Sample records for antisense morpholino mo
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Zhang, Guixin; Jin, Li-qing; Hu, Jianli; Rodemer, William; Selzer, Michael E
2015-01-01
The sea lamprey has been used as a model for the study of axonal regeneration after spinal cord injury. Previous studies have suggested that, unlike developing axons in mammal, the tips of regenerating axons in lamprey spinal cord are simple in shape, packed with neurofilaments (NFs), and contain very little F-actin. Thus it has been proposed that regeneration of axons in the central nervous system of mature vertebrates is not based on the canonical actin-dependent pulling mechanism of growth cones, but involves an internal protrusive force, perhaps generated by the transport or assembly of NFs in the distal axon. In order to assess this hypothesis, expression of NFs was manipulated by antisense morpholino oligonucleotides (MO). A standard, company-supplied MO was used as control. Axon retraction and regeneration were assessed at 2, 4 and 9 weeks after MOs were applied to a spinal cord transection (TX) site. Antisense MO inhibited NF180 expression compared to control MO. The effect of inhibiting NF expression on axon retraction and regeneration was studied by measuring the distance of axon tips from the TX site at 2 and 4 weeks post-TX, and counting the number of reticulospinal neurons (RNs) retrogradely labeled by fluorescently-tagged dextran injected caudal to the injury at 9 weeks post-TX. There was no statistically significant effect of MO on axon retraction at 2 weeks post-TX. However, at both 4 and 9 weeks post-TX, inhibition of NF expression inhibited axon regeneration.
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The HDAC Inhibitor TSA Ameliorates a Zebrafish Model of Duchenne Muscular Dystrophy.
Johnson, Nathan M; Farr, Gist H; Maves, Lisa
2013-09-17
Zebrafish are an excellent model for Duchenne muscular dystrophy. In particular, zebrafish provide a system for rapid, easy, and low-cost screening of small molecules that can ameliorate muscle damage in dystrophic larvae. Here we identify an optimal anti-sense morpholino cocktail that robustly knocks down zebrafish Dystrophin (dmd-MO). We use two approaches, muscle birefringence and muscle actin expression, to quantify muscle damage and show that the dmd-MO dystrophic phenotype closely resembles the zebrafish dmd mutant phenotype. We then show that the histone deacetylase (HDAC) inhibitor TSA, which has been shown to ameliorate the mdx mouse Duchenne model, can rescue muscle fiber damage in both dmd-MO and dmd mutant larvae. Our study identifies optimal morpholino and phenotypic scoring approaches for dystrophic zebrafish, further enhancing the zebrafish dmd model for rapid and cost-effective small molecule screening.
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Nuzzo, Francesca; Radu, Claudia; Baralle, Marco; Spiezia, Luca; Hackeng, Tilman M; Simioni, Paolo; Castoldi, Elisabetta
2013-11-28
Antisense molecules are emerging as a powerful tool to correct splicing defects. Recently, we identified a homozygous deep-intronic mutation (F5 c.1296+268A>G) activating a cryptic donor splice site in a patient with severe coagulation factor V (FV) deficiency and life-threatening bleeding episodes. Here, we assessed the ability of 2 mutation-specific antisense molecules (a morpholino oligonucleotide [MO] and an engineered U7 small nuclear RNA [snRNA]) to correct this splicing defect. COS-1 and HepG2 cells transfected with a F5 minigene construct containing the patient's mutation expressed aberrant messenger RNA (mRNA) in excess of normal mRNA. Treatment with mutation-specific antisense MO (1-5 µM) or a construct expressing antisense U7snRNA (0.25-2 µg) dose-dependently increased the relative amount of correctly spliced mRNA by 1 to 2 orders of magnitude, whereas control MO and U7snRNA were ineffective. Patient-derived megakaryocytes obtained by differentiation of circulating progenitor cells did not express FV, but became positive for FV at immunofluorescence staining after administration of antisense MO or U7snRNA. However, treatment adversely affected cell viability, mainly because of the transfection reagents used to deliver the antisense molecules. Our data provide in vitro and ex vivo proof of principle for the efficacy of RNA therapy in severe FV deficiency, but additional cytotoxicity studies are warranted.
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Gentsch, George E; Spruce, Thomas; Monteiro, Rita S; Owens, Nick D L; Martin, Stephen R; Smith, James C
2018-03-12
Antisense morpholino oligomers (MOs) have been indispensable tools for developmental biologists to transiently knock down (KD) genes rather than to knock them out (KO). Here we report on the implications of genetic KO versus MO-mediated KD of the mesoderm-specifying Brachyury paralogs in the frog Xenopus tropicalis. While both KO and KD embryos fail to activate the same core gene regulatory network, resulting in virtually identical morphological defects, embryos injected with control or target MOs also show a systemic GC content-dependent immune response and many off-target splicing defects. Optimization of MO dosage and increasing incubation temperatures can mitigate, but not eliminate, these MO side effects, which are consistent with the high affinity measured between MO and off-target sequence in vitro. We conclude that while MOs can be useful to profile loss-of-function phenotypes at a molecular level, careful attention must be paid to their immunogenic and off-target side effects. Copyright © 2018 The Francis Crick Institute. Published by Elsevier Inc. All rights reserved.
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Chen, Yau-Hung; Tsai, Huai-Jen
2002-10-01
Myf-5 is a stage-dependent transcription factor associated with somitogenesis. To study its biological functions in zebrafish, we injected the Myf5-morpholinos ZMF-MO (antisense nucleotides 28 to 52) and ZMF-OTHER (antisense nucleotides 3 to 27) into zebrafish embryos to establish a myf-5 gene knockdown. No phenotypic abnormalities were observed following injection with 0.2 ng of ZMF-MO, but defects were displayed in 2 of 118 (1.7%) surviving embryos injected with 1 ng ZMF-MO. Morphological defects became more severe with increased dosages: 105 of 270 (38.9%) surviving embryos injected with 4.5 ng of ZMF-MO displayed such abnormalities as the absence of eyes or brains in addition to the following low-dosage defects in 24 hpf embryos: longitudinal yolk sacs, incomplete epiboly coverage, abnormal and suspended tail buds, diffused somite boundaries, and head shrinkage. Similar results were observed in the 4.5 ng ZMF-OTHER injection group. However, when fish were co-injected with 4.5 ng ZMF-MO and 4.5 ng myf-5 mRNA, abnormality rates decreased from 49.6% to 5.5%. Our results show that the brain krox20 gene was down-regulated at rhombomere 3; the pax2.1 gene was completely down-regulated; myoD was expressed normally; myogenin was substantially down-regulated in whole somites; and desmin was partly inhibited in newly forming somites. Our conclusion is that zebrafish Myf-5 may play important roles in brain formation and in the convergence and extension of shield epiblasts and tail buds during early embryogenesis, in addition to its well-understood role as a muscle regulatory factor in somites.
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Ramirez, Agnese; Crisafulli, Sebastiano G.; Rizzuti, Mafalda; Bresolin, Nereo; Comi, Giacomo P.; Corti, Stefania
2018-01-01
Spinal muscular atrophy (SMA) is an autosomal-recessive childhood motor neuron disease and the main genetic cause of infant mortality. SMA is caused by deletions or mutations in the survival motor neuron 1 (SMN1) gene, which results in SMN protein deficiency. Only one approved drug has recently become available and allows for the correction of aberrant splicing of the paralogous SMN2 gene by antisense oligonucleotides (ASOs), leading to production of full-length SMN protein. We have already demonstrated that a sequence of an ASO variant, Morpholino (MO), is particularly suitable because of its safety and efficacy profile and is both able to increase SMN levels and rescue the murine SMA phenotype. Here, we optimized this strategy by testing the efficacy of four new MO sequences targeting SMN2. Two out of the four new MO sequences showed better efficacy in terms of SMN protein production both in SMA induced pluripotent stem cells (iPSCs) and SMAΔ7 mice. Further, the effect was enhanced when different MO sequences were administered in combination. Our data provide an important insight for MO-based treatment for SMA. Optimization of the target sequence and validation of a treatment based on a combination of different MO sequences could support further pre-clinical studies and the progression toward future clinical trials. PMID:29316633
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Ramirez, Agnese; Crisafulli, Sebastiano G; Rizzuti, Mafalda; Bresolin, Nereo; Comi, Giacomo P; Corti, Stefania; Nizzardo, Monica
2018-01-06
Spinal muscular atrophy (SMA) is an autosomal-recessive childhood motor neuron disease and the main genetic cause of infant mortality. SMA is caused by deletions or mutations in the survival motor neuron 1 ( SMN1 ) gene, which results in SMN protein deficiency. Only one approved drug has recently become available and allows for the correction of aberrant splicing of the paralogous SMN2 gene by antisense oligonucleotides (ASOs), leading to production of full-length SMN protein. We have already demonstrated that a sequence of an ASO variant, Morpholino (MO), is particularly suitable because of its safety and efficacy profile and is both able to increase SMN levels and rescue the murine SMA phenotype. Here, we optimized this strategy by testing the efficacy of four new MO sequences targeting SMN2 . Two out of the four new MO sequences showed better efficacy in terms of SMN protein production both in SMA induced pluripotent stem cells (iPSCs) and SMAΔ7 mice. Further, the effect was enhanced when different MO sequences were administered in combination. Our data provide an important insight for MO-based treatment for SMA. Optimization of the target sequence and validation of a treatment based on a combination of different MO sequences could support further pre-clinical studies and the progression toward future clinical trials.
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Echigoya, Yusuke; Lim, Kenji Rowel Q; Trieu, Nhu; Bao, Bo; Miskew Nichols, Bailey; Vila, Maria Candida; Novak, James S; Hara, Yuko; Lee, Joshua; Touznik, Aleksander; Mamchaoui, Kamel; Aoki, Yoshitsugu; Takeda, Shin'ichi; Nagaraju, Kanneboyina; Mouly, Vincent; Maruyama, Rika; Duddy, William; Yokota, Toshifumi
2017-11-01
Duchenne muscular dystrophy (DMD), the most common lethal genetic disorder, is caused by mutations in the dystrophin (DMD) gene. Exon skipping is a therapeutic approach that uses antisense oligonucleotides (AOs) to modulate splicing and restore the reading frame, leading to truncated, yet functional protein expression. In 2016, the US Food and Drug Administration (FDA) conditionally approved the first phosphorodiamidate morpholino oligomer (morpholino)-based AO drug, eteplirsen, developed for DMD exon 51 skipping. Eteplirsen remains controversial with insufficient evidence of its therapeutic effect in patients. We recently developed an in silico tool to design antisense morpholino sequences for exon skipping. Here, we designed morpholino AOs targeting DMD exon 51 using the in silico tool and quantitatively evaluated the effects in immortalized DMD muscle cells in vitro. To our surprise, most of the newly designed morpholinos induced exon 51 skipping more efficiently compared with the eteplirsen sequence. The efficacy of exon 51 skipping and rescue of dystrophin protein expression were increased by up to more than 12-fold and 7-fold, respectively, compared with the eteplirsen sequence. Significant in vivo efficacy of the most effective morpholino, determined in vitro, was confirmed in mice carrying the human DMD gene. These findings underscore the importance of AO sequence optimization for exon skipping. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.
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Morpholino-mediated Knockdown of DUX4 Toward Facioscapulohumeral Muscular Dystrophy Therapeutics.
Chen, Jennifer Cj; King, Oliver D; Zhang, Yuanfan; Clayton, Nicholas P; Spencer, Carrie; Wentworth, Bruce M; Emerson, Charles P; Wagner, Kathryn R
2016-08-01
Derepression of DUX4 in skeletal muscle has emerged as a likely cause of pathology in facioscapulohumeral muscular dystrophy (FSHD). Here we report on the use of antisense phosphorodiamidate morpholino oligonucleotides to suppress DUX4 expression and function in FSHD myotubes and xenografts. The most effective was phosphorodiamidate morpholino oligonucleotide FM10, which targets the polyadenylation signal of DUX4. FM10 had no significant cell toxicity, and RNA-seq analyses of FSHD and control myotubes revealed that FM10 down-regulated many transcriptional targets of DUX4, without overt off-target effects. Electroporation of FM10 into FSHD patient muscle xenografts in mice also down-regulated DUX4 and DUX4 targets. These findings demonstrate the potential of antisense phosphorodiamidate morpholino oligonucleotides as an FSHD therapeutic option.
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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.
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Comparison of Zebrafish tmem88a mutant and morpholino knockdown phenotypes
Place, Elsie S.; Smith, James C.
2017-01-01
Tmem88a is a transmembrane protein that is thought to be a negative regulator of the Wnt signalling pathway. Several groups have used antisense morpholino oligonucleotides in an effort to characterise the role of tmem88a in zebrafish cardiovascular development, but they have not obtained consistent results. Here, we generate an 8 bp deletion in the coding region of the tmem88a locus using TALENs, and we have gone on to establish a viable homozygous tmem88aΔ8 mutant line. Although tmem88aΔ8 mutants have reduced expression of some key haematopoietic genes, differentiation of erythrocytes and neutrophils is unaffected, contradicting our previous study using antisense morpholino oligonucleotides. We find that expression of the tmem88a paralogue tmem88b is not significantly changed in tmem88aΔ8 mutants and injection of the tmem88a splice-blocking morpholino oligonucleotide into tmem88aΔ8 mutants recapitulates the reduction of erythrocytes observed in morphants using o-Dianisidine. This suggests that there is a partial, but inessential, requirement for tmem88a during haematopoiesis and that morpholino injection exacerbates this phenotype in tmem88a morpholino knockdown embryos. PMID:28192479
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Antisense Oligonucleotide-Mediated Transcript Knockdown in Zebrafish.
Pauli, Andrea; Montague, Tessa G; Lennox, Kim A; Behlke, Mark A; Schier, Alexander F
2015-01-01
Antisense oligonucleotides (ASOs) are synthetic, single-strand RNA-DNA hybrids that induce catalytic degradation of complementary cellular RNAs via RNase H. ASOs are widely used as gene knockdown reagents in tissue culture and in Xenopus and mouse model systems. To test their effectiveness in zebrafish, we targeted 20 developmental genes and compared the morphological changes with mutant and morpholino (MO)-induced phenotypes. ASO-mediated transcript knockdown reproduced the published loss-of-function phenotypes for oep, chordin, dnd, ctnnb2, bmp7a, alk8, smad2 and smad5 in a dosage-sensitive manner. ASOs knocked down both maternal and zygotic transcripts, as well as the long noncoding RNA (lncRNA) MALAT1. ASOs were only effective within a narrow concentration range and were toxic at higher concentrations. Despite this drawback, quantitation of knockdown efficiency and the ability to degrade lncRNAs make ASOs a useful knockdown reagent in zebrafish.
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Smart, Eric J; De Rose, Robert A; Farber, Steven A
2004-03-09
Modulation of cholesterol absorption in the intestine, the primary site of dietary cholesterol uptake in humans, can have profound clinical implications. We have undertaken a reverse genetic approach by disrupting putative cholesterol processing genes in zebrafish larvae by using morpholino (MO) antisense oligonucleotides. By using targeted MO injections and immunoprecipitation (IP) experiments coupled with mass spectrometry, we determined that annexin (ANX)2 complexes with caveolin (CAV)1 in the zebrafish and mouse intestine. The complex is heat stable and unaffected by SDS or reducing conditions. MO targeting of anx2b or cav1, which are both strongly expressed in the larval and adult zebrafish intestinal epithelium, prevents formation of the protein heterocomplex. Furthermore, anx2b MO injection prevents processing of a fluorescent cholesterol reporter and results in reduced sterol mass. Pharmacological treatment of mice with ezetimibe disrupts the heterocomplex in only hypercholesterolemic animals. These data suggest that ANX2 and CAV1 are components of an intestinal sterol transport complex.
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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 at 21 days after fertilization. Moreover, the body cavities of MO-treated and nontreated fish were examined by histology and in situ hybridization, showing gonads which had no germ cells in morphants at various stages (60, 150, and 210 days after fertilization). Taken together, these results report the first known and functional method of sturgeon sterilization. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
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Turning gene function ON and OFF using sense and antisense photo-morpholinos in zebrafish
Tallafuss, Alexandra; Gibson, Dan; Morcos, Paul; Li, Yongfu; Seredick, Steve; Eisen, Judith; Washbourne, Philip
2012-01-01
To understand the molecular mechanisms of development it is essential to be able to turn genes on and off at will and in a spatially restricted fashion. Morpholino oligonucleotides (MOs) are very common tools used in several model organisms with which it is possible to block gene expression. Recently developed photo-activated MOs allow control over the onset of MO activity. However, deactivation of photo-cleavable MO activity has remained elusive. Here, we describe photo-cleavable MOs with which it is possible to activate or de-activate MO function by UV exposure in a temporal and spatial manner. We show, using several different genes as examples, that it is possible to turn gene expression on or off both in the entire zebrafish embryo and in single cells. We use these tools to demonstrate the sufficiency of no tail expression as late as tailbud stage to drive medial precursor cells towards the notochord cell fate. As a broader approach for the use of photo-cleavable MOs, we show temporal control over gal4 function, which has many potential applications in multiple transgenic lines. We demonstrate temporal manipulation of Gal4 transgene expression in only primary motoneurons and not secondary motoneurons, heretofore impossible with conventional transgenic approaches. In another example, we follow and analyze neural crest cells that regained sox10 function after deactivation of a photo-cleavable sox10-MO at different time points. Our results suggest that sox10 function might not be critical during neural crest formation. PMID:22492359
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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 unexplored members, but also demonstrated that fundamental developmental processes including axial patterning and endoderm formation in cnidarians can involve newly evolved (or highly diverged) genes. PMID:25233086
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Lapébie, Pascal; Ruggiero, Antonella; Barreau, Carine; Chevalier, Sandra; Chang, Patrick; Dru, Philippe; Houliston, Evelyn; Momose, Tsuyoshi
2014-09-01
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 unexplored members, but also demonstrated that fundamental developmental processes including axial patterning and endoderm formation in cnidarians can involve newly evolved (or highly diverged) genes.
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Efficient exon skipping of SGCG mutations mediated by phosphorodiamidate morpholino oligomers.
Wyatt, Eugene J; Demonbreun, Alexis R; Kim, Ellis Y; Puckelwartz, Megan J; Vo, Andy H; Dellefave-Castillo, Lisa M; Gao, Quan Q; Vainzof, Mariz; Pavanello, Rita C M; Zatz, Mayana; McNally, Elizabeth M
2018-05-03
Exon skipping uses chemically modified antisense oligonucleotides to modulate RNA splicing. Therapeutically, exon skipping can bypass mutations and restore reading frame disruption by generating internally truncated, functional proteins to rescue the loss of native gene expression. Limb-girdle muscular dystrophy type 2C is caused by autosomal recessive mutations in the SGCG gene, which encodes the dystrophin-associated protein γ-sarcoglycan. The most common SGCG mutations disrupt the transcript reading frame abrogating γ-sarcoglycan protein expression. In order to treat most SGCG gene mutations, it is necessary to skip 4 exons in order to restore the SGCG transcript reading frame, creating an internally truncated protein referred to as Mini-Gamma. Using direct reprogramming of human cells with MyoD, myogenic cells were tested with 2 antisense oligonucleotide chemistries, 2'-O-methyl phosphorothioate oligonucleotides and vivo-phosphorodiamidate morpholino oligomers, to induce exon skipping. Treatment with vivo-phosphorodiamidate morpholino oligomers demonstrated efficient skipping of the targeted exons and corrected the mutant reading frame, resulting in the expression of a functional Mini-Gamma protein. Antisense-induced exon skipping of SGCG occurred in normal cells and those with multiple distinct SGCG mutations, including the most common 521ΔT mutation. These findings demonstrate a multiexon-skipping strategy applicable to the majority of limb-girdle muscular dystrophy 2C patients.
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Early-life glucocorticoids programme behaviour and metabolism in adulthood in zebrafish
Wilson, K S; Tucker, C S; Al-Dujaili, E A S; Holmes, M C; Hadoke, P W F; Kenyon, C J
2016-01-01
Glucocorticoids (GCs) in utero influence embryonic development with consequent programmed effects on adult physiology and pathophysiology and altered susceptibility to cardiovascular disease. However, in viviparous species, studies of these processes are compromised by secondary maternal influences. The zebrafish, being fertilised externally, avoids this problem and has been used here to investigate the effects of transient alterations in GC activity during early development. Embryonic fish were treated either with dexamethasone (a synthetic GC), an antisense GC receptor (GR) morpholino (GR Mo), or hypoxia for the first 120h post fertilisation (hpf); responses were measured during embryonic treatment or later, post treatment, in adults. All treatments reduced cortisol levels in embryonic fish to similar levels. However, morpholino- and hypoxia-treated embryos showed delayed physical development (slower hatching and straightening of head–trunk angle, shorter body length), less locomotor activity, reduced tactile responses and anxiogenic activity. In contrast, dexamethasone-treated embryos showed advanced development and thigmotaxis but no change in locomotor activity or tactile responses. Gene expression changes were consistent with increased (dexamethasone) and decreased (hypoxia, GR Mo) GC activity. In adults, stressed cortisol values were increased with dexamethasone and decreased by GR Mo and hypoxia pre-treatments. Other responses were similarly differentially affected. In three separate tests of behaviour, dexamethasone-programmed fish appeared ‘bolder’ than matched controls, whereas Mo and hypoxia pre-treated fish were unaffected or more reserved. Similarly, the dexamethasone group but not the Mo or hypoxia groups were heavier, longer and had a greater girth than controls. Hyperglycaemia and expression of GC responsive gene (pepck) were also increased in the dexamethasone group. We conclude that GC activity controls many aspects of early-life growth and development in the zebrafish and that, like other species, manipulating GC status pharmacologically, physiologically or genetically in early life leads to programmable metabolic and behavioural traits in adulthood. PMID:27390302
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Twist functions in vertebral column formation in medaka, Oryzias latipes.
Yasutake, Junichi; Inohaya, Keiji; Kudo, Akira
2004-07-01
Medaka twist, a basic helix-loop-helix (bHLH) transcription factor, is expressed in the sclerotome during embryogenesis. We previously established a line of twist-EGFP transgenic medaka, whose EGFP expression is regulated by the twist promoter; therefore, we could observe the behavior of sclerotomal cells in vivo. In the transgenic medaka embryos, EGFP-positive sclerotomal cells migrated dorsally around the notochord and the neural tube, where at a later stage the vertebral column would be formed. This finding strongly suggests that twist-expressing sclerotomal cells participate in vertebral column formation in medaka. To clarify the function of twist gene in the sclerotome, we performed knockdown analysis of twist by using two kinds of morpholino antisense oligonucleotides targeted against twist (MO1 and MO2). Both the MO1 and MO2 morphants exhibited absence of neural arches, which are bilaterally paired, dorsomedially oriented bones on the dorsal aspect of the centrum. In addition, MO2, which blocks translation of only endogenous twist mRNA in the twist-EGFP transgenic medaka, did not affect the migration pattern of EGFP-positive cells, revealing that the migration of sclerotome-derived cells were normal in the absence of twist gene function. These results demonstrate that medaka twist functions in vertebral column formation by regulating the sclerotomal cell differentiation.
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Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins.
O'Connor, Marie N; Salles, Isabelle I; Cvejic, Ana; Watkins, Nicholas A; Walker, Adam; Garner, Stephen F; Jones, Chris I; Macaulay, Iain C; Steward, Michael; Zwaginga, Jaap-Jan; Bray, Sarah L; Dudbridge, Frank; de Bono, Bernard; Goodall, Alison H; Deckmyn, Hans; Stemple, Derek L; Ouwehand, Willem H
2009-05-07
In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)-based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis.
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Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins
O'Connor, Marie N.; Salles, Isabelle I.; Cvejic, Ana; Watkins, Nicholas A.; Walker, Adam; Garner, Stephen F.; Jones, Chris I.; Macaulay, Iain C.; Steward, Michael; Zwaginga, Jaap-Jan; Bray, Sarah L.; Dudbridge, Frank; de Bono, Bernard; Goodall, Alison H.; Stemple, Derek L.; Ouwehand, Willem H.
2009-01-01
In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)–based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis. PMID:19109564
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Using Morpholinos to Probe Gene Networks in Sea Urchin.
Materna, Stefan C
2017-01-01
The control processes that underlie the progression of development can be summarized in maps of gene regulatory networks (GRNs). A critical step in their assembly is the systematic perturbation of network candidates. In sea urchins the most important method for interfering with expression in a gene-specific way is application of morpholino antisense oligonucleotides (MOs). MOs act by binding to their sequence complement in transcripts resulting in a block in translation or a change in splicing and thus result in a loss of function. Despite the tremendous success of this technology, recent comparisons to mutants generated by genome editing have led to renewed criticism and challenged its reliability. As with all methods based on sequence recognition, MOs are prone to off-target binding that may result in phenotypes that are erroneously ascribed to the loss of the intended target. However, the slow progression of development in sea urchins has enabled extremely detailed studies of gene activity in the embryo. This wealth of knowledge paired with the simplicity of the sea urchin embryo enables careful analysis of MO phenotypes through a variety of methods that do not rely on terminal phenotypes. This article summarizes the use of MOs in probing GRNs and the steps that should be taken to assure their specificity.
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Chicken HOXA3 Gene: Its Expression Pattern and Role in Branchial Nerve Precursor Cell Migration
Watari-Goshima, Natsuko; Chisaka, Osamu
2011-01-01
In vertebrates, the proximal and distal sensory ganglia of the branchial nerves are derived from neural crest cells (NCCs) and placodes, respectively. We previously reported that in Hoxa3 knockout mouse embryos, NCCs and placode-derived cells of the glossopharyngeal nerve were defective in their migration. In this report, to determine the cell-type origin for this Hoxa3 knockout phenotype, we blocked the expression of the gene with antisense morpholino oligonucleotides (MO) specifically in either NCCs/neural tube or placodal cells of chicken embryos. Our results showed that HOXA3 function was required for the migration of the epibranchial placode-derived cells and that HOXA3 regulated this cell migration in both NCCs/neural tube and placodal cells. We also report that the expression pattern of chicken HOXA3 was slightly different from that of mouse Hoxa3. PMID:21278919
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PhotoMorphs™: A Novel Light-Activated Reagent for Controlling Gene Expression in Zebrafish
Tomasini, Amber J.; Schuler, Aaron D.; Zebala, John A.; Mayer, Alan N.
2009-01-01
Manipulating gene expression in zebrafish is critical for exploiting the full potential of this vertebrate model organism. Morpholino oligos are the most commonly employed antisense technology for knocking down gene expression. However, morpholinos suffer from a lack of control over the timing and location of knockdown. In this report, we describe a novel light-activatable knockdown reagent called PhotoMorph™. PhotoMorphs can be generated from existing morpholinos by hybridization with a complementary caging strand containing a photocleavable linkage. The caging strand neutralizes the morpholino activity until irradiation of the PhotoMorph with UV light releases the morpholino. We generated PhotoMorphs to target genes encoding enhanced green fluorescent protein (EGFP), No tail, and E-cadherin to illustrate the utility of this approach. Temporal control of gene expression with PhotoMorphs permitted us to circumvent the early lethal phenotype of E-cadherin knockdown. A splice-blocking PhotoMorph directed to the rheb gene showed light-dependent gene knockdown up to 72 hpf. PhotoMorphs thus offer a new class of laboratory reagents suitable for the spatiotemporal control of gene expression in the zebrafish. PMID:19644983
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Chowdhury, Tamjid A; Koceja, Chris; Eisa-Beygi, Shahram; Kleinstiver, Benjamin P; Kumar, Suresh N; Lin, Chien-Wei; Li, Keguo; Prabhudesai, Shubhangi; Joung, J Keith; Ramchandran, Ramani
2018-05-03
Tie1 (tyrosine kinase containing immunoglobulin and epidermal growth factor homology 1), an endothelial and hematopoietic cell-specific receptor tyrosine kinase, is an important regulator of angiogenesis and critical for maintaining vascular integrity. The post-transcriptional regulation of tie1 mRNA expression is not understood, but it might partly explain Tie1's differential expression pattern in endothelium. Following up on our previous work that identified natural antisense transcripts from the tie1 locus- tie1 antisense ( tie1AS ), which regulates tie1 mRNA levels in zebrafish-we attempted to identify the mechanism of this regulation. Through in vitro and in vivo ribonucleoprotein binding studies, we demonstrated that tie1AS long noncoding RNA interacts with an RNA binding protein-embryonic lethal and abnormal vision Drosophila-like 1 (Elavl1)-that regulates tie1 mRNA levels. When we disrupted the interaction between tie1AS and Elavl1 by using constitutively active antisense morpholino oligonucleotides or photoactivatable morpholino oligonucleotides, tie1 mRNA levels increased between 26 and 31 hours post-fertilization, particularly in the head. This increase correlated with dilation of primordial midbrain channels, smaller eyes, and reduced ventricular space. We also observed these phenotypes when we used CRISPR (clustered regularly interspaced short palindromic repeats)-mediated CRISPRi (CRISPR-mediated interference) to knock down tie1AS . Treatment of the morpholino oligonucleotide-injected embryos with a small molecule that decreased tie1 mRNA levels rescued all 3 abnormal phenotypes. We identified a novel mode of temporal and spatial post-transcriptional regulation of tie1 mRNA. It involves long noncoding RNA, tie1AS, and Elavl1 (an interactor of tie1AS ). © 2018 American Heart Association, Inc.
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Characterization of zebrafish dysferlin by morpholino knockdown
DOE Office of Scientific and Technical Information (OSTI.GOV)
Kawahara, Genri; Serafini, Peter R.; Myers, Jennifer A.
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 zebrafishmore » 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.« less
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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.
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Hitting bacteria at the heart of the central dogma: sequence-specific inhibition.
Rasmussen, Louise Carøe Vohlander; Sperling-Petersen, Hans Uffe; Mortensen, Kim Kusk
2007-08-10
An important objective in developing new drugs is the achievement of high specificity to maximize curing effect and minimize side-effects, and high specificity is an integral part of the antisense approach. The antisense techniques have been extensively developed from the application of simple long, regular antisense RNA (asRNA) molecules to highly modified versions conferring resistance to nucleases, stability of hybrid formation and other beneficial characteristics, though still preserving the specificity of the original nucleic acids. These new and improved second- and third-generation antisense molecules have shown promising results. The first antisense drug has been approved and more are in clinical trials. However, these antisense drugs are mainly designed for the treatment of different human cancers and other human diseases. Applying antisense gene silencing and exploiting RNA interference (RNAi) are highly developed approaches in many eukaryotic systems. But in bacteria RNAi is absent, and gene silencing by antisense compounds is not nearly as well developed, despite its great potential and the intriguing possibility of applying antisense molecules in the fight against multiresistant bacteria. Recent breakthrough and current status on the development of antisense gene silencing in bacteria including especially phosphorothioate oligonucleotides (PS-ODNs), peptide nucleic acids (PNAs) and phosphorodiamidate morpholino oligomers (PMOs) will be presented in this review.
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2012-01-01
Background Dysmorphogenesis and multiple organ defects are well known in zebrafish (Danio rerio) embryos with T-box transcription factor 5 (tbx5) deficiencies, mimicking human Holt-Oram syndrome. Methods Using an oligonucleotide-based microarray analysis to study the expression of special genes in tbx5 morphants, we demonstrated that GH and some GH-related genes were markedly downregulated. Zebrafish embryos microinjected with tbx5-morpholino (MO) antisense RNA and mismatched antisense RNA in the 1-cell stage served as controls, while zebrafish embryos co-injected with exogenous growth hormone (GH) concomitant with tbx5-MO comprised the treatment group. Results The attenuating effects of GH in tbx5-MO knockdown embryos were quantified and observed at 24, 30, 48, 72, and 96 h post-fertilization. Though the understanding of mechanisms involving GH in the tbx5 functioning complex is limited, exogenous GH supplied to tbx5 knockdown zebrafish embryos is able to enhance the expression of downstream mediators in the GH and insulin-like growth factor (IGF)-1 pathway, including igf1, ghra, and ghrb, and signal transductors (erk1, akt2), and eventually to correct dysmorphogenesis in various organs including the heart and pectoral fins. Supplementary GH also reduced apoptosis as determined by a TUNEL assay and decreased the expression of apoptosis-related genes and proteins (bcl2 and bad) according to semiquantitative reverse-transcription polymerase chain reaction and immunohistochemical analysis, respectively, as well as improving cell cycle-related genes (p27 and cdk2) and cardiomyogenetic genes (amhc, vmhc, and cmlc2). Conclusions Based on our results, tbx5 knockdown causes a pseudo GH deficiency in zebrafish during early embryonic stages, and supplementation of exogenous GH can partially restore dysmorphogenesis, apoptosis, cell growth inhibition, and abnormal cardiomyogenesis in tbx5 knockdown zebrafish in a paracrine manner. PMID:22776023
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Geller, Bruce L.; Mellbye, Brett; Lane, Douglas; Iversen, Patrick L.; Bavari, Sina
2012-01-01
Targeting bacterial essential genes using antisense phosphorodiamidate morpholino oligomers (PMOs) represents an important strategy in the development of novel antibacterial therapeutics. PMOs are neutral DNA analogues that inhibit gene expression in a sequence-specific manner. In this study, several cationic, membrane-penetrating peptides were conjugated to PMOs (PPMOs) that target 2 bacterial essential genes: acyl carrier protein (acpP) and gyrase A (gyrA). These were tested for their ability to inhibit growth of Bacillus anthracis, a gram-positive spore-forming bacterium and causative agent of anthrax. PPMOs targeted upstream of both target gene start codons and conjugated with the bacterium-permeating peptide (RFF)3R were found to be most effective in inhibiting bacterial growth in vitro. Both of the gene-targeted PPMOs protected macrophages from B. anthracis induced cell death. Subsequent, in vivo testing of the PPMOs resulted in increased survival of mice challenged with the virulent Ames strain of B. anthracis. Together, these studies suggest that PPMOs targeting essential genes have the potential of being used as antisense antibiotics to treat B. anthracis infections. PMID:22978365
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Van Overstraeten-Schlögel, Nancy; Shim, Yong-Ho; Tevel, Virginie; Piel, Géraldine; Piette, Jacques; Dubois, Philippe; Raes, Martine
2012-02-01
Skin carcinomas are among the most commonly diagnosed tumors in the world. In this study, we investigated the transfection of immortalized keratinocytes, used as an in vitro model for skin carcinoma, using the antisense technology and poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA)-based copolymers. In order to improve the transfection efficiency of the classic PDMAEMA polymers, copolymers were synthesized including a poly(N-morpholino)ethylmethacrylate) (PMEMA) moiety for an improved proton-sponge effect, intended to favour the release of the oligonucleotide from the acidic endosome. These copolymers were synthesized either statistically (with alternating PDMAEMA and PMEMA fragments) or in blocks (one PDMAEMA block followed by one PMEMA block). MTT assays were performed using the PDMAEMA-PMEMA copolymers and revealed no significant cytotoxicity of these polymers at an N/P ratio of 7.3. Using fluorescent oligonucleotides and analyzing transfection efficiency by flow cytometry, we noticed no significant differences between the two kinds of copolymers. However copolymers with a higher DMAEMA content and a higher Mn were also those displaying the highest vectorization efficiency. Confocal microscopy showed that these copolymers induced a fine granular distribution of the transfected antisense oligonucleotides inside the cells. We also assessed the functionality of the transfected antisense oligonucleotide by transfecting immortalized GFP expressing keratinocytes with a GFP antisense oligonucleotide using these copolymers. A significant silencing was achieved with a PDMAEMA-PMEMA in block copolymer (Mn=41,000, 89 % PDMAEMA). Together, these results suggest that PDMAEMA-PMEMA copolymers combining low toxicity, vectorization and proton sponge properties, can be efficiently used to transfect immortalized keratinocytes and so open new perspectives in the therapy of skin carcinomas as well as of other skin diseases of genetic or immunological origin. © 2012 Informa Healthcare USA, Inc.
