (AO)–mediated splice correction therapy for Duchenne muscular dystrophy has shown huge promise from recent phase 2b clinical trials, however high doses and costs are required and targeted delivery can lower both of these. We have previously demonstrated the feasibility of targeted delivery of AOs by conjugating a chimeric peptide, consisting of a muscle-specific peptide and a cell-penetrating peptide, to AOs in mdx mice. Although increased uptake in muscle was observed, the majority of peptide–AO conjugate was found in the liver. To search for more effective muscle-homing peptides, we carried out in vitro biopanning in myoblasts and identified a novel 12-mer peptide (M12) showing preferential binding to skeletal muscle compared to the liver. When conjugated to phosphorodiamidate morpholino oligomers, ~25% of normal level of dystrophin expression was achieved in body-wide skeletal muscles in mdx mice with significant recovery in grip strength, whereas <2% in corresponding tissues treated with either muscle-specific peptide–phosphorodiamidate morpholino oligomer or unmodified phosphorodiamidate morpholino oligomer under identical conditions. Our data provide evidences for the first time that a muscle-homing peptide alone can enhance AO delivery to muscle without appreciable toxicity at 75 mg/kg, suggesting M12-phosphorodiamidate morpholino oligomer can be an alternative option to current AOs. PMID:24732757

  3. Knockdown resistance in pyrethroid-resistant horn fly (Diptera: Muscidae) populations in Brazil

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To investigate the kdr (knockdown resistance) resistance-associated gene mutation and determine its frequency in pyrethroid-resistant horn fly (Haematobia irritans) populations, a total of 1,804 horn flies of 37 different populations from all Brazilian regions (North, Northeast, Central-West, Southe...

  4. Overexpression or knockdown of rat tryptophan hyroxylase-2 has opposing effects on anxiety behavior in an estrogen-dependent manner

    PubMed Central

    Hiroi, Ryoko; McDevitt, Ross A.; Morcos, Paul A.; Clark, Michael S.; Neumaier, John F.

    2010-01-01

    Previous studies showed that chronic estrogen treatment increases tryptophan hydroxylase-2 (TpH2) mRNA in the caudal dorsal raphe nucleus (DRN), and this increase was associated with decreased anxiety. The present study explored the interaction of estrogen and targeted, bidirectional manipulation of TpH2 expression in the caudal DRN by knockdown or viral overexpression, to decrease or increase tryptophan hydroxylase expression respectively, on anxiety behavior. Rats were ovariectomized and replaced with empty or estradiol capsules (OVX, OVX/E, respectively). Animals received microinfusions of either antisense TpH2 or control morpholino oligonucleotides into caudal DRN and were later tested in the open field test. A separate group of animals were microinfused with TpH2-GFP or GFP-only herpes simplex viral vectors into caudal DRN and tested in the open field. The bidirectional impact of manipulations on TpH2 expression was confirmed using a combination of quantitative protein and mRNA measurements; TpH2 expression changes were limited to discrete subregions of DRN that were targeted by the manipulations. Estradiol decreased anxiety in all behavioral measures. In the OVX/E group, TpH2 knockdown significantly decreased time spent in the center of the open field, but not in the OVX group, suggesting that TpH2 knockdown reduced the anxiolytic effects of estrogen. Conversely, TpH2 overexpression in the OVX group mimicked the effects of estrogen, as measured by increased time spent in the center of the open field. These results suggest that estrogen and TpH2 in the caudal DRN have a critical interaction in regulating anxiety-like behavior. PMID:21182901

  5. Neurogenic gene regulatory pathways in the sea urchin embryo.

    PubMed

    Wei, Zheng; Angerer, Lynne M; Angerer, Robert C

    2016-01-15

    During embryogenesis the sea urchin early pluteus larva differentiates 40-50 neurons marked by expression of the pan-neural marker synaptotagmin B (SynB) that are distributed along the ciliary band, in the apical plate and pharyngeal endoderm, and 4-6 serotonergic neurons that are confined to the apical plate. Development of all neurons has been shown to depend on the function of Six3. Using a combination of molecular screens and tests of gene function by morpholino-mediated knockdown, we identified SoxC and Brn1/2/4, which function sequentially in the neurogenic regulatory pathway and are also required for the differentiation of all neurons. Misexpression of Brn1/2/4 at low dose caused an increase in the number of serotonin-expressing cells and at higher dose converted most of the embryo to a neurogenic epithelial sphere expressing the Hnf6 ciliary band marker. A third factor, Z167, was shown to work downstream of the Six3 and SoxC core factors and to define a branch specific for the differentiation of serotonergic neurons. These results provide a framework for building a gene regulatory network for neurogenesis in the sea urchin embryo. PMID:26657764

  6. Evidence From Human and Zebrafish That GPC1 Is a Biliary Atresia Susceptibility Gene

    PubMed Central

    Cui, Shuang; Leyva-Vega, Melissa; Tsai, Ellen A.; Eauclaire, Steven F.; Glessner, Joseph T.; Hakonarson, Hakon; Devoto, Marcella; Haber, Barbara A.; Spinner, Nancy B.; Matthews, Randolph P.

    2013-01-01

    BACKGROUND & AIMS Biliary atresia (BA) is a progressive fibroinflammatory disorder of infants involving the extrahepatic and intrahepatic biliary tree. Its etiology is unclear but is believed to involve exposure of a genetically susceptible individual to certain environmental factors. BA occurs exclusively in the neonatal liver, so variants of genes expressed during hepatobiliary development could affect susceptibility. Genome-wide association studies previously identified a potential region of interest at 2q37. We continued these studies to narrow the region and identify BA susceptibility genes. METHODS We searched for copy number variants that were increased among patients with BA (n = 61) compared with healthy individuals (controls; n = 5088). After identifying a candidate gene, we investigated expression patterns of orthologues in zebrafish liver and the effects of reducing expression, with morpholino antisense oligonucleotides, on biliary development, gene expression, and signal transduction. RESULTS We observed a statistically significant increase in deletions at 2q37.3 in patients with BA that resulted in deletion of one copy of GPC1, which encodes glypican 1, a heparan sulfate proteoglycan that regulates Hedgehog signaling and inflammation. Knockdown of gpc1 in zebrafish led to developmental biliary defects. Exposure of the gpc1 morphants to cyclopamine, a Hedgehog antagonist, partially rescued the gpc1-knockdown phenotype. Injection of zebrafish with recombinant Sonic Hedgehog led to biliary defects similar to those of the gpc1 morphants. Liver samples from patients with BA had reduced levels of apical GPC1 in cholangiocytes compared with samples from controls. CONCLUSIONS Based on genetic analysis of patients with BA and zebrafish, GPC1 appears to be a BA susceptibility gene. These findings also support a role for Hedgehog signaling in the pathogenesis of BA. PMID:23336978

  7. Knockdown of Pokemon protein expression inhibits hepatocellular carcinoma cell proliferation by suppression of AKT activity.

    PubMed

    Zhu, Xiaosan; Dai, Yichen; Chen, Zhangxin; Xie, Junpei; Zeng, Wei; Lin, Yuanyuan

    2013-01-01

    Overexpression of Pokemon, which is an erythroid myeloid ontogenic factor protein, occurs in different cancers, including hepatocellular carcinoma (HCC). Pokemon is also reported to have an oncogenic activity in various human cancers. This study investigated the effect of Pokemon knockdown on the regulation of HCC growth. POK shRNA suppressed the expression of Pokemon protein in HepG2 cells compared to the negative control vector-transfected HCC cells. Pokemon knockdown also reduced HCC cell viability and enhanced cisplatin-induced apoptosis in HCC cells. AKT activation and the expression of various cell cycle-related genes were inhibited following Pokemon knockdown. These data demonstrate that Pokemon may play a role in HCC progression, suggesting that inhibition of Pokemon expression using Pokemon shRNA should be further evaluated as a novel target for the control of HCC. PMID:23924858

  8. FTO knockdown in rat ventromedial hypothalamus does not affect energy balance

    PubMed Central

    van Gestel, Margriet A.; Sanders, Loek E.; de Jong, Johannes W.; Luijendijk, Mieneke C. M.; Adan, Roger A. H.

    2014-01-01

    Abstract Single nucleotide polymorphisms (SNPs) clustered in the first intron of the fat mass and obesity‐associated (FTO) gene has been associated with obesity. FTO expression is ubiquitous, with particularly high levels in the hypothalamic area of the brain. To investigate the region‐specific role of FTO, AAV technology was applied to knockdown FTO in the ventromedial hypothalamus (VMH). No effect of FTO knockdown was observed on bodyweight or parameters of energy balance. Animals were exposed twice to an overnight fast, followed by a high‐fat high‐sucrose (HFHS) diet for 1 week. FTO knockdown did not result in a different response to the diets. A region‐specific role for FTO in the VMH in the regulation of energy balance could not be found. PMID:25501432

  9. Association between Three Mutations, F1565C, V1023G and S996P, in the Voltage-Sensitive Sodium Channel Gene and Knockdown Resistance in Aedes aegypti from Yogyakarta, Indonesia.

    PubMed

    Wuliandari, Juli Rochmijati; Lee, Siu Fai; White, Vanessa Linley; Tantowijoyo, Warsito; Hoffmann, Ary Anthony; Endersby-Harshman, Nancy Margaret

    2015-01-01

    Mutations in the voltage-sensitive sodium channel gene (Vssc) have been identified in Aedes aegypti and some have been associated with pyrethroid insecticide resistance. Whether these mutations cause resistance, alone or in combination with other alleles, remains unclear, but must be understood if mutations are to become markers for resistance monitoring. We describe High Resolution Melt (HRM) genotyping assays for assessing mutations found in Ae. aegypti in Indonesia (F1565C, V1023G, S996P) and use them to test for associations with pyrethroid resistance in mosquitoes from Yogyakarta, a city where insecticide use is widespread. Such knowledge is important because Yogyakarta is a target area for releases of Wolbachia-infected mosquitoes with virus-blocking traits for dengue suppression. We identify three alleles across Yogyakarta putatively linked to resistance in previous research. By comparing resistant and susceptible mosquitoes from bioassays, we show that the 1023G allele is associated with resistance to type I and type II pyrethroids. In contrast, F1565C homozygotes were rare and there was only a weak association between individuals heterozygous for the mutation and resistance to a type I pyrethroid. As the heterozygote is expected to be incompletely recessive, it is likely that this association was due to a different resistance mechanism being present. A resistance advantage conferred to V1023G homozygotes through addition of the S996P allele in the homozygous form was suggested for the Type II pyrethroid, deltamethrin. Screening of V1023G and S996P should assist resistance monitoring in Ae. aegypti from Yogyakarta, and these mutations should be maintained in Wolbachia strains destined for release in this city to ensure that these virus-blocking strains of mosquitoes are not disadvantaged, relative to resident populations. PMID:26463408

  10. Association between Three Mutations, F1565C, V1023G and S996P, in the Voltage-Sensitive Sodium Channel Gene and Knockdown Resistance in Aedes aegypti from Yogyakarta, Indonesia

    PubMed Central

    Rochmijati Wuliandari, Juli; Lee, Siu Fai; White, Vanessa Linley; Tantowijoyo, Warsito; Hoffmann, Ary Anthony; Endersby-Harshman, Nancy Margaret

    2015-01-01

    Mutations in the voltage-sensitive sodium channel gene (Vssc) have been identified in Aedes aegypti and some have been associated with pyrethroid insecticide resistance. Whether these mutations cause resistance, alone or in combination with other alleles, remains unclear, but must be understood if mutations are to become markers for resistance monitoring. We describe High Resolution Melt (HRM) genotyping assays for assessing mutations found in Ae. aegypti in Indonesia (F1565C, V1023G, S996P) and use them to test for associations with pyrethroid resistance in mosquitoes from Yogyakarta, a city where insecticide use is widespread. Such knowledge is important because Yogyakarta is a target area for releases of Wolbachia-infected mosquitoes with virus-blocking traits for dengue suppression. We identify three alleles across Yogyakarta putatively linked to resistance in previous research. By comparing resistant and susceptible mosquitoes from bioassays, we show that the 1023G allele is associated with resistance to type I and type II pyrethroids. In contrast, F1565C homozygotes were rare and there was only a weak association between individuals heterozygous for the mutation and resistance to a type I pyrethroid. As the heterozygote is expected to be incompletely recessive, it is likely that this association was due to a different resistance mechanism being present. A resistance advantage conferred to V1023G homozygotes through addition of the S996P allele in the homozygous form was suggested for the Type II pyrethroid, deltamethrin. Screening of V1023G and S996P should assist resistance monitoring in Ae. aegypti from Yogyakarta, and these mutations should be maintained in Wolbachia strains destined for release in this city to ensure that these virus-blocking strains of mosquitoes are not disadvantaged, relative to resident populations. PMID:26463408

  11. The NDST gene family in zebrafish: role of NDST1B in pharyngeal arch formation.

    PubMed

    Filipek-Górniok, Beata; Carlsson, Pernilla; Haitina, Tatjana; Habicher, Judith; Ledin, Johan; Kjellén, Lena

    2015-01-01

    Heparan sulfate (HS) proteoglycans are ubiquitous components of the extracellular matrix and plasma membrane of metazoans. The sulfation pattern of the HS glycosaminoglycan chain is characteristic for each tissue and changes during development. The glucosaminyl N-deacetylase/N-sulfotransferase (NDST) enzymes catalyze N-deacetylation and N-sulfation during HS biosynthesis and have a key role in designing the sulfation pattern. We here report on the presence of five NDST genes in zebrafish. Zebrafish ndst1a, ndst1b, ndst2a and ndst2b represent duplicated mammalian orthologues of NDST1 and NDST2 that arose through teleost specific genome duplication. Interestingly, the single zebrafish orthologue ndst3, is equally similar to tetrapod Ndst3 and Ndst4. It is likely that a local duplication in the common ancestor of lobe-finned fish and tetrapods gave rise to these two genes. All zebrafish Ndst genes showed distinct but partially overlapping expression patterns during embryonic development. Morpholino knockdown of ndst1b resulted in delayed development, craniofacial cartilage abnormalities, shortened body and pectoral fin length, resembling some of the features of the Ndst1 mouse knockout. PMID:25767878

  12. The Ndst Gene Family in Zebrafish: Role of Ndst1b in Pharyngeal Arch Formation

    PubMed Central

    Haitina, Tatjana; Habicher, Judith; Ledin, Johan; Kjellén, Lena

    2015-01-01

    Heparan sulfate (HS) proteoglycans are ubiquitous components of the extracellular matrix and plasma membrane of metazoans. The sulfation pattern of the HS glycosaminoglycan chain is characteristic for each tissue and changes during development. The glucosaminyl N-deacetylase/N-sulfotransferase (NDST) enzymes catalyze N-deacetylation and N-sulfation during HS biosynthesis and have a key role in designing the sulfation pattern. We here report on the presence of five NDST genes in zebrafish. Zebrafish ndst1a, ndst1b, ndst2a and ndst2b represent duplicated mammalian orthologues of NDST1 and NDST2 that arose through teleost specific genome duplication. Interestingly, the single zebrafish orthologue ndst3, is equally similar to tetrapod Ndst3 and Ndst4. It is likely that a local duplication in the common ancestor of lobe-finned fish and tetrapods gave rise to these two genes. All zebrafish Ndst genes showed distinct but partially overlapping expression patterns during embryonic development. Morpholino knockdown of ndst1b resulted in delayed development, craniofacial cartilage abnormalities, shortened body and pectoral fin length, resembling some of the features of the Ndst1 mouse knockout. PMID:25767878

  13. Prediction of siRNA knockdown efficiency using artificial neural network models

    SciTech Connect

    Ge Guangtao . E-mail: guge@eecs.tufts.edu; Wong, G.William . E-mail: wong@wi.mit.edu; Luo Biao . E-mail: bluo@broad.mit.edu

    2005-10-21

    Selective knockdown of gene expression by short interference RNAs (siRNAs) has allowed rapid validation of gene functions and made possible a high throughput, genome scale approach to interrogate gene function. However, randomly designed siRNAs display different knockdown efficiencies of target genes. Hence, various prediction algorithms based on siRNA functionality have recently been constructed to increase the likelihood of selecting effective siRNAs, thereby reducing the experimental cost. Toward this end, we have trained three Back-propagation and Bayesian neural network models, previously not used in this context, to predict the knockdown efficiencies of 180 experimentally verified siRNAs on their corresponding target genes. Using our input coding based primarily on RNA structure thermodynamic parameters and cross-validation method, we showed that our neural network models outperformed most other methods and are comparable to the best predicting algorithm thus far published. Furthermore, our neural network models correctly classified 74% of all siRNAs into different efficiency categories; with a correlation coefficient of 0.43 and receiver operating characteristic curve score of 0.78, thus highlighting the potential utility of this method to complement other existing siRNA classification and prediction schemes.

  14. ETV4 and Myeov knockdown impairs colon cancer cell line proliferation and invasion

    SciTech Connect

    Moss, Alan C. . E-mail: amoss@bidmc.harvard.edu; Lawlor, Garrett; Murray, David; Tighe, Donal; Madden, Stephen F.; Mulligan, Anne-Marie; Keane, Conor O.; Brady, Hugh R.; Doran, Peter P.; MacMathuna, Padraic

    2006-06-23

    We have identified novel colorectal cancer-associated genes using NCBI's UNIGENE cDNA libraries. Colon cancer libraries were examined using Digital Differential Display and disease-associated genes were selected. Among these were ETV4 and MYEOV, novel colorectal cancer-associated genes. Samples of matched normal and neoplastic colon were obtained from human subjects and gene expression was quantified using real-time PCR. ETV4 gene expression was significantly increased in colonic neoplasia in comparison to matched normal colonic tissue (p < 0.05). Myeov expression was also increased in colon neoplasia in comparison to matched normal tissue. The effect of siRNA-mediated knockdown of ETV4 and Myeov on cell proliferation and invasion was assessed. ETV4 knockdown resulted in a 90% decrease in cell proliferation (p < 0.05) and a 67% decrease in cell invasion. Myeov knockdown resulted in a 48% decrease in cell proliferation (p < 0.05) and a 36% decrease in cell invasion. These data suggest that ETV4 and Myeov may provide novel targets for therapeutic intervention.