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2009-05-01
RNA replication. J. Virol. 79:4599–4609. 6. Dolnik, O., L. Kolesnikova, and S. Becker . 2008. Filoviruses: interactions with the host cell. Cell. Mol... dystrophy ) and inhibit murine coronavirus rep- lication in vivo. Biochem. Soc. Trans. 35:826–828. 22. Moulton, H. M., M. H. Nelson, S. A. Hatlevig, M. T
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Xenopus Bicaudal-C Is Required for the Differentiation of the Amphibian Pronephros
Tran, Uyen; Mary Pickney, L.; Duygu Özpolat, B.; Wessely, Oliver
2007-01-01
The RNA-binding molecule Bicaudal-C regulates embryonic development in Drosophila and Xenopus. Interestingly, mouse mutants of Bicaudal-C do not show early patterning defects, but instead develop polycystic kidney disease (PKD). To further investigate the molecular mechanism of Bicaudal-C in kidney development, we analyzed its function in the developing amphibian pronephros. Bicaudal-C mRNA was present in the epithelial structures of the Xenopus pronephros, the tubules and the duct, but not the glomus. Inhibition of the translation of endogenous Bicaudal-C with antisense morpholino oligomers (xBic-C-MO) led to a PKD-like phenotype in Xenopus. Embryos lacking Bicaudal-C developed generalized edemas and dilated pronephric tubules and ducts. This phenotype was caused by impaired differentiation of the pronephros. Molecular markers specifically expressed in the late distal tubule were absent in xBic-C-MO-injected embryos. Furthermore, Bicaudal-C was not required for primary cilia formation, an important organelle affected in PKD. These data support the idea that Bicaudal-C functions downstream or parallel of a cilia-regulated signaling pathway. This pathway is required for terminal differentiation of the late distal tubule of the Xenopus pronephros and regulates renal epithelial cell differentiation, which - when disrupted - results in PKD. PMID:17521625
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Aromatic L-Amino Acid Decarboxylase (AADC) Is Crucial for Brain Development and Motor Functions
Shih, De-Fen; Hsiao, Chung-Der; Min, Ming-Yuan; Lai, Wen-Sung; Yang, Chianne-Wen; Lee, Wang-Tso; Lee, Shyh-Jye
2013-01-01
Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare pediatric neuro-metabolic disease in children. Due to the lack of an animal model, its pathogenetic mechanism is poorly understood. To study the role of AADC in brain development, a zebrafish model of AADC deficiency was generated. We identified an aadc gene homolog, dopa decarboxylase (ddc), in the zebrafish genome. Whole-mount in situ hybridization analysis showed that the ddc gene is expressed in the epiphysis, locus caeruleus, diencephalic catecholaminergic clusters, and raphe nuclei of 36-h post-fertilization (hpf) zebrafish embryos. Inhibition of Ddc by AADC inhibitor NSD-1015 or anti-sense morpholino oligonucleotides (MO) reduced brain volume and body length. We observed increased brain cell apoptosis and loss of dipencephalic catecholaminergic cluster neurons in ddc morphants (ddc MO-injected embryos). Seizure-like activity was also detected in ddc morphants in a dose-dependent manner. ddc morphants had less sensitive touch response and impaired swimming activity that could be rescued by injection of ddc plasmids. In addition, eye movement was also significantly impaired in ddc morphants. Collectively, loss of Ddc appears to result in similar phenotypes as that of ADCC deficiency, thus zebrafish could be a good model for investigating pathogenetic mechanisms of AADC deficiency in children. PMID:23940784
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Aromatic L-amino acid decarboxylase (AADC) is crucial for brain development and motor functions.
Shih, De-Fen; Hsiao, Chung-Der; Min, Ming-Yuan; Lai, Wen-Sung; Yang, Chianne-Wen; Lee, Wang-Tso; Lee, Shyh-Jye
2013-01-01
Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare pediatric neuro-metabolic disease in children. Due to the lack of an animal model, its pathogenetic mechanism is poorly understood. To study the role of AADC in brain development, a zebrafish model of AADC deficiency was generated. We identified an aadc gene homolog, dopa decarboxylase (ddc), in the zebrafish genome. Whole-mount in situ hybridization analysis showed that the ddc gene is expressed in the epiphysis, locus caeruleus, diencephalic catecholaminergic clusters, and raphe nuclei of 36-h post-fertilization (hpf) zebrafish embryos. Inhibition of Ddc by AADC inhibitor NSD-1015 or anti-sense morpholino oligonucleotides (MO) reduced brain volume and body length. We observed increased brain cell apoptosis and loss of dipencephalic catecholaminergic cluster neurons in ddc morphants (ddc MO-injected embryos). Seizure-like activity was also detected in ddc morphants in a dose-dependent manner. ddc morphants had less sensitive touch response and impaired swimming activity that could be rescued by injection of ddc plasmids. In addition, eye movement was also significantly impaired in ddc morphants. Collectively, loss of Ddc appears to result in similar phenotypes as that of ADCC deficiency, thus zebrafish could be a good model for investigating pathogenetic mechanisms of AADC deficiency in children.
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Forkhead transcription factor foxe1 regulates chondrogenesis in zebrafish.
Nakada, Chisako; Iida, Atsumi; Tabata, Yoko; Watanabe, Sumiko
2009-12-15
Forkhead transcription factor (Fox) e1 is a causative gene for Bamforth-Lazarus syndrome, which is characterized by hypothyroidism and cleft palate. Applying degenerate polymerase chain reaction using primers specific for the conserved forkhead domain, we identified zebrafish foxe1 (foxe1). Foxe1 is expressed in the thyroid, pharynx, and pharyngeal skeleton during development; strongly expressed in the gill and weakly expressed in the brain, eye, and heart in adult zebrafish. A loss of function of foxe1 by morpholino antisense oligo (MO) exhibited abnormal craniofacial development, shortening of Meckel's cartilage and the ceratohyals, and suppressed chondrycytic proliferation. However, at 27 hr post fertilization, the foxe1 MO-injected embryos showed normal dlx2, hoxa2, and hoxb2 expression, suggesting that the initial steps of pharyngeal skeletal development, including neural crest migration and specification of the pharyngeal arch occurred normally. In contrast, at 2 dpf, a severe reduction in the expression of sox9a, colIIaI, and runx2b, which play roles in chondrocytic proliferation and differentiation, was observed. Interestingly, fgfr2 was strongly upregulated in the branchial arches of the foxe1 MO-injected embryos. Unlike Foxe1-null mice, normal thyroid development in terms of morphology and thyroid-specific marker expression was observed in foxe1 MO-injected zebrafish embryos. Taken together, our results indicate that Foxe1 plays an important role in chondrogenesis during development of the pharyngeal skeleton in zebrafish, probably through regulation of fgfr2 expression. Furthermore, the roles reported for FOXE1 in mammalian thyroid development may have been acquired during evolution. (c) 2009 Wiley-Liss, Inc.
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Swenson, Dana L; Warfield, Kelly L; Warren, Travis K; Lovejoy, Candace; Hassinger, Jed N; Ruthel, Gordon; Blouch, Robert E; Moulton, Hong M; Weller, Dwight D; Iversen, Patrick L; Bavari, Sina
2009-05-01
Phosphorodiamidate morpholino oligomers (PMOs) are uncharged nucleic acid-like molecules designed to inactivate the expression of specific genes via the antisense-based steric hindrance of mRNA translation. PMOs have been successful at knocking out viral gene expression and replication in the case of acute viral infections in animal models and have been well tolerated in human clinical trials. We propose that antisense PMOs represent a promising class of therapeutic agents that may be useful for combating filoviral infections. We have previously shown that mice treated with a PMO whose sequence is complementary to a region spanning the start codon of VP24 mRNA were protected against lethal Ebola virus challenge. In the present study, we report on the abilities of two additional VP24-specific PMOs to reduce the cell-free translation of a VP24 reporter, to inhibit the in vitro replication of Ebola virus, and to protect mice against lethal challenge when the PMOs are delivered prior to infection. Additionally, structure-activity relationship evaluations were conducted to assess the enhancement of antiviral efficacy associated with PMO chemical modifications that included conjugation with peptides of various lengths and compositions, positioning of conjugated peptides to either the 5' or the 3' terminus, and the conferring of charge modifications by the addition of piperazine moieties. Conjugation with arginine-rich peptides greatly enhanced the antiviral efficacy of VP24-specific PMOs in infected cells and mice during lethal Ebola virus challenge.
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Knockdown of prothrombin in zebrafish.
Day, Kenneth; Krishnegowda, Naveen; Jagadeeswaran, Pudur
2004-01-01
Thrombin is a serine protease generated from its zymogen, prothrombin, and plays a central role in the coagulation cascade. It is also important for mammalian development. The zebrafish has now been established as an excellent genetic model for studies on mammalian hemostasis and development. In this report, we used prothrombin-specific antisense morpholinos to knock down the levels of prothrombin to characterize the effects of prothrombin deficiency in the zebrafish embryo. Prothrombin morpholino-injected zebrafish embryos yielded an early phenotype exhibiting severe abnormalities that later showed occasional bleeding. In a second late phenotype, the embryos had no observable morphological abnormalities in early stages, but showed occasional bleeding at later stages. These phenotypes resembled characteristics shown by prothrombin knockout mice. Laser-induced vascular injury on some of the normal appearing phenotypic larvae showed a prolonged time to occlusion, and recombinant zebrafish prothrombin injected into these larvae restored a normal time to occlusion thus showing the specificity of the morpholino effect. The system developed here should be useful for investigation of the role of thrombin in vertebrate development.
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microRNA-183 is Essential for Hair Cell Regeneration after Neomycin Injury in Zebrafish.
Kim, Chang Woo; Han, Ji Hyuk; Wu, Ling; Choi, Jae Young
2018-01-01
microRNAs (miRNAs) are non-coding RNAs composed of 20 to 22 nucleotides that regulate development and differentiation in various organs by silencing specific RNAs and regulating gene expression. In the present study, we show that the microRNA (miR)-183 cluster is upregulated during hair cell regeneration and that its inhibition reduces hair cell regeneration following neomycin-induced ototoxicity in zebrafish. miRNA expression patterns after neomycin exposure were analyzed using microarray chips. Quantitative polymerase chain reaction was performed to validate miR-183 cluster expression patterns following neomycin exposure (500 μM for 2 h). After injection of an antisense morpholino (MO) to miR-183 (MO-183) immediately after fertilization, hair cell regeneration after neomycin exposure in neuromast cells was evaluated by fluorescent staining (YO-PRO1). The MO-183 effect also was assessed in transgenic zebrafish larvae expressing green fluorescent protein (GFP) in inner ear hair cells. Microarray analysis clearly showed that the miR-183 cluster (miR-96, miR-182, and miR-183) was upregulated after neomycin treatment. We also confirmed upregulated expression of the miR-183 cluster during hair cell regeneration after neomycin-induced ototoxicity. miR-183 inhibition using MO-183 reduced hair cell regeneration in both wild-type and GFP transgenic zebrafish larvae. Our work demonstrates that the miR-183 cluster is essential for the regeneration of hair cells following ototoxic injury in zebrafish larvae. Therefore, regulation of the miR-183 cluster can be a novel target for stimulation of hair cell regeneration. © Copyright: Yonsei University College of Medicine 2018
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Antisense miR-7 impairs insulin expression in developing pancreas and in cultured pancreatic buds.
Nieto, Margarita; Hevia, Pedro; Garcia, Enrique; Klein, Dagmar; Alvarez-Cubela, Silvia; Bravo-Egana, Valia; Rosero, Samuel; Damaris Molano, R; Vargas, Nancy; Ricordi, Camillo; Pileggi, Antonello; Diez, Juan; Domínguez-Bendala, Juan; Pastori, Ricardo L
2012-01-01
MicroRNAs regulate gene expression by inhibiting translation or inducing target mRNA degradation. MicroRNAs regulate organ differentiation and embryonic development, including pancreatic specification and islet function. We showed previously that miR-7 is highly expressed in human pancreatic fetal and adult endocrine cells. Here we determined the expression profile of miR-7 in the mouse-developing pancreas by RT-PCR and in situ hybridization. MiR-7 expression was low between embryonic days e10.5 and e11.5, then began to increase at e13.5 through e14.5, and eventually decreased by e18. In situ hybridization and immunostaining analysis showed that miR-7 colocalizes with endocrine marker Isl1, suggesting that miR-7 is expressed preferentially in endocrine cells. Whole-mount in situ hybridization shows miR-7 highly expressed in the embryonic neural tube. To investigate the role of miR-7 in development of the mouse endocrine pancreas, antisense miR-7 morpholinos (MO) were delivered to the embryo at an early developmental stage (e10.5 days) via intrauterine fetal heart injection. Inhibition of miR-7 during early embryonic life results in an overall downregulation of insulin production, decreased β-cell numbers, and glucose intolerance in the postnatal period. This phenomenon is specific for miR-7 and possibly due to a systemic effect on pancreatic development. On the other hand, the in vitro inhibition of miR-7 in explanted pancreatic buds leads to β-cell death and generation of β-cells expressing less insulin than those in MO control. Therefore, in addition to the potential indirect effects on pancreatic differentiation derived from its systemic downregulation, the knockdown of miR-7 appears to have a β-cell-specific effect as well. These findings suggest that modulation of miR-7 expression could be utilized in the development of stem cell therapies to cure diabetes.
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Abnormal cerebellar development and ataxia in CARP VIII morphant zebrafish.
Aspatwar, Ashok; Tolvanen, Martti E E; Jokitalo, Eija; Parikka, Mataleena; Ortutay, Csaba; Harjula, Sanna-Kaisa E; Rämet, Mika; Vihinen, Mauno; Parkkila, Seppo
2013-02-01
Congenital ataxia and mental retardation are mainly caused by variations in the genes that affect brain development. Recent reports have shown that mutations in the CA8 gene are associated with mental retardation and ataxia in humans and ataxia in mice. The gene product, carbonic anhydrase-related protein VIII (CARP VIII), is predominantly present in cerebellar Purkinje cells, where it interacts with the inositol 1,4,5-trisphosphate receptor type 1, a calcium channel. In this study, we investigated the effects of the loss of function of CARP VIII during embryonic development in zebrafish using antisense morpholino oligonucleotides against the CA8 gene. Knockdown of CA8 in zebrafish larvae resulted in a curved body axis, pericardial edema and abnormal movement patterns. Histologic examination revealed gross morphologic defects in the cerebellar region and in the muscle. Electron microscopy studies showed increased neuronal cell death in developing larvae injected with CA8 antisense morpholinos. These data suggest a pivotal role for CARP VIII during embryonic development. Furthermore, suppression of CA8 expression leads to defects in motor and coordination functions, mimicking the ataxic human phenotype. This work reveals an evolutionarily conserved function of CARP VIII in brain development and introduces a novel zebrafish model in which to investigate the mechanisms of CARP VIII-related ataxia and mental retardation in humans.
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Geller, B. L.; Deere, J. D.; Stein, D. A.; Kroeker, A. D.; Moulton, H. M.; Iversen, P. L.
2003-01-01
Antisense phosphorodiamidate morpholino oligomers (PMOs) were tested for the ability to inhibit gene expression in Escherichia coli. PMOs targeted to either a myc-luciferase reporter gene product or 16S rRNA did not inhibit luciferase expression or growth. However, in a strain with defective lipopolysaccharide (lpxA mutant), which has a leaky outer membrane, PMOs targeted to the myc-luciferase or acyl carrier protein (acpP) mRNA significantly inhibited their targets in a dose-dependent response. A significant improvement was made by covalently joining the peptide (KFF)3KC to the end of PMOs. In strains with an intact outer membrane, (KFF)3KC-myc PMO inhibited luciferase expression by 63%. A second (KFF)3KC-PMO conjugate targeted to lacI mRNA induced β-galactosidase in a dose-dependent response. The end of the PMO to which (KFF)3KC is attached affected the efficiency of target inhibition but in various ways depending on the PMO. Another peptide-lacI PMO conjugate was synthesized with the cationic peptide CRRRQRRKKR and was found not to induce β-galactosidase. We conclude that the outer membrane of E. coli inhibits entry of PMOs and that (KFF)3KC-PMO conjugates are transported across both membranes and specifically inhibit expression of their genetic targets. PMID:14506035
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2006-01-01
Bavari 1* 1 US Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland, United States of America, 2 AVI BioPharma ...used as controls in these experiments. The PMOs were synthesized by AVI BioPharma , (Corvallis, Oregon, United States), as previously described [35]. In...interests. RB, DAS, and PLI have financial interest in AVI BioPharma , the company that supplied the PMO compounds for these studies. & References 1
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Maruyama, Rika; Echigoya, Yusuke; Caluseriu, Oana; Aoki, Yoshitsugu; Takeda, Shin'ichi; Yokota, Toshifumi
2017-01-01
Exon-skipping therapy is an emerging approach that uses synthetic DNA-like molecules called antisense oligonucleotides (AONs) to splice out frame-disrupting parts of mRNA, restore the reading frame, and produce truncated yet functional proteins. Multiple exon skipping utilizing a cocktail of AONs can theoretically treat 80-90% of patients with Duchenne muscular dystrophy (DMD). The success of multiple exon skipping by the systemic delivery of a cocktail of AONs called phosphorodiamidate morpholino oligomers (PMOs) in a DMD dog model has made a significant impact on the development of therapeutics for DMD, leading to clinical trials of PMO-based drugs. Here, we describe the systemic delivery of a cocktail of PMOs to skip multiple exons in dystrophic dogs and the evaluation of the efficacies and toxicity in vivo.
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Antisense antibiotics: a brief review of novel target discovery and delivery.
Bai, Hui; Xue, Xiaoyan; Hou, Zheng; Zhou, Ying; Meng, Jingru; Luo, Xiaoxing
2010-06-01
The nightmare of multi-drug resistant bacteria will still haunt if no panacea is ever found. Efforts on seeking desirable natural products with bactericidal property and screening chemically modified derivatives of traditional antibiotics have lagged behind the emergence of new multi-drug resistant bacteria. The concept of using antisense antibiotics, now as revolutionary as is on threshold has experienced ups and downs in the past decade. In the past five years, however, significant technology advances in the fields of microbial genomics, structural modification of oligonucleotides and efficient delivery system have led to fundamental progress in the research and in vivo application of this paradigm. The wealthy information provided in the microbial genomics era has allowed the identification and/or validation of a number of essential genes that may serve as possible targets for antisense inhibition; antisense oligodeoxynucleotides (ODNs) based on the 3rd generation of modified structures, e.g., peptide nucleic acids (PNAs) and phosphorodiamidate morpholino oligomers (PMOs) have shown great potency in gene expression inhibition in a sequence-specific and dosedependent manner at low micromolar concentrations; and cell penetrating peptide mediated delivery system has enabled the effective display of intracellular antisense inhibition of targeted genes both in vitro and in vivo. The new methods show promise in the discovery of novel gene-specific antisense antibiotics that will be useful in the future battle against drug-resistant bacterial infections. This review describes this promising paradigm, the targets that have been identified and the recent technologies on which it is delivered.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Kubota, Akira, E-mail: akubota@whoi.edu; Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543; Stegeman, John J.
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 viamore » 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 CYP1C prevents mesencephalic circulation failure by TCDD. > Induced CYP1Cs are involved in reduction of mesencephalic vein blood flow by TCDD.« less
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Yahyavi, Mani; Abouzeid, Hana; Gawdat, Ghada; de Preux, Anne-Sophie; Xiao, Tong; Bardakjian, Tanya; Schneider, Adele; Choi, Alex; Jorgenson, Eric; Baier, Herwig; El Sada, Mohamad; Schorderet, Daniel F; Slavotinek, Anne M
2013-08-15
The major active retinoid, all-trans retinoic acid, has long been recognized as critical for the development of several organs, including the eye. Mutations in STRA6, the gene encoding the cellular receptor for vitamin A, in patients with Matthew-Wood syndrome and anophthalmia/microphthalmia (A/M), have previously demonstrated the importance of retinol metabolism in human eye disease. We used homozygosity mapping combined with next-generation sequencing to interrogate patients with anophthalmia and microphthalmia for new causative genes. We used whole-exome and whole-genome sequencing to study a family with two affected brothers with bilateral A/M and a simplex case with bilateral anophthalmia and hypoplasia of the optic nerve and optic chiasm. Analysis of novel sequence variants revealed homozygosity for two nonsense mutations in ALDH1A3, c.568A>G, predicting p.Lys190*, in the familial cases, and c.1165A>T, predicting p.Lys389*, in the simplex case. Both mutations predict nonsense-mediated decay and complete loss of function. We performed antisense morpholino (MO) studies in Danio rerio to characterize the developmental effects of loss of Aldh1a3 function. MO-injected larvae showed a significant reduction in eye size, and aberrant axonal projections to the tectum were noted. We conclude that ALDH1A3 loss of function causes anophthalmia and aberrant eye development in humans and in animal model systems.
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Yahyavi, Mani; Abouzeid, Hana; Gawdat, Ghada; de Preux, Anne-Sophie; Xiao, Tong; Bardakjian, Tanya; Schneider, Adele; Choi, Alex; Jorgenson, Eric; Baier, Herwig; El Sada, Mohamad; Schorderet, Daniel F.; Slavotinek, Anne M.
2013-01-01
The major active retinoid, all-trans retinoic acid, has long been recognized as critical for the development of several organs, including the eye. Mutations in STRA6, the gene encoding the cellular receptor for vitamin A, in patients with Matthew–Wood syndrome and anophthalmia/microphthalmia (A/M), have previously demonstrated the importance of retinol metabolism in human eye disease. We used homozygosity mapping combined with next-generation sequencing to interrogate patients with anophthalmia and microphthalmia for new causative genes. We used whole-exome and whole-genome sequencing to study a family with two affected brothers with bilateral A/M and a simplex case with bilateral anophthalmia and hypoplasia of the optic nerve and optic chiasm. Analysis of novel sequence variants revealed homozygosity for two nonsense mutations in ALDH1A3, c.568A>G, predicting p.Lys190*, in the familial cases, and c.1165A>T, predicting p.Lys389*, in the simplex case. Both mutations predict nonsense-mediated decay and complete loss of function. We performed antisense morpholino (MO) studies in Danio rerio to characterize the developmental effects of loss of Aldh1a3 function. MO-injected larvae showed a significant reduction in eye size, and aberrant axonal projections to the tectum were noted. We conclude that ALDH1A3 loss of function causes anophthalmia and aberrant eye development in humans and in animal model systems. PMID:23591992
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Versatile synthesis and rational design of caged morpholinos.
Ouyang, Xiaohu; Shestopalov, Ilya A; Sinha, Surajit; Zheng, Genhua; Pitt, Cameron L W; Li, Wen-Hong; Olson, Andrew J; Chen, James K
2009-09-23
Embryogenesis is regulated by genetic programs that are dynamically executed in a stereotypic manner, and deciphering these molecular mechanisms requires the ability to control embryonic gene function with similar spatial and temporal precision. Chemical technologies can enable such genetic manipulations, as exemplified by the use of caged morpholino (cMO) oligonucleotides to inactivate genes in zebrafish and other optically transparent organisms with spatiotemporal control. Here we report optimized methods for the design and synthesis of hairpin cMOs incorporating a dimethoxynitrobenzyl (DMNB)-based bifunctional linker that permits cMO assembly in only three steps from commercially available reagents. Using this simplified procedure, we have systematically prepared cMOs with differing structural configurations and investigated how the in vitro thermodynamic properties of these reagents correlate with their in vivo activities. Through these studies, we have established general principles for cMO design and successfully applied them to several developmental genes. Our optimized synthetic and design methodologies have also enabled us to prepare a next-generation cMO that contains a bromohydroxyquinoline (BHQ)-based linker for two-photon uncaging. Collectively, these advances establish the generality of cMO technologies and will facilitate the application of these chemical probes in vivo for functional genomic studies.
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Versatile Synthesis and Rational Design of Caged Morpholinos
2009-01-01
Embryogenesis is regulated by genetic programs that are dynamically executed in a stereotypic manner, and deciphering these molecular mechanisms requires the ability to control embryonic gene function with similar spatial and temporal precision. Chemical technologies can enable such genetic manipulations, as exemplified by the use of caged morpholino (cMO) oligonucleotides to inactivate genes in zebrafish and other optically transparent organisms with spatiotemporal control. Here we report optimized methods for the design and synthesis of hairpin cMOs incorporating a dimethoxynitrobenzyl (DMNB)-based bifunctional linker that permits cMO assembly in only three steps from commercially available reagents. Using this simplified procedure, we have systematically prepared cMOs with differing structural configurations and investigated how the in vitro thermodynamic properties of these reagents correlate with their in vivo activities. Through these studies, we have established general principles for cMO design and successfully applied them to several developmental genes. Our optimized synthetic and design methodologies have also enabled us to prepare a next-generation cMO that contains a bromohydroxyquinoline (BHQ)-based linker for two-photon uncaging. Collectively, these advances establish the generality of cMO technologies and will facilitate the application of these chemical probes in vivo for functional genomic studies. PMID:19708646
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Timme-Laragy, Alicia R; Karchner, Sibel I; Hahn, Mark E
2012-01-01
The zebrafish (Danio rerio) has long been used as a model for developmental biology, making it an excellent model to use also in developmental toxicology. The many advantages of zebrafish include their small size, prolific spawning, rapid development, and transparent embryos. They can be easily manipulated genetically through the use of transgenic technology and gene knockdown via morpholino-modified antisense oligonucleotides (MOs). Knocking down specific genes to assess their role in the response to toxicant exposure provides a way to further our knowledge of how developmental toxicants work on a molecular and mechanistic level while establishing a relationship between these molecular events and morphological, behavioral, and/or physiological effects (i.e., phenotypic anchoring). In this chapter, we address important considerations for using MOs to study developmental toxicology in zebrafish embryos and provide a protocol for their use.
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Timme-Laragy, Alicia R.; Karchner, Sibel I.; Hahn, Mark E.
2014-01-01
Summary The zebrafish (Danio rerio) has long been used as a model for developmental biology, making it an excellent model to use also in developmental toxicology. The many advantages of zebrafish include their small size, prolific spawning, rapid development, and transparent embryos. They can be easily manipulated genetically through the use of transgenic technology and gene knock-down via morpholino-modified antisense oligonucleotides (MOs). Knocking down specific genes to assess their role in the response to toxicant exposure provides a way to further our knowledge of how developmental toxicants work on a molecular and mechanistic level, while establishing a relationship between these molecular events and morphological, behavioral, and/or physiological effects (i.e. phenotypic anchoring). In this chapter we address important considerations for using MOs to study developmental toxicology in zebrafish embryos and provide a protocol for their use. PMID:22669659
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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.
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Orrego, Patricio R.; Olivares, Héctor; Cordero, Esteban M.; Bressan, Albert; Cortez, Mauro; Sagua, Hernán; Neira, Ivan; González, Jorge; da Silveira, José Franco; Yoshida, Nobuko; Araya, Jorge E.
2014-01-01
Parasitological cure for Chagas disease is considered extremely difficult to achieve because of the lack of effective chemotherapeutic agents against Trypanosoma cruzi at different stages of infection. There are currently only two drugs available. These have several limitations and can produce serious side effects. Thus, new chemotherapeutic targets are much sought after. Among T. cruzi components involved in key processes such as parasite proliferation and host cell invasion, Ca2+-dependent molecules play an important role. Calcineurin (CaN) is one such molecule. In this study, we cloned a new isoform of the gene coding for CL strain catalytic subunit CaNA (TcCaNA2) and characterized it molecularly and functionally. There is one copy of the TcCaNA2 gene per haploid genome. It is constitutively transcribed in all T. cruzi developmental forms and is localized predominantly in the cytosol. In the parasite, TcCaNA2 is associated with CaNB. The recombinant protein TcCaNA2 has phosphatase activity that is enhanced by Mn2+/Ni2+. The participation of TcCaNA2 in target cell invasion by metacyclic trypomastigotes was also demonstrated. Metacyclic forms with reduced TcCaNA2 expression following treatment with morpholino antisense oligonucleotides targeted to TcCaNA2 invaded HeLa cells at a lower rate than control parasites treated with morpholino sense oligonucleotides. Similarly, the decreased expression of TcCaNA2 following treatment with antisense morpholino oligonucleotides partially affected the replication of epimastigotes, although to a lesser extent than the decrease in expression following treatment with calcineurin inhibitors. Our findings suggest that the calcineurin activities of TcCaNA2/CaNB and TcCaNA/CaNB, which have distinct cellular localizations (the cytoplasm and the nucleus, respectively), may play a critical role at different stages of T. cruzi development, the former in host cell invasion and the latter in parasite multiplication. PMID:24498455
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Antisense oligonucleotide therapeutics for iron-sulphur cluster deficiency myopathy.
Kollberg, Gittan; Holme, Elisabeth
2009-12-01
Iron-sulphur cluster deficiency myopathy is caused by a deep intronic mutation in ISCU resulting in inclusion of a cryptic exon in the mature mRNA. ISCU encodes the iron-sulphur cluster assembly protein IscU. Iron-sulphur clusters are essential for most basic redox transformations including the respiratory-chain function. Most patients are homozygous for the mutation with a phenotype characterized by a non-progressive myopathy with childhood onset of early fatigue, dyspnoea and palpitation on trivial exercise. A more severe phenotype with early onset of a slowly progressive severe muscle weakness, severe exercise intolerance and cardiomyopathy is caused by a missense mutation in compound with the intronic mutation. Treatment of cultured fibroblasts derived from three homozygous patients with an antisense phosphorodiamidate morpholino oligonucleotide for 48 h resulted in 100% restoration of the normal splicing pattern. The restoration was stable and after 21 days the correctly spliced mRNA still was the dominating RNA species.
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Histone deacetylase 1 is required for the development of the zebrafish inner ear
He, Yingzi; Tang, Dongmei; Li, Wenyan; Chai, Renjie; Li, Huawei
2016-01-01
Histone deacetylase 1 (HDAC1) has been reported to be important for multiple aspects of normal embryonic development, but little is known about its function in the development of mechanosensory organs. Here, we first confirmed that HDAC1 is expressed in the developing otic vesicles of zebrafish by whole-mount in situ hybridization. Knockdown of HDAC1 using antisense morpholino oligonucleotides in zebrafish embryos induced smaller otic vesicles, abnormal otoliths, malformed or absent semicircular canals, and fewer sensory hair cells. HDAC1 loss of function also caused attenuated expression of a subset of key genes required for otic vesicle formation during development. Morpholino-mediated knockdown of HDAC1 resulted in decreased expression of members of the Fgf family in the otic vesicles, suggesting that HDAC1 is involved in the development of the inner ear through regulation of Fgf signaling pathways. Taken together, our results indicate that HDAC1 plays an important role in otic vesicle formation. PMID:26832938
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The G-protein-coupled receptor, GPR84, is important for eye development in Xenopus laevis.
Perry, Kimberly J; Johnson, Verity R; Malloch, Erica L; Fukui, Lisa; Wever, Jason; Thomas, Alvin G; Hamilton, Paul W; Henry, Jonathan J
2010-11-01
G-protein-coupled receptors (GPCRs) represent diverse, multifamily groups of cell signaling receptors involved in many cellular processes. We identified Xenopus laevis GPR84 as a member of the A18 subfamily of GPCRs. During development, GPR84 is detected in the embryonic lens placode, differentiating lens fiber cells, retina, and cornea. Anti-sense morpholino oligonucleotide-mediated knockdown and RNA rescue experiments demonstrate GPR84's importance in lens, cornea, and retinal development. Examination of cell proliferation using an antibody against histone H3 S10P reveals significant increases in the lens and retina following GPR84 knockdown. Additionally, there was also an increase in apoptosis in the retina and lens, as revealed by TUNEL assay. Reciprocal transplantation of the presumptive lens ectoderm between uninjected controls and morpholino-injected embryos demonstrates that GPR84 is necessary in the retina for proper development of the retina, as well as other eye tissues including the lens and cornea. © 2010 Wiley-Liss, Inc.
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The G-protein-coupled receptor, GPR84, is important for eye development in Xenopus laevis
Perry, Kimberly J.; Johnson, Verity R.; Malloch, Erica L.; Fukui, Lisa; Wever, Jason; Thomas, Alvin G.; Hamilton, Paul W.; Henry, Jonathan J.
2010-01-01
G-protein-coupled receptors (GPCRs) represent diverse, multifamily groups of cell signaling receptors involved in many cellular processes. We identified Xenopus laevis GPR84 as a member of the A18 subfamily of GPCRs. During development, GPR84 is detected in the embryonic lens placode, differentiating lens fiber cells, retina and cornea. Anti-sense morpholino oligonucleotide-mediated knockdown and RNA rescue experiments demonstrate GPR84’s importance in lens, cornea and retinal development. Examination of cell proliferation using an antibody against histone H3 S10P reveals significant increases in the lens and retina following GPR84 knockdown. Additionally, there was also an increase in apoptosis in the retina and lens, as revealed by TUNEL assay. Reciprocal transplantation of the presumptive lens ectoderm between uninjected controls and morpholino injected embryos demonstrates that GPR84 is necessary in the retina for proper development of the retina, as well as other eye tissues including the lens and cornea. PMID:20925114
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Sun, Zhi-Hui; Zhou, Li; Li, Zhi; Liu, Xiao-Chun; Li, Shui-Sheng; Wang, Yang; Gui, Jian-Fang
2017-06-01
Dead end (dnd), vertebrate-specific germ cell marker, had been demonstrated to be essential for primordial germ cell (PGC) migration and survival, and the link between PGC number and sex change had been revealed in some teleost species, but little is known about dnd in hermaphroditic vertebrates. In the present study, a protogynous hermaphroditic orange-spotted grouper (Epinephelus coioides) dnd homologue (Ecdnd) was identified and characterized. Quantitative real-time PCR and in situ hybridization analysis revealed a dynamic and sexually dimorphic expression pattern in PGCs and germ cells of gonads. During sex changing, the Ecdnd transcript sharply increased in early transitional gonad, reached the highest level at late transitional gonad stage, and decreased after testis maturation. Visualization of zebrafish PGCs by injecting with RFP-Ecdnd-3'UTR RNA and GFP-zfnanos3-3'UTR RNA confirmed importance of Ecdnd 3'UTR for the PGC distribution. In addition, knockdown of EcDnd by using antisense morpholinos (MO) caused the ablation of PGCs in orange-spotted grouper. Therefore, the current data indicate that Ecdnd is essential for PGCs survival and may serve as a useful germ cell marker during gametogenesis in hermaphroditic grouper. Copyright © 2017 Elsevier Inc. All rights reserved.
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Neuman, Benjamin W.; Stein, David A.; Kroeker, Andrew D.; Churchill, Michael J.; Kim, Alice M.; Kuhn, Peter; Dawson, Philip; Moulton, Hong M.; Bestwick, Richard K.; Iversen, Patrick L.; Buchmeier, Michael J.
2005-01-01
The recently emerged severe acute respiratory syndrome coronavirus (SARS-CoV) is a potent pathogen of humans and is capable of rapid global spread. Peptide-conjugated antisense morpholino oligomers (P-PMO) were designed to bind by base pairing to specific sequences in the SARS-CoV (Tor2 strain) genome. The P-PMO were tested for their capacity to inhibit production of infectious virus as well as to probe the function of conserved viral RNA motifs and secondary structures. Several virus-targeted P-PMO and a random-sequence control P-PMO showed low inhibitory activity against SARS coronavirus. Certain other virus-targeted P-PMO reduced virus-induced cytopathology and cell-to-cell spread as a consequence of decreasing viral amplification. Active P-PMO were effective when administered at any time prior to peak viral synthesis and exerted sustained antiviral effects while present in culture medium. P-PMO showed low nonspecific inhibitory activity against translation of nontargeted RNA or growth of the arenavirus lymphocytic choriomeningitis virus. Two P-PMO targeting the viral transcription-regulatory sequence (TRS) region in the 5′ untranslated region were the most effective inhibitors tested. After several viral passages in the presence of a TRS-targeted P-PMO, partially drug-resistant SARS-CoV mutants arose which contained three contiguous base point mutations at the binding site of a TRS-targeted P-PMO. Those partially resistant viruses grew more slowly and formed smaller plaques than wild-type SARS-CoV. These results suggest PMO compounds have powerful therapeutic and investigative potential toward coronavirus infection. PMID:16014928
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Shabanpoor, Fazel; Hammond, Suzan M; Abendroth, Frank; Hazell, Gareth; Wood, Matthew J.A.
2017-01-01
Splice-switching antisense oligonucleotides are emerging treatments for neuromuscular diseases, with several splice-switching oligonucleotides (SSOs) currently undergoing clinical trials such as for Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA). However, the development of systemically delivered antisense therapeutics has been hampered by poor tissue penetration and cellular uptake, including crossing of the blood–brain barrier (BBB) to reach targets in the central nervous system (CNS). For SMA application, we have investigated the ability of various BBB-crossing peptides for CNS delivery of a splice-switching phosphorodiamidate morpholino oligonucleotide (PMO) targeting survival motor neuron 2 (SMN2) exon 7 inclusion. We identified a branched derivative of the well-known ApoE (141–150) peptide, which as a PMO conjugate was capable of exon inclusion in the CNS following systemic administration, leading to an increase in the level of full-length SMN2 transcript. Treatment of newborn SMA mice with this peptide-PMO (P-PMO) conjugate resulted in a significant increase in the average lifespan and gains in weight, muscle strength, and righting reflexes. Systemic treatment of adult SMA mice with this newly identified P-PMO also resulted in small but significant increases in the levels of SMN2 pre-messenger RNA (mRNA) exon inclusion in the CNS and peripheral tissues. This work provides proof of principle for the ability to select new peptide paradigms to enhance CNS delivery and activity of a PMO SSO through use of a peptide-based delivery platform for the treatment of SMA potentially extending to other neuromuscular and neurodegenerative diseases. PMID:28118087
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Penn, Andrew C.; Balik, Ales; Greger, Ingo H.