  15. The functional genetic link of NLGN4X knockdown and neurodevelopment in neural stem cells

    PubMed Central

    Shi, Lingling; Chang, Xiao; Zhang, Peilin; Coba, Marcelo P.; Lu, Wange; Wang, Kai

    2013-01-01

    Genetic mutations in NLGN4X (neuroligin 4), including point mutations and copy number variants (CNVs), have been associated with susceptibility to autism spectrum disorders (ASDs). However, it is unclear how mutations in NLGN4X result in neurodevelopmental defects. Here, we used neural stem cells (NSCs) as in vitro models to explore the impacts of NLGN4X knockdown on neurodevelopment. Using two shRNAmir-based vectors targeting NLGN4X and one control shRNAmir vector, we modulated NLGN4X expression and differentiated these NSCs into mature neurons. We monitored the neurodevelopmental process at Weeks 0, 0.5, 1, 2, 4 and 6, based on morphological analysis and whole-genome gene expression profiling. At the cellular level, in NSCs with NLGN4X knockdown, we observed increasingly delayed neuronal development and compromised neurite formation, starting from Week 2 through Week 6 post differentiation. At the molecular level, we identified multiple pathways, such as neurogenesis, neuron differentiation and muscle development, which are increasingly disturbed in cells with NLGN4X knockdown. Notably, several postsynaptic genes, including DLG4, NLGN1 and NLGN3, also have decreased expression. Based on in vitro models, NLGN4X knockdown directly impacts neurodevelopmental process during the formation of neurons and their connections. Our functional genomics study highlights the utility of NSCs models in understanding the functional roles of CNVs in affecting neurodevelopment and conferring susceptibility to neurodevelopmental diseases. PMID:23710042

  16. A Simple Retroelement Based Knock-Down System in Dictyostelium: Further Insights into RNA Interference Mechanisms

    PubMed Central

    Schuster, Isabelle; Nellen, Wolfgang

    2015-01-01

    Characteristics of DIRS-1 Mediated Knock-Downs We have previously shown that the most abundant Dictyostelium discoideum retroelement DIRS-1 is suppressed by RNAi mechanisms. Here we provide evidence that both inverted terminal repeats have strong promoter activity and that bidirectional expression apparently generates a substrate for Dicer. A cassette containing the inverted terminal repeats and a fragment of a gene of interest was sufficient to activate the RNAi response, resulting in the generation of ~21 nt siRNAs, a reduction of mRNA and protein expression of the respective endogene. Surprisingly, no transitivity was observed on the endogene. This was in contrast to previous observations, where endogenous siRNAs caused spreading on an artificial transgene. Knock-down was successful on seven target genes that we examined. In three cases a phenotypic analysis proved the efficiency of the approach. One of the target genes was apparently essential because no knock-out could be obtained; the RNAi mediated knock-down, however, resulted in a very slow growing culture indicating a still viable reduction of gene expression. Advantages of the DIRS-1–RNAi System The knock-down system required a short DNA fragment (~400 bp) of the target gene as an initial trigger. Further siRNAs were generated by RdRPs since we have shown some siRNAs with a 5’-triphosphate group. Extrachromosomal vectors facilitate the procedure and allowed for molecular and phenotypic analysis within one week. The system provides an efficient and rapid method to reduce protein levels including those of essential genes. PMID:26110905

  17. Specific functions of the Wnt signaling system in gene regulatory networks throughout the early sea urchin embryo

    PubMed Central

    Cui, Miao; Siriwon, Natnaree; Li, Enhu; Davidson, Eric H.; Peter, Isabelle S.

    2014-01-01

    Wnt signaling affects cell-fate specification processes throughout embryonic development. Here we take advantage of the well-studied gene regulatory networks (GRNs) that control pregastrular sea urchin embryogenesis to reveal the gene regulatory functions of the entire Wnt-signaling system. Five wnt genes, three frizzled genes, two secreted frizzled-related protein 1 genes, and two Dickkopf genes are expressed in dynamic spatial patterns in the pregastrular embryo of Strongylocentrotus purpuratus. We present a comprehensive analysis of these genes in each embryonic domain. Total functions of the Wnt-signaling system in regulatory gene expression throughout the embryo were studied by use of the Porcupine inhibitor C59, which interferes with zygotic Wnt ligand secretion. Morpholino-mediated knockdown of each expressed Wnt ligand demonstrated that individual Wnt ligands are functionally distinct, despite their partially overlapping spatial expression. They target specific embryonic domains and affect particular regulatory genes. The sum of the effects of blocking expression of individual wnt genes is shown to equal C59 effects. Remarkably, zygotic Wnt-signaling inputs are required for only three general aspects of embryonic specification: the broad activation of endodermal GRNs, the regional specification of the immediately adjacent stripe of ectoderm, and the restriction of the apical neurogenic domain. All Wnt signaling in this pregastrular embryo is short range (and/or autocrine). Furthermore, we show that the transcriptional drivers of wnt genes execute important specification functions in the embryonic domains targeted by the ligands, thus connecting the expression and function of wnt genes by encoded cross-regulatory interactions within the specific regional GRNs. PMID:25385617

  18. The zebrafish dog-eared mutation disrupts eya1, a gene required for cell survival and differentiation in the inner ear and lateral line.

    PubMed

    Kozlowski, David J; Whitfield, Tanya T; Hukriede, Neil A; Lam, Wai K; Weinberg, Eric S

    2005-01-01

    To understand the molecular basis of sensory organ development and disease, we have cloned and characterized the zebrafish mutation dog-eared (dog) that is defective in formation of the inner ear and lateral line sensory systems. The dog locus encodes the eyes absent-1 (eya1) gene and single point mutations were found in three independent dog alleles, each prematurely truncating the expressed protein within the Eya domain. Moreover, morpholino-mediated knockdown of eya1 gene function phenocopies the dog-eared mutation. In zebrafish, the eya1 gene is widely expressed in placode-derived sensory organs during embryogenesis but Eya1 function appears to be primarily required for survival of sensory hair cells in the developing ear and lateral line neuromasts. Increased levels of apoptosis occur in the migrating primordia of the posterior lateral line in dog embryos and as well as in regions of the developing otocyst that are mainly fated to give rise to sensory cells of the cristae. Importantly, mutation of the EYA1 or EYA4 gene causes hereditary syndromic deafness in humans. Determination of eya gene function during zebrafish organogenesis will facilitate understanding the molecular etiology of human vestibular and hearing disorders. PMID:15572137

  19. Hunchback knockdown induces supernumerary segment formation in Bombyx.

    PubMed

    Nakao, Hajime

    2016-05-15

    Insect segment number within species appears to be fixed irrespective of germ types: long vs. short/intermediate. The present study showed induction of supernumerary segment formation by the knockdown of Bombyx hunchback (Bm-hb), presumably by terminal segment addition, a short/intermediate-like-segmentation mode that is not observed in normal Bombyx embryogenesis. This suggests that Bm-hb suppresses segmentation. The results obtained also suggest that the gap gene Bm-Kr (Bombyx Krüppel) provides a permissive environment for the progression of segmentation by suppressing the expression Bm-hb, which terminates segmentation. This indicates a novel mechanism by which the gap gene is involved in segmentation. It appears that Bm-Kr and Bm-hb are involved in segment counting and their interplay contributes to the correct number of segments being formed in Bombyx. Similar mechanisms may be operating in insects that employ the non-Drosophilan mode of segmentation such as in short/intermediate-germ insects. PMID:27016505

  20. Inhibition of Hepatitis E Virus Replication by Peptide-Conjugated Morpholino Oligomers

    PubMed Central

    Nan, Yuchen; Ma, Zexu; Kannan, Harilakshmi; Stein, David A.; Iversen, Patrick I.; Meng, Xiang-Jin; Zhang, Yan-Jin

    2015-01-01

    Hepatitis E virus (HEV) infection is a cause of hepatitis in humans worldwide and has been associated with a mortality rate of up to 30% in pregnant women. Recently, persistent and chronic HEV infections have been recognized as a serious clinical problem, especially in immunocompromised individuals. To date, there are no FDA-approved HEV-specific antiviral drugs. In this study, we evaluated antisense peptide-conjugated morpholino oligomers (PPMO) designed against HEV genomic sequences as potential HEV-specific antiviral compounds. Two genetically-distinct strains of human HEV, genotype 1 Sar55 and genotype 3 Kernow-C1, isolated from patients with acute and chronic hepatitis, respectively, were used to evaluate inhibition of viral replication by PPMO in liver cells. The anti-HEV PPMO produced a significant reduction in the levels of HEV RNA and capsid protein, indicating effective inhibition of HEV replication. PPMO HP1, which targets a highly conserved sequence in the start site region of ORF1, was also effective against the genotype 3 Kernow-C1 strain in stably-infected HepG2/C3A liver cells. The antiviral activity observed was specific, dose-responsive and potent, suggesting that further exploration of PPMO HP1 as a potential HEV-specific antiviral agent is warranted. PMID:26086884

  1. Capacitive Monitoring of Morpholino-DNA Surface Hybridization: Experimental and Theoretical Analysis

    PubMed Central

    Tercero, Napoleon; Wang, Kang; Levicky, Rastislav

    2010-01-01

    Impedance and cyclic voltammetry methods, complemented by Poisson-Boltzmann (PB) modeling, are used to study hybridization of DNA analyte strands to monolayers of morpholino oligomers (MOs) immobilized by one end to mercaptopropanol-passivated gold electrodes. MOs, like peptide nucleic acids (PNAs), are uncharged molecules that recognize nucleic acids following conventional base-pairing rules. The capacitive response to hybridization, determined from real-time impedance measurements, is analyzed with emphasis on understanding the underlying structural changes and on providing a foundation for label-free diagnostics. The capacitive response is correlated with the instantaneous surface molecular populations by labeling DNA and MO strands with ferrocene tags and using cyclic voltammetry to monitor their respective coverages in real-time. This approach allows analysis of hybridization-induced changes in interfacial capacitance as a function of duplex coverage, the DC bias used for readout, buffer molarity, and probe coverage. The results indicate that unhybridized MO layers exist in a compact state on the solid support. For hybridized layers, the intrinsic signal per hybridization event is strongly enhanced at low ionic strengths but, interestingly, does not depend on the readout bias in the sampled range negative of the capacitive minimum. A PB model incorporating an effective medium description of the hybridizing films is used to establish how hybridization-derived changes in dielectric composition and charge distribution at the surface translate into experimentally-observed variations in interfacial capacitance. PMID:20690772