2013-01-01
Adenosine-to-Inosine (A-to-I) RNA editing is a post-transcriptional mechanism, evolved to diversify the transcriptome in metazoa. In addition to wide-spread editing in non-coding regions protein recoding by RNA editing allows for fine tuning of protein function. Functional consequences are only known for some editing sites and the combinatorial effect between multiple sites (functional epistasis) is currently unclear. Similarly, the interplay between RNA editing and splicing, which impacts on post-transcriptional gene regulation, has not been resolved. Here, we describe a versatile antisense approach, which will aid resolving these open questions. We have developed and characterized morpholino oligos targeting the most efficiently edited site—the AMPA receptor GluA2 Q/R site. We show that inhibition of editing closely correlates with intronic editing efficiency, which is linked to splicing efficiency. In addition to providing a versatile tool our data underscore the unique efficiency of a physiologically pivotal editing site. PMID:23172291
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Lu-Nguyen, Ngoc B; Jarmin, Susan A; Saleh, Amer F; Popplewell, Linda; Gait, Michael J; Dickson, George
2015-08-01
The fatal X-linked Duchenne muscular dystrophy (DMD), characterized by progressive muscle wasting and muscle weakness, is caused by mutations within the DMD gene. The use of antisense oligonucleotides (AOs) modulating pre-mRNA splicing to restore the disrupted dystrophin reading frame, subsequently generating a shortened but functional protein has emerged as a potential strategy in DMD treatment. AO therapy has recently been applied to induce out-of-frame exon skipping of myostatin pre-mRNA, knocking-down expression of myostatin protein, and such an approach is suggested to enhance muscle hypertrophy/hyperplasia and to reduce muscle necrosis. Within this study, we investigated dual exon skipping of dystrophin and myostatin pre-mRNAs using phosphorodiamidate morpholino oligomers conjugated with an arginine-rich peptide (B-PMOs). Intraperitoneal administration of B-PMOs was performed in neonatal mdx males on the day of birth, and at weeks 3 and 6. At week 9, we observed in treated mice (as compared to age-matched, saline-injected controls) normalization of muscle mass, a recovery in dystrophin expression, and a decrease in muscle necrosis, particularly in the diaphragm. Our data provide a proof of concept for antisense therapy combining dystrophin restoration and myostatin inhibition for the treatment of DMD.
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Gne depletion during zebrafish development impairs skeletal muscle structure and function.
Daya, Alon; Vatine, Gad David; Becker-Cohen, Michal; Tal-Goldberg, Tzukit; Friedmann, Adam; Gothilf, Yoav; Du, Shao Jun; Mitrani-Rosenbaum, Stella
2014-07-01
GNE Myopathy is a rare recessively inherited neuromuscular disorder caused by mutations in the GNE gene, which codes for the key enzyme in the metabolic pathway of sialic acid synthesis. The process by which GNE mutations lead to myopathy is not well understood. By in situ hybridization and gne promoter-driven fluorescent transgenic fish generation, we have characterized the spatiotemporal expression pattern of the zebrafish gne gene and have shown that it is highly conserved compared with the human ortholog. We also show the deposition of maternal gne mRNA and maternal GNE protein at the earliest embryonic stage, emphasizing the critical role of gne in embryonic development. Injection of morpholino (MO)-modified antisense oligonucleotides specifically designed to knockdown gne, into one-cell embryos lead to a variety of phenotypic severity. Characterization of the gne knockdown morphants showed a significantly reduced locomotor activity as well as distorted muscle integrity, including a reduction in the number of muscle myofibers, even in mild or intermediate phenotype morphants. These findings were further confirmed by electron microscopy studies, where large gaps between sarcolemmas were visualized, although normal sarcomeric structures were maintained. These results demonstrate a critical novel role for gne in embryonic development and particularly in myofiber development, muscle integrity and activity. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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Iversen, Patrick L.; Warren, Travis K.; Wells, Jay B.; Garza, Nicole L.; Mourich, Dan V.; Welch, Lisa S.; Panchal, Rekha G.; Bavari, Sina
2012-01-01
There are no currently approved treatments for filovirus infections. In this study we report the discovery process which led to the development of antisense Phosphorodiamidate Morpholino Oligomers (PMOs) AVI-6002 (composed of AVI-7357 and AVI-7539) and AVI-6003 (composed of AVI-7287 and AVI-7288) targeting Ebola virus and Marburg virus respectively. The discovery process involved identification of optimal transcript binding sites for PMO based RNA-therapeutics followed by screening for effective viral gene target in mouse and guinea pig models utilizing adapted viral isolates. An evolution of chemical modifications were tested, beginning with simple Phosphorodiamidate Morpholino Oligomers (PMO) transitioning to cell penetrating peptide conjugated PMOs (PPMO) and ending with PMOplus containing a limited number of positively charged linkages in the PMO structure. The initial lead compounds were combinations of two agents targeting separate genes. In the final analysis, a single agent for treatment of each virus was selected, AVI-7537 targeting the VP24 gene of Ebola virus and AVI-7288 targeting NP of Marburg virus, and are now progressing into late stage clinical development as the optimal therapeutic candidates. PMID:23202506
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Fletcher, Sue; Honeyman, Kaite; Fall, Abbie M; Harding, Penny L; Johnsen, Russell D; Steinhaus, Joshua P; Moulton, Hong M; Iversen, Patrick L; Wilton, Stephen D
2007-09-01
Duchenne and Becker muscular dystrophies are allelic disorders arising from mutations in the dystrophin gene. Duchenne muscular dystrophy is characterized by an absence of functional protein, whereas Becker muscular dystrophy, commonly caused by in-frame deletions, shows synthesis of partially functional protein. Anti-sense oligonucleotides can induce specific exon removal during processing of the dystrophin primary transcript, while maintaining or restoring the reading frame, and thereby overcome protein-truncating mutations. The mdx mouse has a non-sense mutation in exon 23 of the dystrophin gene that precludes functional dystrophin production, and this model has been used in the development of treatment strategies for dystrophinopathies. A phosphorodiamidate morpholino oligomer (PMO) has previously been shown to exclude exon 23 from the dystrophin gene transcript and induce dystrophin expression in the mdxmouse, in vivo and in vitro. In this report, a cell-penetrating peptide (CPP)-conjugated oligomer targeted to the mouse dystrophin exon 23 donor splice site was administered to mdxmice by intraperitoneal injection. We demonstrate dystrophin expression and near-normal muscle architecture in all muscles examined, except for cardiac muscle. The CPP greatly enhanced uptake of the PMO, resulting in widespread dystrophin expression.
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Iversen, Patrick L; Warren, Travis K; Wells, Jay B; Garza, Nicole L; Mourich, Dan V; Welch, Lisa S; Panchal, Rekha G; Bavari, Sina
2012-11-06
There are no currently approved treatments for filovirus infections. In this study we report the discovery process which led to the development of antisense Phosphorodiamidate Morpholino Oligomers (PMOs) AVI-6002 (composed of AVI-7357 and AVI-7539) and AVI-6003 (composed of AVI-7287 and AVI-7288) targeting Ebola virus and Marburg virus respectively. The discovery process involved identification of optimal transcript binding sites for PMO based RNA-therapeutics followed by screening for effective viral gene target in mouse and guinea pig models utilizing adapted viral isolates. An evolution of chemical modifications were tested, beginning with simple Phosphorodiamidate Morpholino Oligomers (PMO) transitioning to cell penetrating peptide conjugated PMOs (PPMO) and ending with PMOplus containing a limited number of positively charged linkages in the PMO structure. The initial lead compounds were combinations of two agents targeting separate genes. In the final analysis, a single agent for treatment of each virus was selected, AVI-7537 targeting the VP24 gene of Ebola virus and AVI-7288 targeting NP of Marburg virus, and are now progressing into late stage clinical development as the optimal therapeutic candidates.
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Nakada, Chisako; Satoh, Shinya; Tabata, Yoko; Arai, Ken-ichi; Watanabe, Sumiko
2006-10-01
We identified zebra fish forkhead transcription factor l1 (zfoxl1) as a gene strongly expressed in neural tissues such as midbrain, hindbrain, and the otic vesicle at the early embryonic stage. Loss of the function of zfoxl1 effected by morpholino antisense oligonucleotide resulted in defects in midbrain and eye development, and in that of formation of the pectoral fins. Interestingly, ectopic expression of shh in the midbrain and elevated pax2a expression in the optic stalk were observed in foxl1 MO-injected embryos. In contrast, expression of pax6a, which is negatively regulated by shh, was suppressed in the thalamus and pretectum regions, supporting the idea of augmentation of the shh signaling pathway by suppression of foxl1. Expression of zfoxl1-EnR (repressing) rather than zfoxl1-VP16 (activating) resulted in a phenotype similar to that induced by foxl1-mRNA, suggesting that foxl1 may act as a transcriptional repressor of shh in zebra fish embryos. Supporting this notion, foxl1 suppressed isolated 2.7-kb shh promoter activity in PC12 cells, and the minimal region of foxl1 required for its transcriptional repressor activity showed strong homology with the groucho binding motif, which is found in genes encoding various homeodomain proteins. In view of all of our data taken together, we propose zfoxl1 to be a novel regulator of neural development that acts by suppressing shh expression.
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Nakada, Chisako; Satoh, Shinya; Tabata, Yoko; Arai, Ken-ichi; Watanabe, Sumiko
2006-01-01
We identified zebra fish forkhead transcription factor l1 (zfoxl1) as a gene strongly expressed in neural tissues such as midbrain, hindbrain, and the otic vesicle at the early embryonic stage. Loss of the function of zfoxl1 effected by morpholino antisense oligonucleotide resulted in defects in midbrain and eye development, and in that of formation of the pectoral fins. Interestingly, ectopic expression of shh in the midbrain and elevated pax2a expression in the optic stalk were observed in foxl1 MO-injected embryos. In contrast, expression of pax6a, which is negatively regulated by shh, was suppressed in the thalamus and pretectum regions, supporting the idea of augmentation of the shh signaling pathway by suppression of foxl1. Expression of zfoxl1-EnR (repressing) rather than zfoxl1-VP16 (activating) resulted in a phenotype similar to that induced by foxl1-mRNA, suggesting that foxl1 may act as a transcriptional repressor of shh in zebra fish embryos. Supporting this notion, foxl1 suppressed isolated 2.7-kb shh promoter activity in PC12 cells, and the minimal region of foxl1 required for its transcriptional repressor activity showed strong homology with the groucho binding motif, which is found in genes encoding various homeodomain proteins. In view of all of our data taken together, we propose zfoxl1 to be a novel regulator of neural development that acts by suppressing shh expression. PMID:16980626
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The Molecular Biology of Frog Virus 3 and other Iridoviruses Infecting Cold-Blooded Vertebrates
Chinchar, V. Gregory; Yu, Kwang H.; Jancovich, James K.
2011-01-01
Frog virus 3 (FV3) is the best characterized member of the family Iridoviridae. FV3 study has provided insights into the replication of other family members, and has served as a model of viral transcription, genome replication, and virus-mediated host-shutoff. Although the broad outlines of FV3 replication have been elucidated, the precise roles of most viral proteins remain unknown. Current studies using knock down (KD) mediated by antisense morpholino oligonucleotides (asMO) and small, interfering RNAs (siRNA), knock out (KO) following replacement of the targeted gene with a selectable marker by homologous recombination, ectopic viral gene expression, and recombinant viral proteins have enabled researchers to systematically ascertain replicative- and virulence-related gene functions. In addition, the application of molecular tools to ecological studies is providing novel ways for field biologists to identify potential pathogens, quantify infections, and trace the evolution of ecologically important viral species. In this review, we summarize current studies using not only FV3, but also other iridoviruses infecting ectotherms. As described below, general principles ascertained using FV3 served as a model for the family, and studies utilizing other ranaviruses and megalocytiviruses have confirmed and extended our understanding of iridovirus replication. Collectively, these and future efforts will elucidate molecular events in viral replication, intrinsic and extrinsic factors that contribute to disease outbreaks, and the role of the host immune system in protection from disease. PMID:22069524
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Augmenter of Liver Regeneration (alr) Promotes Liver Outgrowth during Zebrafish Hepatogenesis
Li, Yan; Farooq, Muhammad; Sheng, Donglai; Chandramouli, Chanchal; Lan, Tian; Mahajan, Nilesh K.; Kini, R. Manjunatha; Hong, Yunhan; Lisowsky, Thomas; Ge, Ruowen
2012-01-01
Augmenter of Liver Regeneration (ALR) is a sulfhydryl oxidase carrying out fundamental functions facilitating protein disulfide bond formation. In mammals, it also functions as a hepatotrophic growth factor that specifically stimulates hepatocyte proliferation and promotes liver regeneration after liver damage or partial hepatectomy. Whether ALR also plays a role during vertebrate hepatogenesis is unknown. In this work, we investigated the function of alr in liver organogenesis in zebrafish model. We showed that alr is expressed in liver throughout hepatogenesis. Knockdown of alr through morpholino antisense oligonucleotide (MO) leads to suppression of liver outgrowth while overexpression of alr promotes liver growth. The small-liver phenotype in alr morphants results from a reduction of hepatocyte proliferation without affecting apoptosis. When expressed in cultured cells, zebrafish Alr exists as dimer and is localized in mitochondria as well as cytosol but not in nucleus or secreted outside of the cell. Similar to mammalian ALR, zebrafish Alr is a flavin-linked sulfhydryl oxidase and mutation of the conserved cysteine in the CxxC motif abolishes its enzymatic activity. Interestingly, overexpression of either wild type Alr or enzyme-inactive AlrC131S mutant promoted liver growth and rescued the liver growth defect of alr morphants. Nevertheless, alr C131S is less efficacious in both functions. Meantime, high doses of alr MOs lead to widespread developmental defects and early embryonic death in an alr sequence-dependent manner. These results suggest that alr promotes zebrafish liver outgrowth using mechanisms that are dependent as well as independent of its sulfhydryl oxidase activity. This is the first demonstration of a developmental role of alr in vertebrate. It exemplifies that a low-level sulfhydryl oxidase activity of Alr is essential for embryonic development and cellular survival. The dose-dependent and partial suppression of alr expression through MO-mediated knockdown allows the identification of its late developmental role in vertebrate liver organogenesis. PMID:22292055
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Combinatorial Wnt control of zebrafish midbrain-hindbrain boundary formation.
Buckles, Gerri R; Thorpe, Christopher J; Ramel, Marie-Christine; Lekven, Arne C
2004-05-01
Wnt signaling is known to be required for the normal development of the vertebrate midbrain and hindbrain, but genetic loss of function analyses in the mouse and zebrafish yield differing results regarding the relative importance of specific Wnt loci. In the zebrafish, Wnt1 and Wnt10b functionally overlap in their control of gene expression in the ventral midbrain-hindbrain boundary (MHB), but they are not required for the formation of the MHB constriction. Whether other wnt loci are involved in zebrafish MHB development is unclear, although the expression of at least two wnts, wnt3a and wnt8b, is maintained in wnt1/wnt10b mutants. In order to address the role of wnt3a in zebrafish, we have isolated a full length cDNA and examined its expression and function via knockdown by morpholino antisense oligonucleotide (MO)-mediated knockdown. The expression pattern of wnt3a appears to be evolutionarily conserved between zebrafish and mouse, and MO knockdown shows that Wnt3a, while not uniquely required for MHB development, is required in the absence of Wnt1 and Wnt10b for the formation of the MHB constriction. In zebrafish embryos lacking Wnt3a, Wnt1 and Wnt10b, the expression of engrailed orthologs, pax2a and fgf8 is not maintained after mid-somitogenesis. In contrast to acerebellar and no isthmus mutants, in which midbrain and hindbrain cells acquire new fates but cell number is not significantly affected until late in embryogenesis, zebrafish embryos lacking Wnt3a, Wnt1 and Wnt10b undergo extensive apoptosis in the midbrain and cerebellum anlagen beginning in mid-somitogenesis, which results in the absence of a significant portion of the midbrain and cerebellum. Thus, the requirement for Wnt signaling in forming the MHB constriction is evolutionarily conserved in vertebrates and it is possible in zebrafish to dissect the relative impact of multiple Wnt loci in midbrain and hindbrain development.
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Zebrafish fgf3 and fgf8 encode redundant functions required for otic placode induction.
Phillips, B T; Bolding, K; Riley, B B
2001-07-15
Members of the fibroblast growth factor (FGF) family of peptide ligands have been implicated in otic placode induction in several vertebrate species. Here, we have functionally analyzed the roles of fgf3 and fgf8 in zebrafish otic development. The role of fgf8 was assessed by analyzing acerebellar (ace) mutants. fgf3 function was disrupted by injecting embryos with antisense morpholino oligomers (MO) specifically designed to block translation of fgf3 transcripts. Disruption of either fgf3 or fgf8 causes moderate reduction in the size of the otic vesicle. Injection of fgf3-MO into ace/ace mutants causes much more severe reduction or complete loss of otic tissue. Moreover, preplacode cells fail to express pax8 and pax2.1, indicating disruption of early stages of otic induction in fgf3-depleted ace/ace mutants. Both fgf3 and fgf8 are normally expressed in the germring by 50% epiboly and are induced in the primordium of rhombomere 4 by 80% epibloy. In addition, fgf3 is expressed during the latter half of gastrulation in the prechordal plate and paraxial cephalic mesendoderm, tissues that either pass beneath or persist near the prospective otic ectoderm. Conditions that alter the pattern of expression of fgf3 and/or fgf8 cause corresponding changes in otic induction. Loss of maternal and zygotic one-eyed pinhead (oep) does not alter expression of fgf3 or fgf8 in the hindbrain, but ablates mesendodermal sources of fgf signaling and delays otic induction by several hours. Conversely, treatment of wild-type embryos with retinoic acid greatly expands the periotic domains of expression of fgf3, fgf8, and pax8 and leads to formation of supernumerary and ectopic otic vesicles. These data support the hypothesis that fgf3 and fgf8 cooperate during the latter half of gastrulation to induce differentiation of otic placodes. Copyright 2001 Academic Press.
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Function of the two Xenopus smad4s in early frog development.
Chang, Chenbei; Brivanlou, Ali H; Harland, Richard M
2006-10-13
Signals from the transforming growth factor beta family members are transmitted in the cell through specific receptor-activated Smads and a common partner Smad4. Two Smad4 genes (alpha and beta/10, or smad4 and smad4.2) have been isolated from Xenopus, and conflicting data are reported for Smad4beta/10 actions in mesodermal and neural induction. To further understand the functions of the Smad4s in early frog development, we analyzed their activities in detail. We report that Smad10 is a mutant form of Smad4beta that harbors a missense mutation of a conserved arginine to histidine in the MH1 domain. The mutation results in enhanced association of Smad10 with the nuclear transcription corepressor Ski and leads to its neural inducing activity through inhibition of bone morphogenetic protein (BMP) signaling. In contrast to Smad10, both Smad4alpha and Smad4beta enhanced BMP signals in ectodermal explants. Using antisense morpholino oligonucleotides (MOs) to knockdown endogenous Smad4 protein levels, we discovered that Smad4beta was required for both activin- and BMP-mediated mesodermal induction in animal caps, whereas Smad4alpha affected only the BMP signals. Neither Smad4 was involved directly in neural induction. Expression of Smad4beta-MO in early frog embryos resulted in reduction of mesodermal markers and defects in axial structures, which were rescued by either Smad4alpha or Smad4beta. Smad4alpha-MO induced only minor deficiency at late stages. As Smad4beta, but not Smad4alpha, is expressed at high levels maternally and during early gastrulation, our data suggest that although Smad4alpha and Smad4beta may have similar activities, they are differentially utilized during frog embryogenesis, with only Smad4beta being essential for mesoderm induction.
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Wang, Mingxing; Wu, Bo; Tucker, Jason D; Bollinger, Lauren E; Lu, Peijuan; Lu, Qilong
2016-01-01
A series of poly(esteramine)s (PEAs) constructed from low molecular weight polyethyleneimine (LPEI) and Pluronic were evaluated for the delivery of antisense oligonuclotides (AOs), 2′-O-methyl phosphorothioate RNA (2′-OMePS) and phosphorodiamidate morpholino oligomer (PMO) in cell culture and dystrophic mdx mice. Improved exon-skipping efficiency of both 2′-OMePS and PMO was observed in the C2C12E50 cell line with all PEA polymers compared with PEI 25k or LF-2k. The degree of efficiency was found in the order of PEA 01, PEA 04 > PEA 05 > others. The in vivo study in mdx mice demonstrated enhanced exon-skipping of 2′-OMePS with the order of PEA 06 > PEA 04, PEA 07 > PEA 03 > PEA 01 > others, and much higher than PEI 25k formulated 2′-OMePS. Exon-skipping efficiency of PMO in formulation with the PEAs were significantly enhanced in the order of PEA 02 > PEA 10 > PEA 01, PEA 03 > PEA 05, PEA 07, PEA 08 > others, with PEA 02 reaching fourfold of Endo-porter formulated PMO. PEAs improve PMO delivery more effectively than 2′-OMePS delivery in vivo, and the systemic delivery evaluation further highlight the efficiency of PEA for PMO delivery in all skeletal muscle. The results suggest that the flexibility of PEA polymers could be explored for delivery of different AO chemistries, especially for antisense therapy. PMID:27483024
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Genome editing in sea urchin embryos by using a CRISPR/Cas9 system.
Lin, Che-Yi; Su, Yi-Hsien
2016-01-15
Sea urchin embryos are a useful model system for investigating early developmental processes and the underlying gene regulatory networks. Most functional studies using sea urchin embryos rely on antisense morpholino oligonucleotides to knockdown gene functions. However, major concerns related to this technique include off-target effects, variations in morpholino efficiency, and potential morpholino toxicity; furthermore, such problems are difficult to discern. Recent advances in genome editing technologies have introduced the prospect of not only generating sequence-specific knockouts, but also providing genome-engineering applications. Two genome editing tools, zinc-finger nuclease (ZFN) and transcription activator-like effector nucleases (TALENs), have been utilized in sea urchin embryos, but the resulting efficiencies are far from satisfactory. The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9) system serves as an easy and efficient method with which to edit the genomes of several established and emerging model organisms in the field of developmental biology. Here, we apply the CRISPR/Cas9 system to the sea urchin embryo. We designed six guide RNAs (gRNAs) against the well-studied nodal gene and discovered that five of the gRNAs induced the expected phenotype in 60-80% of the injected embryos. In addition, we developed a simple method for isolating genomic DNA from individual embryos, enabling phenotype to be precisely linked to genotype, and revealed that the mutation rates were 67-100% among the sequenced clones. Of the two potential off-target sites we examined, no off-target effects were observed. The detailed procedures described herein promise to accelerate the usage of CRISPR/Cas9 system for genome editing in sea urchin embryos. Copyright © 2015 Elsevier Inc. All rights reserved.
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Warren, Travis K; Whitehouse, Chris A; Wells, Jay; Welch, Lisa; Heald, Alison E; Charleston, Jay S; Sazani, Pete; Reid, St Patrick; Iversen, Patrick L; Bavari, Sina
2015-02-10
Ebola viruses (EBOV) cause severe disease in humans and nonhuman primates with high mortality rates and continue to emerge in new geographic locations, including several countries in West Africa, the site of a large ongoing outbreak. Phosphorodiamidate morpholino oligomers (PMOs) are synthetic antisense molecules that are able to target mRNAs in a sequence-specific fashion and suppress translation through steric hindrance. We previously showed that the use of PMOs targeting a combination of VP35 and VP24 protected rhesus monkeys from lethal EBOV infection. Surprisingly, the present study revealed that a PMOplus compound targeting VP24 alone was sufficient to confer protection from lethal EBOV infection but that a PMOplus targeting VP35 alone resulted in no protection. This study further substantiates recent data demonstrating that VP24 may be a key virulence factor encoded by EBOV and suggests that VP24 is a promising target for the development of effective anti-EBOV countermeasures. Several West African countries are currently being ravaged by an outbreak of Ebola virus (EBOV) that has become a major epidemic affecting not only these African countries but also Europe and the United States. A better understanding of the mechanism of virulence of EBOV is important for the development of effective treatments, as no licensed treatments or vaccines for EBOV disease are currently available. This study of phosphorodiamidate morpholino oligomers (PMOs) targeting the mRNAs of two different EBOV proteins, alone and in combination, demonstrated that targeting a single protein was effective at conferring a significant survival benefit in an EBOV lethal primate model. Future development of PMOs with efficacy against EBOV will be simplified if only one PMO is required instead of a combination, particularly in terms of regulatory approval. Copyright © 2015 Warren et al.
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Frodo proteins: modulators of Wnt signaling in vertebrate development.
Brott, Barbara K; Sokol, Sergei Y
2005-09-01
The Frodo/dapper (Frd) proteins are recently discovered signaling adaptors, which functionally and physically interact with Wnt and Nodal signaling pathways during vertebrate development. The Frd1 and Frd2 genes are expressed in dynamic patterns in early embryos, frequently in cells undergoing epithelial-mesenchymal transition. The Frd proteins function in multiple developmental processes, including mesoderm and neural tissue specification, early morphogenetic cell movements, and organogenesis. Loss-of-function studies using morpholino antisense oligonucleotides demonstrate that the Frd proteins regulate Wnt signal transduction in a context-dependent manner and may be involved in Nodal signaling. The identification of Frd-associated factors and cellular targets of the Frd proteins should shed light on the molecular mechanisms underlying Frd functions in embryonic development and in cancer.
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Antisense pre-treatment increases gene therapy efficacy in dystrophic muscles.
Peccate, Cécile; Mollard, Amédée; Le Hir, Maëva; Julien, Laura; McClorey, Graham; Jarmin, Susan; Le Heron, Anita; Dickson, George; Benkhelifa-Ziyyat, Sofia; Piétri-Rouxel, France; Wood, Matthew J; Voit, Thomas; Lorain, Stéphanie
2016-08-15
In preclinical models for Duchenne muscular dystrophy, dystrophin restoration during adeno-associated virus (AAV)-U7-mediated exon-skipping therapy was shown to decrease drastically after six months in treated muscles. This decline in efficacy is strongly correlated with the loss of the therapeutic AAV genomes, probably due to alterations of the dystrophic myofiber membranes. To improve the membrane integrity of the dystrophic myofibers at the time of AAV-U7 injection, mdx muscles were pre-treated with a single dose of the peptide-phosphorodiamidate morpholino (PPMO) antisense oligonucleotides that induced temporary dystrophin expression at the sarcolemma. The PPMO pre-treatment allowed efficient maintenance of AAV genomes in mdx muscles and enhanced the AAV-U7 therapy effect with a ten-fold increase of the protein level after 6 months. PPMO pre-treatment was also beneficial to AAV-mediated gene therapy with transfer of micro-dystrophin cDNA into muscles. Therefore, avoiding vector genome loss after AAV injection by PPMO pre-treatment would allow efficient long-term restoration of dystrophin and the use of lower and thus safer vector doses for Duchenne patients. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
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Cadherin-17 is required to maintain pronephric duct integrity during zebrafish development.
Horsfield, Julia; Ramachandran, Anassuya; Reuter, Katja; LaVallie, Edward; Collins-Racie, Lisa; Crosier, Kathryn; Crosier, Philip
2002-07-01
We have isolated a zebrafish cadherin that is orthologous to human LI-cadherin (CDH17). Zebrafish cdh17 is expressed exclusively in the pronephric ducts during embryogenesis, and in the mesonephros during larval development and adulthood. Like its mammalian ortholog, cdh17 is also expressed in liver and intestine in adult zebrafish. We show that cdh17-positive mesodermal cells do not contribute to the hematopoietic system. Consistent with a cell adhesion role for Cdh17, depletion of Cdh17 function using antisense morpholino oligonucleotides compromised cell cohesion during pronephric duct formation. Our results indicate that Cdh17 is necessary for maintaining the integrity of the pronephric ducts during zebrafish embryogenesis. This finding contrasts with the role of mammalian CDH17, which does not appear to be involved in nephric development.
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Roles of microRNA-34a in the pathogenesis of placenta accreta.
Umemura, Kota; Ishioka, Shin-Ichi; Endo, Toshiaki; Ezaka, Yoshiaki; Takahashi, Madoka; Saito, Tsuyoshi
2013-01-01
MicroRNA-34a (miR-34a) is associated with invasion and metastasis of various cancers. The trophoblastic cells of placenta accreta invade into the myometrium in a similar way to the invasion of cancers. We studied the roles of miR-34a in the pathogenesis of placenta accreta. The human choriocarcinoma cell line JAR was used for in vitro experiments as a model of trophoblasts, and placental tissues from the operative specimen of patients with or without placenta accreta were used for experiments in vivo. Morpholino antisense oligomer against miR-34a (miR-34a Morpho/AS) was added to JAR, and the expression of miR-34a and plasminogen activator inhibitor-1 (PAI-1) was determined by real time PCR. The effects of antisense, interleukin (IL)-6 and IL-8 in the process of invasion were studied with an invasion assay. Expression of miR-34a in vivo was studied with the use of fluorescent in situ hybridization (FISH). Expression of miR-34a was inhibited by 65% with the administration of antisense, and a slight increase in miR-34a expression was observed with the addition of IL-6 and IL-8. PAI-1 expression decreased with the addition of IL-6 and IL-8, and increased with the administration of antisense. There was an increase in invasive capacity through the inhibition of miR-34a expression. Strong FISH expression of miR-34a was observed in trophoblast cells of non-placenta accreta, and a clear decrease in miR-34a expression was observed in those of placenta accreta. Expression of miR-34a was downregulated in placenta accreta. In vitro experiments also showed that the invasive potential of JAR increased by suppressing miR-34a, probably through the expression of PAI-1. © 2012 The Authors. Journal of Obstetrics and Gynaecology Research © 2012 Japan Society of Obstetrics and Gynecology.
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Zhang, Chengjin; Boa-Amponsem, Oswald; Cole, Gregory J
2017-08-01
This study was undertaken to ascertain whether defined markers of early zebrafish brain development are affected by chronic ethanol exposure or morpholino knockdown of agrin, sonic hedgehog, retinoic acid, and fibroblast growth factors, four signaling molecules that are suggested to be ethanol sensitive. Zebrafish embryos were exposed to 2% ethanol from 6 to 24 hpf or injected with agrin, shha, aldh1a3, or fgf8a morpholinos. In situ hybridization was employed to analyze otx2, pax6a, epha4a, krx20, pax2a, fgf8a, wnt1, and eng2b expression during early brain development. Our results showed that pax6a mRNA expression was decreased in eye, forebrain, and hindbrain of both chronic ethanol exposed and select MO treatments. Epha4a expression in rhombomere R1 boundary was decreased in chronic ethanol exposure and aldh1a3 morphants, lost in fgf8a morphants, but largely unaffected in agrin and shha morphants. Ectopic pax6a and epha4a expression in midbrain was only found in fgf8a morphants. These results suggest that while chronic ethanol induces obvious morphological change in brain architecture, many molecular markers of these brain structures are relatively unaffected by ethanol exposure.
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Sawada, Rie; Kamei, Hiroyasu; Hakuno, Fumihiko; Takahashi, Shin-Ichiro; Shimizu, Toshiaki
2015-02-01
Congenital loss of the SHOX gene is considered to be a genetic cause of short stature phenotype in Turner syndrome and Leri-Weill dyschondrosteosis patients. Though SHOX expression initiates during early fetal development, little is known about the embryonic roles of SHOX. The evolutionary conservation of the zebrafish shox gene and the convenience of the early developmental stages for analyses make zebrafish a preferred model. Here, we characterized structure, expression, and developmental roles of zebrafish shox through a loss-of-function approach. We found a previously undiscovered Shox protein that has both a homeodomain and an OAR-domain in zebrafish. The shox transcript emerged during the segmentation period and it increased in later stages. The predominant domains of shox expression were mandibular arch, pectoral fin, anterior notochord, rhombencephalon, and mesencephalon, suggesting that Shox is involved in bone and neural development. Translational blockade of Shox mRNA by an antisense morpholino oligo delayed embryonic growth, which was restored by the co-overexpression of morpholino-resistant Shox mRNA. At later stages, impaired Shox expression markedly delayed the calcification process in the anterior vertebral column and craniofacial bones. Our data demonstrate evolutionarily conserved Shox plays roles in early embryonic growth and in later bone formation. © 2014 Wiley Periodicals, Inc.
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Coordinated activation of the secretory pathway during notochord formation in the Xenopus embryo.
Tanegashima, Kosuke; Zhao, Hui; Rebbert, Martha L; Dawid, Igor B
2009-11-01
We compared the transcriptome in the developing notochord of Xenopus laevis embryos with that of other embryonic regions. A coordinated and intense activation of a large set of secretory pathway genes was observed in the notochord, but not in notochord precursors in the axial mesoderm at early gastrula stage. The genes encoding Xbp1 and Creb3l2 were also activated in the notochord. These two transcription factors are implicated in the activation of secretory pathway genes during the unfolded protein response, where cells react to the stress of a build-up of unfolded proteins in their endoplasmic reticulum. Xbp1 and Creb3l2 are differentially expressed but not differentially activated in the notochord. Reduction of expression of Xbp1 or Creb3l2 by injection of antisense morpholinos led to strong deficits in notochord but not somitic muscle development. In addition, the expression of some, but not all, genes encoding secretory proteins was inhibited by injection of xbp1 morpholinos. Furthermore, expression of activated forms of Xbp1 or Creb3l2 in animal explants could activate a similar subset of secretory pathway genes. We conclude that coordinated activation of a battery of secretory pathway genes mediated by Xbp1 and Creb/ATF factors is a characteristic and necessary feature of notochord formation.
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Glycogen Reduction in Myotubes of Late-Onset Pompe Disease Patients Using Antisense Technology.
Goina, Elisa; Peruzzo, Paolo; Bembi, Bruno; Dardis, Andrea; Buratti, Emanuele
2017-09-06
Glycogen storage disease type II (GSDII) is a lysosomal disorder caused by the deficient activity of acid alpha-glucosidase (GAA) enzyme, leading to the accumulation of glycogen within the lysosomes. The disease has been classified in infantile and late-onset forms. Most late-onset patients share a splicing mutation c.-32-13T > G in intron 1 of the GAA gene that prevents efficient recognition of exon 2 by the spliceosome. In this study, we have mapped the splicing silencers of GAA exon 2 and developed antisense morpholino oligonucleotides (AMOs) to inhibit those regions and rescue normal splicing in the presence of the c.-32-13T > G mutation. Using a minigene approach and patient fibroblasts, we successfully increased inclusion of exon 2 in the mRNA and GAA enzyme production by targeting a specific silencer with a combination of AMOs. Most importantly, the use of these AMOs in patient myotubes results in a decreased accumulation of glycogen. To our knowledge, this is the only therapeutic approach resulting in a decrease of glycogen accumulation in patient tissues beside enzyme replacement therapy (ERT) and TFEB overexpression. As a result, it may represent a highly novel and promising therapeutic line for GSDII. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.
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Concerted action of two dlx paralogs in sensory placode formation.
Solomon, Keely S; Fritz, Andreas
2002-07-01
Sensory placodes are ectodermal thickenings that give rise to elements of the vertebrate cranial sensory nervous system, including the inner ear and nose. Although mutations have been described in humans, mice and zebrafish that perturb ear and nose development, no mutation is known to prevent sensory placode formation. Thus, it has been postulated that a functional redundancy exists in the genetic mechanisms that govern sensory placode development. We describe a zebrafish deletion mutation, b380, which results in a lack of both otic and olfactory placodes. The b380 deletion removes several known genes and expressed sequence tags, including dlx3 and dlx7, two transcription factors that share a homoeobox domain similar in sequence to the Drosophila Distal-less gene. dlx3 and dlx7 are expressed in an overlapping pattern in the regions that produce the otic and olfactory placodes in zebrafish. We present evidence suggesting that it is specifically the removal of these two genes that leads to the otic and olfactory phenotype of b380 mutants. Using morpholinos, antisense oligonucleotides that effectively block translation of target genes, we find that functional reduction of both dlx genes contributes to placode loss. Expression patterns of the otic marker pax2.1, olfactory marker anxV and eya1, a marker of both placodes, in morpholino-injected embryos recapitulate the reduced expression of these genes seen in b380 mutants. We also examine expression of dlx3 and dlx7 in the morpholino-injected embryos and present evidence for existence of auto- and cross-regulatory control of expression among these genes. We demonstrate that dlx3 is necessary and sufficient for proper otic and olfactory placode development. However, our results indicate that dlx3 and dlx7 act in concert and their importance in placode formation is only revealed by inactivating both paralogs.
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Janghra, Narinder; Mitrpant, Chalermchai; Dickinson, Rachel L.; Anthony, Karen; Price, Loren; Eperon, Ian C.; Wilton, Stephen D.; Morgan, Jennifer
2013-01-01
Abstract In the search for the most efficacious antisense oligonucleotides (AOs) aimed at inducing SMN2 exon 7 inclusion, we systematically assessed three AOs, PMO25 (−10, −34), PMO18 (−10, −27), and PMO20 (−10, −29), complementary to the SMN2 intron 7 splicing silencer (ISS-N1). PMO25 was the most efficacious in augmenting exon 7 inclusion in vitro in spinal muscular atrophy (SMA) patient fibroblasts and in vitro splicing assays. PMO25 and PMO18 were compared further in a mouse model of severe SMA. After a single intracerebroventricular (ICV) injection in neonatal mice, PMO25 increased the life span of severe SMA mice up to 30-fold, with average survival greater by 3-fold compared with PMO18 at a dose of 20 μg/g and 2-fold at 40 μg/g. Exon 7 inclusion was increased in the CNS but not in peripheral tissues. Systemic delivery of PMO25 at birth achieved a similar outcome and produced increased exon 7 inclusion both in the CNS and peripherally. Systemic administration of a 10-μg/g concentration of PMO25 conjugated to an octaguanidine dendrimer (VMO25) increased the life span only 2-fold in neonatal type I SMA mice, although it prevented tail necrosis in mild SMA mice. Higher doses and ICV injection of VMO25 were associated with toxicity. We conclude that (1) the 25-mer AO is more efficient than the 18-mer and 20-mer in modifying SMN2 splicing in vitro; (2) it is more efficient in prolonging survival in SMA mice; and (3) naked Morpholino oligomers are more efficient and safer than the Vivo-Morpholino and have potential for future SMA clinical applications. PMID:23339722
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Imai, Kaoru S; Satoh, Nori; Satou, Yutaka
2002-04-01
In early Ciona savignyi embryos, nuclear localization of beta-catenin is the first step of endodermal cell specification, and triggers the activation of various target genes. A cDNA for Cs-FGF4/6/9, a gene activated downstream of beta-catenin signaling, was isolated and shown to encode an FGF protein with features of both FGF4/6 and FGF9/20. The early embryonic expression of Cs-FGF4/6/9 was transient and the transcript was seen in endodermal cells at the 16- and 32-cell stages, in notochord and muscle cells at the 64-cell stage, and in nerve cord and muscle cells at the 110-cell stage; the gene was then expressed again in cells of the nervous system after neurulation. When the gene function was suppressed with a specific antisense morpholino oligo, the differentiation of mesenchyme cells was completely blocked, and the fate of presumptive mesenchyme cells appeared to change into that of muscle cells. The inhibition of mesenchyme differentiation was abrogated by coinjection of the morpholino oligo and synthetic Cs-FGF4/6/9 mRNA. Downregulation of beta-catenin nuclear localization resulted in the absence of mesenchyme cell differentiation due to failure of the formation of signal-producing endodermal cells. Injection of synthetic Cs-FGF4/6/9 mRNA in beta-catenin-downregulated embryos evoked mesenchyme cell differentiation. These results strongly suggest that Cs-FGF4/6/9 produced by endodermal cells acts an inductive signal for the differentiation of mesenchyme cells. On the other hand, the role of Cs-FGF4/6/9 in the induction of notochord cells is partial; the initial process of the induction was inhibited by Cs-FGF4/6/9 morpholino oligo, but notochord-specific genes were expressed later to form a partial notochord.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Teraoka, Hiroki; Kubota, Akira; Dong, Wu
2009-01-01
Previously, we reported that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) evoked developmental toxicity required activation of aryl hydrocarbon receptor type 2 (AHR2), using zebrafish embryos. However, the downstream molecular targets of AHR2 activation are largely unknown and are the focus of the present investigation. TCDD induces cyclooxygenase 2 (COX2), a rate-limiting enzyme for prostaglandin synthesis in certain cells. In the present study, we investigated the role of the COX2-thromboxane pathway in causing a specific endpoint of TCDD developmental toxicity in the zebrafish embryo, namely, a decrease in regional blood flow in the dorsal midbrain. It was found that the TCDD-induced reduction in mesencephalic veinmore » blood flow was markedly inhibited by selective COX2 inhibitors, NS-398 and SC-236, and by a general COX inhibitor, indomethacin, but not by a selective COX1 inhibitor, SC-560. Gene knock-down of COX2 by two different types of morpholino antisense oligonucleotides, but not by their negative homologs, also protected the zebrafish embryos from mesencephalic vein circulation failure caused by TCDD. This inhibitory effect of TCDD on regional blood flow in the dorsal midbrain was also blocked by selective antagonists of the thromboxane receptor (TP). Treatment of control zebrafish embryos with a TP agonist also caused a reduction in mesencephalic vein blood flow and it too was blocked by a TP antagonist, without any effect on trunk circulation. Finally, gene knock-down of thromboxane A synthase 1 (TBXS) with morpholinos but not by the morpholinos' negative homologs provided significant protection against TCDD-induced mesencephalic circulation failure. Taken together, these results point to a role of the prostanoid synthesis pathway via COX2-TBXS-TP in the local circulation failure induced by TCDD in the dorsal midbrain of the zebrafish embryo.« less
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Participation of Xenopus Elr-type Proteins in Vegetal mRNA Localization during Oogenesis*
Arthur, Patrick K.; Claussen, Maike; Koch, Susanne; Tarbashevich, Katsiaryna; Jahn, Olaf; Pieler, Tomas
2009-01-01
Directional transport of specific mRNAs is of primary biological relevance. In Xenopus oocytes, mRNA localization to the vegetal pole is important for germ layer formation and germ cell development. Using a biochemical approach, we identified Xenopus Elr-type proteins, homologs of the Hu/ELAV proteins, as novel components of the vegetal mRNA localization machinery. They bind specifically to the localization elements of several different vegetally localizing Xenopus mRNAs, and they are part of one RNP together with other localization proteins, such as Vg1RBP and XStaufen 1. Blocking Elr-type protein binding by either localization element mutagenesis or antisense morpholino oligonucleotide-mediated masking of their target RNA structures, as well as overexpression of wild type and mutant ElrB proteins, interferes with vegetal localization in Xenopus oocytes. PMID:19458392
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Lessons learned from vivo-morpholinos: How to avoid vivo-morpholino toxicity
Ferguson, David P.; Dangott, Lawrence J.; Lightfoot, J. Timothy
2014-01-01
Vivo-morpholinos are a promising tool for gene silencing. These oligonucleotide analogs transiently silence genes by blocking either translation or pre-mRNA splicing. Little to no toxicity has been reported for vivo-morpholino treatment. However, in a recent study conducted in our lab, treatment of mice with vivo-morpholinos resulted in high mortality rates. We hypothesized that the deaths were the result of oligonucleotide hybridization, causing an increased cationic charge associated with the dendrimer delivery moiety of the vivo-morpholino. The cationic charge increased blood clot formation in whole blood treated with vivo-morpholinos, suggesting that clotting could have caused cardiac arrest in the deceased mice. Therefore, we investigate the mechanism by which some vivo-morpholinos increase mortality rates and propose techniques to alleviate vivo-morpholino toxicity. PMID:24806225
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Modulation of Stat3 Alternative Splicing in Breast Cancer
2010-09-01
using morpholino oligonucleotides covalently linked to an octaguanidine dendrimer (vivo- morpholinos) [54]. Since delivery of vivo-morpholino oligos...Li, and S. Jiang, Vivo-Morpholinos: a non-peptide transporter delivers Morpholinos into a wide array of mouse tissues. Biotechniques, 2008. 45(6
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A zebrafish sox9 gene required for cartilage morphogenesis.
Yan, Yi-Lin; Miller, Craig T; Nissen, Robert M; Singer, Amy; Liu, Dong; Kirn, Anette; Draper, Bruce; Willoughby, John; Morcos, Paul A; Amsterdam, Adam; Chung, Bon-Chu; Westerfield, Monte; Haffter, Pascal; Hopkins, Nancy; Kimmel, Charles; Postlethwait, John H; Nissen, Robert
2002-11-01
The molecular genetic mechanisms of cartilage construction are incompletely understood. Zebrafish embryos homozygous for jellyfish (jef) mutations show craniofacial defects and lack cartilage elements of the neurocranium, pharyngeal arches, and pectoral girdle similar to humans with campomelic dysplasia. We show that two alleles of jef contain mutations in sox9a, one of two zebrafish orthologs of the human transcription factor SOX9. A mutation induced by ethyl nitrosourea changed a conserved nucleotide at a splice junction and severely reduced splicing of sox9a transcript. A retrovirus insertion into sox9a disrupted its DNA-binding domain. Inhibiting splicing of the sox9a transcript in wild-type embryos with splice site-directed morpholino antisense oligonucleotides produced a phenotype like jef mutant larvae, and caused sox9a transcript to accumulate in the nucleus; this accumulation can serve as an assay for the efficacy of a morpholino independent of phenotype. RNase-protection assays showed that in morpholino-injected animals, the percent of splicing inhibition decreased from 80% at 28 hours post fertilization to 45% by 4 days. Homozygous mutant embryos had greatly reduced quantities of col2a1 message, the major collagen of cartilage. Analysis of dlx2 expression showed that neural crest specification and migration was normal in jef (sox9a) embryos. Confocal images of living embryos stained with BODIPY-ceramide revealed at single-cell resolution the formation of precartilage condensations in mutant embryos. Besides the lack of overt cartilage differentiation, pharyngeal arch condensations in jef (sox9a) mutants lacked three specific morphogenetic behaviors: the stacking of chondrocytes into orderly arrays, the individuation of pharyngeal cartilage organs and the proper shaping of individual cartilages. Despite the severe reduction of cartilages, analysis of titin expression showed normal muscle patterning in jef (sox9a) mutants. Likewise, calcein labeling revealed that early bone formation was largely unaffected in jef (sox9a) mutants. These studies show that jef (sox9a) is essential for both morphogenesis of condensations and overt cartilage differentiation.
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Yeh, Lung-Kun; Liu, Chia-Yang; Kao, Winston W.-Y.; Huang, Chang-Jen; Hu, Fung-Rong; Chien, Chung-Liang; Wang, I-Jong
2010-01-01
The lumican gene (lum), which encodes one of the major keratan sulfate proteoglycans (KSPGs) in the vertebrate cornea and sclera, has been linked to axial myopia in humans. In this study, we chose zebrafish (Danio rerio) as an animal model to elucidate the role of lumican in the development of axial myopia. The zebrafish lumican gene (zlum) spans ∼4.6 kb of the zebrafish genome. Like human (hLUM) and mouse (mlum), zlum consists of three exons, two introns, and a TATA box-less promoter at the 5′-flanking region of the transcription initiation site. Sequence analysis of the cDNA predicts that zLum encodes 344 amino acids. zLum shares 51% amino acid sequence identity with human lumican. Similar to hLUM and mlum, zlum mRNA is expressed in the eye and many other tissues, such as brain, muscle, and liver as well. Transgenic zebrafish harboring an enhanced GFP reporter gene construct downstream of a 1.7-kb zlum 5′-flanking region displayed enhanced GFP expression in the cornea and sclera, as well as throughout the body. Down-regulation of zlum expression by antisense zlum morpholinos manifested ocular enlargement resembling axial myopia due to disruption of the collagen fibril arrangement in the sclera and resulted in scleral thinning. Administration of muscarinic receptor antagonists, e.g. atropine and pirenzepine, effectively subdued the ocular enlargement caused by morpholinos in in vivo zebrafish larvae assays. The observation suggests that zebrafish can be used as an in vivo model for screening compounds in treating myopia. PMID:20551313
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nlz gene family is required for hindbrain patterning in the zebrafish.
Hoyle, Jacqueline; Tang, Yixin P; Wiellette, Elizabeth L; Wardle, Fiona C; Sive, Hazel
2004-04-01
This study describes the conserved nlz gene family whose members encode unusual zinc finger proteins. In the zebrafish neurectoderm, both nlz1 and the newly isolated nlz2 are expressed in the presumptive hindbrain and midbrain/hindbrain boundary, where expression of nlz1 is dependent on pax2a. In addition, nlz2 is uniquely expressed more anteriorly, in the presumptive midbrain and diencephalon. Overexpression of Nlz proteins during gastrula stages inhibits hindbrain development. In particular, ectopically expressed Nlz1 inhibits formation of future rhombomeres 2 and 3 (r2, r3), whereas neighboring r1 and r4 are not affected. Conversely, simultaneous reduction of Nlz1 and Nlz2 protein function by expression of antisense morpholino-modified oligomers leads to expansion of future r3 and r5, with associated loss of r4. These data indicate that one function of the nlz gene family is to specify or maintain r4 identity, and to limit r3 and r5 during hindbrain formation. Copyright 2004 Wiley-Liss, Inc.
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Long-term consequences of Sox9 depletion on inner ear development
Park, Byung-Yong; Saint-Jeannet, Jean-Pierre
2010-01-01
The transcription factor Sox9 has been implicated in inner ear formation in several species. To investigate the long-term consequences of Sox9 depletion on inner ear development we analyzed the inner ear architecture of Sox9-depleted Xenopus tadpoles generated by injection of increasing amounts of Sox9 morpholino antisense oligonucleotides. We found that Sox9-depletion resulted in major defects in the development of vestibular structures, semicircular canals and utricle, while the ventrally located saccule was less severely affected in these embryos. Consistent with this phenotype we observed a specific loss of the dorsal expression of Wnt3a expression in the otic vesicle of Sox9 morphants, associated with an increase in cell death and a reduction in cell proliferation in the region of the presumptive otic epithelium. We propose that in addition to its early role in placode specification, Sox9 is also required for the maintenance of progenitors in the otic epithelium. PMID:20201105
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Whitley, Dexter S.; Yu, Kwang; Sample, Robert C.
2010-09-30
Although previous work identified 12 complementation groups with possible roles in virus assembly, currently only one frog virus 3 protein, the major capsid protein (MCP), has been linked with virion formation. To identify other proteins required for assembly, we used an antisense morpholino oligonucleotide to target 53R, a putative myristoylated membrane protein, and showed that treatment resulted in marked reductions in 53R levels and a 60% drop in virus titers. Immunofluorescence assays confirmed knock down and showed that 53R was found primarily within viral assembly sites, whereas transmission electron microscopy detected fewer mature virions and, in some cells, dense granularmore » bodies that may represent unencapsidated DNA-protein complexes. Treatment with a myristoylation inhibitor (2-hydroxymyristic acid) resulted in an 80% reduction in viral titers. Collectively, these data indicate that 53R is an essential viral protein that is required for replication in vitro and suggest it plays a critical role in virion formation.« less
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Negishi, Yoichi; Ishii, Yuko; Nirasawa, Kei; Sasaki, Eri; Endo-Takahashi, Yoko; Suzuki, Ryo; Maruyama, Kazuo
2018-01-01
Duchenne muscular dystrophy (DMD) is a genetic disorder characterized by progressive muscle degeneration, caused by nonsense or frameshift mutations in the dystrophin (DMD) gene. Antisense oligonucleotides can be used to induce specific exon skipping; recently, a phosphorodiamidate morpholino oligomer (PMO) has been approved for clinical use in DMD. However, an efficient PMO delivery strategy is required to improve the therapeutic efficacy in DMD patients. We previously developed polyethylene glycol (PEG)-modified liposomes containing ultrasound contrast gas, "Bubble liposomes" (BLs), and found that the combination of BLs with ultrasound exposure is a useful gene delivery tool. Here, we describe an efficient PMO delivery strategy using the combination of BLs and ultrasound exposure to treat muscles in a DMD mouse model (mdx). This ultrasound-mediated BL technique can increase the PMO-mediated exon-skipping efficiency, leading to significantly increased dystrophin expression. Thus, the combination of BLs and ultrasound exposure may be a feasible PMO delivery method to improve therapeutic efficacy and reduce the PMO dosage for DMD treatment.
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Marchese, Maria; Pappalardo, Andrea; Baldacci, Jacopo; Verri, Tiziano; Doccini, Stefano; Cassandrini, Denise; Bruno, Claudio; Fiorillo, Chiara; Garcia-Gil, Mercedes; Bertini, Enrico; Pitto, Letizia; Santorelli, Filippo M
2016-08-12
Defective dolichol-phosphate mannose synthase (DPMS) complex is a rare cause of congenital muscular dystrophy associated with hypoglycosylation of alpha-dystroglycan (α-DG) in skeletal muscle. We used the zebrafish (Danio rerio) to model muscle abnormalities due to defects in the subunits of DPMS. The three zebrafish ortholog subunits (encoded by the dpm1, dpm2 and dpm3 genes, respectively) showed high similarity to the human proteins, and their expression displayed localization in the midbrain/hindbrain area and somites. Antisense morpholino oligonucleotides targeting each subunit were used to transiently deplete the dpm genes. The resulting morphant embryos showed early death, muscle disorganization, low DPMS complex activity, and increased levels of apoptotic nuclei, together with hypoglycosylated α-DG in muscle fibers, thus recapitulating most of the characteristics seen in patients with mutations in DPMS. Our results in zebrafish suggest that DPMS plays a role in stabilizing muscle structures and in apoptotic cell death. Copyright © 2016 Elsevier Inc. All rights reserved.
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The zebrafish dorsal axis is apparent at the four-cell stage.
Gore, Aniket V; Maegawa, Shingo; Cheong, Albert; Gilligan, Patrick C; Weinberg, Eric S; Sampath, Karuna
2005-12-15
A central question in the development of multicellular organisms pertains to the timing and mechanisms of specification of the embryonic axes. In many organisms, specification of the dorsoventral axis requires signalling by proteins of the Transforming growth factor-beta and Wnt families. Here we show that maternal transcripts of the zebrafish Nodal-related morphogen, Squint (Sqt), can localize to two blastomeres at the four-cell stage and predict the dorsal axis. Removal of cells containing sqt transcripts from four-to-eight-cell embryos or injection of antisense morpholino oligonucleotides targeting sqt into oocytes can cause a loss of dorsal structures. Localization of sqt transcripts is independent of maternal Wnt pathway function and requires a highly conserved sequence in the 3' untranslated region. Thus, the dorsoventral axis is apparent by early cleavage stages and may require the maternally encoded morphogen Sqt and its associated factors. Because the 3' untranslated region of the human nodal gene can also localize exogenous sequences to dorsal cells, this mechanism may be evolutionarily conserved.
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The Xenopus Tgfbi is required for embryogenesis through regulation of canonical Wnt signalling.
Wang, Feng; Hu, Wanzhou; Xian, Jian; Ohnuma, Shin-ichi; Brenton, James D
2013-07-01
Tgfbi, a fasciclin family extracellular matrix protein, has various roles in human diseases from corneal dystrophies to cancer. However, the molecular mechanisms that underlie its functions are poorly understood. Here, we studied the role of Tgfbi during Xenopus embryogenesis. During gastrulation and immediately after, Xtgfbi is expressed at developmentally important signaling centers including the dorsal marginal zone, notochord and floorplate. Xtgfbi knockdown by anti-sense morpholinos causes defective organizer induction, patterning and differentiation of muscle, neuron and neural crests, similar to suppression of canonical Wnt signaling. In Xenopus embryos and animal caps as well as DLD-1 cells, we show that Tgfbi is strongly required for the full activation of the canonical Wnt pathway by promoting phosphorylation of GSK3β and consequently enhancing the stabilization and nuclear localization of β-catenin. Further analysis shows that Tgfbi is likely to promote GSK3β phosphorylation through integrin-linked kinase. Copyright © 2013 Elsevier Inc. All rights reserved.
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Senturker, Sema; Thomas, John Terrig; Mateshaytis, Jennifer; Moos, Malcolm
2012-01-01
Background Subtilisin-like Proprotein Convertase 7 (SPC7) is a member of the subtilisin/kexin family of pro-protein convertases. It cleaves many pro-proteins to release their active proteins, including members of the bone morphogenetic protein (BMP) family of signaling molecules. Other SPCs are known to be required during embryonic development but corresponding data regarding SPC7 have not been reported previously. Methodology/Principal Findings We demonstrated that Xenopus SPC7 (SPC7) was expressed predominantly in the developing brain and eye, throughout the neural plate initially, then more specifically in the lens and retina primordia as development progressed. Since no prior functional information has been reported for SPC7, we used gain- and loss-of-function experiments to investigate the possibility that it may also convey patterning or tissue specification information similarly to Furin, SPC4, and SPC6. Overexpression of SPC7 was without effect. In contrast, injection of SPC7 antisense morpholino oligonucleotides (MO) into a single blastomere at the 2- or 4-cell stage produced marked disruption of head structures; anophthalmia was salient. Bilateral injections suppressed head and eye formation completely. In parallel with suppression of eye and brain development by SPC7 knockdown, expression of early anterior neural markers (Sox2, Otx2, Rx2, and Pax6) and late eye-specific markers (β-Crystallin and Opsin), and of BMP target genes such as Tbx2 and Tbx3, was reduced or eliminated. Taken together, these findings suggest a critical role for SPC7–perhaps, at least in part, due to activation of one or more BMPs–in early patterning of the anterior neural plate and its derivatives. Conclusion/Significance SPC7 is required for normal development of the eye and brain, possibly through processing BMPs, though other potential substrates cannot be excluded. PMID:22761776
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Zebrafish Lmx1b.1 and Lmx1b.2 are required for maintenance of the isthmic organizer.
O'Hara, F Patrick; Beck, Ernestine; Barr, Lauren K; Wong, Lily L; Kessler, Daniel S; Riddle, Robert D
2005-07-01
The mesencephalic and metencephalic region (MMR) of the vertebrate central nervous system develops in response to signals produced by the isthmic organizer (IsO). We have previously reported that the LIM homeobox transcription factor Lmx1b is expressed within the chick IsO, where it is sufficient to maintain expression of the secreted factor wnt1. In this paper, we show that zebrafish express two Lmx1b orthologs, lmx1b.1 and lmx1b.2, in the rostral IsO, and demonstrate that these genes are necessary for key aspects of MMR development. Simultaneous knockdown of Lmx1b.1 and Lmx1b.2 using morpholino antisense oligos results in a loss of wnt1, wnt3a, wnt10b, pax8 and fgf8 expression at the IsO, leading ultimately to programmed cell death and the loss of the isthmic constriction and cerebellum. Single morpholino knockdown of either Lmx1b.1 or Lmx1b.2 has no discernible effect on MMR development. Maintenance of lmx1b.1 and lmx1b.2 expression at the isthmus requires the function of no isthmus/pax2.1, as well as Fgf signaling. Transient misexpression of Lmx1b.1 or Lmx1b.2 during early MMR development induces ectopic wnt1 and fgf8 expression in the MMR, as well as throughout much of the embryo. We propose that Lmx1b.1- and Lmx1b.2-mediated regulation of wnt1, wnt3a, wnt10b, pax8 and fgf8 maintains cell survival in the isthmocerebellar region.
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Essner, Jeffrey J; Amack, Jeffrey D; Nyholm, Molly K; Harris, Erin B; Yost, H Joseph
2005-03-01
Monocilia have been proposed to establish the left-right (LR) body axis in vertebrate embryos by creating a directional fluid flow that triggers asymmetric gene expression. In zebrafish, dorsal forerunner cells (DFCs) express a conserved ciliary dynein gene (left-right dynein-related1, lrdr1) and form a ciliated epithelium inside a fluid-filled organ called Kupffer's vesicle (KV). Here, videomicroscopy demonstrates that cilia inside KV are motile and create a directional fluid flow just prior to the onset of asymmetric gene expression in lateral cells. Laser ablation of DFCs and surgical disruption of KV provide direct evidence that ciliated KV cells are required during early somitogenesis for subsequent LR patterning in the brain, heart and gut. Antisense morpholinos against lrdr1 disrupt KV fluid flow and perturb LR development. Furthermore, lrdr1 morpholinos targeted to DFC/KV cells demonstrate that Lrdr1 functions in these ciliated cells to control LR patterning. This provides the first direct evidence, in any vertebrate, that impairing cilia function in derivatives of the dorsal organizer, and not in other cells that express ciliogenic genes, alters LR development. Finally, genetic analysis reveals novel roles for the T-box transcription factor no tail and the Nodal signaling pathway as upstream regulators of lrdr1 expression and KV morphogenesis. We propose that KV is a transient embryonic 'organ of asymmetry' that directs LR development by establishing a directional fluid flow. These results suggest that cilia are an essential component of a conserved mechanism that controls the transition from bilateral symmetry to LR asymmetry in vertebrates.
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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.
2014-01-01
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 HdhQ7/Q150 knock-in HD mice. These data demonstrate the potential of PMOs as an approach to suppressing the expression of mutant HTT. PMID:25035419
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Ezzat, Kariem; Aoki, Yoshitsugu; Koo, Taeyoung; McClorey, Graham; Benner, Leif; Coenen-Stass, Anna; O'Donovan, Liz; Lehto, Taavi; Garcia-Guerra, Antonio; Nordin, Joel; Saleh, Amer F; Behlke, Mark; Morris, John; Goyenvalle, Aurelie; Dugovic, Branislav; Leumann, Christian; Gordon, Siamon; Gait, Michael J; El-Andaloussi, Samir; Wood, Matthew J A
2015-07-08
Antisense oligonucleotides (ASOs) have the potential to revolutionize medicine due to their ability to manipulate gene function for therapeutic purposes. ASOs are chemically modified and/or incorporated within nanoparticles to enhance their stability and cellular uptake, however, a major challenge is the poor understanding of their uptake mechanisms, which would facilitate improved ASO designs with enhanced activity and reduced toxicity. Here, we study the uptake mechanism of three therapeutically relevant ASOs (peptide-conjugated phosphorodiamidate morpholino (PPMO), 2'Omethyl phosphorothioate (2'OMe), and phosphorothioated tricyclo DNA (tcDNA) that have been optimized to induce exon skipping in models of Duchenne muscular dystrophy (DMD). We show that PPMO and tcDNA have high propensity to spontaneously self-assemble into nanoparticles. PPMO forms micelles of defined size and their net charge (zeta potential) is dependent on the medium and concentration. In biomimetic conditions and at low concentrations, PPMO obtains net negative charge and its uptake is mediated by class A scavenger receptor subtypes (SCARAs) as shown by competitive inhibition and RNAi silencing experiments in vitro. In vivo, the activity of PPMO was significantly decreased in SCARA1 knockout mice compared to wild-type animals. Additionally, we show that SCARA1 is involved in the uptake of tcDNA and 2'OMe as shown by competitive inhibition and colocalization experiments. Surface plasmon resonance binding analysis to SCARA1 demonstrated that PPMO and tcDNA have higher binding profiles to the receptor compared to 2'OMe. These results demonstrate receptor-mediated uptake for a range of therapeutic ASO chemistries, a mechanism that is dependent on their self-assembly into nanoparticles.
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Knowlton, Michelle N; Li, Tongbin; Ren, Yongliang; Bill, Brent R; Ellis, Lynda Bm; Ekker, Stephen C
2008-01-07
The zebrafish is a powerful model vertebrate amenable to high throughput in vivo genetic analyses. Examples include reverse genetic screens using morpholino knockdown, expression-based screening using enhancer trapping and forward genetic screening using transposon insertional mutagenesis. We have created a database to facilitate web-based distribution of data from such genetic studies. The MOrpholino DataBase is a MySQL relational database with an online, PHP interface. Multiple quality control levels allow differential access to data in raw and finished formats. MODBv1 includes sequence information relating to almost 800 morpholinos and their targets and phenotypic data regarding the dose effect of each morpholino (mortality, toxicity and defects). To improve the searchability of this database, we have incorporated a fixed-vocabulary defect ontology that allows for the organization of morpholino affects based on anatomical structure affected and defect produced. This also allows comparison between species utilizing Phenotypic Attribute Trait Ontology (PATO) designated terminology. MODB is also cross-linked with ZFIN, allowing full searches between the two databases. MODB offers users the ability to retrieve morpholino data by sequence of morpholino or target, name of target, anatomical structure affected and defect produced. MODB data can be used for functional genomic analysis of morpholino design to maximize efficacy and minimize toxicity. MODB also serves as a template for future sequence-based functional genetic screen databases, and it is currently being used as a model for the creation of a mutagenic insertional transposon database.
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Fyn/Yes and non-canonical Wnt signalling converge on RhoA in vertebrate gastrulation cell movements
Jopling, Chris; den Hertog, Jeroen
2005-01-01
Convergent extension (CE) cell movements during gastrulation mediate extension of the anterior–posterior body axis of vertebrate embryos. Non-canonical Wnt5 and Wnt11 signalling is essential for normal CE movements in vertebrate gastrulation. Here, we show that morpholino (MO)-mediated double knock-down of the Fyn and Yes tyrosine kinases in zebrafish embryos impaired normal CE cell movements, resembling the silberblick and pipetail mutants, caused by mutations in wnt11 and wnt5, respectively. Co-injection of Fyn/Yes- and Wnt11- or Wnt5-MO was synergistic, but wnt11 or wnt5 RNA did not rescue the Fyn/Yes knockdown or vice versa. Remarkably, active RhoA rescued the Fyn/Yes knockdown as well as the Wnt11 knockdown, indicating that Fyn/Yes and Wnt11 signalling converged on RhoA. Our results show that Fyn and Yes act together with non-canonical Wnt signalling via RhoA in CE cell movements during gastrulation. PMID:15815683
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Schubert, Michael; Yu, Jr-Kai; Holland, Nicholas D; Escriva, Hector; Laudet, Vincent; Holland, Linda Z
2005-01-01
In the invertebrate chordate amphioxus, as in vertebrates, retinoic acid (RA) specifies position along the anterior/posterior axis with elevated RA signaling in the middle third of the endoderm setting the posterior limit of the pharynx. Here we show that AmphiHox1 is also expressed in the middle third of the developing amphioxus endoderm and is activated by RA signaling. Knockdown of AmphiHox1 function with an antisense morpholino oligonucleotide shows that AmphiHox1 mediates the role of RA signaling in setting the posterior limit of the pharynx by repressing expression of pharyngeal markers in the posterior foregut/midgut endoderm. The spatiotemporal expression of these endodermal genes in embryos treated with RA or the RA antagonist BMS009 indicates that Pax1/9, Pitx and Notch are probably more upstream than Otx and Nodal in the hierarchy of genes repressed by RA signaling. This work highlights the potential of amphioxus, a genomically simple, vertebrate-like invertebrate chordate, as a paradigm for understanding gene hierarchies similar to the more complex ones of vertebrates.
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MANF regulates dopaminergic neuron development in larval zebrafish.
Chen, Y-C; Sundvik, M; Rozov, S; Priyadarshini, M; Panula, P
2012-10-15
Mesencephalic astrocyte derived neurotrophic factor (MANF) is recognized as a dopaminergic neurotrophic factor, which can protect dopaminergic neurons from neurotoxic damage. However, little is known about the function of MANF during the vertebrate development. Here, we report that MANF expression is widespread during embryonic development and in adult organs analyzed by qPCR and in situ hybridization in zebrafish. Knockdown of MANF expression with antisense splice-blocking morpholino oligonucleotides resulted in no apparent abnormal phenotype. Nevertheless, the dopamine level of MANF morphants was lower than that of the wild type larvae, the expression levels of the two tyrosine hydroxylase gene transcripts were decreased and a decrease in neuron number in certain groups of th1 and th2 cells in the diencephalon region in MANF morphants was observed. These defects were rescued by injection of exogenous manf mRNA. Strikingly, manf mRNA could partly restore the decrease of th1 positive cells in Nr4a2-deficient larvae. These results suggest that MANF is involved in the regulation of the development of dopaminergic system in zebrafish. Copyright © 2012 Elsevier Inc. All rights reserved.
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The Wnt signaling regulator R-spondin 3 promotes angioblast and vascular development.
Kazanskaya, Olga; Ohkawara, Bisei; Heroult, Melanie; Wu, Wei; Maltry, Nicole; Augustin, Hellmut G; Niehrs, Christof
2008-11-01
The vertebrate embryonic vasculature develops from angioblasts, which are specified from mesodermal precursors and develop in close association with blood cells. The signals that regulate embryonic vasculogenesis and angiogenesis are incompletely understood. Here, we show that R-spondin 3 (Rspo3), a member of a novel family of secreted proteins in vertebrates that activate Wnt/beta-catenin signaling, plays a key role in these processes. In Xenopus embryos, morpholino antisense knockdown of Rspo3 induces vascular defects because Rspo3 is essential for regulating the balance between angioblast and blood cell specification. In mice, targeted disruption of Rspo3 leads to embryonic lethality caused by vascular defects. Specifically in the placenta, remodeling of the vascular plexus is impaired. In human endothelial cells, R-spondin signaling promotes proliferation and sprouting angiogenesis in vitro, indicating that Rspo3 can regulate endothelial cells directly. We show that vascular endothelial growth factor is an immediate early response gene and a mediator of R-spondin signaling. The results identify Rspo3 as a novel, evolutionarily conserved angiogenic factor in embryogenesis.
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The hypoxia factor Hif-1α controls neural crest chemotaxis and epithelial to mesenchymal transition
Barriga, Elias H.; Maxwell, Patrick H.
2013-01-01
One of the most important mechanisms that promotes metastasis is the stabilization of Hif-1 (hypoxia-inducible transcription factor 1). We decided to test whether Hif-1α also was required for early embryonic development. We focused our attention on the development of the neural crest, a highly migratory embryonic cell population whose behavior has been likened to cancer metastasis. Inhibition of Hif-1α by antisense morpholinos in Xenopus laevis or zebrafish embryos led to complete inhibition of neural crest migration. We show that Hif-1α controls the expression of Twist, which in turn represses E-cadherin during epithelial to mesenchymal transition (EMT) of neural crest cells. Thus, Hif-1α allows cells to initiate migration by promoting the release of cell–cell adhesions. Additionally, Hif-1α controls chemotaxis toward the chemokine SDF-1 by regulating expression of its receptor Cxcr4. Our results point to Hif-1α as a novel and key regulator that integrates EMT and chemotaxis during migration of neural crest cells. PMID:23712262
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Kowalski, Madzia P.; Baylis, Howard A.; Krude, Torsten
2015-01-01
ABSTRACT Stem bulge RNAs (sbRNAs) are a family of small non-coding stem-loop RNAs present in Caenorhabditis elegans and other nematodes, the function of which is unknown. Here, we report the first functional characterisation of nematode sbRNAs. We demonstrate that sbRNAs from a range of nematode species are able to reconstitute the initiation of chromosomal DNA replication in the presence of replication proteins in vitro, and that conserved nucleotide sequence motifs are essential for this function. By functionally inactivating sbRNAs with antisense morpholino oligonucleotides, we show that sbRNAs are required for S phase progression, early embryonic development and the viability of C. elegans in vivo. Thus, we demonstrate a new and essential role for sbRNAs during the early development of C. elegans. sbRNAs show limited nucleotide sequence similarity to vertebrate Y RNAs, which are also essential for the initiation of DNA replication. Our results therefore establish that the essential function of small non-coding stem-loop RNAs during DNA replication extends beyond vertebrates. PMID:25908866
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Chen, Hua-Ling; Yuh, Chiou-Hwa; Wu, Kenneth K
2010-02-19
Nestin is expressed in neural progenitor cells (NPC) of developing brain. Despite its wide use as an NPC marker, the function of nestin in embryo development is unclear. As nestin is conserved in zebrafish and its predicted sequence is clustered with the mammalian nestin orthologue, we used zebrafish as a model to investigate its role in embryogenesis. Injection of nestin morpholino (MO) into fertilized eggs induced time- and dose-dependent brain and eye developmental defects. Nestin morphants exhibited characteristic morphological changes including small head, small eyes and hydrocephalus. Histological examinations show reduced hind- and mid-brain size, dilated ventricle, poorly organized retina and underdeveloped lens. Injection of control nestin MO did not induce brain or eye changes. Nestin MO injection reduced expression of ascl1b (achaete-scute complex-like 1b), a marker of NPCs, without affecting its distribution. Nestin MO did not influence Elavl3/4 (Embryonic lethal, abnormal vision, Drosophila-like 3/4) (a neuronal marker), or otx2 (a midbrain neuronal marker), but severely perturbed cranial motor nerve development and axon distribution. To determine whether the developmental defects are due to excessive NPC apoptosis and/or reduced NPC proliferation, we analyzed apoptosis by TUNEL assay and acridine orange staining and proliferation by BrdU incorporation, pcna and mcm5 expressions. Excessive apoptosis was noted in hindbrain and midbrain cells. Apoptotic signals were colocalized with ascl1b. Proliferation markers were not significantly altered by nestin MO. These results suggest that nestin is essential for zebrafish brain and eye development probably through control of progenitor cell apoptosis.
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Targeted self-assembly of functionalized carbon nanotubes on tumors
Scheinberg, David A.; McDevitt, Michael R.; Villa, Carlos H.; Mulvey, J. Justin
2018-05-22
Provided herein are methods for delivering a molecule in situ to a cell and for treating a cancer via the in situ delivery. The methods comprise contacting or administering to the cell, as two separate components, a morpholino oligonucleotide comprising a targeting moiety followed by a single wall nanotube construct comprising second morpholino oligonucleotides complementary to the first morpholino oligonucleotides and one or both of a therapeutic or diagnostic payload molecule linked to the single wall nanotube construct. Upon self-assembly of a single wall nanotube complex via hybridization of the first morpholino and second complementary morpholino oligonucleotides at the cell, the payload molecule is delivered. Also provided is the two component self-assembly single wall nanotube system and the single wall nanotube construct comprising the second component.
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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 for...
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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 for...
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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 for...
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Li, Xi; He, Jiangyan; Hu, Wei; Yin, Zhan
2009-06-01
Ghrelin, a multifunctional hormone, including potent GH stimulation activity, has been suggested to be important during embryonic development. Expression of ghrelin has been confirmed in the zebrafish pancreas during embryonic stages. Interfering with ghrelin function using two specific antisense morpholino oligonucleotides causes defects during zebrafish embryonic development. In ghrelin morphants the expression of GH was abolished in zebrafish somatotropes, whereas the expression patterns of the other key molecules involved in hypothalamic-pituitary development and distinct pituitary hormones genes remain largely intact at the appropriate time during zebrafish adenohypophysis development. Effective rescue of the ghrelin morphants with exogenous ghrelin mRNA showed that the correct gene had been targeted. Moreover, by analyzing the efficiencies of the ghrelin morphants rescue experiments with various forms of exogenous mutant ghrelin mRNAs, we also demonstrated the essentiality of the form acyl-ghrelin on GH stimulation during zebrafish adenohypophysis development. Our in vivo experiments, for the first time, also provided evidence of the existence of functional obestatin in the C-terminal part of zebrafish proghrelin peptides. Our research here has demonstrated that zebrafish is a unique model for functional studies of endogenous ghrelin, especially during embryonic development.