  2. Sustained Dystrophin Expression Induced by Peptide-conjugated Morpholino Oligomers in the Muscles of mdx Mice

    PubMed Central

    Jearawiriyapaisarn, Natee; Moulton, Hong M; Buckley, Brian; Roberts, Jennifer; Sazani, Peter; Fucharoen, Suthat; Iversen, Patrick L; Kole, Ryszard

    2009-01-01

    Cell-penetrating peptides (CPPs), containing arginine (R), 6-aminohexanoic acid (X), and/or β-alanine (B) conjugated to phosphorodiamidate morpholino oligomers (PMOs), enhance their delivery in cell culture. In this study, the potency, functional biodistribution, and toxicity of these conjugates were evaluated in vivo, in EGFP-654 transgenic mice that ubiquitously express the aberrantly spliced EGFP-654 pre-mRNA reporter. Correct splicing and enhanced green fluorescence protein (EGFP) upregulation serve as a positive readout for peptide-PMO (PPMO) entry into cells and access to EGFP-654 pre-mRNA in the nucleus. Intraperitoneal injections of a series of PPMOs, A-N (12 mg/kg), administered once a day for four successive days resulted in splicing correction in numerous tissues. PPMO-B was highly potent in the heart, diaphragm, and quadriceps, which are key muscles in the treatment of Duchenne muscular dystrophy. We therefore investigated PPMO M23D-B, designed to force skipping of stop-codon containing dystrophin exon 23, in an mdx mouse model of the disease. Systemic delivery of M23D-B yielded persistent exon 23 skipping, yielding high and sustained dystrophin protein expression in body-wide muscles, including cardiac muscle, without detectable toxicity. The rescued dystrophin reduced serum creatinine kinase to near-wild-type levels, indicating improvement in muscle integrity. This is the first report of oligonucleotide-mediated exon skipping and dystrophin protein induction in the heart of treated animals. PMID:18545222

  3. Morpholino-mediated SOD1 reduction ameliorates an amyotrophic lateral sclerosis disease phenotype

    PubMed Central

    Nizzardo, M.; Simone, C.; Rizzo, F.; Ulzi, G.; Ramirez, A.; Rizzuti, M.; Bordoni, A.; Bucchia, M.; Gatti, S.; Bresolin, N.; Comi, G. P.; Corti, S.

    2016-01-01

    Neurotoxicity due to the accumulation of mutant proteins is thought to drive pathogenesis in neurodegenerative diseases. Mutations in superoxide dismutase 1 (SOD1) are linked to familial amyotrophic lateral sclerosis (fALS); these mutations result in progressive motor neuron death through one or more acquired toxicities. Interestingly, SOD1 is not only responsible for fALS but may also play a significant role in sporadic ALS; therefore, SOD1 represents a promising therapeutic target. Here, we report slowed disease progression, improved neuromuscular function, and increased survival in an in vivo ALS model following therapeutic delivery of morpholino oligonucleotides (MOs) designed to reduce the synthesis of human SOD1. Neuropathological analysis demonstrated increased motor neuron and axon numbers and a remarkable reduction in astrogliosis and microgliosis. To test this strategy in a human model, we treated human fALS induced pluripotent stem cell (iPSC)-derived motor neurons with MOs; these cells exhibited increased survival and reduced expression of apoptotic markers. Our data demonstrated the efficacy of MO-mediated therapy in mouse and human ALS models, setting the stage for human clinical trials. PMID:26878886

  4. Polyethylenimine-modified pluronics (PCMs) improve morpholino oligomer delivery in cell culture and dystrophic mdx mice.

    PubMed

    Wang, Mingxing; Wu, Bo; Lu, Peijuan; Cloer, Caryn; Tucker, Jay D; Lu, Qilong

    2013-01-01

    We investigated a series of small-sized polyethylenimine (PEI, 0.8/1.2 k)-conjugated pluronic copolymers (PCMs) for their potential to enhance delivery of an antisense phosphorodiamidate morpholino oligomer (PMO) in vitro and in dystrophic mdx mice. PCM polymers containing pluronics of molecular weight (Mw) ranging 2-6 k, with hydrophilic-lipophilic balance (HLB) 7-23, significantly enhanced PMO-induced exon-skipping in a green fluorescent protein (GFP) reporter-based myoblast culture system. Application of optimized formulations of PCMs with PMO targeted to dystrophin exon 23 demonstrated a significant increase in exon-skipping efficiency in dystrophic mdx mice. Consistent with our observations in vitro, optimization of molecular size and the HLB of pluronics are important factors for PCMs to achieve enhanced PMO delivery in vivo. Observed cytotoxicity of the PCMs was lower than Endo-porter and PEI 25 k. Tissue toxicity of PCMs in muscle was not clearly detected with the concentrations used, indicating the potential of the PCMs as effective and safe PMO carriers for treating diseases such as muscular dystrophy. PMID:23164938

  5. In vivo knockdown of basal forebrain p75 neurotrophin receptor stimulates choline acetyltransferase activity in the mature hippocampus.

    PubMed

    Barrett, Graham L; Naim, Timur; Trieu, Jennifer; Huang, Mengjie

    2016-05-01

    This study seeks to determine whether knockdown of basal forebrain p75 neurotrophin receptor (p75(NTR) ) expression elicits increased hippocampal choline acetyltransferase (ChAT) activity in mature animals. Antisense (AS) oligonucleotides (oligos) targeting p75(NTR) were infused into the medial septal area of mature rats continuously for 4 weeks. In all rats, the cannula outlet was placed equidistant between the left and the right sides of the vertical diagonal band of Broca. We tested phosphorothioate (PS), morpholino (Mo), and gapmer (mixed PS/RNA) oligos. Gapmer AS infusions of 7.5 and 22 μg/day decreased septal p75(NTR) mRNA by 34% and 48%, respectively. The same infusions increased hippocampal ChAT activity by 41% and 55%. Increased hippocampal ChAT activity correlated strongly with septal p75(NTR) downregulation in individual rats. Infusions of PS and Mo AS oligos did not downregulate p75(NTR) mRNA or stimulate ChAT activity. These results demonstrate that p75(NTR) can dynamically regulate hippocampal ChAT activity in the mature CNS. They also reveal the different efficacies of three diverse AS oligo chemistries when infused intracerebrally. Among the three types, gapmer oligos worked best. PMID:26864466

  6. Knockdown of the T-box transcription factor Brachyury increases sensitivity of adenoid cystic carcinoma cells to chemotherapy and radiation in vitro: Implications for a new therapeutic principle

    PubMed Central

    KOBAYASHI, YOSUKE; SUGIURA, TSUYOSHI; IMAJYO, IKUMI; SHIMODA, MIYUKI; ISHII, KOTARO; AKIMOTO, NAONARI; YOSHIHAMA, NAOYA; MORI, YOSHIHIDE

    2014-01-01

    Adenoid cystic carcinoma (AdCC) is highly metastatic and resistant to chemotherapy and radiotherapy. Recently, we reported that the T-box transcription factor Brachyury is a potential regulator of cancer stem cells (CSCs). Specifically, growth of CSCs was found to be controlled by Brachyury knockdown in AdCC. Since CSCs are resistant to chemotherapy and radiotherapy, this finding provides a new principle for therapies targeting CSCs. In the present study, we established that Brachyury knockdown suppresses chemoresistance and radioresistance in vitro. Brachyury was knocked down by transfecting Brachyury short hairpin RNA (shRNA) into the AdCC CSC cell line ACCS-M GFP. Brachyury knockdown significantly inhibited cell migration and invasion and suppressed chemoresistance. A quantitative PCR array of drug transporter genes revealed that knockdown of Brachyury caused downregulation of ATP-binding cassette transporter genes. Furthermore, ACCS-M GFP radioresistance was significantly suppressed by Brachyury knockdown. Knockdown of Brachyury significantly sensitized ACCS-M GFP cells to chemoradiotherapy. This study demonstrates that Brachyury knockdown reduces invasiveness and chemoresistance and radioresistance of CSCs in vivo. Therefore, Brachyury knockdown may be a useful therapeutic tool for sensitizing CSCs to conventional chemoradiotherapy. PMID:24504414