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Dash, Surjya Narayan; Lehtonen, Eero; Wasik, Anita A; Schepis, Antonino; Paavola, Jere; Panula, Pertti; Nelson, W James; Lehtonen, Sanna
2014-04-01
The conserved septin family of filamentous small GTPases plays important roles in mitosis, cell migration and cell morphogenesis by forming scaffolds and diffusion barriers. Recent studies in cultured cells in vitro indicate that a septin complex of septin 2, 7 and 9 is required for ciliogenesis and cilia function, but septin function in ciliogenesis in vertebrate organs in vivo is not understood. We show that sept7b is expressed in ciliated cells in different tissues during early zebrafish development. Knockdown of sept7b by using morpholino antisense oligonucleotides caused misorientation of basal bodies and cilia, reduction of apical actin and the shortening of motile cilia in Kupffer's vesicle and pronephric tubules. This resulted in pericardial and yolk sac edema, body axis curvature and hydrocephaly. Notably, in sept7b morphants we detected strong left-right asymmetry defects in the heart and lateral plate mesoderm (situs inversus), reduced fluid flow in the kidney, the formation of kidney cysts and loss of glomerular filtration barrier function. Thus, sept7b is essential during zebrafish development for pronephric function and ciliogenesis, and loss of expression of sept7b results in defects that resemble human ciliopathies.
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Transient translational quiescence in primordial germ cells
Oulhen, Nathalie; Swartz, S. Zachary; Laird, Jessica; Mascaro, Alexandra
2017-01-01
Stem cells in animals often exhibit a slow cell cycle and/or low transcriptional activity referred to as quiescence. Here, we report that the translational activity in the primordial germ cells (PGCs) of the sea urchin embryo (Strongylocentrotus purpuratus) is quiescent. We measured new protein synthesis with O-propargyl-puromycin and L-homopropargylglycine Click-iT technologies, and determined that these cells synthesize protein at only 6% the level of their adjacent somatic cells. Knockdown of translation of the RNA-binding protein Nanos2 by morpholino antisense oligonucleotides, or knockout of the Nanos2 gene by CRISPR/Cas9 resulted in a significant, but partial, increase (47%) in general translation specifically in the PGCs. We found that the mRNA of the translation factor eEF1A is excluded from the PGCs in a Nanos2-dependent manner, a consequence of a Nanos/Pumilio response element (PRE) in its 3′UTR. In addition to eEF1A, the cytoplasmic pH of the PGCs appears to repress translation and simply increasing the pH also significantly restores translation selectively in the PGCs. We conclude that the PGCs of this sea urchin institute parallel pathways to quiesce translation thoroughly but transiently. PMID:28235822
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Giacomotto, J; Carroll, A P; Rinkwitz, S; Mowry, B; Cairns, M J; Becker, T S
2016-01-01
The neurodevelopmentally regulated microRNA miR-137 was strongly implicated as risk locus for schizophrenia in the most recent genome wide association study coordinated by the Psychiatric Genome Consortium (PGC). This molecule is highly conserved in vertebrates enabling the investigation of its function in the developing zebrafish. We utilized this model system to achieve overexpression and suppression of miR-137, both transiently and stably through transgenesis. While miR-137 overexpression was not associated with an observable specific phenotype, downregulation by antisense morpholino and/or transgenic expression of miR-sponge RNA induced significant impairment of both embryonic and larval touch-sensitivity without compromising overall anatomical development. We observed miR-137 expression and activity in sensory neurons including Rohon–Beard neurons and dorsal root ganglia, two neuronal cell types that confer touch-sensitivity in normal zebrafish, suggesting a role of these cell types in the observed phenotype. The lack of obvious anatomical or histological pathology in these cells, however, suggested that subtle axonal network defects or a change in synaptic function and neural connectivity might be responsible for the behavioral phenotype rather than a change in the cellular morphology or neuroanatomy. PMID:27219344
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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 during vertebrate embryonic development.
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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 during vertebrate embryonic development. PMID:25855985
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PCSK9 Inhibitors: Novel Therapeutic Strategies for Lowering LDL-Cholesterol.
Liu, Zhao-Peng; Wang, Yan
2018-04-22
Statins are currently the major therapeutic strategies to lower low-density lipoprotein cholesterol (LDL-C) levels. However, a number of hypercholesterolemia patients still have a residual cardiovascular disease (CVD) risk despite taking the maximum-tolerated dose of statins. Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to low-density lipoprotein receptor (LDLR), inducing its degradation in the lysosome and inhibiting LDLR recirculating to the cell membranes. The gain-of-function mutations in PCSK9 elevate the LDL-C levels in plasma. Therefore, PCSK9 inhibitors become novel therapeutic approaches in the treatment of hypercholesterolemia. Several PCSK9 inhibitors have been under investigation, and much progress has been made in clinical trials, especially for monoclonal antibodies (MoAbs). Two MoAbs, evolocumab and alirocumab, are now in clinical use. In this review, we summarize the development of PCSK9 inhibitors, including antisense oligonucleotides (ASOs), small interfering RNA (siRNA), small molecule inhibitor, MoAbs, mimetic peptides and adnectins, and the related safety issues. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.
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An indicator gene to demonstrate intracellular transposition of defective retroviruses.
Heidmann, T; Heidmann, O; Nicolas, J F
1988-01-01
An indicator gene for detection and quantitation of RNA-mediated transposition was constructed (neoRT). It was inserted into a Moloney murine leukemia provirus (Mo-MLV) deleted for the envelope gene to test for intracellular transposition of defective retroviruses [Mo-MLV(neo)RT]. NeoRT contains the selectable neo gene (which confers resistance to the drug G418), inactivated by a polyadenylylation sequence inserted between the neo promotor and coding sequence. The polyadenylylation sequence is flanked (on the antisense strand of the DNA) by a donor and an acceptor splice site so as to be removed upon passage of the provirus through an RNA intermediate. 3T3 cells transfected with the defective Mo-MLV(neo)RT provirus are sensitive to G418. After trans-complementation with Mo-MLV, viral transcripts confer resistance to G418 upon infection of test cells. In the resistant cells, the polyadenylylation sequence has been removed, as a result in most cases of precise splicing of the intronic domain. Retrotransposition of the defective Mo-MLV(neo)RT provirus was demonstrated by submitting transfected G418-sensitive clones to selection. Between 1 and 10 G418-resistant clones were obtained per 10(7) cells. Several possess additional copies, with evidence for precise removal of the intronic domain. By using target test cells in coculture experiments, extracellular intermediates of retrotransposition could not be detected. Images PMID:2832848
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Deas, Tia S; Binduga-Gajewska, Iwona; Tilgner, Mark; Ren, Ping; Stein, David A; Moulton, Hong M; Iversen, Patrick L; Kauffman, Elizabeth B; Kramer, Laura D; Shi, Pei-Yong
2005-04-01
RNA elements within flavivirus genomes are potential targets for antiviral therapy. A panel of phosphorodiamidate morpholino oligomers (PMOs), whose sequences are complementary to RNA elements located in the 5'- and 3'-termini of the West Nile (WN) virus genome, were designed to anneal to important cis-acting elements and potentially to inhibit WN infection. A novel Arg-rich peptide was conjugated to each PMO for efficient cellular delivery. These PMOs exhibited various degrees of antiviral activity upon incubation with a WN virus luciferase-replicon-containing cell line. Among them, PMOs targeting the 5'-terminal 20 nucleotides (5'End) or targeting the 3'-terminal element involved in a potential genome cyclizing interaction (3'CSI) exhibited the greatest potency. When cells infected with an epidemic strain of WN virus were treated with the 5'End or 3'CSI PMO, virus titers were reduced by approximately 5 to 6 logs at a 5 muM concentration without apparent cytotoxicity. The 3'CSI PMO also inhibited mosquito-borne flaviviruses other than WN virus, and the antiviral potency correlated with the conservation of the targeted 3'CSI sequences of specific viruses. Mode-of-action analyses showed that the 5'End and 3'CSI PMOs suppressed viral infection through two distinct mechanisms. The 5'End PMO inhibited viral translation, whereas the 3'CSI PMO did not significantly affect viral translation but suppressed RNA replication. The results suggest that antisense PMO-mediated blocking of cis-acting elements of flavivirus genomes can potentially be developed into an anti-flavivirus therapy. In addition, we report that although a full-length WN virus containing a luciferase reporter (engineered at the 3' untranslated region of the genome) is not stable, an early passage of this reporting virus can be used to screen for inhibitors against any step of the virus life cycle.
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Echigoya, Yusuke; Mouly, Vincent; Garcia, Luis; Yokota, Toshifumi; Duddy, William
2015-01-01
The use of antisense ‘splice-switching’ oligonucleotides to induce exon skipping represents a potential therapeutic approach to various human genetic diseases. It has achieved greatest maturity in exon skipping of the dystrophin transcript in Duchenne muscular dystrophy (DMD), for which several clinical trials are completed or ongoing, and a large body of data exists describing tested oligonucleotides and their efficacy. The rational design of an exon skipping oligonucleotide involves the choice of an antisense sequence, usually between 15 and 32 nucleotides, targeting the exon that is to be skipped. Although parameters describing the target site can be computationally estimated and several have been identified to correlate with efficacy, methods to predict efficacy are limited. Here, an in silico pre-screening approach is proposed, based on predictive statistical modelling. Previous DMD data were compiled together and, for each oligonucleotide, some 60 descriptors were considered. Statistical modelling approaches were applied to derive algorithms that predict exon skipping for a given target site. We confirmed (1) the binding energetics of the oligonucleotide to the RNA, and (2) the distance in bases of the target site from the splice acceptor site, as the two most predictive parameters, and we included these and several other parameters (while discounting many) into an in silico screening process, based on their capacity to predict high or low efficacy in either phosphorodiamidate morpholino oligomers (89% correctly predicted) and/or 2’O Methyl RNA oligonucleotides (76% correctly predicted). Predictions correlated strongly with in vitro testing for sixteen de novo PMO sequences targeting various positions on DMD exons 44 (R2 0.89) and 53 (R2 0.89), one of which represents a potential novel candidate for clinical trials. We provide these algorithms together with a computational tool that facilitates screening to predict exon skipping efficacy at each position of a target exon. PMID:25816009
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Boron nitride nanotubes for gene silencing.
Şen, Özlem; Çobandede, Zehra; Emanet, Melis; Bayrak, Ömer Faruk; Çulha, Mustafa
2017-09-01
Non-viral gene delivery is increasingly investigated as an alternative to viral vectors due to low toxicity and immunogenicity, easy preparation, tissue specificity, and ability to transfer larger sizes of genes. In this study, boron nitride nanotubes (BNNTs) are functionalized with oligonucleotides (oligo-BNNTs). The morpholinos complementary to the oligonucleotides attached to the BNNTs (morpholino/oligo-BNNTs) are hybridized to silence the luciferase gene. The morpholino/oligo-BNNTs conjugates are administered to luciferase-expressing cells (MDA-MB-231-luc2) and the luciferase activity is monitored. The luciferase activity is decreased when MDA-MB-231-luc2 cells were treated with morpholino/oligo-BNNTs. The study suggests that BNNTs can be used as a potential vector to transfect cells. BNNTs are potential new nanocarriers for gene delivery applications. Copyright © 2017 Elsevier B.V. All rights reserved.
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Egr1 gene knockdown affects embryonic ocular development in zebrafish.
Hu, Chao-Yu; Yang, Chang-Hao; Chen, Wei-Yu; Huang, Chiu-Ju; Huang, Hsing-Yen; Chen, Muh-Shy; Tsai, Huai-Jen
2006-10-26
To identify the changes in zebrafish embryonic ocular development after early growth response factor 1 (Egr1) gene knockdown by Egr1-specific translation inhibitor, morpholino oligonucleotides (MO). Two kinds of Egr1-MO were microinjected separately with various dosages into one to four celled zebrafish embryos to find an optimal dose generating an acceptable mortality rate and high frequency of specific phenotype. Chordin-MO served as the positive control; a 5 mismatch MO of Egr1-MO1 and a nonspecific MO served as negative controls. We graded the Egr1 morphants according to their gross abnormalities, and measured their ocular dimensions accordingly. Western blot analysis and synthetic Egr1 mRNA rescue experiments confirmed whether the deformities were caused by Egr1 gene knockdown. Histological examination and three kinds of immunohistochemical staining were applied to identify glutamate receptor one expression in retinal ganglion cells and amacrine cells, to recognize acetylated alpha-tubulin expression which indicated axonogenesis, and to label photoreceptor cells with zpr-1 antibody. After microinjection of 8 ng Egr1-MO1 or 2 ng Egr1-MO2, 81.8% and 97.3% of larvae at 72 h postfertilization had specific defects, respectively. The gross phenotype included string-like heart, flat head, and deformed tail. The more severely deformed larvae had smaller eyes and pupils. Co-injection of 8 ng Egr1-MO1 and supplementary 12 pg synthetic Egr1 mRNA reduced the gross abnormality rate from 84.4% to 29.7%, and decreased the severity of deformities. Egr1 protein appeared in the wildtype and rescued morphants, but was lacking in the Egr1 morphants with specific deformities. Lenses of Egr1 morphants were smaller and had some residual nucleated lens fiber cells. Morphants' retinal cells arranged disorderly and compactly with thin plexiform layers. Immunohistochemical studies showed that morphants had a markedly decreased number of mature retinal ganglion cells, amacrine cells, and photoreceptor cells. Retinal axonogenesis was prominently reduced in morphants. The Egr1 gene plays an important role in zebrafish embryonic oculogenesis. Ocular structures including lens and retina were primitive and lacked appropriate differentiation. Such arrested retinal and lenticular development in Egr1 morphants resulted in microphthalmos.
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Hinits, Yaniv; Pan, Luyuan; Walker, Charline; Dowd, John; Moens, Cecilia B.; Hughes, Simon M.
2013-01-01
Summary Mef2 transcription factors have been strongly linked with early heart development. D-mef2 is required for heart formation in Drosophila, but whether Mef2 is essential for vertebrate cardiomyocyte (CM) differentiation is unclear. In mice, although Mef2c is expressed in all CMs, targeted deletion of Mef2c causes lethal loss of second heart field (SHF) derivatives and failure of cardiac looping, but first heart field CMs can differentiate. Here we examine Mef2 function in early heart development in zebrafish. Two Mef2c genes exist in zebrafish, mef2ca and mef2cb. Both are expressed similarly in the bilateral heart fields but mef2cb is strongly expressed in the heart poles at the primitive heart tube stage. By using fish mutants for mef2ca and mef2cb and antisense morpholinos to knock down either or both Mef2cs, we show that Mef2ca and Mef2cb have essential but redundant roles in myocardial differentiation. Loss of both Mef2ca and Mef2cb function does not interfere with early cardiogenic markers such as nkx2.5, gata4 and hand2 but results in a dramatic loss of expression of sarcomeric genes and myocardial markers such as bmp4, nppa, smyd1b and late nkx2.5 mRNA. Rare residual CMs observed in mef2ca;mef2cb double mutants are ablated by a morpholino capable of knocking down other Mef2s. Mef2cb over-expression activates bmp4 within the cardiogenic region, but no ectopic CMs are formed. Surprisingly, anterior mesoderm and other tissues become skeletal muscle. Mef2ca single mutants have delayed heart development, but form an apparently normal heart. Mef2cb single mutants have a functional heart and are viable adults. Our results show that the key role of Mef2c in myocardial differentiation is conserved throughout the vertebrate heart. PMID:22750409
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Brooks, Kelsey; Burns, Gregory; Spencer, Thomas E
2015-08-01
In sheep, the elongating conceptus synthesizes and secretes interferon tau (IFNT) as well as prostaglandins (PGs) and cortisol. The enzymes, hydroxysteroid (11-beta) dehydrogenase 1 (HSD11B1) and HSD11B2 interconvert cortisone and cortisol. In sheep, HSD11B1 is expressed and active in the conceptus trophectoderm as well as in the endometrial luminal epithelia; in contrast, HSD11B2 expression is most abundant in conceptus trophectoderm. Cortisol is a biologically active glucocorticoid and ligand for the glucocorticoid receptor (NR3C1 or GR) and mineralocorticoid receptor (NR3C2 or MR). Expression of MR is not detectable in either the ovine endometrium or conceptus during early pregnancy. In tissues that do not express MR, HSD11B2 protects cells from the growth-inhibiting and/or proapoptotic effects of cortisol, particularly during embryonic development. In study one, an in utero loss-of-function analysis of HSD11B1 and HSD11B2 was conducted in the conceptus trophectoderm using morpholino antisense oligonucleotides (MAOs) that inhibit mRNA translation. Elongating, filamentous conceptuses were recovered on Day 14 from ewes infused with control morpholino or HSD11B2 MAO. In contrast, HSD11B1 MAO resulted in severely growth-retarded conceptuses or conceptus fragments with apoptotic trophectoderm. In study two, clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome editing was used to determine the role of GR in conceptus elongation and development. Elongating, filamentous-type conceptuses (12-14 cm in length) were recovered from ewes gestating control embryos (n = 7/7) and gestating GR-edited embryos (n = 6/7). These results support the idea that the effects of HSD11B1-derived cortisol on conceptus elongation are indirectly mediated by the endometrium and are not directly mediated through GR in the trophectoderm. © 2015 by the Society for the Study of Reproduction, Inc.
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The transcriptional activator ZNF143 is essential for normal development in zebrafish
2012-01-01
Background ZNF143 is a sequence-specific DNA-binding protein that stimulates transcription of both small RNA genes by RNA polymerase II or III, or protein-coding genes by RNA polymerase II, using separable activating domains. We describe phenotypic effects following knockdown of this protein in developing Danio rerio (zebrafish) embryos by injection of morpholino antisense oligonucleotides that target znf143 mRNA. Results The loss of function phenotype is pleiotropic and includes a broad array of abnormalities including defects in heart, blood, ear and midbrain hindbrain boundary. Defects are rescued by coinjection of synthetic mRNA encoding full-length ZNF143 protein, but not by protein lacking the amino-terminal activation domains. Accordingly, expression of several marker genes is affected following knockdown, including GATA-binding protein 1 (gata1), cardiac myosin light chain 2 (cmlc2) and paired box gene 2a (pax2a). The zebrafish pax2a gene proximal promoter contains two binding sites for ZNF143, and reporter gene transcription driven by this promoter in transfected cells is activated by this protein. Conclusions Normal development of zebrafish embryos requires ZNF143. Furthermore, the pax2a gene is probably one example of many protein-coding gene targets of ZNF143 during zebrafish development. PMID:22268977
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The transcriptional activator ZNF143 is essential for normal development in zebrafish.
Halbig, Kari M; Lekven, Arne C; Kunkel, Gary R
2012-01-23
ZNF143 is a sequence-specific DNA-binding protein that stimulates transcription of both small RNA genes by RNA polymerase II or III, or protein-coding genes by RNA polymerase II, using separable activating domains. We describe phenotypic effects following knockdown of this protein in developing Danio rerio (zebrafish) embryos by injection of morpholino antisense oligonucleotides that target znf143 mRNA. The loss of function phenotype is pleiotropic and includes a broad array of abnormalities including defects in heart, blood, ear and midbrain hindbrain boundary. Defects are rescued by coinjection of synthetic mRNA encoding full-length ZNF143 protein, but not by protein lacking the amino-terminal activation domains. Accordingly, expression of several marker genes is affected following knockdown, including GATA-binding protein 1 (gata1), cardiac myosin light chain 2 (cmlc2) and paired box gene 2a (pax2a). The zebrafish pax2a gene proximal promoter contains two binding sites for ZNF143, and reporter gene transcription driven by this promoter in transfected cells is activated by this protein. Normal development of zebrafish embryos requires ZNF143. Furthermore, the pax2a gene is probably one example of many protein-coding gene targets of ZNF143 during zebrafish development.
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Cui, Miao; Lin, Che-Yi; Su, Yi-Hsien
2017-09-01
Studies on the gene regulatory networks (GRNs) of sea urchin embryos have provided a basic understanding of the molecular mechanisms controlling animal development. The causal links in GRNs have been verified experimentally through perturbation of gene functions. Microinjection of antisense morpholino oligonucleotides (MOs) into the egg is the most widely used approach for gene knockdown in sea urchin embryos. The modification of MOs into a membrane-permeable form (vivo-MOs) has allowed gene knockdown at later developmental stages. Recent advances in genome editing tools, such as zinc-finger nucleases, transcription activator-like effector-based nucleases and the clustered regularly interspaced short palindromic repeat/clustered regularly interspaced short palindromic repeat-associated protein 9 (CRISPR/Cas9) system, have provided methods for gene knockout in sea urchins. Here, we review the use of vivo-MOs and genome editing tools in sea urchin studies since the publication of its genome in 2006. Various applications of the CRISPR/Cas9 system and their potential in studying sea urchin development are also discussed. These new tools will provide more sophisticated experimental methods for studying sea urchin development. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.
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HBEGF, SRA1, and IK: Three cosegregating genes as determinants of cardiomyopathy.
Friedrichs, Frauke; Zugck, Christian; Rauch, Gerd-Jörg; Ivandic, Boris; Weichenhan, Dieter; Müller-Bardorff, Margit; Meder, Benjamin; El Mokhtari, Nour Eddine; Regitz-Zagrosek, Vera; Hetzer, Roland; Schäfer, Arne; Schreiber, Stefan; Chen, Jian; Neuhaus, Isaac; Ji, Ruiru; Siemers, Nathan O; Frey, Norbert; Rottbauer, Wolfgang; Katus, Hugo A; Stoll, Monika
2009-03-01
Human dilated cardiomyopathy (DCM), a disorder of the cardiac muscle, causes considerable morbidity and mortality and is one of the major causes of sudden cardiac death. Genetic factors play a role in the etiology and pathogenesis of DCM. Disease-associated genetic variations identified to date have been identified in single families or single sporadic patients and explain a minority of the etiology of DCM. We show that a 600-kb region of linkage disequilibrium (LD) on 5q31.2-3, harboring multiple genes, is associated with cardiomyopathy in three independent Caucasian populations (combined P-value = 0.00087). Functional assessment in zebrafish demonstrates that at least three genes, orthologous to loci in this LD block, HBEGF, IK, and SRA1, result independently in a phenotype of myocardial contractile dysfunction when their expression is reduced with morpholino antisense reagents. Evolutionary analysis across multiple vertebrate genomes suggests that this heart failure-associated LD block emerged by a series of genomic rearrangements across amphibian, avian, and mammalian genomes and is maintained as a cluster in mammals. Taken together, these observations challenge the simple notion that disease phenotypes can be traced to altered function of a single locus within a haplotype and suggest that a more detailed assessment of causality can be necessary.
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HBEGF, SRA1, and IK: Three cosegregating genes as determinants of cardiomyopathy
Friedrichs, Frauke; Zugck, Christian; Rauch, Gerd-Jörg; Ivandic, Boris; Weichenhan, Dieter; Müller-Bardorff, Margit; Meder, Benjamin; El Mokhtari, Nour Eddine; Regitz-Zagrosek, Vera; Hetzer, Roland; Schäfer, Arne; Schreiber, Stefan; Chen, Jian; Neuhaus, Isaac; Ji, Ruiru; Siemers, Nathan O.; Frey, Norbert; Rottbauer, Wolfgang; Katus, Hugo A.; Stoll, Monika
2009-01-01
Human dilated cardiomyopathy (DCM), a disorder of the cardiac muscle, causes considerable morbidity and mortality and is one of the major causes of sudden cardiac death. Genetic factors play a role in the etiology and pathogenesis of DCM. Disease-associated genetic variations identified to date have been identified in single families or single sporadic patients and explain a minority of the etiology of DCM. We show that a 600-kb region of linkage disequilibrium (LD) on 5q31.2-3, harboring multiple genes, is associated with cardiomyopathy in three independent Caucasian populations (combined P-value = 0.00087). Functional assessment in zebrafish demonstrates that at least three genes, orthologous to loci in this LD block, HBEGF, IK, and SRA1, result independently in a phenotype of myocardial contractile dysfunction when their expression is reduced with morpholino antisense reagents. Evolutionary analysis across multiple vertebrate genomes suggests that this heart failure-associated LD block emerged by a series of genomic rearrangements across amphibian, avian, and mammalian genomes and is maintained as a cluster in mammals. Taken together, these observations challenge the simple notion that disease phenotypes can be traced to altered function of a single locus within a haplotype and suggest that a more detailed assessment of causality can be necessary. PMID:19064678
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Yamada, Shigehiro; Ueno, Naoto; Satoh, Nori; Takahashi, Hiroki
2011-01-01
Brachyury plays a pivotal role in the notochord formation in ascidian embryos. Ciona intestinalis Noto4 (Ci-Noto4) was isolated as a gene downstream of Ci-Bra. This gene encodes a 307 amino-acid protein with a C-terminal phosphotyrosine interaction domain (PTB/PID). Expression of Ci-Noto4 commences at the neural plate stage and is specific to notochord cells. Suppression of Ci-Noto4 levels with specific antisense morpholino oligonucleotides resulted in the formation of two rows of notochord cells owing to a lack of midline intercalation between the bilateral populations of progenitor cells. In contrast, overexpression of Ci-Noto4 by injection of a Ci-Bra(promoter):Ci-Noto4-EGFP construct into fertilized eggs disrupted the localization of notochord cells. Ci-Noto4 overexpression did not affect cellular differentiation in the notochord, muscle, mesenchyme, or nervous system. Analysis of Ci-Noto4 regions that are responsible for its function suggested significant roles for the PTB/PID and a central region, an area with no obvious sequence similarity to other known proteins. These results suggested that PTB/PID-containing Ci-Noto4 is essential for midline intercalation of notochord cells in chordate embryos.
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The Midblastula Transition Defines the Onset of Y RNA-Dependent DNA Replication in Xenopus laevis ▿
Collart, Clara; Christov, Christo P.; Smith, James C.; Krude, Torsten
2011-01-01
Noncoding Y RNAs are essential for the initiation of chromosomal DNA replication in mammalian cell extracts, but their role in this process during early vertebrate development is unknown. Here, we use antisense morpholino nucleotides (MOs) to investigate Y RNA function in Xenopus laevis and zebrafish embryos. We show that embryos in which Y RNA function is inhibited by MOs develop normally until the midblastula transition (MBT) but then fail to replicate their DNA and die before gastrulation. Consistent with this observation, Y RNA function is not required for DNA replication in Xenopus egg extracts but is required for replication in a post-MBT cell line. Y RNAs do not bind chromatin in karyomeres before MBT, but they associate with interphase nuclei after MBT in an origin recognition complex (ORC)-dependent manner. Y RNA-specific MOs inhibit the association of Y RNAs with ORC, Cdt1, and HMGA1a proteins, suggesting that these molecular associations are essential for Y RNA function in DNA replication. The MBT is thus a transition point between Y RNA-independent and Y RNA-dependent control of vertebrate DNA replication. Our data suggest that in vertebrates Y RNAs function as a developmentally regulated layer of control over the evolutionarily conserved eukaryotic DNA replication machinery. PMID:21791613
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Li, Mei; Andersson-Lendahl, Monika; Sejersen, Thomas; Arner, Anders
2013-03-01
Skeletal muscle was examined in zebrafish larvae in order to address questions related to the function of the intermediate filament protein desmin and its role in the pathogenesis of human desminopathy. A novel approach including mechanical and structural studies of 4-6-d-old larvae was applied. Morpholino antisense oligonucleotides were used to knock down desmin. Expression was assessed using messenger RNA and protein analyses. Histology and synchrotron light-based small angle x-ray diffraction were applied. Functional properties were analyzed with in vivo studies of swimming behavior and with in vitro mechanical examinations of muscle. The two desmin genes normally expressed in zebrafish could be knocked down by ~50%. This resulted in a phenotype with disorganized muscles with altered attachments to the myosepta. The knockdown larvae were smaller and had diminished swimming activity. Active tension was lowered and muscles were less vulnerable to acute stretch-induced injury. X-ray diffraction revealed wider interfilament spacing. In conclusion, desmin intermediate filaments are required for normal active force generation and affect vulnerability during eccentric work. This is related to the role of desmin in anchoring sarcomeres for optimal force transmission. The results also show that a partial lack of desmin, without protein aggregates, is sufficient to cause muscle pathology resembling that in human desminopathy.
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Martin, Elodie; Schüle, Rebecca; Smets, Katrien; Rastetter, Agnès; Boukhris, Amir; Loureiro, José L.; Gonzalez, Michael A.; Mundwiller, Emeline; Deconinck, Tine; Wessner, Marc; Jornea, Ludmila; Oteyza, Andrés Caballero; Durr, Alexandra; Martin, Jean-Jacques; Schöls, Ludger; Mhiri, Chokri; Lamari, Foudil; Züchner, Stephan; De Jonghe, Peter; Kabashi, Edor; Brice, Alexis; Stevanin, Giovanni
2013-01-01
Spastic paraplegia 46 refers to a locus mapped to chromosome 9 that accounts for a complicated autosomal-recessive form of hereditary spastic paraplegia (HSP). With next-generation sequencing in three independent families, we identified four different mutations in GBA2 (three truncating variants and one missense variant), which were found to cosegregate with the disease and were absent in controls. GBA2 encodes a microsomal nonlysosomal glucosylceramidase that catalyzes the conversion of glucosylceramide to free glucose and ceramide and the hydrolysis of bile acid 3-O-glucosides. The missense variant was also found at the homozygous state in a simplex subject in whom no residual glucocerebrosidase activity of GBA2 could be evidenced in blood cells, opening the way to a possible measurement of this enzyme activity in clinical practice. The overall phenotype was a complex HSP with mental impairment, cataract, and hypogonadism in males associated with various degrees of corpus callosum and cerebellar atrophy on brain imaging. Antisense morpholino oligonucleotides targeting the zebrafish GBA2 orthologous gene led to abnormal motor behavior and axonal shortening/branching of motoneurons that were rescued by the human wild-type mRNA but not by applying the same mRNA containing the missense mutation. This study highlights the role of ceramide metabolism in HSP pathology. PMID:23332916
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Smad4 is required for the development of cardiac and skeletal muscle in zebrafish.
Yang, Jie; Wang, Junnai; Zeng, Zhen; Qiao, Long; Zhuang, Liang; Jiang, Lijun; Wei, Juncheng; Ma, Quanfu; Wu, Mingfu; Ye, Shuangmei; Gao, Qinglei; Ma, Ding; Huang, Xiaoyuan
Transforming growth factor-beta (TGF-beta) regulates cellular functions and plays key roles in development and carcinogenesis. Smad4 is the central intracellular mediator of TGF-beta signaling and plays crucial roles in tissue regeneration, cell differentiation, embryonic development, regulation of the immune system and tumor progression. To clarify the role of smad4 in development, we examined both the pattern of smad4 expression in zebrafish embryos and the effect of smad4 suppression on embryonic development using smad4-specific antisense morpholino-oligonucleotides. We show that smad4 is expressed in zebrafish embryos at all developmental stages examined and that embryonic knockdown of smad4 results in pericardial edema, decreased heartbeat and defects in the trunk structure. Additionally, these phenotypes were associated with abnormal expression of the two heart-chamber markers, cmlc2 and vmhc, as well as abnormal expression of three makers of myogenic terminal differentiation, mylz2, smyhc1 and mck. Furthermore, a notable increase in apoptosis was apparent in the smad4 knockdown embryos, while no obvious reduction in cell proliferation was observed. Collectively, these data suggest that smad4 plays an important role in heart and skeletal muscle development. Copyright © 2016 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.
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The Ca2+-induced methyltransferase xPRMT1b controls neural fate in amphibian embryo.
Batut, Julie; Vandel, Laurence; Leclerc, Catherine; Daguzan, Christiane; Moreau, Marc; Néant, Isabelle
2005-10-18
We have previously shown that an increase in intracellular Ca2+ is both necessary and sufficient to commit ectoderm to a neural fate in Xenopus embryos. However, the relationship between this Ca2+ increase and the expression of early neural genes has yet to be defined. Using a subtractive cDNA library between untreated and caffeine-treated animal caps, i.e., control ectoderm and ectoderm induced toward a neural fate by a release of Ca2+, we have isolated the arginine N-methyltransferase, xPRMT1b, a Ca2+-induced target gene, which plays a pivotal role in this process. First, we show in embryo and in animal cap that xPRMT1b expression is Ca2+-regulated. Second, overexpression of xPRMT1b induces the expression of early neural genes such as Zic3. Finally, in the whole embryo, antisense approach with morpholino oligonucleotide against xPRMT1b impairs neural development and in animal caps blocks the expression of neural markers induced by a release of internal Ca2+. Our results implicate an instructive role of an enzyme, an arginine methyltransferase protein, in the embryonic choice of determination between epidermal and neural fate. The results presented provide insights by which a Ca2+ increase induces neural fate.
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Synaptic Glutamate Spillover Due to Impaired Glutamate Uptake Mediates Heroin Relapse
Scofield, Michael D.; Boger, Heather; Hensley, Megan; Kalivas, Peter W.
2014-01-01
Reducing the enduring vulnerability to relapse is a therapeutic goal in treating drug addiction. Studies with animal models of drug addiction show a marked increase in extrasynaptic glutamate in the core subcompartment of the nucleus accumbens (NAcore) during reinstated drug seeking. However, the synaptic mechanisms linking drug-induced changes in extrasynaptic glutamate to relapse are poorly understood. Here, we discovered impaired glutamate elimination in rats extinguished from heroin self-administration that leads to spillover of synaptically released glutamate into the nonsynaptic extracellular space in NAcore and investigated whether restoration of glutamate transport prevented reinstated heroin seeking. Through multiple functional assays of glutamate uptake and analyzing NMDA receptor-mediated currents, we show that heroin self-administration produced long-lasting downregulation of glutamate uptake and surface expression of the transporter GLT-1. This downregulation was associated with spillover of synaptic glutamate to extrasynaptic NMDA receptors within the NAcore. Ceftriaxone restored glutamate uptake and prevented synaptic glutamate spillover and cue-induced heroin seeking. Ceftriaxone-induced inhibition of reinstated heroin seeking was blocked by morpholino-antisense targeting GLT-1 synthesis. These data reveal that the synaptic glutamate spillover in the NAcore results from reduced glutamate transport and is a critical pathophysiological mechanism underling reinstated drug seeking in rats extinguished from heroin self-administration. PMID:24741055
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Cdc42 controls primary mesenchyme cell morphogenesis in the sea urchin embryo.
Sepúlveda-Ramírez, Silvia P; Toledo-Jacobo, Leslie; Henson, John H; Shuster, Charles B
2018-05-15
In the sea urchin embryo, gastrulation is characterized by the ingression and directed cell migration of primary mesenchyme cells (PMCs), as well as the primary invagination and convergent extension of the endomesoderm. Like all cell shape changes, individual and collective cell motility is orchestrated by Rho family GTPases and their modulation of the actomyosin cytoskeleton. And while endomesoderm specification has been intensively studied in echinoids, much less is known about the proximate regulators driving cell motility. Toward these ends, we employed anti-sense morpholinos, mutant alleles and pharmacological inhibitors to assess the role of Cdc42 during sea urchin gastrulation. While inhibition of Cdc42 expression or activity had only mild effects on PMC ingression, PMC migration, alignment and skeletogenesis were disrupted in the absence of Cdc42, as well as elongation of the archenteron. PMC migration and patterning of the larval skeleton relies on the extension of filopodia, and Cdc42 was required for filopodia in vivo as well as in cultured PMCs. Lastly, filopodial extension required both Arp2/3 and formin actin-nucleating factors, supporting models of filopodial nucleation observed in other systems. Together, these results suggest that Cdc42 plays essential roles during PMC cell motility and organogenesis. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.
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Tumor suppressor Lzap regulates cell cycle progression, doming and zebrafish epiboly
Liu, Dan; Wang, Wen-Der; Melville, David B.; Cha, Yong I.; Yin, Zhirong; Issaeva, Natalia; Knapik, Ela W.; Yarbrough, Wendell G.