  7. Systems genetics identifies Sestrin 3 as a regulator of a proconvulsant gene network in human epileptic hippocampus

    PubMed Central

    Johnson, Michael R.; Rossetti, Tiziana; Speed, Doug; Srivastava, Prashant K.; Chadeau-Hyam, Marc; Hajji, Nabil; Dabrowska, Aleksandra; Rotival, Maxime; Razzaghi, Banafsheh; Kovac, Stjepana; Wanisch, Klaus; Grillo, Federico W.; Slaviero, Anna; Langley, Sarah R.; Shkura, Kirill; Roncon, Paolo; De, Tisham; Mattheisen, Manuel; Niehusmann, Pitt; O’Brien, Terence J.; Petrovski, Slave; von Lehe, Marec; Hoffmann, Per; Eriksson, Johan; Coffey, Alison J.; Cichon, Sven; Walker, Matthew; Simonato, Michele; Danis, Bénédicte; Mazzuferi, Manuela; Foerch, Patrik; Schoch, Susanne; De Paola, Vincenzo; Kaminski, Rafal M.; Cunliffe, Vincent T.; Becker, Albert J.; Petretto, Enrico

    2015-01-01

    Gene-regulatory network analysis is a powerful approach to elucidate the molecular processes and pathways underlying complex disease. Here we employ systems genetics approaches to characterize the genetic regulation of pathophysiological pathways in human temporal lobe epilepsy (TLE). Using surgically acquired hippocampi from 129 TLE patients, we identify a gene-regulatory network genetically associated with epilepsy that contains a specialized, highly expressed transcriptional module encoding proconvulsive cytokines and Toll-like receptor signalling genes. RNA sequencing analysis in a mouse model of TLE using 100 epileptic and 100 control hippocampi shows the proconvulsive module is preserved across-species, specific to the epileptic hippocampus and upregulated in chronic epilepsy. In the TLE patients, we map the trans-acting genetic control of this proconvulsive module to Sestrin 3 (SESN3), and demonstrate that SESN3 positively regulates the module in macrophages, microglia and neurons. Morpholino-mediated Sesn3 knockdown in zebrafish confirms the regulation of the transcriptional module, and attenuates chemically induced behavioural seizures in vivo. PMID:25615886

  8. Molecular characterization and expression pattern of a germ cell marker gene dnd in gibel carp (Carassius gibelio).

    PubMed

    Li, Shi-Zhu; Liu, Wei; Li, Zhi; Wang, Yang; Zhou, Li; Yi, Mei-Sheng; Gui, Jian-Fang

    2016-10-10

    As a germ cell marker gene, Dead end (dnd) has been identified and characterized in many vertebrates. Recently, we created a complete germ cell-depleted gonad model by the dnd-specific morpholino-mediated knockdown approach, and revealed sex-biased gene expression alteration through utilizing unisexual gynogenetic superiority in polyploid gibel carp. However, dnd and its expression pattern are still unclear in the gibel carp. In this study, we further analyzed molecular characterization of gibel carp dnd and its dynamic expression pattern during gametogenesis and embryogenesis. Similar to other homologs in vertebrates, gibel carp dnd contains a conserved RRM motif and five other motifs, and is highly evolutionary conserved in genomic organization and neighborhood gene synteny. RT-PCR and Western blot analyses showed its gonad-specific expression intensively in testis and ovary. Section in situ hybridization (SISH) and immunofluorescence localization revealed its dynamic expression pattern specific to oogenic cells and spermatogenetic cells during oogenesis and spermatogenesis. Moreover, its temporal and spatial distribution specific to PGCs were also demonstrated by RT-PCR and whole mount in situ hybridization (WISH) during embryogenesis. Therefore, gibel carp Dnd is a conserved germ cell marker during gametogenesis, and its maternal transcript is also a useful marker for tracing PGC specification and migration. PMID:27418526

  9. Inhibition of SULT4A1 expression induces up-regulation of phototransduction gene expression in 72-hour postfertilization zebrafish larvae.

    PubMed

    Crittenden, Frank; Thomas, Holly; Ethen, Cheryl M; Wu, Zhengliang L; Chen, Dongquan; Kraft, Timothy W; Parant, John M; Falany, Charles N

    2014-05-01

    Sulfotransferase (SULT) 4A1 is an orphan enzyme that shares distinct structure and sequence similarities with other cytosolic SULTs. SULT4A1 is primarily expressed in neuronal tissue and is also the most conserved SULT, having been identified in every vertebrate investigated to date. Certain haplotypes of the SULT4A1 gene are correlated with higher baseline psychopathology in schizophrenic patients, but no substrate or function for SULT4A1 has yet been identified despite its high level of sequence conservation. In this study, deep RNA sequencing was used to search for alterations in gene expression in 72-hour postfertilization zebrafish larvae following transient SULT4A1 knockdown (KD) utilizing splice blocking morpholino oligonucleotides. This study demonstrates that transient inhibition of SULT4A1 expression in developing zebrafish larvae results in the up-regulation of several genes involved in phototransduction. SULT4A1 KD was verified by immunoblot analysis and quantitative real-time polymerase chain reaction (qPCR). Gene regulation changes identified by deep RNA sequencing were validated by qPCR. This study is the first identification of a cellular process whose regulation appears to be associated with SULT4A1 expression. PMID:24553382

  10. Inhibition of SULT4A1 Expression Induces Up-Regulation of Phototransduction Gene Expression in 72-Hour Postfertilization Zebrafish Larvae

    PubMed Central

    Crittenden, Frank; Thomas, Holly; Ethen, Cheryl M.; Wu, Zhengliang L.; Chen, Dongquan; Kraft, Timothy W.; Parant, John M.

    2014-01-01

    Sulfotransferase (SULT) 4A1 is an orphan enzyme that shares distinct structure and sequence similarities with other cytosolic SULTs. SULT4A1 is primarily expressed in neuronal tissue and is also the most conserved SULT, having been identified in every vertebrate investigated to date. Certain haplotypes of the SULT4A1 gene are correlated with higher baseline psychopathology in schizophrenic patients, but no substrate or function for SULT4A1 has yet been identified despite its high level of sequence conservation. In this study, deep RNA sequencing was used to search for alterations in gene expression in 72-hour postfertilization zebrafish larvae following transient SULT4A1 knockdown (KD) utilizing splice blocking morpholino oligonucleotides. This study demonstrates that transient inhibition of SULT4A1 expression in developing zebrafish larvae results in the up-regulation of several genes involved in phototransduction. SULT4A1 KD was verified by immunoblot analysis and quantitative real-time polymerase chain reaction (qPCR). Gene regulation changes identified by deep RNA sequencing were validated by qPCR. This study is the first identification of a cellular process whose regulation appears to be associated with SULT4A1 expression. PMID:24553382

  11. Identification of mutations in the MYO9A gene in patients with congenital myasthenic syndrome

    PubMed Central

    O’Connor, Emily; Töpf, Ana; Müller, Juliane S.; Cox, Daniel; Evangelista, Teresinha; Colomer, Jaume; Abicht, Angela; Senderek, Jan; Hasselmann, Oswald; Yaramis, Ahmet; Laval, Steven H.

    2016-01-01

    Congenital myasthenic syndromes are a group of rare and genetically heterogenous disorders resulting from defects in the structure and function of the neuromuscular junction. Patients with congenital myasthenic syndrome exhibit fatigable muscle weakness with a variety of accompanying phenotypes depending on the protein affected. A cohort of patients with a clinical diagnosis of congenital myasthenic syndrome that lacked a genetic diagnosis underwent whole exome sequencing in order to identify genetic causation. Missense biallelic mutations in the MYO9A gene, encoding an unconventional myosin, were identified in two unrelated families. Depletion of MYO9A in NSC-34 cells revealed a direct effect of MYO9A on neuronal branching and axon guidance. Morpholino-mediated knockdown of the two MYO9A orthologues in zebrafish, myo9aa/ab, demonstrated a requirement for MYO9A in the formation of the neuromuscular junction during development. The morphants displayed shortened and abnormally branched motor axons, lack of movement within the chorion and abnormal swimming in response to tactile stimulation. We therefore conclude that MYO9A deficiency may affect the presynaptic motor axon, manifesting in congenital myasthenic syndrome. These results highlight the involvement of unconventional myosins in motor axon functionality, as well as the need to look outside traditional neuromuscular junction-specific proteins for further congenital myasthenic syndrome candidate genes. PMID:27259756

  12. Identification of mutations in the MYO9A gene in patients with congenital myasthenic syndrome.

    PubMed

    O'Connor, Emily; Töpf, Ana; Müller, Juliane S; Cox, Daniel; Evangelista, Teresinha; Colomer, Jaume; Abicht, Angela; Senderek, Jan; Hasselmann, Oswald; Yaramis, Ahmet; Laval, Steven H; Lochmüller, Hanns

    2016-08-01

    Congenital myasthenic syndromes are a group of rare and genetically heterogenous disorders resulting from defects in the structure and function of the neuromuscular junction. Patients with congenital myasthenic syndrome exhibit fatigable muscle weakness with a variety of accompanying phenotypes depending on the protein affected. A cohort of patients with a clinical diagnosis of congenital myasthenic syndrome that lacked a genetic diagnosis underwent whole exome sequencing in order to identify genetic causation. Missense biallelic mutations in the MYO9A gene, encoding an unconventional myosin, were identified in two unrelated families. Depletion of MYO9A in NSC-34 cells revealed a direct effect of MYO9A on neuronal branching and axon guidance. Morpholino-mediated knockdown of the two MYO9A orthologues in zebrafish, myo9aa/ab, demonstrated a requirement for MYO9A in the formation of the neuromuscular junction during development. The morphants displayed shortened and abnormally branched motor axons, lack of movement within the chorion and abnormal swimming in response to tactile stimulation. We therefore conclude that MYO9A deficiency may affect the presynaptic motor axon, manifesting in congenital myasthenic syndrome. These results highlight the involvement of unconventional myosins in motor axon functionality, as well as the need to look outside traditional neuromuscular junction-specific proteins for further congenital myasthenic syndrome candidate genes. PMID:27259756

  13. 1-phenyl-2-decanoylamino-3-morpholino-1-propanol chemosensitizes neuroblastoma cells for taxol and vincristine.

    PubMed

    Sietsma, H; Veldman, R J; Kolk, D; Ausema, B; Nijhof, W; Kamps, W; Vellenga, E; Kok, J W

    2000-03-01

    In this study, we show that an inhibitor of glycosphin-golipid biosynthesis, D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), increases the chemosensitivity of neuroblastoma tumor cells for Taxol and vincristine. At noneffective low doses of Taxol or vincristine, the addition of a noneffective dose of PDMP resulted in 70% cytotoxicity, indicating synergy. Such an effect was not observed for etoposide (VP16). PDMP caused an early (6 h) increase in ceramide (Cer) levels, but the excess Cer was metabolically removed in the long-term (96 h). However, upon incubation with PDMP in combination with Taxol, but not with etoposide, Cer levels remained elevated at 96 h. These results suggest that neuroblastoma cells are normally able to metabolically remove excess Cer, but lose this capacity upon exposure to microtubule modulating anticancer agents (Taxol or vincristine). In addition, PDMP treatment resulted in a decreased efflux of [14C]Taxol and [3H]vincristine from neuroblastoma cells, similar to treatment with PSC833 or MK571, suggesting an effect of PDMP on the transporter proteins P-glycoprotein and/or multidrug resistance protein. PDMP did not further reduce [14C]Taxol or [3H]vincristine efflux in PSC833-treated cells, although it did further diminish cell survival under these conditions. We conclude that a combined administration of nontoxic concentrations of PDMP and either Taxol or vincristine results in highly sensitized neuroblastoma cells. This appears to involve a sustained elevation of Cer levels, possibly in concert with increased drug accumulation. PMID:10741719

  14. Selective Neuromuscular Denervation in Taiwanese Severe SMA Mouse Can Be Reversed by Morpholino Antisense Oligonucleotides

    PubMed Central

    Lin, Te-Lin; Chen, Tai-Heng; Hsu, Ya-Yun; Cheng, Yu-Hua; Juang, Bi-Tzen; Jong, Yuh-Jyh

    2016-01-01

    Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease caused by deficiency of the survival of motor neuron (SMN) protein, which leads to synaptic defects and spinal motor neuron death. Neuromuscular junction (NMJ) abnormalities have been found to be involved in SMA pathogenesis in the SMNΔ7 SMA mouse model. However, whether similar NMJ pathological findings present in another commonly used mouse model, the Taiwanese SMA mouse, has not been fully investigated. To examine the NMJs of the Taiwanese severe SMA mouse model (Smn-/-; SMN2tg/0), which is characterized by severe phenotype and death before postnatal day (P) 9, we investigated 25 axial and appendicular muscles from P1 to P9. We labelled the muscles with anti-neurofilament and anti-synaptophysin antibodies for nerve terminals and α-bungarotoxin for acetylcholine receptors (AChRs). We found that severe NMJ denervation (<50% fully innervated endplates) selectively occurred in the flexor digitorum brevis 2 and 3 (FDB-2/3) muscles from P5, and an increased percentage of fully denervated endplates correlated with SMA progression. Furthermore, synaptophysin signals were absent at the endplate compared to control littermate mice, suggesting that vesicle transport might only be affected at the end stage. Subsequently, we treated the Taiwanese severe SMA mice with morpholino (MO) antisense oligonucleotides (80 μg/g) via subcutaneous injection at P0. We found that MO significantly reversed the NMJ denervation in FDB-2/3 muscles and extended the survival of Taiwanese severe SMA mice. We conclude that early NMJ denervation in the FDB-2/3 muscles of Taiwanese severe SMA mice can be reversed by MO treatment. The FDB-2/3 muscles of Taiwanese severe SMA mice provide a very sensitive platform for assessing the effectiveness of drug treatments in SMA preclinical studies. PMID:27124114

  15. CRISPR/Cas9-based generation of knockdown mice by intronic insertion of artificial microRNA using longer single-stranded DNA.

    PubMed

    Miura, Hiromi; Gurumurthy, Channabasavaiah B; Sato, Takehito; Sato, Masahiro; Ohtsuka, Masato

    2015-01-01

    Knockdown mouse models, where gene dosages can be modulated, provide valuable insights into gene function. Typically, such models are generated by embryonic stem (ES) cell-based targeted insertion, or pronuclear injection, of the knockdown expression cassette. However, these methods are associated with laborious and time-consuming steps, such as the generation of large constructs with elements needed for expression of a functional RNAi-cassette, ES-cell handling, or screening for mice with the desired knockdown effect. Here, we demonstrate that reliable knockdown models can be generated by targeted insertion of artificial microRNA (amiRNA) sequences into a specific locus in the genome [such as intronic regions of endogenous eukaryotic translation elongation factor 2 (eEF-2) gene] using the Clustered Regularly Interspaced Short Palindromic Repeats/Crispr associated 9 (CRISPR/Cas9) system. We used in vitro synthesized single-stranded DNAs (about 0.5-kb long) that code for amiRNA sequences as repair templates in CRISPR/Cas9 mutagenesis. Using this approach we demonstrate that amiRNA cassettes against exogenous (eGFP) or endogenous [orthodenticle homeobox 2 (Otx2)] genes can be efficiently targeted to a predetermined locus in the genome and result in knockdown of gene expression. We also provide a strategy to establish conditional knockdown models with this method. PMID:26242611

  16. Design, synthesis, and evaluation of water-soluble morpholino-decorated paclitaxel prodrugs with remarkably decreased toxicity.

    PubMed

    Feng, Siliang; Chen, Kuncheng; Wang, Chenhong; Jiang, Xifeng; Dong, Huajin; Gong, Zehui; Liu, Keliang

    2016-08-01

    Novel water-soluble paclitaxel prodrugs were designed and synthesized by introducing morpholino groups through different linkers. These derivatives showed 400-20,000-times greater water solubility than paclitaxel as well as comparable activity in MCF-7 and HeLa cell lines. The prodrug PM4 was tested in the S-180 tumor mouse model, with paclitaxel as the positive control. The results showed that PM4 had comparable antitumor activity as paclitaxel, with tumor inhibition of 54% versus 56%, and remarkably decreased toxicity. The survival rate of treated mice was 8/8 in the PM4 group, compared to 3/8 in the paclitaxel group. PMID:27311893

  17. Knockdown of Human TCF4 Affects Multiple Signaling Pathways Involved in Cell Survival, Epithelial to Mesenchymal Transition and Neuronal Differentiation

    PubMed Central

    Forrest, Marc P.; Waite, Adrian J.; Martin-Rendon, Enca; Blake, Derek J.

    2013-01-01

    Haploinsufficiency of TCF4 causes Pitt-Hopkins syndrome (PTHS): a severe form of mental retardation with phenotypic similarities to Angelman, Mowat-Wilson and Rett syndromes. Genome-wide association studies have also found that common variants in TCF4 are associated with an increased risk of schizophrenia. Although TCF4 is transcription factor, little is known about TCF4-regulated processes in the brain. In this study we used genome-wide expression profiling to determine the effects of acute TCF4 knockdown on gene expression in SH-SY5Y neuroblastoma cells. We identified 1204 gene expression changes (494 upregulated, 710 downregulated) in TCF4 knockdown cells. Pathway and enrichment analysis on the differentially expressed genes in TCF4-knockdown cells identified an over-representation of genes involved in TGF-β signaling, epithelial to mesenchymal transition (EMT) and apoptosis. Among the most significantly differentially expressed genes were the EMT regulators, SNAI2 and DEC1 and the proneural genes, NEUROG2 and ASCL1. Altered expression of several mental retardation genes such as UBE3A (Angelman Syndrome), ZEB2 (Mowat-Wilson Syndrome) and MEF2C was also found in TCF4-depleted cells. These data suggest that TCF4 regulates a number of convergent signaling pathways involved in cell differentiation and survival in addition to a subset of clinically important mental retardation genes. PMID:24058414

  18. Enhanced toxic cloud knockdown spray system for decontamination applications

    DOEpatents

    Betty, Rita G.; Tucker, Mark D.; Brockmann, John E.; Lucero, Daniel A.; Levin, Bruce L.; Leonard, Jonathan

    2011-09-06

    Methods and systems for knockdown and neutralization of toxic clouds of aerosolized chemical or biological warfare (CBW) agents and toxic industrial chemicals using a non-toxic, non-corrosive aqueous decontamination formulation.