2012-01-01
Initial stages of embryonic development rely on rapid, synchronized cell divisions of the fertilized egg followed by a set of morphogenetic movements collectively called epiboly and gastrulation. Lzap is a putative tumor suppressor whose expression is lost in 30% of head and neck squamous cell carcinomas. Lzap activities include regulation of cell cycle progression and response to therapeutic agents. Here we explore developmental roles of the lzap gene during zebrafish morphogenesis. Lzap is highly conserved among vertebrates and is maternally deposited. Expression is initially ubiquitous during gastrulation, and later becomes more prominent in the pharyngeal arches, digestive tract and brain. Antisense morpholino-mediated depletion of Lzap resulted in delayed cell divisions and apoptosis during blastomere formation, resulting in fewer, larger cells. Cell cycle analysis suggested that Lzap loss in early embryonic cells resulted in a G2/M arrest. Furthermore, the Lzap-deficient embryos failed to initiate epiboly – the earliest morphogenetic movement in animal development – which has been shown to be dependent on cell adhesion and migration of epithelial sheets. Our results strongly implicate Lzap in regulation of cell cycle progression, adhesion and migratory activity of epithelial cell sheets during early development. These functions provide further insight into Lzap activity that may contribute not only to development, but also to tumor formation. PMID:21523853
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Kcnh1 Voltage-gated Potassium Channels Are Essential for Early Zebrafish Development*
Stengel, Rayk; Rivera-Milla, Eric; Sahoo, Nirakar; Ebert, Christina; Bollig, Frank; Heinemann, Stefan H.; Schönherr, Roland; Englert, Christoph
2012-01-01
The Kcnh1 gene encodes a voltage-gated potassium channel highly expressed in neurons and involved in tumor cell proliferation, yet its physiological roles remain unclear. We have used the zebrafish as a model to analyze Kcnh1 function in vitro and in vivo. We found that the kcnh1 gene is duplicated in teleost fish (i.e. kcnh1a and kcnh1b) and that both genes are maternally expressed during early development. In adult zebrafish, kcnh1a and kcnh1b have distinct expression patterns but share expression in brain and testis. Heterologous expression of both genes in Xenopus oocytes revealed a strong conservation of characteristic functional properties between human and fish channels, including a unique sensitivity to intracellular Ca2+/calmodulin and modulation of voltage-dependent gating by extracellular Mg2+. Using a morpholino antisense approach, we demonstrate a strong kcnh1 loss-of-function phenotype in developing zebrafish, characterized by growth retardation, delayed hindbrain formation, and embryonic lethality. This late phenotype was preceded by transcriptional up-regulation of known cell-cycle inhibitors (p21, p27, cdh2) and down-regulation of pro-proliferative factors, including cyclin D1, at 70% epiboly. These results reveal an unanticipated basic activity of kcnh1 that is crucial for early embryonic development and patterning. PMID:22927438
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Changing Hydrozoan Bauplans by Silencing Hox-Like Genes
Jakob, Wolfgang; Schierwater, Bernd
2007-01-01
Regulatory genes of the Antp class have been a major factor for the invention and radiation of animal bauplans. One of the most diverse animal phyla are the Cnidaria, which are close to the root of metazoan life and which often appear in two distinct generations and a remarkable variety of body forms. Hox-like genes have been known to be involved in axial patterning in the Cnidaria and have been suspected to play roles in the genetic control of many of the observed bauplan changes. Unfortunately RNAi mediated gene silencing studies have not been satisfactory for marine invertebrate organisms thus far. No direct evidence supporting Hox-like gene induced bauplan changes in cnidarians have been documented as of yet. Herein, we report a protocol for RNAi transfection of marine invertebrates and demonstrate that knock downs of Hox-like genes in Cnidaria create substantial bauplan alterations, including the formation of multiple oral poles (“heads”) by Cnox-2 and Cnox-3 inhibition, deformation of the main body axis by Cnox-5 inhibition and duplication of tentacles by Cnox-1 inhibition. All phenotypes observed in the course of the RNAi studies were identical to those obtained by morpholino antisense oligo experiments and are reminiscent of macroevolutionary bauplan changes. The reported protocol will allow routine RNAi studies in marine invertebrates to be established. PMID:17668071
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Functional rescue of dystrophin-deficient mdx mice by a chimeric peptide-PMO.
Yin, Haifang; Moulton, Hong M; Betts, Corinne; Merritt, Thomas; Seow, Yiqi; Ashraf, Shirin; Wang, Qingsong; Boutilier, Jordan; Wood, Matthew Ja
2010-10-01
Splice modulation using antisense oligonucleotides (AOs) has been shown to yield targeted exon exclusion to restore the open reading frame and generate truncated but partially functional dystrophin protein. This has been successfully demonstrated in dystrophin-deficient mdx mice and in Duchenne muscular dystrophy (DMD) patients. However, DMD is a systemic disease; successful therapeutic exploitation of this approach will therefore depend on effective systemic delivery of AOs to all affected tissues. We have previously shown the potential of a muscle-specific/arginine-rich chimeric peptide-phosphorodiamidate morpholino (PMO) conjugate, but its long-term activity, optimized dosing regimen, capacity for functional correction and safety profile remain to be established. Here, we report the results of this chimeric peptide-PMO conjugate in the mdx mouse using low doses (3 and 6 mg/kg) administered via a 6 biweekly systemic intravenous injection protocol. We show 100% dystrophin-positive fibers and near complete correction of the dystrophin transcript defect in all peripheral muscle groups, with restoration of 50% dystrophin protein over 12 weeks, leading to correction of the DMD pathological phenotype and restoration of muscle function in the absence of detectable toxicity or immune response. Chimeric muscle-specific/cell-penetrating peptides therefore represent highly promising agents for systemic delivery of splice-correcting PMO oligomers for DMD therapy.
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NASA Astrophysics Data System (ADS)
Srinivasa, Hosapalya Thimmaiah; Harishkumar, Hosanagara Narayana; Palakshamurthy, Bandrehalli Siddagangappa
2017-03-01
New set of trifluromethyl, diethylamino and morpholino derived coumarin compounds were prepared by reacting various coumarin 3-carboxylic acids with various phenyl esters with peripheral alkyl, ester and polar cyano moieties in the presence of EDC.HCl/DMAP as esterification agent. The chemical structures of new coumarin derivatives were confirmed by standard spectroscopic techniques and mesomorphic behaviours were established by polarised optical microscopy (POM) and differential scanning calorimetry (DSC). Trifluoromethane and morpholino derivatives show SmA/Nematic phase, while diethylamino derivatives did not show liquid crystalline property.
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Angiomodulin is a specific marker of vasculature and regulates VEGF-A dependent neo-angiogenesis
Hooper, Andrea T.; Shmelkov, Sergey V.; Gupta, Sunny; Milde, Till; Bambino, Kathryn; Gillen, Kelly; Goetz, Mollie; Chavala, Sai; Baljevic, Muhamed; Murphy, Andrew J.; Valenzuela, David M.; Gale, Nicholas W.; Thurston, Gavin; Yancopoulos, George D.; Vahdat, Linda; Evans, Todd; Rafii, Shahin
2010-01-01
Blood vessel formation is controlled by the balance between pro- and anti-angiogenic pathways. Although much is known about the factors that drive sprouting of neovessels, the factors that stabilize and pattern neovessels are undefined. The expression of angiomodulin (AGM), a VEGF-A binding protein, was increased in the vasculature of several human tumors as compared to normal tissue, raising the hypothesis that AGM may modulate VEGF-A-dependent vascular patterning. To elucidate the expression pattern of AGM, we developed an AGM knockin reporter mouse (AGMlacZ/+) wherein we demonstrate that AGM is predominantly expressed in the vasculature of developing embryos and adult organs. During physiological and pathological angiogenesis, AGM is upregulated in the angiogenic vasculature. Using the zebrafish model, we found that AGM is restricted to developing vasculature by 17-22 hpf. Blockade of AGM activity with morpholino oligomers (MO) results in prominent angiogenesis defects in vascular sprouting and remodeling. Concurrent knockdown of both AGM and VEGF-A results in synergistic angiogenesis defects. When VEGF-A is overexpressed, the compensatory induction of the VEGF-A receptor, VEGFR-2/flk-1, is blocked by the simultaneous injection of AGM MO. These results demonstrate that the vascular-specific marker AGM modulates vascular remodeling in part by temporizing the pro-angiogenic effects of VEGF-A. PMID:19542015
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Angiopoietin-like 3 regulates hepatocyte proliferation and lipid metabolism in zebrafish
DOE Office of Scientific and Technical Information (OSTI.GOV)
Lee, So-Hyun; Department of Biology, Chungnam National University, Daejeon; So, Ju-Hoon
2014-04-18
Highlights: • angptl3 is specifically expressed in the liver of developing zebrafish. • Knockdown of Angptl3 decreases liver size in developing zebrafish. • Knockdown of zebrafish Angptl3 elicits a hypocholesterolemia phenotype. - Abstract: Loss-of-function mutations in angiopoietin-like 3 (ANGPTL3) cause familial hypobetalipoproteinemia type 2 (FHBL2) in humans. ANGPTL3 belongs to the angiopoietin-like family, the vascular endothelial growth factor family that is structurally similar to angiopoietins and is known for a regulator of lipid and glucose metabolism, although it is unclear how mutations in ANGPTL3 lead to defect in liver development in the vertebrates. We report here that angptl3 is primarilymore » expressed in the zebrafish developing liver and that morpholino (MO) knockdown of Angptl3 reduces the size of the developing liver, which is caused by suppression of cell proliferation, but not by enhancement of apoptosis. However, MO knockdown of Angptl3 did not alter angiogenesis in the developing liver. Additionally, disruption of zebrafish Angptl3 elicits the hypocholesterolemia phenotype that is characteristic of FHBL2 in humans. Together, our findings propose a novel role for Angptl3 in liver cell proliferation and maintenance during zebrafish embryogenesis. Finally, angptl3 morphants will serve as a good model for understanding the pathophysiology of FHBL2.« less
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Marivin, E; Mourot, B; Loyer, P; Rime, H; Bobe, J; Fostier, A
2015-09-15
Over-expression or inhibition of gene expression can be efficiently used to analyse the functions and/or regulation of target genes. Modulation of gene expression can be achieved through transfection of exogenous nucleic acids into target cells. Such techniques require the development of specific protocols to transfect cell cultures with nucleic acids. The aim of this study was to develop a method of transfection suitable for rainbow trout granulosa cells in primary culture. After the isolation of rainbow trout granulosa cells, chemical transfection of cells with a fluorescent morpholino oligonucleotide (MO) was tested using FuGENE HD at 12 °C. Electroporation was also employed to transfect these cells with either a plasmid or MO. Transfection was more efficient using electroporation (with the following settings: 1200 V/40 ms/1p) than chemical transfection, but electroporation by itself was deleterious, resulting in a decrease of the steroidogenic capacity of the cells, measured via estradiol production from its androgenic substrate. The disturbance of cell biology induced by the transfection method per se should be taken into account in data interpretation when investigating the effects of under- or over-expression of candidate genes. Copyright © 2015 Elsevier Inc. All rights reserved.
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Curado, Silvia; Ober, Elke A.; Walsh, Susan; Cortes-Hernandez, Paulina; Verkade, Heather; Koehler, Carla M.; Stainier, Didier Y. R.
2010-01-01
SUMMARY Understanding liver development should lead to greater insights into liver diseases and improve therapeutic strategies. In a forward genetic screen for genes regulating liver development in zebrafish, we identified a mutant – oliver – that exhibits liver-specific defects. In oliver mutants, the liver is specified, bile ducts form and hepatocytes differentiate. However, the hepatocytes die shortly after their differentiation, and thus the resulting mutant liver consists mainly of biliary tissue. We identified a mutation in the gene encoding translocase of the outer mitochondrial membrane 22 (Tomm22) as responsible for this phenotype. Mutations in tomm genes have been associated with mitochondrial dysfunction, but most studies on the effect of defective mitochondrial protein translocation have been carried out in cultured cells or unicellular organisms. Therefore, the tomm22 mutant represents an important vertebrate genetic model to study mitochondrial biology and hepatic mitochondrial diseases. We further found that the temporary knockdown of Tomm22 levels by morpholino antisense oligonucleotides causes a specific hepatocyte degeneration phenotype that is reversible: new hepatocytes repopulate the liver as Tomm22 recovers to wild-type levels. The specificity and reversibility of hepatocyte ablation after temporary knockdown of Tomm22 provides an additional model to study liver regeneration, under conditions where most hepatocytes have died. We used this regeneration model to analyze the signaling commonalities between hepatocyte development and regeneration. PMID:20483998
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Shigetani, Yasuyo; Howard, Sara; Guidato, Sonia; Furushima, Kenryo; Abe, Takaya; Itasaki, Nobue
2008-07-15
While most cranial ganglia contain neurons of either neural crest or placodal origin, neurons of the trigeminal ganglion derive from both populations. The Wnt signaling pathway is known to be required for the development of neural crest cells and for trigeminal ganglion formation, however, migrating neural crest cells do not express any known Wnt ligands. Here we demonstrate that Wise, a Wnt modulator expressed in the surface ectoderm overlying the trigeminal ganglion, play a role in promoting the assembly of placodal and neural crest cells. When overexpressed in chick, Wise causes delamination of ectodermal cells and attracts migrating neural crest cells. Overexpression of Wise is thus sufficient to ectopically induce ganglion-like structures consisting of both origins. The function of Wise is likely synergized with Wnt6, expressed in an overlapping manner with Wise in the surface ectoderm. Electroporation of morpholino antisense oligonucleotides against Wise and Wnt6 causes decrease in the contact of neural crest cells with the delaminated placode-derived cells. In addition, targeted deletion of Wise in mouse causes phenotypes that can be explained by a decrease in the contribution of neural crest cells to the ophthalmic lobe of the trigeminal ganglion. These data suggest that Wise is able to function cell non-autonomously on neural crest cells and promote trigeminal ganglion formation.
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Neuropilin asymmetry mediates a left-right difference in habenular connectivity.
Kuan, Yung-Shu; Yu, Hung-Hsiang; Moens, Cecilia B; Halpern, Marnie E
2007-03-01
The medial habenular nuclei of the zebrafish diencephalon, which lie bilateral to the pineal complex, exhibit left-right differences in their neuroanatomy, gene expression profiles and axonal projections to the unpaired midbrain target--the interpeduncular nucleus (IPN). Efferents from the left habenula terminate along the entire dorsoventral extent of the IPN, whereas axons from the right habenula project only to the ventral IPN. How this left-right difference in connectivity is established and the factors involved in differential target recognition are unknown. Prior to IPN innervation, we find that only the left habenula expresses the zebrafish homologue of Neuropilin1a (Nrp1a), a receptor for class III Semaphorins (Sema3s). Directional asymmetry of nrp1a expression relies on Nodal signaling and the presence of the left-sided parapineal organ. Loss of Nrp1a, through parapineal ablation or depletion by antisense morpholinos, prevents left habenular neurons from projecting to the dorsal IPN. Selective depletion of Sema3D, but not of other Sema family members, similarly disrupts innervation of the dorsal IPN. Conversely, Sema3D overexpression results in left habenular projections that extend to the dorsal IPN, as well as beyond the target. The results indicate that Sema3D acts in concert with Nrp1a to guide neurons on the left side of the brain to innervate the target nucleus differently than those on the right side.
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Hazell, Gareth; Shabanpoor, Fazel; Saleh, Amer F.; Bowerman, Melissa; Meijboom, Katharina E.; Zhou, Haiyan; Muntoni, Francesco; Talbot, Kevin; Gait, Michael J.; Wood, Matthew J. A.
2016-01-01
The development of antisense oligonucleotide therapy is an important advance in the identification of corrective therapy for neuromuscular diseases, such as spinal muscular atrophy (SMA). Because of difficulties of delivering single-stranded oligonucleotides to the CNS, current approaches have been restricted to using invasive intrathecal single-stranded oligonucleotide delivery. Here, we report an advanced peptide-oligonucleotide, Pip6a-morpholino phosphorodiamidate oligomer (PMO), which demonstrates potent efficacy in both the CNS and peripheral tissues in severe SMA mice following systemic administration. SMA results from reduced levels of the ubiquitously expressed survival motor neuron (SMN) protein because of loss-of-function mutations in the SMN1 gene. Therapeutic splice-switching oligonucleotides (SSOs) modulate exon 7 splicing of the nearly identical SMN2 gene to generate functional SMN protein. Pip6a-PMO yields SMN expression at high efficiency in peripheral and CNS tissues, resulting in profound phenotypic correction at doses an order-of-magnitude lower than required by standard naked SSOs. Survival is dramatically extended from 12 d to a mean of 456 d, with improvement in neuromuscular junction morphology, down-regulation of transcripts related to programmed cell death in the spinal cord, and normalization of circulating insulin-like growth factor 1. The potent systemic efficacy of Pip6a-PMO, targeting both peripheral as well as CNS tissues, demonstrates the high clinical potential of peptide-PMO therapy for SMA. PMID:27621445
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Christiansen, Helena E; Lang, Michael R; Pace, James M; Parichy, David M
2009-12-29
Fibrillar collagens are well known for their links to human diseases, with which all have been associated except for the two most recently identified fibrillar collagens, type XXIV collagen and type XXVII collagen. To assess functions and potential disease phenotypes of type XXVII collagen, we examined its roles in zebrafish embryonic and post-embryonic development. We identified two type XXVII collagen genes in zebrafish, col27a1a and col27a1b. Both col27a1a and col27a1b were expressed in notochord and cartilage in the embryo and early larva. To determine sites of type XXVII collagen function, col27a1a and col27a1b were knocked down using morpholino antisense oligonucleotides. Knockdown of col27a1a singly or in conjunction with col27a1b resulted in curvature of the notochord at early stages and formation of scoliotic curves as well as dysmorphic vertebrae at later stages. These defects were accompanied by abnormal distributions of cells and protein localization in the notochord, as visualized by transmission electron microscopy, as well as delayed vertebral mineralization as detected histologically. Together, our findings indicate a key role for type XXVII collagen in notochord morphogenesis and axial skeletogenesis and suggest a possible human disease phenotype.
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Calcium signals act through histone deacetylase to mediate pronephric kidney morphogenesis.
Rothschild, Sarah C; Lee, Hunter J; Ingram, Sarah R; Mohammadi, Daniel K; Walsh, Gregory S; Tombes, Robert M
2018-06-01
Autosomal dominant polycystic kidney disease is the most common monogenetic kidney disorder and is linked to mutations in PKD1 and PKD2. PKD2, a Ca 2+ -conducting TRP channel enriched in ciliated cells and gated by extracellular signals, is necessary to activate the multifunctional Ca 2+/ calmodulin-dependent protein kinase type 2 (CaMK-II), enabling kidney morphogenesis and cilia stability. In this study, antisense morpholino oligonucleotides and pharmacological compounds were employed to investigate the roles of class II HDAC family members (HDAC 4, 5, and 6) in Zebrafish kidney development. While all three class II HDAC genes were expressed throughout the embryo during early development, HDAC5-morphant embryos exhibited anterior cysts and destabilized cloacal cilia, similar to PKD2 and CaMK-II morphants. In contrast, HDAC4-morphant embryos exhibited elongated cloacal cilia and lacked anterior kidney defects. Suppression of HDAC4 partially reversed the cilia shortening and anterior convolution defects caused by CaMK-II deficiency, whereas HDAC5 loss exacerbated these defects. EGFP-HDAC4, but not EGFP-HDAC5, translocated into the nucleus upon CaMK-II suppression in pronephric kidney cells. These results support a model by which activated CaMK-II sequesters HDAC4 in the cytosol to enable primary cilia formation and kidney morphogenesis. Developmental Dynamics 247:807-817, 2018. © 2018 Wiley Periodicals, Inc. © 2018 Wiley Periodicals, Inc.
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Isthmin is a novel secreted angiogenesis inhibitor that inhibits tumour growth in mice
Xiang, Wei; Ke, Zhiyuan; Zhang, Yong; Ho-Yuet Cheng, Grace; Irwan, Ishak Darryl; Sulochana, K N; Potturi, Padma; Wang, Zhengyuan; Yang, He; Wang, Jingyu; Zhuo, Lang; Kini, R Manjunatha; Ge, Ruowen
2011-01-01
Abstract Anti-angiogenesis represents a promising therapeutic strategy for the treatment of various malignancies. Isthmin (ISM) is a gene highly expressed in the isthmus of the midbrain–hindbrain organizer in Xenopus with no known functions. It encodes a secreted 60 kD protein containing a thrombospondin type 1 repeat domain in the central region and an adhesion-associated domain in MUC4 and other proteins (AMOP) domain at the C-terminal. In this work, we demonstrate that ISM is a novel angiogenesis inhibitor. Recombinant mouse ISM inhibited endothelial cell (EC) capillary network formation on Matrigel through its C-terminal AMOP domain. It also suppressed vascular endothelial growth factor (VEGF)-basic fibroblast growth factor (bFGF) induced in vivo angiogenesis in mouse. It mitigated VEGF-stimulated EC proliferation without affecting EC migration. Furthermore, ISM induced EC apoptosis in the presence of VEGF through a caspase-dependent pathway. ISM binds to αvβ5 integrin on EC surface and supports EC adhesion. Overexpression of ISM significantly suppressed mouse B16 melanoma tumour growth through inhibition of tumour angiogenesis without affecting tumour cell proliferation. Knockdown of isthmin in zebrafish embryos using morpholino antisense oligonucleotides led to disorganized intersegmen-tal vessels in the trunk. Our results demonstrate that ISM is a novel endogenous angiogenesis inhibitor with functions likely in physiological as well as pathological angiogenesis. PMID:19874420
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Martin, Elodie; Schüle, Rebecca; Smets, Katrien; Rastetter, Agnès; Boukhris, Amir; Loureiro, José L; Gonzalez, Michael A; Mundwiller, Emeline; Deconinck, Tine; Wessner, Marc; Jornea, Ludmila; Oteyza, Andrés Caballero; Durr, Alexandra; Martin, Jean-Jacques; Schöls, Ludger; Mhiri, Chokri; Lamari, Foudil; Züchner, Stephan; De Jonghe, Peter; Kabashi, Edor; Brice, Alexis; Stevanin, Giovanni
2013-02-07
Spastic paraplegia 46 refers to a locus mapped to chromosome 9 that accounts for a complicated autosomal-recessive form of hereditary spastic paraplegia (HSP). With next-generation sequencing in three independent families, we identified four different mutations in GBA2 (three truncating variants and one missense variant), which were found to cosegregate with the disease and were absent in controls. GBA2 encodes a microsomal nonlysosomal glucosylceramidase that catalyzes the conversion of glucosylceramide to free glucose and ceramide and the hydrolysis of bile acid 3-O-glucosides. The missense variant was also found at the homozygous state in a simplex subject in whom no residual glucocerebrosidase activity of GBA2 could be evidenced in blood cells, opening the way to a possible measurement of this enzyme activity in clinical practice. The overall phenotype was a complex HSP with mental impairment, cataract, and hypogonadism in males associated with various degrees of corpus callosum and cerebellar atrophy on brain imaging. Antisense morpholino oligonucleotides targeting the zebrafish GBA2 orthologous gene led to abnormal motor behavior and axonal shortening/branching of motoneurons that were rescued by the human wild-type mRNA but not by applying the same mRNA containing the missense mutation. This study highlights the role of ceramide metabolism in HSP pathology. Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.
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Widespread antisense transcription of Populus genome under drought.
Yuan, Yinan; Chen, Su
2018-06-06
Antisense transcription is widespread in many genomes and plays important regulatory roles in gene expression. The objective of our study was to investigate the extent and functional relevance of antisense transcription in forest trees. We employed Populus, a model tree species, to probe the antisense transcriptional response of tree genome under drought, through stranded RNA-seq analysis. We detected nearly 48% of annotated Populus gene loci with antisense transcripts and 44% of them with co-transcription from both DNA strands. Global distribution of reads pattern across annotated gene regions uncovered that antisense transcription was enriched in untranslated regions while sense reads were predominantly mapped in coding exons. We further detected 1185 drought-responsive sense and antisense gene loci and identified a strong positive correlation between the expression of antisense and sense transcripts. Additionally, we assessed the antisense expression in introns and found a strong correlation between intronic expression and exonic expression, confirming antisense transcription of introns contributes to transcriptional activity of Populus genome under drought. Finally, we functionally characterized drought-responsive sense-antisense transcript pairs through gene ontology analysis and discovered that functional groups including transcription factors and histones were concordantly regulated at both sense and antisense transcriptional level. Overall, our study demonstrated the extensive occurrence of antisense transcripts of Populus genes under drought and provided insights into genome structure, regulation pattern and functional significance of drought-responsive antisense genes in forest trees. Datasets generated in this study serve as a foundation for future genetic analysis to improve our understanding of gene regulation by antisense transcription.
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Popik, Waldemar; Khatua, Atanu; Hildreth, James E K; Lee, Benjamin; Alcendor, Donald J
2018-06-01
Zika virus (ZIKV) infection has been associated with microcephaly in infants. Currently there is no treatment or vaccine. Here we explore the use of a morpholino oligonucleotide targeted to the 5' untranslated region (5'-UTR) of the ZIKV RNA to prevent ZIKV replication. Morpholino DWK-1 inhibition of ZIKV replication in human glomerular podocytes was examined by qRT-PCR, reduction in ZIKV genome copy number, western blot analysis, immunofluorescence and proinflammatory cytokine gene expression. Podocytes pretreated with DWK-1 showed reduced levels of both viral mRNA and ZIKV E protein expression compared to controls. We observed suppression in proinflammatory gene expression for IFN-β (interferon β) RANTES (regulated on activation, normal T cell expressed and secreted), MIP-1α (macrophage inflammatory protein-1α), TNF-α (tumor necrosis factor-α) and IL1-α (interleukin 1-α) in ZIKV-infected podocytes pretreated with DWK-1. Morpholino DWK-1 targeting the ZIKV 5'-UTR effectively inhibits ZIKV replication and suppresses ZIKV-induced proinflammatory gene expression. Copyright © 2018 Elsevier Inc. All rights reserved.
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Effect of Tbx1 knock-down on cardiac performance in zebrafish.
Zhang, Li-feng; Gui, Yong-hao; Wang, Yue-xiang; Jiang, Qiu; Song, Hou-yan
2010-05-05
Tbx1 is the major candidate gene for DiGeorge syndrome (DGS). Similar to defects observed in DGS patients, the structures disrupted in Tbx1(-/-) animal models are derived from the neural crest cells during development. Although the morphological phenotypes of some Tbx1 knock-down animal models have been well described, analysis of the cardiac performance is limited. Therefore, myocardial performance was explored in Tbx1 morpholino injected zebrafish embryos. To elucidate these issues, Tbx1 specific morpholino was used to reduce the function of Tbx1 in zebrafish. The differentiation of the myocardial cells was observed using whole mount in situ hybridization. Heart rates were observed and recorded under the microscope from 24 to 72 hours post fertilization (hpf). The cardiac performance was analyzed by measuring ventricular shortening fraction and atrial shortening fraction. Tbx1 morpholino injected embryos were characterized by defects in the pharyngeal arches, otic vesicle, aortic arches and thymus. In addition, Tbx1 knock down reduced the amount of pharyngeal neural crest cells in zebrafish. Abnormal cardiac morphology was visible in nearly 20% of the Tbx1 morpholino injected embryos. The hearts in these embryos did not loop or loop incompletely. Importantly, cardiac performance and heart rate were reduced in Tbx1 morpholino injected embryos. Tbx1 might play an essential role in the development of pharyngeal neural crest cells in zebrafish. Cardiac performance is impaired by Tbx1 knock down in zebrafish.
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Lin, Che-Yi; Tsai, Ming-Yuan; Liu, Yu-Hsiu; Lu, Yu-Fen; Chen, Yi-Chung; Lai, Yun-Ren; Liao, Hsin-Chi; Lien, Huang-Wei; Yang, Chung-Hsiang; Huang, Chang-Jen; Hwang, Sheng-Ping L
2017-07-17
Although vertebrates are bilaterally symmetric organisms, their internal organs are distributed asymmetrically along a left-right axis. Disruption of left-right axis asymmetric patterning often occurs in human genetic disorders. In zebrafish embryos, Kupffer's vesicle, like the mouse node, breaks symmetry by inducing asymmetric expression of the Nodal-related gene, spaw, in the left lateral plate mesoderm (LPM). Spaw then stimulates transcription of itself and downstream genes, including lft1, lft2, and pitx2, specifically in the left side of the diencephalon, heart and LPM. This developmental step is essential to establish subsequent asymmetric organ positioning. In this study, we evaluated the role of krüppel-like factor 8 (klf8) in regulating left-right asymmetric patterning in zebrafish embryos. Zebrafish klf8 expression was disrupted by both morpholino antisense oligomer-mediated knockdown and a CRISPR-Cas9 system. Whole-mount in situ hybridization was conducted to evaluate gene expression patterns of Nodal signalling components and the positions of heart and visceral organs. Dorsal forerunner cell number was evaluated in Tg(sox17:gfp) embryos and the length and number of cilia in Kupffer's vesicle were analyzed by immunocytochemistry using an acetylated tubulin antibody. Heart jogging, looping and visceral organ positioning were all defective in zebrafish klf8 morphants. At the 18-22 s stages, klf8 morphants showed reduced expression of genes encoding Nodal signalling components (spaw, lft1, lft2, and pitx2) in the left LPM, diencephalon, and heart. Co-injection of klf8 mRNA with klf8 morpholino partially rescued spaw expression. Furthermore, klf8 but not klf8△zf overexpressing embryos showed dysregulated bilateral expression of Nodal signalling components at late somite stages. At the 10s stage, klf8 morphants exhibited reductions in length and number of cilia in Kupffer's vesicle, while at 75% epiboly, fewer dorsal forerunner cells were observed. Interestingly, klf8 mutant embryos, generated by a CRISPR-Cas9 system, showed bilateral spaw expression in the LPM at late somite stages. This observation may be partly attributed to compensatory upregulation of klf12b, because klf12b knockdown reduced the percentage of klf8 mutants exhibiting bilateral spaw expression. Our results demonstrate that zebrafish Klf8 regulates left-right asymmetric patterning by modulating both Kupffer's vesicle morphogenesis and spaw expression in the left LPM.
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Miles, Lee B; Darido, Charbel; Kaslin, Jan; Heath, Joan K; Jane, Stephen M; Dworkin, Sebastian
2017-12-14
The grainyhead-like (grhl) transcription factors play crucial roles in craniofacial development, epithelial morphogenesis, neural tube closure, and dorso-ventral patterning. By utilising the zebrafish to differentially regulate expression of family members grhl2b and grhl3, we show that both genes regulate epithelial migration, particularly convergence-extension (CE) type movements, during embryogenesis. Genetic deletion of grhl3 via CRISPR/Cas9 results in failure to complete epiboly and pre-gastrulation embryonic rupture, whereas morpholino (MO)-mediated knockdown of grhl3 signalling leads to aberrant neural tube morphogenesis at the midbrain-hindbrain boundary (MHB), a phenotype likely due to a compromised overlying enveloping layer (EVL). Further disruptions of grhl3-dependent pathways (through co-knockdown of grhl3 with target genes spec1 and arhgef19) confirm significant MHB morphogenesis and neural tube closure defects. Concomitant MO-mediated disruption of both grhl2b and grhl3 results in further extensive CE-like defects in body patterning, notochord and somite morphogenesis. Interestingly, over-expression of either grhl2b or grhl3 also leads to numerous phenotypes consistent with disrupted cellular migration during gastrulation, including embryo dorsalisation, axial duplication and impaired neural tube migration leading to cyclopia. Taken together, our study ascribes novel roles to the Grhl family in the context of embryonic development and morphogenesis.
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NASA Technical Reports Server (NTRS)
Reinsch, S. S.; Conway, G. C.
2003-01-01
After fertilization Zebrafish embryos undergo synchronous cleavage to form a blastula of cells sitting upon a single multinucleate yolk cell. At the beginning of gastrulation these cells undergo extensive cell migrations to form the major body axes. We have discovered a gene, G12, which is required for cell migrations and positioning of nuclei in the large syncytial yolk cell. Overexpression of a G12-GFP fusion protein is not toxic and shows that the protein localizes inside the yolk cell to the yolk nuclei, microtubules, and to the margin between the blastomeres and the large yolk cell. Morpholino (MO) injection into the 1-cell embryo or into just the yolk syncytium conipletely inhibits cell migrations, doming of the yolk cell, and positioning of nuclei around the margin. This effect can be partially rescued by injection of G12-GFP encoding RNA. Given the known role of microtubules in nuclear positioning of yolk nuclei in Zebrafish, we investigated the microtubules in morpholiiio injected and rescued embryos. We find that microtubules are sparse and disorganized in MO-injected embryos and are restored to normal organization upon G12-GFP rescue. G12 plays a pivotal role in organization of inicrotubules during early development. G12 is highly conserved in vertebrates and two homologues exist in the human genome. One of the human hoinologues is amplified in aggressive breast tumors.
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Shabanpoor, Fazel; Gait, Michael J
2013-11-11
We describe a general methodology for fluorescent labelling of peptide conjugates of phosphorodiamidate morpholino oligonucleotides (PMOs) by alkyne functionalization of peptides, subsequent conjugation to PMOs and labelling with a fluorescent compound (Cy5-azide). Two peptide-PMO (PPMO) examples are shown. No detrimental effect of such labelled PMOs was seen in a biological assay.
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Zebrafish CiA interneurons are late-born primary neurons.
Yeo, Sang-Yeob
2009-12-11
Pax2 is a neural-related transcription factor downstream of Notch signaling and is expressed in the developing spinal cord of zebrafish, including in CiA interneurons. However, the characteristics of pax2-positive neurons are largely unknown. The goal of this study was to characterize Pax2-positive neurons by examining their expression in embryos in which Notch function had been knocked down by mutation or injection of a morpholino or mRNA. I found that Pax2-positive CiA interneurons were late-differentiating primary neurons. pax2.1 was expressed in CoPA commissural neurons and CiA interneurons at 26 hpf. The number of pax2.1-positive cells increased in mind bomb mutant embryos or embryos injected with Su(H)1-MO, but not in cells injected with Xenopus Delta or Delta(stu) mRNA. These observations imply that Notch signaling plays a role in regulating the number of CiA neurons by preventing uncommitted precursors from acquiring a neuronal fate during vertebrate development.
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Identification of sequence motifs significantly associated with antisense activity.
McQuisten, Kyle A; Peek, Andrew S
2007-06-07
Predicting the suppression activity of antisense oligonucleotide sequences is the main goal of the rational design of nucleic acids. To create an effective predictive model, it is important to know what properties of an oligonucleotide sequence associate significantly with antisense activity. Also, for the model to be efficient we must know what properties do not associate significantly and can be omitted from the model. This paper will discuss the results of a randomization procedure to find motifs that associate significantly with either high or low antisense suppression activity, analysis of their properties, as well as the results of support vector machine modelling using these significant motifs as features. We discovered 155 motifs that associate significantly with high antisense suppression activity and 202 motifs that associate significantly with low suppression activity. The motifs range in length from 2 to 5 bases, contain several motifs that have been previously discovered as associating highly with antisense activity, and have thermodynamic properties consistent with previous work associating thermodynamic properties of sequences with their antisense activity. Statistical analysis revealed no correlation between a motif's position within an antisense sequence and that sequences antisense activity. Also, many significant motifs existed as subwords of other significant motifs. Support vector regression experiments indicated that the feature set of significant motifs increased correlation compared to all possible motifs as well as several subsets of the significant motifs. The thermodynamic properties of the significantly associated motifs support existing data correlating the thermodynamic properties of the antisense oligonucleotide with antisense efficiency, reinforcing our hypothesis that antisense suppression is strongly associated with probe/target thermodynamics, as there are no enzymatic mediators to speed the process along like the RNA Induced Silencing Complex (RISC) in RNAi. The independence of motif position and antisense activity also allows us to bypass consideration of this feature in the modelling process, promoting model efficiency and reducing the chance of overfitting when predicting antisense activity. The increase in SVR correlation with significant features compared to nearest-neighbour features indicates that thermodynamics alone is likely not the only factor in determining antisense efficiency.
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HIV-1-encoded antisense RNA suppresses viral replication for a prolonged period
2012-01-01
Background Recent evidence proposes a novel concept that mammalian natural antisense RNAs play important roles in cellular homeostasis by regulating the expression of several genes. Identification and characterization of retroviral antisense RNA would provide new insights into mechanisms of replication and pathogenesis. HIV-1 encoded-antisense RNAs have been reported, although their structures and functions remain to be studied. We have tried to identify and characterize antisense RNAs of HIV-1 and their function in viral infection. Results Characterization of transcripts of HEK293T cells that were transiently transfected with an expression plasmid with HIV-1NL4–3 DNA in the antisense orientation showed that various antisense transcripts can be expressed. By screening and characterizing antisense RNAs in HIV-1NL4–3-infected cells, we defined the primary structure of a major form of HIV-1 antisense RNAs, which corresponds to a variant of previously reported ASP mRNA. This 2.6 kb RNA was transcribed from the U3 region of the 3′ LTR and terminated at the env region in acutely or chronically infected cell lines and acutely infected human peripheral blood mononuclear cells. Reporter assays clearly demonstrated that the HIV-1 LTR harbours promoter activity in the reverse orientation. Mutation analyses suggested the involvement of NF-κΒ binding sites in the regulation of antisense transcription. The antisense RNA was localized in the nuclei of the infected cells. The expression of this antisense RNA suppressed HIV-1 replication for more than one month. Furthermore, the specific knockdown of this antisense RNA enhanced HIV-1 gene expression and replication. Conclusions The results of the present study identified an accurate structure of the major form of antisense RNAs expressed from the HIV-1NL4–3 provirus and demonstrated its nuclear localization. Functional studies collectively demonstrated a new role of the antisense RNA in viral replication. Thus, we suggest a novel viral mechanism that self-limits HIV-1 replication and provides new insight into the viral life cycle. PMID:22569184
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Antisense technology: an emerging platform for cardiovascular disease therapeutics.
Lee, Richard G; Crosby, Jeff; Baker, Brenda F; Graham, Mark J; Crooke, Rosanne M
2013-12-01
Antisense oligonucleotides and small interfering RNAs, which suppress the translation of specific mRNA target proteins, are emerging as important therapeutic modalities for the treatment of cardiovascular disease. Over the last 25 years, the advances in all aspects of antisense technology, as well as a detailed understanding of the mechanism of action of antisense drugs, have enabled their use as therapeutic agents. These advancements culminated in the FDA approval of the first chronically administered cardiovascular antisense therapeutic, mipomersen, which targets hepatic apolipoprotein B mRNA. This review provides a brief history of antisense technology, highlights the progression of mipomersen from preclinical studies to multiple Phase III registration trials, and gives an update on the status of other cardiovascular antisense therapeutics currently in the clinic.