  19. RNAi-mediated knockdown of IKK1 in transgenic mice using a transgenic construct containing the human H1 promoter.

    PubMed

    Moreno-Maldonado, Rodolfo; Murillas, Rodolfo; Navarro, Manuel; Page, Angustias; Suarez-Cabrera, Cristian; Alameda, Josefa P; Bravo, Ana; Casanova, M Llanos; Ramirez, Angel

    2014-01-01

    Inhibition of gene expression through siRNAs is a tool increasingly used for the study of gene function in model systems, including transgenic mice. To achieve perdurable effects, the stable expression of siRNAs by an integrated transgenic construct is necessary. For transgenic siRNA expression, promoters transcribed by either RNApol II or III (such as U6 or H1 promoters) can be used. Relatively large amounts of small RNAs synthesis are achieved when using RNApol III promoters, which can be advantageous in knockdown experiments. To study the feasibility of H1 promoter-driven RNAi-expressing constructs for protein knockdown in transgenic mice, we chose IKK1 as the target gene. Our results indicate that constructs containing the H1 promoter are sensitive to the presence of prokaryotic sequences and to transgene position effects, similar to RNApol II promoters-driven constructs. We observed variable expression levels of transgenic siRNA among different tissues and animals and a reduction of up to 80% in IKK1 expression. Furthermore, IKK1 knockdown led to hair follicle alterations. In summary, we show that constructs directed by the H1 promoter can be used for knockdown of genes of interest in different organs and for the generation of animal models complementary to knockout and overexpression models. PMID:24523631

  20. An improved method for covalently conjugating morpholino oligomers to antitumor antibodies.

    PubMed

    He, Jiang; Liu, Guozheng; Dou, Shuping; Gupta, Suresh; Rusckowski, Mary; Hnatowich, Donald

    2007-01-01

    Whether for conventional pretargeting, amplification pretargeting, or affinity enhancement pretargeting, it will be necessary to conjugate an antitumor antibody as the first injectate. This laboratory is investigating phosphorodiamidate morpholinos (MORFs) for pretargeting, and accordingly we are examining methods of attaching MORFs to antitumor antibodies that provide at least one group per molecule (gpm) without adversely influencing antibody properties. The aim of this investigation was to evaluate the commercial Hydralink for the conjugation of the anti-CEA MN14 antibody with an 18 mer amine-derivatized MORF. The conjugation was approached in both directions by first reacting MN14 with the NHS derivatives of 4-hydrozinonicotinate acetone hydrazone (SANH) or 4-formylbenzoate (SFB) and then combining with MORF that was previously reacted with SFB or SANH to yield MN14(SANH)-MORF and MN14(SFB)-MORF respectively. The storage stability, immunoreactive fraction, and the biodistribution in normal mice were compared for both conjugates. Thereafter, MN14(SANH)-MORF was used in a pretargeting study in tumored nude mice, and the results were compared to that obtained historically with MN14-MORF prepared by carbodiimide (EDC) coupling. Both new methods of conjugation provided between 1 and 2 gpm compared to 0.2 achieved previously by EDC. Furthermore, by repeat SE HPLC with and without CEA, both showed an unimpaired immunoreactive fraction. MN14(SANH)-MORF tolerated long-term storage best. More importantly, when labeled by hybridization with 99mTc-labeled complementary MORF (99mTc-cMORF), the biodistribution of MN14(SANH)-MORF was more favorable than that of MN14(SFB)-MORF in normal mice with lower liver (5.7 vs 9.4 %ID/g at 18 h) and spleen (3.5 vs 8.4 %ID/g) accumulations and higher blood levels (4.8 vs 3.4 %ID/g). Accordingly, only MN14(SANH)-MORF was used in a pretargeting study in tumored mice. When targeted with 99mTc-cMORF and at 2 days postinjection of antibody

  1. Pretargeting in tumored mice with radiolabeled morpholino oligomer showing low kidney uptake.

    PubMed

    Liu, Guozheng; He, Jiang; Dou, Shuping; Gupta, Suresh; Vanderheyden, Jean-Luc; Rusckowski, Mary; Hnatowich, Donald J

    2004-03-01

    We have recently shown that accumulation in mouse kidneys of technetium-99m labeled phosphorodiamidate morpholinos (MORFs) increases with the number of cytosines in the base sequence. To improve tumor/kidney ratios in tumored mice, pretargeting studies were performed with a cytosine-free MORF. An 18-mer MORF (5'-TCTTCTACTTCACAACTA) was conjugated to the anti-CEA antibody MN14 (Immunomedics) and administered to nude mice bearing LS174T tumors. Thereafter, the (99m)Tc-labeled cytosine-free cMORF (5'-TAGTTGTGAAGTAGAAGA-amide-MAG(3)) was administered. For comparison, the identical study was repeated but with our original pair of 18-mer MORFs (5'-GGGTGTACGTCACAACTA-conjugated MN14 and (99m)Tc-labeled 5'-TAGTTGTGACGTACACCC-amide-MAG(3)). Surface plasmon resonance was used to show that the hybridization affinities of the original and the modified pair of MORFs were essentially equal. Hybridization of the cytosine-free cMORF-(99m)Tc to MN14-MORF was demonstrated in vitro by size-exclusion high-performance liquid chromatography. At 3 h, kidney levels in normal mice were 2.0%ID/organ for the modified cMORF vs. 4.1%ID/organ for the original cMORF sequence, while at 24 h, these values were 0.9% vs 1.8%ID/organ. Pretargeting studies in tumored mice receiving 25 microg of conjugated antibody, 0.5 microg of labeled cMORF 48 h later, followed by imaging and sacrifice at 3 h showed that kidney levels were reduced using the cytosine-free cMORF. Moreover, tumor accumulation was about 3.6%ID/g and was independent of sequence. The whole-body images clearly reflected the improved tumor to kidney ratios. By choosing a cytosine-free base sequence for pretargeting studies, kidney accumulation of cMORF-(99m)Tc was reduced without adversely influencing tumor accumulation. The lowering of kidney radioactivity levels in this way may be important to reduce toxicity to this organ in connection with pretargeting radiotherapy studies. PMID:14691611

  2. The Zebrafish Orthologue of the Dyslexia Candidate Gene DYX1C1 Is Essential for Cilia Growth and Function

    PubMed Central

    Chandrasekar, Gayathri; Vesterlund, Liselotte; Hultenby, Kjell; Tapia-Páez, Isabel; Kere, Juha

    2013-01-01

    DYX1C1, a susceptibility gene for dyslexia, encodes a tetratricopeptide repeat domain containing protein that has been implicated in neuronal migration in rodent models. The developmental role of this gene remains unexplored. To understand the biological function(s) of zebrafish dyx1c1 during embryonic development, we cloned the zebrafish dyx1c1 and used morpholino-based knockdown strategy. Quantitative real-time PCR analysis revealed the presence of dyx1c1 transcripts in embryos, early larval stages and in a wide range of adult tissues. Using mRNA in situ hybridization, we show here that dyx1c1 is expressed in many ciliated tissues in zebrafish. Inhibition of dyx1c1 produced pleiotropic phenotypes characteristically associated with cilia defects such as body curvature, hydrocephalus, situs inversus and kidney cysts. We also demonstrate that in dyx1c1 morphants, cilia length is reduced in several organs including Kupffer’s vesicle, pronephros, spinal canal and olfactory placode. Furthermore, electron microscopic analysis of cilia in dyx1c1 morphants revealed loss of both outer (ODA) and inner dynein arms (IDA) that have been shown to be required for cilia motility. Considering all these results, we propose an essential role for dyx1c1 in cilia growth and function. PMID:23650548

  3. Knockdown of Amyloid Precursor Protein in Zebrafish Causes Defects in Motor Axon Outgrowth

    PubMed Central

    Song, Ping; Pimplikar, Sanjay W.

    2012-01-01

    Amyloid precursor protein (APP) plays a pivotal role in Alzheimer’s disease (AD) pathogenesis, but its normal physiological functions are less clear. Combined deletion of the APP and APP-like protein 2 (APLP2) genes in mice results in post-natal lethality, suggesting that APP performs an essential, if redundant, function during embryogenesis. We previously showed that injection of antisense morpholino to reduce APP levels in zebrafish embryos caused convergent-extension defects. Here we report that a reduction in APP levels causes defective axonal outgrowth of facial branchiomotor and spinal motor neurons, which involves disorganized axonal cytoskeletal elements. The defective outgrowth is caused in a cell-autonomous manner and both extracellular and intracellular domains of human APP are required to rescue the defective phenotype. Interestingly, wild-type human APP rescues the defective phenotype but APPswe mutation, which causes familial AD, does not. Our results show that the zebrafish model provides a powerful system to delineate APP functions in vivo and to study the biological effects of APP mutations. PMID:22545081

  4. The function of DrPax1b gene in the embryonic development of zebrafish.

    PubMed

    Liu, Xin; Wang, Hua; Li, Guang; Huang, Hui-Zhe; Wang, Yi-Quan

    2013-01-01

    Vertebrate Pax1 gene is a member of Pax gene family and encodes a transcription factor associated with crucial roles in the development of pharyngeal pouch, scletrotome and limb bud. In zebrafish, the genome contains two Pax1 paralogs, DrPax1a and DrPax1b, which share high sequence similarity with other Pax1 genes. To elucidate the function of zebrafish DrPax1b gene, we first examined the gene expression pattern and found that it was mainly expressed in the endodermal pharyngeal pouch, caudal somites, notochord, and fin bud. Then, we performed knockdown experiments using antisense morpholino oligonucleotides, which lead to the defects in the vertebral column, tail, pharyngeal skeleton, and pectoral fin. Additionally, we also found that the mouse MmPax1 mRNA, but not the amphioxus AmphiPax1/9 mRNA, could rescue the MO-induced defects. Furthermore, sequence alignment revealed that the N-terminal region of vertebrate Pax1 and amphioxus Pax1/9 were highly conserved, whereas their C-terminal regions were relatively divergent. However, the chimeric Am(N)Dr(C)Pax1, Mm(N)Dr(C)Pax1 and Dr(N)Mm(C)Pax1 mRNA could partially rescue the defects, while the Dr(N)Am(C)Pax1 mRNA could not. In conclusion, our data demonstrate a conserved function of DrPax1b in the development of the vertebral column, pectoral fin and pharyngeal skeleton formation in zebrafish and also provide critical insight into the functional evolution of Pax1 gene by changing its C-terminal sequence. PMID:24463529