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JÄNICKE, MARTINA; RENISCH, BJÖRN; HAMMERSCHMIDT, MATTHIAS
2012-01-01
Grainyhead/CP2 transcription factor family members are widely conserved among the animal kingdom and have been implicated in different developmental processes. Thus far, nothing has been known about their roles in zebrafish. Here we identify seven zebrafish grainyhead-like (grhl) / cp2 genes, with focus on grhl1, which is expressed in the periderm and in epidermal ionocyte progenitors, but downregulated when ionocytes differentiate. In addition, expression was detected in other “non-keratinocyte” cell types of the epidermis, such as pvalb8-expressing cells, which according to our lineage tracing experiments are derived from the same pool of progenitor cells like keratinocytes and ionocytes. Antisense morpholino oligonucleotide-based loss-of-function analysis revealed that grhl1 is dispensable for the development and function of all investigated epidermal cell types, but required as a negative regulator of its own transcription during ionocyte differentiation. Knockdown of the transcription factor Foxi3a, which is expressed in a subset of the grhl1 population, caused a loss of ionocytes and a corresponding increase in the number of pvalb8-expressing cells, while leaving the number of grhl1-positive cells unaltered. We propose that grhl1 is a novel common marker of all or most “non-keratinocyte” epidermal progenitors, and that the sub-functionalisation of these cells is regulated by differential positive and negative effects of Foxi3 factors. PMID:19757382
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Isthmin is a novel secreted angiogenesis inhibitor that inhibits tumour growth in mice.
Xiang, Wei; Ke, Zhiyuan; Zhang, Yong; Cheng, Grace Ho-Yuet; Irwan, Ishak Darryl; Sulochana, K N; Potturi, Padma; Wang, Zhengyuan; Yang, He; Wang, Jingyu; Zhuo, Lang; Kini, R Manjunatha; Ge, Ruowen
2011-02-01
Anti-angiogenesis represents a promising therapeutic strategy for the treatment of various malignancies. Isthmin (ISM) is a gene highly expressed in the isthmus of the midbrain-hindbrain organizer in Xenopus with no known functions. It encodes a secreted 60 kD protein containing a thrombospondin type 1 repeat domain in the central region and an adhesion-associated domain in MUC4 and other proteins (AMOP) domain at the C-terminal. In this work, we demonstrate that ISM is a novel angiogenesis inhibitor. Recombinant mouse ISM inhibited endothelial cell (EC) capillary network formation on Matrigel through its C-terminal AMOP domain. It also suppressed vascular endothelial growth factor (VEGF)-basic fibroblast growth factor (bFGF) induced in vivo angiogenesis in mouse. It mitigated VEGF-stimulated EC proliferation without affecting EC migration. Furthermore, ISM induced EC apoptosis in the presence of VEGF through a caspase-dependent pathway. ISM binds to αvβ(5) integrin on EC surface and supports EC adhesion. Overexpression of ISM significantly suppressed mouse B16 melanoma tumour growth through inhibition of tumour angiogenesis without affecting tumour cell proliferation. Knockdown of isthmin in zebrafish embryos using morpholino antisense oligonucleotides led to disorganized intersegmental vessels in the trunk. Our results demonstrate that ISM is a novel endogenous angiogenesis inhibitor with functions likely in physiological as well as pathological angiogenesis. © 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.
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Virgilio, Maria; Narla, Anupama; Sun, Hong; Levine, Michelle; Paw, Barry H.; Berliner, Nancy; Look, A. Thomas; Ebert, Benjamin L.
2012-01-01
Haploinsufficiency of ribosomal proteins (RPs) has been proposed to be the common basis for the anemia observed in Diamond-Blackfan anemia (DBA) and myelodysplastic syndrome with loss of chromosome 5q [del(5q) MDS]. We have modeled DBA and del(5q) MDS in zebrafish using antisense morpholinos to rps19 and rps14, respectively, and have demonstrated that, as in humans, haploinsufficient levels of these proteins lead to a profound anemia. To address the hypothesis that RP loss results in impaired mRNA translation, we treated Rps19 and Rps14-deficient embryos with the amino acid L-leucine, a known activator of mRNA translation. This resulted in a striking improvement of the anemia associated with RP loss. We confirmed our findings in primary human CD34+ cells, after shRNA knockdown of RPS19 and RPS14. Furthermore, we showed that loss of Rps19 or Rps14 activates the mTOR pathway, and this is accentuated by L-leucine in both Rps19 and Rps14 morphants. This effect could be abrogated by rapamycin suggesting that mTOR signaling may be responsible for the improvement in anemia associated with L-leucine. Our studies support the rationale for ongoing clinical trials of L-leucine as a therapeutic agent for DBA, and potentially for patients with del(5q) MDS. PMID:22734070
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Jahrling, Peter B; Hensley, Lisa E; Barrett, Kevin; Lane, Henry Clifford; Davey, Richard T
2015-10-01
The ongoing outbreak of Ebola in West Africa has raised a general awareness that at present there are no Ebola-specific medical countermeasures (MCMs) with proven effectiveness. This paper recapitulates discussions held at the 6th International Filovirus Symposium in March 2014 as well as the subsequent design of a randomized clinical trial design for treating Ebola virus-infected patients evacuated from West Africa to the United States. A number of different drugs or biologics were critically reviewed and 3 different postexposure strategies were identified as being farthest along in development; passive immunotherapy with monoclonal antibodies, postexposure vaccination with constructs involving viral vectors (such as vesicular stomatitis virus), and antisense compounds directly targeting the viral genome such as modified phosphorodiamidate morpholino oligomer-based compounds and small interfering RNA products. At the time of the meetings, there were no investigational new drugs (INDs) in place for the candidate MCMs. Developers and sponsors of these candidate products were strongly encouraged to prepare pre-IND packets and submit pre-IND meeting requests to the Food and Drug Administration. Some of these investigational products have already been used under emergency authorizations to treat patients in Africa as well as patients evacuated to the United States or Western Europe. Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.
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Saito, Taiju; Goto-Kazeto, Rie; Arai, Katsutoshi; Yamaha, Etsuro
2008-01-01
Primordial germ cells (PGCs) are the only cells in developing embryos with the potential to transmit genetic information to the next generation. PGCs therefore have the potential to be of value for gene banking and cryopreservation, particularly via the production of donor gametes with germ-line chimeras. Currently, it is not clear how many PGCs are required for germ-line differentiation and formation of gonadal structures. In the present study, we achieved complete germ-line replacement between two related teleost species, the pearl danio (Danio albolineatus) and the zebrafish (Danio rerio), with transplantation of a single PGC into each host embryo. We isolated and transplanted a single PGC into each blastula-stage, zebrafish embryo. Development of host germ-line cells was prevented by an antisense dead end morpholino oligonucleotide. In many host embryos, the transplanted donor PGC successfully migrated toward the gonadal anlage without undergoing cell division. At the gonadal anlage, the PGC differentiated to form one normally sized gonad rather than the pair of gonads usually present. Offspring were obtained from natural spawning of these chimeras. Analyses of morphology and DNA showed that the offspring were of donor origin. We extended our study to confirm that transplanted single PGCs of goldfish (Carassius auratus) and loach (Misgurnus anguillicaudatus) can similarly differentiate into sperm in zebrafish host embryos. Our results show that xenogenesis is realistic and practical across species, genus, and family barriers and can be achieved by the transplantation of a single PGC from a donor species.
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Natural Antisense Transcripts: Molecular Mechanisms and Implications in Breast Cancers
Latgé, Guillaume; Poulet, Christophe; Bours, Vincent; Jerusalem, Guy
2018-01-01
Natural antisense transcripts are RNA sequences that can be transcribed from both DNA strands at the same locus but in the opposite direction from the gene transcript. Because strand-specific high-throughput sequencing of the antisense transcriptome has only been available for less than a decade, many natural antisense transcripts were first described as long non-coding RNAs. Although the precise biological roles of natural antisense transcripts are not known yet, an increasing number of studies report their implication in gene expression regulation. Their expression levels are altered in many physiological and pathological conditions, including breast cancers. Among the potential clinical utilities of the natural antisense transcripts, the non-coding|coding transcript pairs are of high interest for treatment. Indeed, these pairs can be targeted by antisense oligonucleotides to specifically tune the expression of the coding-gene. Here, we describe the current knowledge about natural antisense transcripts, their varying molecular mechanisms as gene expression regulators, and their potential as prognostic or predictive biomarkers in breast cancers. PMID:29301303
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Natural Antisense Transcripts: Molecular Mechanisms and Implications in Breast Cancers.
Latgé, Guillaume; Poulet, Christophe; Bours, Vincent; Josse, Claire; Jerusalem, Guy
2018-01-02
Natural antisense transcripts are RNA sequences that can be transcribed from both DNA strands at the same locus but in the opposite direction from the gene transcript. Because strand-specific high-throughput sequencing of the antisense transcriptome has only been available for less than a decade, many natural antisense transcripts were first described as long non-coding RNAs. Although the precise biological roles of natural antisense transcripts are not known yet, an increasing number of studies report their implication in gene expression regulation. Their expression levels are altered in many physiological and pathological conditions, including breast cancers. Among the potential clinical utilities of the natural antisense transcripts, the non-coding|coding transcript pairs are of high interest for treatment. Indeed, these pairs can be targeted by antisense oligonucleotides to specifically tune the expression of the coding-gene. Here, we describe the current knowledge about natural antisense transcripts, their varying molecular mechanisms as gene expression regulators, and their potential as prognostic or predictive biomarkers in breast cancers.
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Antisense Therapy in Neurology
Lee, Joshua J.A.; Yokota, Toshifumi
2013-01-01
Antisense therapy is an approach to fighting diseases using short DNA-like molecules called antisense oligonucleotides. Recently, antisense therapy has emerged as an exciting and promising strategy for the treatment of various neurodegenerative and neuromuscular disorders. Previous and ongoing pre-clinical and clinical trials have provided encouraging early results. Spinal muscular atrophy (SMA), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy (DMD), Fukuyama congenital muscular dystrophy (FCMD), dysferlinopathy (including limb-girdle muscular dystrophy 2B; LGMD2B, Miyoshi myopathy; MM, and distal myopathy with anterior tibial onset; DMAT), and myotonic dystrophy (DM) are all reported to be promising targets for antisense therapy. This paper focuses on the current progress of antisense therapies in neurology. PMID:25562650
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Homann, M; Tzortzakaki, S; Rittner, K; Sczakiel, G; Tabler, M
1993-01-01
The catalytic domain of a hammerhead ribozyme was incorporated into a 413 nucleotides long antisense RNA directed against the 5'-leader/gag region of the human immunodeficiency virus type 1 (HIV-1) (pos. +222 to +634). The resulting catalytic antisense RNA was shown to cleave its target RNA in vitro specifically at physiological ion strength and temperature. We compared the antiviral effectiveness of this catalytic antisense RNA with that of the corresponding unmodified antisense RNA and with a mutated catalytic antisense RNA, which did not cleave the substrate RNA in vitro. Each of these RNAs was co-transfected into human SW480 cells together with infectious complete proviral HIV-1 DNA, followed by analysis of HIV-1 replication. The presence of the catalytically active domain resulted in 4 to 7 fold stronger inhibition of HIV-1 replication as compared to the parental antisense RNA and the inactive mutant. Kinetic and structural studies performed in vitro indicated that the ability for double strand formation was not changed in catalytic antisense RNA versus parental antisense RNA. Together, these data suggest that the ability to cleave target RNA is a crucial prerequisite for the observed increase of inhibition of the replication of HIV-1. Images PMID:8332489
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Ludwig, Linda B; Ambrus, Julian L; Krawczyk, Kristie A; Sharma, Sanjay; Brooks, Stephen; Hsiao, Chiu-Bin; Schwartz, Stanley A
2006-01-01
Background While viruses have long been shown to capitalize on their limited genomic size by utilizing both strands of DNA or complementary DNA/RNA intermediates to code for viral proteins, it has been assumed that human retroviruses have all their major proteins translated only from the plus or sense strand of RNA, despite their requirement for a dsDNA proviral intermediate. Several studies, however, have suggested the presence of antisense transcription for both HIV-1 and HTLV-1. More recently an antisense transcript responsible for the HTLV-1 bZIP factor (HBZ) protein has been described. In this study we investigated the possibility of an antisense gene contained within the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). Results Inspection of published sequences revealed a potential transcription initiator element (INR) situated downstream of, and in reverse orientation to, the usual HIV-1 promoter and transcription start site. This antisense initiator (HIVaINR) suggested the possibility of an antisense gene responsible for RNA and protein production. We show that antisense transcripts are generated, in vitro and in vivo, originating from the TAR DNA of the HIV-1 LTR. To test the possibility that protein(s) could be translated from this novel HIV-1 antisense RNA, recombinant HIV antisense gene-FLAG vectors were designed. Recombinant protein(s) were produced and isolated utilizing carboxy-terminal FLAG epitope (DYKDDDDK) sequences. In addition, affinity-purified antisera to an internal peptide derived from the HIV antisense protein (HAP) sequences identified HAPs from HIV+ human peripheral blood lymphocytes. Conclusion HIV-1 contains an antisense gene in the U3-R regions of the LTR responsible for both an antisense RNA transcript and proteins. This antisense transcript has tremendous potential for intrinsic RNA regulation because of its overlap with the beginning of all HIV-1 sense RNA transcripts by 25 nucleotides. The novel HAPs are encoded in a region of the LTR that has already been shown to be deleted in some HIV-infected long-term survivors and represent new potential targets for vaccine development. PMID:17090330
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Pharmacology of Antisense Drugs.
Bennett, C Frank; Baker, Brenda F; Pham, Nguyen; Swayze, Eric; Geary, Richard S
2017-01-06
Recent studies have led to a greater appreciation of the diverse roles RNAs play in maintaining normal cellular function and how they contribute to disease pathology, broadening the number of potential therapeutic targets. Antisense oligonucleotides are the most direct means to target RNA in a selective manner and have become an established platform technology for drug discovery. There are multiple molecular mechanisms by which antisense oligonucleotides can be used to modulate RNAs in cells, including promoting the degradation of the targeted RNA or modulating RNA function without degradation. Antisense drugs utilizing various antisense mechanisms are demonstrating therapeutic potential for the treatment of a broad variety of diseases. This review focuses on some of the advances that have taken place in translating antisense technology from the bench to the clinic.
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Zhu, Lin; Zhu, Jian; Liu, Zhixue; Wang, Zhengyi; Zhou, Cheng; Wang, Hong
2017-09-26
Magnaporthe oryzae is a devastating plant pathogen, which has a detrimental impact on rice production worldwide. Despite its agronomical importance, some newly-emerging pathotypes often overcome race-specific disease resistance rapidly. It is thus desirable to develop a novel strategy for the long-lasting resistance of rice plants to ever-changing fungal pathogens. Brome mosaic virus (BMV)-induced RNA interference (RNAi) has emerged as a useful tool to study host-resistance genes for rice blast protection. Planta-generated silencing of targeted genes inside biotrophic pathogens can be achieved by expression of M. oryzae -derived gene fragments in the BMV-mediated gene silencing system, a technique termed host-induced gene silencing (HIGS). In this study, the effectiveness of BMV-mediated HIGS in M. oryzae was examined by targeting three predicted pathogenicity genes, MoABC1, MoMAC1 and MoPMK1 . Systemic generation of fungal gene-specific small interfering RNA (siRNA) molecules induced by inoculation of BMV viral vectors inhibited disease development and reduced the transcription of targeted fungal genes after subsequent M. oryzae inoculation. Combined introduction of fungal gene sequences in sense and antisense orientation mediated by the BMV silencing vectors significantly enhanced the efficiency of this host-generated trans-specific RNAi, implying that these fungal genes played crucial roles in pathogenicity. Collectively, our results indicated that BMV-HIGS system was a great strategy for protecting host plants against the invasion of pathogenic fungi.
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Burki, Umar; Straub, Volker
2017-01-01
Determining the concentration of oligonucleotide in biological samples such as tissue lysate and serum is essential for determining the biodistribution and pharmacokinetic profile, respectively. ELISA-based assays have shown far greater sensitivities compared to other methods such as HPLC and LC/MS. Here, we describe a novel ultrasensitive hybridization-based ELISA method for quantitating morpholino oligonucleotides in mouse tissue lysate and serum samples. The assay has a linear detection range of 5-250 pM (R2 > 0.99).
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Farr, Susan A; Erickson, Michelle A; Niehoff, Michael L; Banks, William A; Morley, John E
2014-01-01
Alzheimer's disease (AD) is a progressive neurodegenerative disease. Currently, there are no therapies to stop or reverse the symptoms of AD. We have developed an antisense oligonucleotide (OL-1) against the amyloid-β protein precursor (AβPP) that can decrease AβPP expression and amyloid-β protein (Aβ) production. This antisense rapidly crosses the blood-brain barrier, reverses learning and memory impairments, reduces oxidative stress, and restores brain-to-blood efflux of Aβ in SAMP8 mice. Here, we examined the effects of this AβPP antisense in the Tg2576 mouse model of AD. We administered the OL-1 antisense into the lateral ventricle 3 times at 2week intervals. Seventy-two hours after the third injection, we tested learning and memory in T-maze foot shock avoidance. In the second study, we injected the mice with OL-1 antisense 3 times at 2-week intervals via the tail vein. Seventy-two hours later, we tested learning and memory T-maze, novel object recognition, and elevated plus maze. At the end of behavioral testing, brain tissue was collected. OL-1 antisense administered centrally improved acquisition and retention of T-maze foot shock avoidance. OL-1 antisense administered via tail vein improved learning and memory in both T-maze foot shock avoidance and novel object-place recognition. In the elevated plus maze, the mice which received OL-1 antisense spent less time in the open arms and had fewer entries into the open arms indicating reduced disinhibitation. Biochemical analyses reveal significant reduction of AβPP signal and a reduction of measures of neuroinflammation. The current findings support the therapeutic potential of OL-1 AβPP antisense.
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Erickson, Michelle A; Niehoff, Michael L; Farr, Susan A; Morley, John E; Dillman, Lucy A; Lynch, Kristin M; Banks, William A
2012-01-01
The senescence accelerated mouse-prone 8 (SAMP8) mouse model of Alzheimer's disease has a natural mutation leading to age-related increases in the amyloid-β protein precursor (AβPP) and amyloid-β (Aβ) in the brain, memory impairment, and deficits in Aβ removal from the brain. Previous studies show that centrally administered antisense oligonucleotide directed against AβPP can decrease AβPP expression and Aβ production in the brains of aged SAMP8 mice, and improve memory. The same antisense crosses the blood-brain barrier and reverses memory deficits when injected intravenously. Here, we give 6 μg of AβPP or control antisense 3 times over 2 week intervals to 12 month old SAMP8 mice. Object recognition test was done 48 hours later, followed by removal of whole brains for immunoblot analysis of AβPP, low-density lipoprotein-related protein-1 (LRP-1), p-glycoprotein (Pgp), receptor for advanced glycation endproducts (RAGE), or ELISA of soluble Aβ(40). Our results show that AβPP antisense completely reverses a 30% age-associated increase in AβPP signal (p < 0.05 versus untreated 4 month old SAMP8). Soluble Aβ(40) increased with age, but was not reversed by antisense. LRP-1 large and small subunits increased significantly with age (147.7%, p < 0.01 and 123.7%, p < 0.05 respectively), and AβPP antisense completely reversed these increases (p < 0.05). Pgp and RAGE were not significantly altered with age or antisense. Antisense also caused improvements in memory (p < 0.001). Together, these data support the therapeutic potential of AβPP antisense and show a unique association between AβPP and LRP-1 expression in the SAMP8 mouse.
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Epithelin/Granulin Precursor Expression in Human Breast Carcinoma
1998-09-01
antisense RNA as an inhibitor of oncogenic protein production (13). The development of stable transfected clones with antisense cDNA is advantageous...in that it allows a continuous supply of antisense RNA to disrupt protein synthesis, and it is well suited for in vivo tumorigenic assays. Our...processed form epithelin 1 in normal mammary epithelial cells and mammary carcinoma cells. 3- Effect of inhibition of PCDGF expression ( antisense
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Daam1 regulates fascin for actin assembly in mouse oocyte meiosis.
Lu, Yujie; Zhang, Yu; Pan, Meng-Hao; Kim, Nam-Hyung; Sun, Shao-Chen; Cui, Xiang-Shun
2017-07-18
As a formin protein, Daam1 (Dishevelled-associated activator of morphogenesis 1) is reported to regulate series of cell processes like endocytosis, cell morphology and migration via its effects on actin assembly in mitosis. However, whether Daam1 plays roles in female meiosis remains uncertain. In this study, we investigated the expression and functions of Daam1 during mouse oocyte meiosis. Our results indicated that Daam1 localized at the cortex of oocytes, which was similar with actin filaments. After Daam1 morpholino (MO) microinjection, the expression of Daam1 significantly decreased, which resulted in the failure of oocyte polar body extrusion. These results might be due to the defects of actin assembly, since the decreased fluorescence intensity of actin filaments in oocyte cortex and cytoplasm were observed. However, Daam1 knockdown seemed not to affect the meiotic spindle movement. In addition, we found that fascin might be the down effector of Daam1, since the protein expression of fascin decreased after Daam1 knockdown. Thus, our data suggested that Daam1 affected actin assembly during oocyte meiotic division via the regulation of fascin expression.
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Perez, J R; Higgins-Sochaski, K A; Maltese, J Y; Narayanan, R
1994-01-01
The NF-kappa B transcription factor is a pleiotropic activator that participates in the induction of a wide variety of cellular genes. Antisense oligomer inhibition of the RelA subunit of NF-kappa B results in a block of cellular adhesion and inhibition of tumor cell growth. Investigation of the molecular basis for these effects showed that in vitro inhibition of the growth of transformed fibroblasts by relA antisense oligonucleotides can be reversed by the parental-cell-conditioned medium. Cytokine profile analysis of these cells treated with relA antisense oligonucleotides revealed inhibition of transforming growth factor beta 1 (TGF-beta 1 to the transformed fibroblasts reversed the inhibitory effects of relA antisense oligomers on soft agar colony formation and cell adhesion to the substratum. Direct inhibition of TGF-beta 1 expression by antisense phosphorothioates to TGF-beta 1 mimicked the in vitro effects of blocking cell adhesion that are elicited by antisense relA oligomers. These results may explain the in vitro effects of relA antisense oligomers on fibrosarcoma cell growth and adhesion. Images PMID:8035811
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An in vivo and in silico approach to study cis-antisense: a short cut to higher order response
NASA Astrophysics Data System (ADS)
Courtney, Colleen; Varanasi, Usha; Chatterjee, Anushree
2014-03-01
Antisense interactions are present in all domains of life. Typically sense, antisense RNA pairs originate from overlapping genes with convergent face to face promoters, and are speculated to be involved in gene regulation. Recent studies indicate the role of transcriptional interference (TI) in regulating expression of genes in convergent orientation. Modeling antisense, TI gene regulation mechanisms allows us to understand how organisms control gene expression. We present a modeling and experimental framework to understand convergent transcription that combines the effects of transcriptional interference and cis-antisense regulation. Our model shows that combining transcriptional interference and antisense RNA interaction adds multiple-levels of regulation which affords a highly tunable biological output, ranging from first order response to complex higher-order response. To study this system we created a library of experimental constructs with engineered TI and antisense interaction by using face-to-face inducible promoters separated by carefully tailored overlapping DNA sequences to control expression of a set of fluorescent reporter proteins. Studying this gene expression mechanism allows for an understanding of higher order behavior of gene expression networks.
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Wilson, K S; Baily, J; Tucker, C S; Matrone, G; Vass, S; Moran, C; Chapman, K E; Mullins, J J; Kenyon, C; Hadoke, P W F; Denvir, M A
2015-10-15
Transient early-life perturbations in glucocorticoids (GC) are linked with cardiovascular disease risk in later life. Here the impact of early life manipulations of GC on adult heart structure, function and gene expression were assessed. Zebrafish embryos were incubated in dexamethasone (Dex) or injected with targeted glucocorticoid receptor (GR) morpholino knockdown (GR Mo) over the first 120 h post fertilisation (hpf); surviving embryos (>90%) were maintained until adulthood under normal conditions. Cardiac function, heart histology and cardiac genes were assessed in embryonic (120 hpf) and adult (120 days post fertilisation (dpf)) hearts. GR Mo embryos (120 hpf) had smaller hearts with fewer cardiomyocytes, less mature striation pattern, reduced cardiac function and reduced levels of vmhc and igf mRNA compared with controls. GR Mo adult hearts were smaller with diminished trabecular network pattern, reduced expression of vmhc and altered echocardiographic Doppler flow compared to controls. Dex embryos had larger hearts at 120 hpf (Dex 107.2 ± 3.1 vs. controls 90.2 ± 1.1 μm, p < 0.001) with a more mature trabecular network and larger cardiomyocytes (1.62 ± 0.13 cells/μm vs control 2.18 ± 0.13 cells/μm, p < 0.05) and enhanced cardiac performance compared to controls. Adult hearts were larger (1.02 ± 0.07 μg/mg vs controls 0.63 ± 0.06 μg/mg, p = 0.0007), had increased vmhc and gr mRNA levels. Perturbations in GR activity during embryonic development results in short and long-term alterations in the heart. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.
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Lin, Chia-Hao; Hu, Huei-Jyun; Hwang, Pung-Pung
2016-02-15
In mammals, sodium/hydrogen exchanger (NHE) and sodium-chloride cotransporter (NCC) are expressed in renal tubules, and exhibit functional redundancy and mutual compensation in Na(+) uptake. In teleosts, the gills of the adult and skin of the embryonic stage function as external kidneys, and ionocytes are responsible for ionoregulation in these tissues. NHE- and NCC-expressing ionocytes mutually cooperate to adjust Na(+) uptake, which is analogous to the activity of the mammalian kidney. Cortisol is a hormone that controls Na(+) uptake through regulating NCC expression and activity in mammals; however, cortisol-mediated control of NCC expression is little understood in non-mammalian vertebrates, such as teleosts. It is essential for our understanding of the evolution of such regulation to determine whether cortisol has a conserved effect on NCC in vertebrates. In the present study, we treated zebrafish embryos with low Na(+) medium (LNa, 0.04 mM Na(+)) for 3 d to stimulate the mRNA expression of nhe3b, ncc, and cyp11b1 (a cortisol-synthesis enzyme) and whole body cortisol level. Exogenous cortisol treatment (20 mg/l, 3 d) resulted in an elevation of whole-body Na(+) content, ncc expression, and the density of ncc-expressing cells in zebrafish larvae. In loss-of-function experiments, microinjection of glucocorticoid receptor (gr) morpholino (MO) suppressed sodium content, ncc expression, and the density of ncc-expressing cells, but injection of mr MO had no such effects. In addition, exogenous cortisol treatment and gr MO injection also altered ncc expression and the density of ncc-expressing cells in gcm2 morphant larvae. Taken together, cortisol and GR appear to regulate Na(+) absorption through stimulating ncc expression and the differentiation of ncc-expressing ionocytes, providing new insights into the actions of cortisol on Na(+) uptake. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
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Wilson, K.S.; Baily, J.; Tucker, C.S.; Matrone, G.; Vass, S.; Moran, C.; Chapman, K.E.; Mullins, J.J.; Kenyon, C.; Hadoke, P.W.F.; Denvir, M.A.
2015-01-01
Background Transient early-life perturbations in glucocorticoids (GC) are linked with cardiovascular disease risk in later life. Here the impact of early life manipulations of GC on adult heart structure, function and gene expression were assessed. Methods and results Zebrafish embryos were incubated in dexamethasone (Dex) or injected with targeted glucocorticoid receptor (GR) morpholino knockdown (GR Mo) over the first 120 h post fertilisation (hpf); surviving embryos (>90%) were maintained until adulthood under normal conditions. Cardiac function, heart histology and cardiac genes were assessed in embryonic (120 hpf) and adult (120 days post fertilisation (dpf)) hearts. GR Mo embryos (120 hpf) had smaller hearts with fewer cardiomyocytes, less mature striation pattern, reduced cardiac function and reduced levels of vmhc and igf mRNA compared with controls. GR Mo adult hearts were smaller with diminished trabecular network pattern, reduced expression of vmhc and altered echocardiographic Doppler flow compared to controls. Dex embryos had larger hearts at 120 hpf (Dex 107.2 ± 3.1 vs. controls 90.2 ± 1.1 μm, p < 0.001) with a more mature trabecular network and larger cardiomyocytes (1.62 ± 0.13 cells/μm vs control 2.18 ± 0.13 cells/μm, p < 0.05) and enhanced cardiac performance compared to controls. Adult hearts were larger (1.02 ± 0.07 μg/mg vs controls 0.63 ± 0.06 μg/mg, p = 0.0007), had increased vmhc and gr mRNA levels. Conclusion Perturbations in GR activity during embryonic development results in short and long-term alterations in the heart. PMID:26219824
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miRNA-dependent gene silencing involving Ago2-mediated cleavage of a circular antisense RNA
Hansen, Thomas B; Wiklund, Erik D; Bramsen, Jesper B; Villadsen, Sune B; Statham, Aaron L; Clark, Susan J; Kjems, Jørgen
2011-01-01
MicroRNAs (miRNAs) are ∼22 nt non-coding RNAs that typically bind to the 3′ UTR of target mRNAs in the cytoplasm, resulting in mRNA destabilization and translational repression. Here, we report that miRNAs can also regulate gene expression by targeting non-coding antisense transcripts in human cells. Specifically, we show that miR-671 directs cleavage of a circular antisense transcript of the Cerebellar Degeneration-Related protein 1 (CDR1) locus in an Ago2-slicer-dependent manner. The resulting downregulation of circular antisense has a concomitant decrease in CDR1 mRNA levels, independently of heterochromatin formation. This study provides the first evidence for non-coding antisense transcripts as functional miRNA targets, and a novel regulatory mechanism involving a positive correlation between mRNA and antisense circular RNA levels. PMID:21964070
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Ningappa, M; Ashokkumar, C; Higgs, B W; Sun, Q; Jaffe, R; Mazariegos, G; Li, D; Weeks, D E; Subramaniam, S; Ferrell, R; Hakonarson, H; Sindhi, R
2016-02-01
T cell suppression prevents acute cellular rejection but causes life-threatening infections and malignancies. Previously, liver transplant (LTx) rejection in children was associated with the single-nucleotide polymorphism (SNP) rs9296068 upstream of the HLA-DOA gene. HLA-DOA inhibits B cell presentation of antigen, a potentially novel antirejection drug target. Using archived samples from 122 white pediatric LTx patients (including 77 described previously), we confirmed the association between rs9296068 and LTx rejection (p = 0.001, odds ratio [OR] 2.55). Next-generation sequencing revealed that the putative transcription factor (CCCTC binding factor [CTCF]) binding SNP locus rs2395304, in linkage disequilibrium with rs9296068 (D' 0.578, r(2) = 0.4), is also associated with LTx rejection (p = 0.008, OR 2.34). Furthermore, LTx rejection is associated with enhanced B cell presentation of donor antigen relative to HLA-nonidentical antigen in a novel cell-based assay and with a downregulated HLA-DOA gene in a subset of these children. In lymphoblastoid B (Raji) cells, rs2395304 coimmunoprecipitates with CTCF, and CTCF knockdown with morpholino antisense oligonucleotides enhances alloantigen presentation and downregulates the HLA-DOA gene, reproducing observations made with HLA-DOA knockdown and clinical rejection. Alloantigen presentation is suppressed by inhibitors of methylation and histone deacetylation, reproducing observations made during resolution of rejection. Enhanced donor antigen presentation by B cells and its epigenetic dysregulation via the HLA-DOA gene represent novel opportunities for surveillance and treatment of transplant rejection. © Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.
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Tailhades, Julien; Takizawa, Hotake; Gait, Michael J.; Wellings, Don A.; Wade, John D.; Aoki, Yoshitsugu; Shabanpoor, Fazel
2017-01-01
Antisense oligonucleotide (ASO)-based drug development is gaining significant momentum following the recent FDA approval of Eteplirsen (an ASO based on phosphorodiamidate morpholino) and Spinraza (2′-O-methoxyethyl-phosphorothioate) in late 2016. Their attractiveness is mainly due to the backbone modifications which have improved the in vivo characteristics of oligonucleotide drugs. Another class of ASO, based on peptide nucleic acid (PNA) chemistry, is also gaining popularity as a platform for development of gene-specific therapy for various disorders. However, the chemical synthesis of long PNAs, which are more target-specific, remains an ongoing challenge. Most of the reported methodology for the solid-phase synthesis of PNA suffer from poor coupling efficiency which limits production to short PNA sequences of less than 15 residues. Here, we have studied the effect of backbone modifications with Hmb (2-hydroxy-4-methoxybenzyl) and Dmb (2,4-dimethoxybenzyl) to ameliorate difficult couplings and reduce “on-resin” aggregation. We firstly synthesized a library of PNA dimers incorporating either Hmb or Dmb and identified that Hmb is superior to Dmb in terms of its ease of removal. Subsequently, we used Hmb backbone modification to synthesize a 22-mer purine-rich PNA, targeting dystrophin RNA splicing, which could not be synthesized by standard coupling methodology. Hmb backbone modification allowed this difficult PNA to be synthesized as well as to be continued to include a cell-penetrating peptide on the same solid support. This approach provides a novel and straightforward strategy for facile solid-phase synthesis of difficult purine-rich PNA sequences. PMID:29094037
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Tailhades, Julien; Takizawa, Hotake; Gait, Michael J; Wellings, Don A; Wade, John D; Aoki, Yoshitsugu; Shabanpoor, Fazel
2017-01-01
Antisense oligonucleotide (ASO)-based drug development is gaining significant momentum following the recent FDA approval of Eteplirsen (an ASO based on phosphorodiamidate morpholino) and Spinraza (2'- O -methoxyethyl-phosphorothioate) in late 2016. Their attractiveness is mainly due to the backbone modifications which have improved the in vivo characteristics of oligonucleotide drugs. Another class of ASO, based on peptide nucleic acid (PNA) chemistry, is also gaining popularity as a platform for development of gene-specific therapy for various disorders. However, the chemical synthesis of long PNAs, which are more target-specific, remains an ongoing challenge. Most of the reported methodology for the solid-phase synthesis of PNA suffer from poor coupling efficiency which limits production to short PNA sequences of less than 15 residues. Here, we have studied the effect of backbone modifications with Hmb (2-hydroxy-4-methoxybenzyl) and Dmb (2,4-dimethoxybenzyl) to ameliorate difficult couplings and reduce "on-resin" aggregation. We firstly synthesized a library of PNA dimers incorporating either Hmb or Dmb and identified that Hmb is superior to Dmb in terms of its ease of removal. Subsequently, we used Hmb backbone modification to synthesize a 22-mer purine-rich PNA, targeting dystrophin RNA splicing, which could not be synthesized by standard coupling methodology. Hmb backbone modification allowed this difficult PNA to be synthesized as well as to be continued to include a cell-penetrating peptide on the same solid support. This approach provides a novel and straightforward strategy for facile solid-phase synthesis of difficult purine-rich PNA sequences.
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Nie, Shuyi; Bronner, Marianne E.
2015-01-01
Aims Ets1 is an important transcription factor that is expressed in both the cardiac neural crest (NC) and heart mesoderm of vertebrate embryos. Moreover, Ets1 deletion in humans results in congenital heart abnormalities. To clarify the functional contributions of Ets1 in cardiac NC vs. heart mesoderm, we performed tissue-targeted loss-of-function analysis to compare the relative roles of Ets1 in these two tissues during heart formation using Xenopus embryos as a model system. Methods and results We confirmed by in situ hybridization analysis that Ets1 is expressed in NC and heart mesoderm during embryogenesis. Using a translation-blocking antisense morpholino to knockdown Ets1 protein selectively in the NC, we observed defects in NC delamination from the neural tube, collective cell migration, as well as segregation of NC streams in the cranial and cardiac regions. Many cardiac NC cells failed to reach their destination in the heart, resulting in defective aortic arch artery formation. A different set of defects was noted when Ets1 knockdown was targeted to heart mesoderm. The formation of the primitive heart tube was dramatically delayed and the endocardial tissue appeared depleted. As a result, the conformation of the heart was severely disrupted. In addition, the outflow tract septum was missing, and trabeculae formation in the ventricle was abolished. Conclusion Our study shows that Ets1 is required in both the cardiac NC and heart mesoderm, albeit for different aspects of heart formation. Our results reinforce the suggestion that proper interaction between these tissues is critical for normal heart development. PMID:25691536
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Panchal, Rekha G.; Ulrich, Ricky L.; Bradfute, Steven B.; Lane, Douglas; Ruthel, Gordon; Kenny, Tara A.; Iversen, Patrick L.; Anderson, Arthur O.; Gussio, Rick; Raschke, William C.; Bavari, Sina
2009-01-01
The modulation of cellular processes by small molecule inhibitors, gene inactivation, or targeted knockdown strategies combined with phenotypic screens are powerful approaches to delineate complex cellular pathways and to identify key players involved in disease pathogenesis. Using chemical genetic screening, we tested a library of known phosphatase inhibitors and identified several compounds that protected Bacillus anthracis infected macrophages from cell death. The most potent compound was assayed against a panel of sixteen different phosphatases of which CD45 was found to be most sensitive to inhibition. Testing of a known CD45 inhibitor and antisense phosphorodiamidate morpholino oligomers targeting CD45 also protected B. anthracis-infected macrophages from cell death. However, reduced CD45 expression did not protect anthrax lethal toxin (LT) treated macrophages, suggesting that the pathogen and independently added LT may signal through distinct pathways. Subsequent, in vivo studies with both gene-targeted knockdown of CD45 and genetically engineered mice expressing reduced levels of CD45 resulted in protection of mice after infection with the virulent Ames B. anthracis. Intermediate levels of CD45 expression were critical for the protection, as mice expressing normal levels of CD45 or disrupted CD45 phosphatase activity or no CD45 all succumbed to this pathogen. Mechanism-based studies suggest that the protection provided by reduced CD45 levels results from regulated immune cell homeostasis that may diminish the impact of apoptosis during the infection. To date, this is the first report demonstrating that reduced levels of host phosphatase CD45 modulate anthrax pathogenesis. PMID:19269962
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scaRNAs regulate splicing and vertebrate heart development.