  5. Identification and functional analysis of endothelial tip cell–enriched genes

    PubMed Central

    del Toro, Raquel; Prahst, Claudia; Mathivet, Thomas; Siegfried, Geraldine; Kaminker, Joshua S.; Larrivee, Bruno; Breant, Christiane; Duarte, Antonio; Takakura, Nobuyuki; Fukamizu, Akiyoshi; Penninger, Josef

    2010-01-01

    Sprouting of developing blood vessels is mediated by specialized motile endothelial cells localized at the tips of growing capillaries. Following behind the tip cells, endothelial stalk cells form the capillary lumen and proliferate. Expression of the Notch ligand Delta-like-4 (Dll4) in tip cells suppresses tip cell fate in neighboring stalk cells via Notch signaling. In DLL4+/− mouse mutants, most retinal endothelial cells display morphologic features of tip cells. We hypothesized that these mouse mutants could be used to isolate tip cells and so to determine their genetic repertoire. Using transcriptome analysis of retinal endothelial cells isolated from DLL4+/− and wild-type mice, we identified 3 clusters of tip cell–enriched genes, encoding extracellular matrix degrading enzymes, basement membrane components, and secreted molecules. Secreted molecules endothelial-specific molecule 1, angiopoietin 2, and apelin bind to cognate receptors on endothelial stalk cells. Knockout mice and zebrafish morpholino knockdown of apelin showed delayed angiogenesis and reduced proliferation of stalk cells expressing the apelin receptor APJ. Thus, tip cells may regulate angiogenesis via matrix remodeling, production of basement membrane, and release of secreted molecules, some of which regulate stalk cell behavior. PMID:20705756

  6. Specific in vivo knockdown of protein function by intrabodies

    PubMed Central

    Marschall, Andrea LJ; Dübel, Stefan; Böldicke, Thomas

    2015-01-01

    Intracellular antibodies (intrabodies) are recombinant antibody fragments that bind to target proteins expressed inside of the same living cell producing the antibodies. The molecules are commonly used to study the function of the target proteins (i.e., their antigens). The intrabody technology is an attractive alternative to the generation of gene-targeted knockout animals, and complements knockdown techniques such as RNAi, miRNA and small molecule inhibitors, by-passing various limitations and disadvantages of these methods. The advantages of intrabodies include very high specificity for the target, the possibility to knock down several protein isoforms by one intrabody and targeting of specific splice variants or even post-translational modifications. Different types of intrabodies must be designed to target proteins at different locations, typically either in the cytoplasm, in the nucleus or in the endoplasmic reticulum (ER). Most straightforward is the use of intrabodies retained in the ER (ER intrabodies) to knock down the function of proteins passing the ER, which disturbs the function of members of the membrane or plasma proteomes. More effort is needed to functionally knock down cytoplasmic or nuclear proteins because in this case antibodies need to provide an inhibitory effect and must be able to fold in the reducing milieu of the cytoplasm. In this review, we present a broad overview of intrabody technology, as well as applications both of ER and cytoplasmic intrabodies, which have yielded valuable insights in the biology of many targets relevant for drug development, including α-synuclein, TAU, BCR-ABL, ErbB-2, EGFR, HIV gp120, CCR5, IL-2, IL-6, β-amyloid protein and p75NTR. Strategies for the generation of intrabodies and various designs of their applications are also reviewed. PMID:26252565

  7. miRNA-embedded shRNAs for Lineage-specific BCL11A Knockdown and Hemoglobin F Induction

    PubMed Central

    Guda, Swaroopa; Brendel, Christian; Renella, Raffaele; Du, Peng; Bauer, Daniel E; Canver, Matthew C; Grenier, Jennifer K; Grimson, Andrew W; Kamran, Sophia C; Thornton, James; de Boer, Helen; Root, David E; Milsom, Michael D; Orkin, Stuart H; Gregory, Richard I; Williams, David A

    2015-01-01

    RNA interference (RNAi) technology using short hairpin RNAs (shRNAs) expressed via RNA polymerase (pol) III promoters has been widely exploited to modulate gene expression in a variety of mammalian cell types. For certain applications, such as lineage-specific knockdown, embedding targeting sequences into pol II-driven microRNA (miRNA) architecture is required. Here, using the potential therapeutic target BCL11A, we demonstrate that pol III-driven shRNAs lead to significantly increased knockdown but also increased cytotoxcity in comparison to pol II-driven miRNA adapted shRNAs (shRNAmiR) in multiple hematopoietic cell lines. We show that the two expression systems yield mature guide strand sequences that differ by a 4 bp shift. This results in alternate seed sequences and consequently influences the efficacy of target gene knockdown. Incorporating a corresponding 4 bp shift into the guide strand of shRNAmiRs resulted in improved knockdown efficiency of BCL11A. This was associated with a significant de-repression of the hemoglobin target of BCL11A, human γ-globin or the murine homolog Hbb-y. Our results suggest the requirement for optimization of shRNA sequences upon incorporation into a miRNA backbone. These findings have important implications in future design of shRNAmiRs for RNAi-based therapy in hemoglobinopathies and other diseases requiring lineage-specific expression of gene silencing sequences. PMID:26080908

  8. Identification of Genetic Suppressors of the Sin3A Knockdown Wing Phenotype

    PubMed Central

    Fox, Stephanie; Gammouh, Sarah; Pile, Lori A.

    2012-01-01

    The role of the Sin3A transcriptional corepressor in regulating the cell cycle is established in various metazoans. Little is known, however, about the signaling pathways that trigger or are triggered by Sin3A function. To discover genes that work in similar or opposing pathways to Sin3A during development, we have performed an unbiased screen of deficiencies of the Drosophila third chromosome. Additionally, we have performed a targeted loss of function screen to identify cell cycle genes that genetically interact with Sin3A. We have identified genes that encode proteins involved in regulation of gene expression, signaling pathways and cell cycle that can suppress the curved wing phenotype caused by the knockdown of Sin3A. These data indicate that Sin3A function is quite diverse and impacts a wide variety of cellular processes. PMID:23166712

  9. Hematopoietic knockdown of PPARδ reduces atherosclerosis in LDLR −/− mice

    PubMed Central

    Li, G; Chen, C; Laing, SD; Ballard, C; Biju, KC; Reddick, RL; Clark, RA; Li, S

    2016-01-01

    PPARδ (peroxisome proliferator-activated receptor δ) mediates inflammation in response to lipid accumulation. Systemic administration of a PPARδ agonist can ameliorate atherosclerosis. Paradoxically, genetic deletion of PPARδ in hematopoietic cells led to a reduction of atherosclerosis in murine models, suggesting that downregulation of PPARδ expression in these cells may mitigate atherogenesis. To advance this finding forward to potential clinical translation through hematopoietic stem cell transplantation-based gene therapy, we employed a microRNA (miRNA) approach to knock down PPARδ expression in bone marrow cells followed by transplantation of the cells into LDLR −/− mice. We found that knockdown of PPARδ expression in the hematopoietic system caused a dramatic reduction in aortic atherosclerotic lesions. In macrophages, a key component in atherogenesis, knockdown of PPARδ led to decreased expression of multiple pro-inflammatory factors, including monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-1β and IL-6. Expression of CCR2, a receptor for MCP-1, was also decreased. The downregulation of pro-inflammatory factors is consistent with significant reduction of macrophage presence in the lesions, which may also be attributable to elevation of ABCA1 (ATP-binding cassette, subfamily A, member 1) and depression of adipocyte differentiate-related protein. Furthermore, the abundance of both MCP-1 and matrix metalloproteinase-9 proteins was reduced in plaque areas. Our results demonstrate that miRNA-mediated PPARδ knockdown in hematopoietic cells is able to ameliorate atherosclerosis. PMID:26204499

  10. Akt3 knockdown induces mitochondrial dysfunction in human cancer cells.

    PubMed

    Kim, Minjee; Kim, Young Yeon; Jee, Hye Jin; Bae, Sun Sik; Jeong, Na Young; Um, Jee-Hyun; Yun, Jeanho

    2016-05-01

    Akt/PKB plays a pivotal role in cell proliferation and survival. However, the isotype-specific roles of Akt in mitochondrial function have not been fully addressed. In this study, we explored the role of Akt in mitochondrial function after stable knockdown of the Akt isoforms in EJ human bladder cancer cells. We found that the mitochondrial mass was significantly increased in the Akt1- and Akt3-knockdown cells, and this increase was accompanied by an increase in TFAM and NRF1. Akt2 knockdown did not cause a similar effect. Interestingly, Akt3 knockdown also led to severe structural defects in the mitochondria, an increase in doxorubicin-induced senescence, and impairment of cell proliferation in galactose medium. Consistent with these observations, the mitochondrial oxygen consumption rate was significantly reduced in the Akt3-knockdown cells. An Akt3 deficiency-induced decrease in mitochondrial respiration was also observed in A549 lung cancer cells. Collectively, these results suggest that the Akt isoforms play distinct roles in mitochondrial function and that Akt3 is critical for proper mitochondrial respiration in human cancer cells. PMID:26972278