Patil, Prakash; Kibiryeva, Nataliya; Uechi, Tamayo; Marshall, Jennifer; O'Brien, James E; Artman, Michael; Kenmochi, Naoya; Bittel, Douglas C
2015-08-01
Alternative splicing (AS) plays an important role in regulating mammalian heart development, but a link between misregulated splicing and congenital heart defects (CHDs) has not been shown. We reported that more than 50% of genes associated with heart development were alternatively spliced in the right ventricle (RV) of infants with tetralogy of Fallot (TOF). Moreover, there was a significant decrease in the level of 12 small cajal body-specific RNAs (scaRNAs) that direct the biochemical modification of specific nucleotides in spliceosomal RNAs. We sought to determine if scaRNA levels influence patterns of AS and heart development. We used primary cells derived from the RV of infants with TOF to show a direct link between scaRNA levels and splice isoforms of several genes that regulate heart development (e.g., GATA4, NOTCH2, DAAM1, DICER1, MBNL1 and MBNL2). In addition, we used antisense morpholinos to knock down the expression of two scaRNAs (scarna1 and snord94) in zebrafish and saw a corresponding disruption of heart development with an accompanying alteration in splice isoforms of cardiac regulatory genes. Based on these combined results, we hypothesize that scaRNA modification of spliceosomal RNAs assists in fine tuning the spliceosome for dynamic selection of mRNA splice isoforms. Our results are consistent with disruption of splicing patterns during early embryonic development leading to insufficient communication between the first and second heart fields, resulting in conotruncal misalignment and TOF. Our findings represent a new paradigm for determining the mechanisms underlying congenital cardiac malformations. Copyright © 2015. Published by Elsevier B.V.
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NASA Astrophysics Data System (ADS)
Tailhades, Julien; Takizawa, Hotake; Gait, Michael J.; Wellings, Don A.; Wade, John D.; Aoki, Yoshitsugu; Shabanpoor, Fazel
2017-10-01
Antisense oligonucleotide (ASO)-based drug development is gaining significant momentum following the recent FDA approval of Eteplirsen (an ASO based on phosphorodiamidate morpholino) and Spinraza (2’-O-methoxyethyl-phosphorothioate) in late 2016. Their attractiveness is mainly due to the backbone modifications which have improved the in vivo characteristics of oligonucleotide drugs. Another class of ASO, based on peptide nucleic acid (PNA) chemistry, is also gaining popularity as a platform for development of gene-specific therapy for various disorders. However, the chemical synthesis of long PNAs, which are more target-specific, remains an ongoing challenge. Most of the reported methodology for the solid-phase synthesis of PNA suffer from poor coupling efficiency which limits production to short PNA sequences of less than 15 residues. Here we have studied the effect of backbone modifications with Hmb (2-hydroxy-4-methoxybenzyl) and Dmb (2,4-dimethoxybenzyl) to ameliorate difficult couplings and reduce “on-resin” aggregation. We firstly synthesized a library of PNA dimers incorporating either Hmb or Dmb and identified that Hmb is superior to Dmb in terms of its ease of removal. Subsequently, we used Hmb backbone modification to synthesize a 22-mer purine-rich PNA, targeting dystrophin RNA splicing, which could not be synthesized by standard coupling methodology. Hmb backbone modification allowed this difficult PNA to be synthesized as well as to be continued to include a cell-penetrating peptide on the same solid support. This approach provides a novel and straightforward strategy for facile solid-phase synthesis of difficult purine-rich PNA sequences.
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Busch-Nentwich, Elisabeth; Söllner, Christian; Roehl, Henry; Nicolson, Teresa
2004-02-01
Over 30 genes responsible for human hereditary hearing loss have been identified during the last 10 years. The proteins encoded by these genes play roles in a diverse set of cellular functions ranging from transcriptional regulation to K(+) recycling. In a few cases, the genes are novel and do not give much insight into the cellular or molecular cause for the hearing loss. Among these poorly understood deafness genes is DFNA5. How the truncation of the encoded protein DFNA5 leads to an autosomal dominant form of hearing loss is not clear. In order to understand the biological role of Dfna5, we took a reversegenetic approach in zebrafish. Here we show that morpholino antisense nucleotide knock-down of dfna5 function in zebrafish leads to disorganization of the developing semicircular canals and reduction of pharyngeal cartilage. This phenotype closely resembles previously isolated zebrafish craniofacial mutants including the mutant jekyll. jekyll encodes Ugdh [uridine 5'-diphosphate (UDP)-glucose dehydrogenase], an enzyme that is crucial for production of the extracellular matrix component hyaluronic acid (HA). In dfna5 morphants, expression of ugdh is absent in the developing ear and pharyngeal arches, and HA levels are strongly reduced in the outgrowing protrusions of the developing semicircular canals. Previous studies suggest that HA is essential for differentiating cartilage and directed outgrowth of the epithelial protrusions in the developing ear. We hypothesize that the reduction of HA production leads to uncoordinated outgrowth of the canal columns and impaired facial cartilage differentiation.
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Nesterova, Maria V; Cho-Chung, Yoon S
2004-07-01
There are two types of cyclic AMP (cAMP)-dependent protein kinase (PKA), type I (PKA-I) and type II (PKA-II), which share a common catalytic (C) subunit but contain distinct regulatory (R) subunits, RI versus RII, respectively. Evidence suggests that increased expression of PKA-I and its regulatory subunit (RIalpha) correlates with tumorigenesis and tumor growth. We investigated the effect of sequence-specific inhibition of RIalpha gene expression at the initial phase of 7,12-dimethylbenz(alphaa)anthracene (DMBA)-induced mammary carcinogenesis. Antisense RIalpha oligodeoxynucleotide (ODN) targeted against PKA RIalpha was administered (0.1 mg/day/rat, i.p.) 1 day before DMBA intubation and during the first 9 days post-DMBA intubation to determine the anticarcinogenic effects. Antisense RIalpha, in a sequence-specific manner, inhibited the tumor production. At 90 days after DMBA intubation, untreated controls and RIalpha-antisense-treated rats exhibited an average mean number of tumors per rat of 4.2 and 1.8, respectively, and 90% of control and 45% of antisense-treated animals had tumors. The antisense also delayed the first tumor appearance. An increase in RIalpha and PKA-I levels in the mammary gland and liver preceded DMBA-induced tumor production, and antisense down-regulation of RIalpha restored normal levels of PKA-I and PKA-II in these tissues. Antisense RIalpha in the liver induced the phase II enzymes, glutathione S-transferase and quinone oxidoreductase, c-fos protein, and activator protein 1 (AP-1)- and cAMP response element (CRE)-directed transcription. In the mammary glands, antisense RIalpha promoted DNA repair processes. In contrast, the CRE transcription-factor decoy could not mimic these effects of antisense RIalpha. The results demonstrate that RIalpha antisense produces dual anticarcinogenic effects: (a) increasing DMBA detoxification in the liver by increasing phase II enzyme activities, increasing CRE-binding-protein phosphorylation and enhancing CRE- and Ap-1-directed transcription; and (b) activating DNA repair processes in the mammary gland by down-regulating PKA-I.
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Richardson, Casey R.; Luo, Qing-Jun; Gontcharova, Viktoria; Jiang, Ying-Wen; Samanta, Manoj; Youn, Eunseog; Rock, Christopher D.
2010-01-01
Background MicroRNAs (miRNAs) and trans-acting small-interfering RNAs (tasi-RNAs) are small (20–22 nt long) RNAs (smRNAs) generated from hairpin secondary structures or antisense transcripts, respectively, that regulate gene expression by Watson-Crick pairing to a target mRNA and altering expression by mechanisms related to RNA interference. The high sequence homology of plant miRNAs to their targets has been the mainstay of miRNA prediction algorithms, which are limited in their predictive power for other kingdoms because miRNA complementarity is less conserved yet transitive processes (production of antisense smRNAs) are active in eukaryotes. We hypothesize that antisense transcription and associated smRNAs are biomarkers which can be computationally modeled for gene discovery. Principal Findings We explored rice (Oryza sativa) sense and antisense gene expression in publicly available whole genome tiling array transcriptome data and sequenced smRNA libraries (as well as C. elegans) and found evidence of transitivity of MIRNA genes similar to that found in Arabidopsis. Statistical analysis of antisense transcript abundances, presence of antisense ESTs, and association with smRNAs suggests several hundred Arabidopsis ‘orphan’ hypothetical genes are non-coding RNAs. Consistent with this hypothesis, we found novel Arabidopsis homologues of some MIRNA genes on the antisense strand of previously annotated protein-coding genes. A Support Vector Machine (SVM) was applied using thermodynamic energy of binding plus novel expression features of sense/antisense transcription topology and siRNA abundances to build a prediction model of miRNA targets. The SVM when trained on targets could predict the “ancient” (deeply conserved) class of validated Arabidopsis MIRNA genes with an accuracy of 84%, and 76% for “new” rapidly-evolving MIRNA genes. Conclusions Antisense and smRNA expression features and computational methods may identify novel MIRNA genes and other non-coding RNAs in plants and potentially other kingdoms, which can provide insight into antisense transcription, miRNA evolution, and post-transcriptional gene regulation. PMID:20520764
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Identification of antisense long noncoding RNAs that function as SINEUPs in human cells.
Schein, Aleks; Zucchelli, Silvia; Kauppinen, Sakari; Gustincich, Stefano; Carninci, Piero
2016-09-20
Mammalian genomes encode numerous natural antisense long noncoding RNAs (lncRNAs) that regulate gene expression. Recently, an antisense lncRNA to mouse Ubiquitin carboxyl-terminal hydrolase L1 (Uchl1) was reported to increase UCHL1 protein synthesis, representing a new functional class of lncRNAs, designated as SINEUPs, for SINE element-containing translation UP-regulators. Here, we show that an antisense lncRNA to the human protein phosphatase 1 regulatory subunit 12A (PPP1R12A), named as R12A-AS1, which overlaps with the 5' UTR and first coding exon of the PPP1R12A mRNA, functions as a SINEUP, increasing PPP1R12A protein translation in human cells. The SINEUP activity depends on the aforementioned sense-antisense interaction and a free right Alu monomer repeat element at the 3' end of R12A-AS1. In addition, we identify another human antisense lncRNA with SINEUP activity. Our results demonstrate for the first time that human natural antisense lncRNAs can up-regulate protein translation, suggesting that endogenous SINEUPs may be widespread and present in many mammalian species.
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Voltage-gated calcium channel and antisense oligonucleotides thereto
NASA Technical Reports Server (NTRS)
Friedman, Peter A. (Inventor); Duncan, Randall L. (Inventor); Hruska, Keith A. (Inventor); Barry, Elizabeth L. R. (Inventor)
1998-01-01
An antisense oligonucleotide of 10 to 35 nucleotides in length that can hybridize with a region of the .alpha..sub.1 subunit of the SA-Cat channel gene DNA or mRNA is provided, together with pharmaceutical compositions containing and methods utilizing such antisense oligonucleotide.
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Antiviral effects of herpes simplex virus specific anti-sense nucleic acids.
Cantin, E M; Podsakoff, G; Willey, D E; Openshaw, H
1992-01-01
We have targeted mRNA sequences encompassing the translation initiation codon of the essential herpes simplex virus type 1 (HSV-1) IE3 gene with three kinds of anti-sense molecule. Addition of a 15mer oligodeoxyribonucleoside methylphosphonate to tissue culture cells resulted in suppression of viral replication. HSV-1 replication was also inhibited in cultured cells containing anti-sense vectors expressing transcripts complementary to the IE3 mRNA. We have also constructed a ribozyme which upon base pairing with the target IE3 mRNA induces cleavage at the predicted GUC site. A major obstacle to anti-sense studies in animals is drug delivery of preformed antisense molecules to ganglionic neurons, the site of HSV latency and reactivation. We speculate as to how this may be accomplished through carrier compounds which are taken up by nerve terminals and transported by retrograde axoplasmic flow. By the same route, HSV itself may be used as an anti-sense vector.
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Structural insights of a PI3K/mTOR dual inhibitor with the morpholino-triazine scaffold
NASA Astrophysics Data System (ADS)
Takeda, Takako; Wang, Yanli; Bryant, Stephen H.
2016-04-01
Stimulation of the PI3K/Akt/mTOR pathway, which controls cell proliferation and growth, is often observed in cancer cell. Inhibiting both PI3K and mTOR in this pathway can switch off Akt activation and hence, plays a powerful role for modulating this pathway. PKI-587, a drug containing the structure of morpholino-triazines, shows a dual and nano-molar inhibition activity and is currently in clinical trial. To provide an insight into the mechanism of this dual inhibition, pharmacophore and QSAR models were developed in this work using compounds based on the morpholino-triazines scaffold, followed by a docking study. Pharmacophore model suggested the mechanism of the inhibition of PI3Kα and mTOR by the compounds were mostly the same, which was supported by the docking study showing similar docking modes. The analysis also suggested the importance of the flat plane shape of the ligands, the space surrounding the ligands in the binding pocket, and the slight difference in the shape of the binding sites between PI3Kα and mTOR.
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Targeting Cancer with Antisense Oligomers
DOE Office of Scientific and Technical Information (OSTI.GOV)
Hnatowich, DJ
With financial assistance from the Department of Energy, we have shown definitively that radiolabeled antisense DNAs and other oligomers will accumulate in target cancer cells in vitro and in vivo by an antisense mechanism. We have also shown that the number of mRNA targets for our antisense oligomers in the cancer cell types that we have investigated so far is sufficient to provide and antisense image and/or radiotherapy of cancer in mice. These studies have been reported in about 10 publications. However our observation over the past several years has shown that radiolabeled antisense oligomers administered intravenously in their nativemore » and naked form will accumulate and be retained in target xenografts by an antisense mechanism but will also accumulate at high levels in normal organs such as liver, spleen and kidneys. We have investigated unsuccessfully several commercially available vectors. Thus the use of radiolabeled antisense oligomers for the imaging of cancer must await novel approaches to delivery. This laboratory has therefore pursued two new paths, optical imaging of tumor and Auger radiotherapy. We are developing a novel method of optical imaging tumor using antisense oligomers with a fluorophore is administered while hybridized with a shorter complementary oligomer with an inhibitor. In culture and in tumored mice that the duplex remains intact and thus nonfluorescent until it encounters its target mRNA at which time it dissociates and the antisense oligomer binds along with its fluorophore to the target. Simultaneous with the above, we have also observed, as have others, that antisense oligomers migrate rapidly and quantitatively to the nucleus upon crossing cell membranes. The Auger electron radiotherapy path results from this observation since the nuclear migration properties could be used effectively to bring and to retain in the nucleus an Auger emitting radionuclide such as 111In or 125I bound to the antisense oligomer. Since the object becomes radiotherapy rather than imaging, the delivery problem may be obviated by attaching the antisense oligomer to an antitumor antibody to improve delivery following intravenous administration. Since many antibodies are trapped in endosomes following internalization, a cell penetrating peptide such as tat will also be included to ensure transport of the complex without entrapment. Rather than covalent conjugation of the three entities, we are using streptavidin as linker after biotinylated each component. Our recent efforts have concentrated on establishing the influence of the streptavidin linker on the properties of each component within the delivery nanoparticle. Thus, we have shown that the Herceptin antibody, when linked to a labeled oligomer via streptavidin, remains capable of directing the label oligomer to Her2+ tumor cells in vitro and Her2+ tumor xenografts in mice. In addition, we have demonstrated that a labeled antisense oligomer within the nanoparticle remains capable of migrating to the nucleus and binding to its target mRNA in vitro and in vivo. We have shown that the tat peptide also preserves its properties of cell transport when incubated as one component of the nanoparticle. Most recently, we have addressed another of our concerns, namely whether the streptavidin would adversely effect the biodistribution of the antisense oligomer. We were pleased to find that the 99mTc-labeled antisense MORF within the Herceptin three component and two component nanoparticles accumulated and was retained in tumor in a manner suggestive of radiolabeled Herceptin itself. Thus the preserved properties within the streptavidin delivery nanoparticle of the Herceptin antibody, the tat peptide and the 111In labeled antisense MORF oligomer will explain why we have successfully demonstrated an Auger electron-mediated, antisense-mediated radiotherapy in cells in culture. One remaining concern is that the delivery nanoparticle may deliver the Auger electron emitting radionuclide to the nucleus of normal cells as well as tumor cells. We have now performed tumored mice studies of the three component delivery nanoparticle with the antisense MORF labeled with Cy3 so that tissue slices could be examined by immunohistology for evidence of MORF accumulations in the nuclei of both tumor and normal tissues. Microscopic examination shows nuclear staining in approximately 20% of the tumor cells in animals injected with the antisense nanoparticle and 10% of the tumor cells in animals receiving the sense nanoparticle, whereas no nuclear staining is seen in the tumor cells of mice given the PBS injection as another control. No nuclear staining was observed in all sections from all normal organs. Finally, my colleagues and I wish to express our gratitude to the DOE for their generous support of our research at a time when the NIH was unwilling to fund what they believed to be a risky« less
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Larcher, J C; Basseville, M; Vayssiere, J L; Cordeau-Lossouarn, L; Croizat, B; Gros, F
1992-06-30
Antisense oligodeoxynucleotides were found to be stable in the culture medium containing fetal calf serum (heat-inactivated 30 minutes at 65 degrees C) and in cells. Antisense oligomer treatment causes cessation of mitoses, but does not lead to morphological differentiation. Under antisense conditions, we have observed an increase in the amount of two neurospecific protein, namely peripherin and gamma-enolase. Comparison of the results obtained with chemical inducers and antisense oligodeoxynucleotides allows us to postulate three phases in N1E-115 differentiation: the first correspond to the arrest of mitosis, the second to the expression of a limited neuronal program, and the third to the morphological and electrophysiological differentiation.
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Antisense therapy and emerging applications for the management of dyslipidemia.
Toth, Peter P
2011-01-01
Because a significant percentage of patients who require high-dose statin therapy for dyslipidemia experience treatment-related muscle symptoms and an inconsistent clinical response, alternative or adjunctive approaches to the management of dyslipidemia are needed. One alternative approach, antisense therapy, may offer an effective and well-tolerated option for patients not satisfactorily responsive to or intolerant to standard pharmacologic dyslipidemia therapies. This review provides an overview of antisense technology and its potential role in the management of dyslipidemia. Source material was obtained primarily from the published literature identified through a search of the PubMed database. Antisense technology is an evolving approach to therapy that has gone through a series of refinements to enhance molecular stability, potency, and tolerability. Mipomersen is an antisense molecule capable of producing clinically meaningful reductions in low-density lipoprotein cholesterol in patients with severe familial hypercholesterolemia. Further long-term clinical studies are required to more clearly quantify its impact on risk for cardiovascular events and establish whether it increases risk for hepatosteatosis. Antisense therapy represents a potentially effective and well-tolerated emerging treatment modality for numerous diseases. In the treatment of hypercholesterolemia, the antisense therapy mipomersen may provide a possible treatment option for patients with treatment-resistant dyslipidemia. Copyright © 2011 National Lipid Association. Published by Elsevier Inc. All rights reserved.
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Xiong, Changyan; Li, Xuejiao; Liu, Juanli; Zhao, Xin; Xu, Shungao; Huang, Xinxiang
2018-01-01
Antisense RNAs from complementary strands of protein coding genes regulate the expression of genes involved in many cellular processes. Using deep sequencing analysis of the Salmonella enterica serovar Typhi ( S. Typhi) transcriptome, a novel antisense RNA encoded on the strand complementary to the rpoH gene was revealed. In this study, the molecular features of this antisense RNA were assessed using northern blotting and rapid amplification of cDNA ends. The 3,508 nt sequence of RNA was identified as the antisense RNA of the rpoH gene and was named ArpH. ArpH was found to attenuate the invasion of HeLa cells by S. Typhi by regulating the expression of SPI-1 genes. In an rpoH mutant strain, the invasive capacity of S. Typhi was increased, whereas overexpression of ArpH positively regulates rpoH mRNA levels. Results of this study suggest that the cis -encoded antisense RNA ArpH is likely to affect the invasive capacity of S. Typhi by regulating the expression of rpoH .
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Dadzie, Isaac; Xu, Shungao; Ni, Bin; Zhang, Xiaolei; Zhang, Haifang; Sheng, Xiumei; Xu, Huaxi; Huang, Xinxiang
2013-01-01
Antisense RNAs that originate from the complementary strand of protein coding genes are involved in the regulation of gene expression in all domains of life. In bacteria, some of these antisense RNAs are transcriptional noise whiles others play a vital role to adapt the cell to changing environmental conditions. By deep sequencing analysis of transcriptome of Salmonella enterica serovar Typhi, a partial RNA sequence encoded in-cis to the dnaA gene was revealed. Northern blot and RACE analysis confirmed the transcription of this antisense RNA which was expressed mostly in the stationary phase of the bacterial growth and also under iron limitation and osmotic stress. Pulse expression analysis showed that overexpression of the antisense RNA resulted in a significant increase in the mRNA levels of dnaA, which will ultimately enhance their translation. Our findings have revealed that antisense RNA of dnaA is indeed transcribed not merely as a by-product of the cell's transcription machinery but plays a vital role as far as stability of dnaA mRNA is concerned. PMID:23637809
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Cis-encoded non-coding antisense RNAs in streptococci and other low GC Gram (+) bacterial pathogens
Cho, Kyu Hong; Kim, Jeong-Ho
2015-01-01
Due to recent advances of bioinformatics and high throughput sequencing technology, discovery of regulatory non-coding RNAs in bacteria has been increased to a great extent. Based on this bandwagon, many studies searching for trans-acting small non-coding RNAs in streptococci have been performed intensively, especially in the important human pathogen, group A and B streptococci. However, studies for cis-encoded non-coding antisense RNAs in streptococci have been scarce. A recent study shows antisense RNAs are involved in virulence gene regulation in group B streptococcus, S. agalactiae. This suggests antisense RNAs could have important roles in the pathogenesis of streptococcal pathogens. In this review, we describe recent discoveries of chromosomal cis-encoded antisense RNAs in streptococcal pathogens and other low GC Gram (+) bacteria to provide a guide for future studies. PMID:25859258
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Liu, Feng; Du, JinTao; Xian, Junming; Liu, Yafeng; Liu, Shixi; Lin, Yan
2015-01-01
The tumor suppressor p14(ARF) and proto-oncogene epidermal growth factor receptor (EGFR) play important roles in the development of laryngeal squamous cell carcinoma (LSCC). This study was aimed to determine whether combining recombinant p14(ARF) with antisense complementary DNA of EGFR could improve the therapeutic effectiveness in LSCC. After human larynx cancer cells (Hep-2) were infected with recombinant adenoviruses (Ad-p14(ARF) and Ad-antisense EGFR) together or alone in vitro, the proliferation and cell cycle distribution of Hep-2 cells were detected by MTT assay and flow cytometer analysis, respectively. Furthermore, the antitumor effects of recombinant adenoviruses together or alone on Hep-2 xenografts were examined in vivo. The levels of p14(ARF) and EGFR expressed in Hep-2 cells and xenografts were determined by western blot assay. Ad-p14(ARF) combining with Ad-antisense EGFR markedly inhibited the Hep-2 proliferation compared with alone (P=0.001, P=0.002 respectively). Combination of Ad-p14(ARF) and Ad-antisense EGFR led to the proportion of Hep-2 cells in G0/G1 phases increased by up to 86.9%. The down-expression of EGFR protein and overexpression of p14(ARF) protein were observed in vitro and in vivo, and this effect was preserved when Ad-p14(ARF) was combined with Ad-antisense EGFR. Besides, Ad-p14(ARF) plus Ad-antisense EGFR significantly (P<0.05) increased the antitumor activity against Hep-2 tumor xenografts comparing with Ad-p14(ARF) or Ad-antisense EGFR alone. Combination Ad-p14(ARF) with Ad-antisense EGFR significantly increased the antitumor responses in LSCC. An effectively potential gene therapy to prevent proliferation of LSCC was provided. Copyright © 2015 Elsevier Inc. All rights reserved.
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Saberi, Fatemeh; Kamali, Mehdi; Najafi, Ali; Yazdanparast, Alavieh; Moghaddam, Mehrdad Moosazadeh
2016-01-01
Naturally occurring antisense RNAs are small, diffusible, untranslated transcripts that pair to target RNAs at specific regions of complementarity to control their biological function by regulating gene expression at the post-transcriptional level. This review focuses on known cases of antisense RNA control in prokaryotes and provides an overview of some natural RNA-based mechanisms that bacteria use to modulate gene expression, such as mRNA sensors, riboswitches and antisense RNAs. We also highlight recent advances in RNA-based technology. The review shows that studies on both natural and synthetic systems are reciprocally beneficial.
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Kayim, M; Ceccardi, T L; Berretta, M J G; Barthe, G A; Derrick, K S
2004-11-01
The protein p12 accumulates in leaves of trees with citrus blight (CB), a serious decline of unknown cause. The function of p12 is not known, but sequence analysis indicates it may be related to expansins. In studies to determine the function of p12, sense and antisense constructs were used to make transgenic Carrizo citrange using an Agrobacterium-mediated transformation system. Homogeneous beta-glucuronidase+ (GUS+) sense and antisense transgenic shoots were regenerated using kanamycin as a selective agent. Twenty-five sense and 45 antisense transgenic shoots were in vivo grafted onto Carrizo citrange for further analyses. In addition, 20 sense and 18 antisense shoots were rooted. The homogeneous GUS+ plants contained either the p12 sense or antisense gene (without the intron associated with the gene in untransformed citrus) as shown by PCR and Southern blotting. Northern blots showed the expected RNA in the sense and antisense plants. A protein of identical size and immunoreactivity was observed in seven of nine sense plants but not in nine antisense or non-transgenic plants. At the current stage of growth, there are no visual phenotypic differences between the transgenic and non-transgenic plants. Selected plants will be budded with sweet orange for field evaluation for resistance or susceptibility to CB and general rootstock performance.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Sorscher, E.J.; Kirk, K.L.; Weaver, M.L.
The authors have tested the hypothesis that the cystic fibrosis (CF) gene product, called the CF transmembrane conductance regulator (CFTR), mediates anion transport in normal human sweat duct cells. Sweat duct cells in primary culture were treated with oligodeoxynucleotides that were antisense to the CFTR gene transcript in order to block the expression of the wild-type CFTR. Anion transport in CFTR transcript antisense-treated cells was then assessed with a halide-specific dye, 6-methoxy-N-(3-sulfopropryl)quinolinium, and fluorescent digital imaging microscopy to monitor halide influx and efflux from single sweat duct cells. Antisense oligodeoxynucleotide treatment for 24 hr virtually abolished Cl{sup {minus}} transport inmore » sweat duct cells compared with untreated cells or control cells treated with sense oligodeoxynucleotides. Br{sup {minus}} uptake into sweat duct cells was also blocked after a 24-hr CFTR transcript antisense treatments, but not after treatments for only 4 hr. Lower concentrations of antisense oligodeoxynucleotides were less effective at inhibiting Cl{sup {minus}} transport. These results indicate that oligodeoxynucleotides that are antisense to CFTR transcript inhibit sweat duct Cl{sup {minus}} permeability in both a time-dependent and dose-dependent manner. This approach provides evidence that inhibition of the expression of the wild-type CFTR gene in a normal, untransfected epithelial cell results in an inhibition of Cl{sup {minus}} permeability.« less
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Zeniya, Satoshi; Kuwahara, Hiroya; Daizo, Kaiichi; Watari, Akihiro; Kondoh, Masuo; Yoshida-Tanaka, Kie; Kaburagi, Hidetoshi; Asada, Ken; Nagata, Tetsuya; Nagahama, Masahiro; Yagi, Kiyohito; Yokota, Takanori
2018-05-17
Within the field of RNA therapeutics, antisense oligonucleotide-based therapeutics are a potentially powerful means of treating intractable diseases. However, if these therapeutics are used for the treatment of neurological disorders, safe yet efficient methods of delivering antisense oligonucleotides across the blood-brain barrier to the central nervous system must be developed. Here, we examined the use of angubindin-1, a binder to the tricellular tight junction, to modulate paracellular transport between brain microvascular endothelial cells in the blood-brain barrier for the delivery of antisense oligonucleotides to the central nervous system. This proof-of-concept study demonstrated that intravenously injected angubindin-1 increased the permeability of the blood-brain barrier and enabled transient delivery of subsequently administered antisense oligonucleotides into the mouse brain and spinal cord, leading to silencing of a target RNA without any overt adverse effects. We also found that two bicellular tight junction modulators did not produce such a silencing effect, suggesting that the tricellular tight junction is likely a better target for the delivery of antisense oligonucleotides than the bicellular tight junction. Our delivery strategy of modulating the tricellular tight junction in the blood-brain barrier via angubindin-1 provides a novel avenue of research for the development of antisense oligonucleotide-based therapeutics for the treatment of neurological disorders. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.
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Bierhoff, H; Schmitz, K; Maass, F; Ye, J; Grummt, I
2010-01-01
Alternative transcription of the same gene in sense and antisense orientation regulates expression of protein-coding genes. Here we show that noncoding RNA (ncRNA) in sense and antisense orientation also controls transcription of rRNA genes (rDNA). rDNA exists in two types of chromatin--a euchromatic conformation that is permissive to transcription and a heterochromatic conformation that is transcriptionally silent. Silencing of rDNA is mediated by NoRC, a chromatin-remodeling complex that triggers heterochromatin formation. NoRC function requires RNA that is complementary to the rDNA promoter (pRNA). pRNA forms a DNA:RNA triplex with a regulatory element in the rDNA promoter, and this triplex structure is recognized by DNMT3b. The results imply that triplex-mediated targeting of DNMT3b to specific sequences may be a common pathway in epigenetic regulation. We also show that rDNA is transcribed in antisense orientation. The level of antisense RNA (asRNA) is down-regulated in cancer cells and up-regulated in senescent cells. Ectopic asRNA triggers trimethylation of histone H4 at lysine 20 (H4K20me3), suggesting that antisense transcripts guide the histone methyltransferase Suv4-20 to rDNA. The results reveal that noncoding RNAs in sense and antisense orientation are important determinants of the epigenetic state of rDNA.
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Pituitary adenylate cyclase-activating polypeptide (PACAP) in zebrafish models of nephrotic syndrome
van den Heuvel, Lambertus P.; Khodaparast, Laleh; Khodaparast, Ladan; van Geet, Chris; Freson, Kathleen
2017-01-01
Pituitary adenylate cyclase-activating polypeptide (PACAP) is an inhibitor of megakaryopoiesis and platelet function. Recently, PACAP deficiency was observed in children with nephrotic syndrome (NS), associated with increased platelet count and aggregability and increased risk of thrombosis. To further study PACAP deficiency in NS, we used transgenic Tg(cd41:EGFP) zebrafish with GFP-labeled thrombocytes. We generated two models for congenital NS, a morpholino injected model targeting nphs1 (nephrin), which is mutated in the Finnish-type congenital NS. The second model was induced by exposure to the nephrotoxic compound adriamycin. Nephrin RNA expression was quantified and zebrafish embryos were live-screened for proteinuria and pericardial edema as evidence of renal impairment. Protein levels of PACAP and its binding-protein ceruloplasmin were measured and GFP-labeled thrombocytes were quantified. We also evaluated the effects of PACAP morpholino injection and the rescue effects of PACAP-38 peptide in both congenital NS models. Nephrin downregulation and pericardial edema were observed in both nephrin morpholino injected and adriamycin exposed congenital NS models. However, PACAP deficiency was demonstrated only in the adriamycin exposed condition. Ceruloplasmin levels and the number of GFP-labeled thrombocytes remained unchanged in both models. PACAP morpholino injections worsened survival rates and the edema phenotype in both congenital NS models while injection with human PACAP-38 could only rescue the adriamycin exposed model. We hereby report, for the first time, PACAP deficiency in a NS zebrafish model as a consequence of adriamycin exposure. However, distinct from the human congenital NS, both zebrafish models retained normal levels of ceruloplasmin and thrombocytes. We further extend the renoprotective effects of the PACAP-38 peptide against adriamycin toxicity in zebrafish. PMID:28759637
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USDA-ARS?s Scientific Manuscript database
Endogenous mRNA-antisense transcripts are involved in regulation of a wide range of biological processes including muscle development and quality traits of farm animals. Standard RNA-Seq can be used to identify sense-antisense transcripts. However, strand-specific RNA-Seq is required to resolve ambi...
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ERIC Educational Resources Information Center
Canal, Clinton E.; Chang, Qing; Gold, Paul E.
2008-01-01
Infusions of CREB antisense into the amygdala prior to training impair memory for aversive tasks, suggesting that the antisense may interfere with CRE-mediated gene transcription and protein synthesis important for the formation of new memories within the amygdala. However, the amygdala also appears to modulate memory formation in distributed…
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Wnt1 and wnt10b function redundantly at the zebrafish midbrain-hindbrain boundary.
Lekven, Arne C; Buckles, Gerri R; Kostakis, Nicholas; Moon, Randall T
2003-02-15
Wnt signals have been shown to be involved in multiple steps of vertebrate neural patterning, yet the relative contributions of individual Wnts to the process of brain regionalization is poorly understood. Wnt1 has been shown in the mouse to be required for the formation of the midbrain and the anterior hindbrain, but this function of wnt1 has not been explored in other model systems. Further, wnt1 is part of a Wnt cluster conserved in all vertebrates comprising wnt1 and wnt10b, yet the function of wnt10b during embryogenesis has not been explored. Here, we report that in zebrafish wnt10b is expressed in a pattern overlapping extensively with that of wnt1. We have generated a deficiency allele for these closely linked loci and performed morpholino antisense oligo knockdown to show that wnt1 and wnt10b provide partially redundant functions in the formation of the midbrain-hindbrain boundary (MHB). When both loci are deleted, the expression of pax2.1, en2, and her5 is lost in the ventral portion of the MHB beginning at the 8-somite stage. However, wnt1 and wnt10b are not required for the maintenance of fgf8, en3, wnt8b, or wnt3a expression. Embryos homozygous for the wnt1-wnt10b deficiency display a mild MHB phenotype, but are sensitized to reductions in either Pax2.1 or Fgf8; that is, in combination with mutant alleles of either of these loci, the morphological MHB is lost. Thus, wnt1 and wnt10b are required to maintain threshold levels of Pax2.1 and Fgf8 at the MHB. Copyright 2003 Elsevier Science (USA)
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Ito, Yusuke; Kobayashi, Sayako; Nakamura, Nobuhiro; Miyagi, Hisako; Esaki, Masahiro; Hoshijima, Kazuyuki; Hirose, Shigehisa
2013-01-01
Freshwater (FW) fishes actively absorb salt from their environment to tolerate low salinities. We previously reported that vacuolar-type H+-ATPase/mitochondrion-rich cells (H-MRCs) on the skin epithelium of zebrafish larvae (Danio rerio) are primary sites for Na+ uptake. In this study, in an attempt to clarify the mechanism for the Na+ uptake, we performed a systematic analysis of gene expression patterns of zebrafish carbonic anhydrase (CA) isoforms and found that, of 12 CA isoforms, CA2a and CA15a are highly expressed in H-MRCs at larval stages. The ca2a and ca15a mRNA expression were salinity-dependent; they were upregulated in 0.03 mM Na+ water whereas ca15a but not ca2a was down-regulated in 70 mM Na+ water. Immunohistochemistry demonstrated cytoplasmic distribution of CA2a and apical membrane localization of CA15a. Furthermore, cell surface immunofluorescence staining revealed external surface localization of CA15a. Depletion of either CA2a or CA15a expression by Morpholino antisense oligonucleotides resulted in a significant decrease in Na+ accumulation in H-MRCs. An in situ proximity ligation assay demonstrated a very close association of CA2a, CA15a, Na+/H+ exchanger 3b (Nhe3b), and Rhcg1 ammonia transporter in H-MRC. Our findings suggest that CA2a, CA15a, and Rhcg1 play a key role in Na+uptake under FW conditions by forming a transport metabolon with Nhe3b. PMID:23565095
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Devotta, Arun; Juraver-Geslin, Hugo; Gonzalez, Jose Antonio; Hong, Chang-Soo; Saint-Jeannet, Jean-Pierre
2016-01-01
Mandibulofacial dysostosis (MFD) is a human developmental disorder characterized by defects of the facial bones. It is the second most frequent craniofacial malformation after cleft lip and palate. Nager syndrome combines many features of MFD with a variety of limb defects. Mutations in SF3B4 (splicing factor 3b, subunit 4) gene, which encodes a component of the pre-mRNA spliceosomal complex, were recently identified as a cause for Nager syndrome, accounting for 60% of affected individuals. Nothing is known about the cellular pathogenesis underlying Nager type MFD. Here we describe the first animal model for Nager syndrome, generated by knocking down Sf3b4 function in Xenopus laevis embryos, using morpholino antisense oligonucleotides. Our results indicate that Sf3b4-depleted embryos show reduced expression of the neural crest genes sox10, snail2 and twist at the neural plate border, associated with a broadening of the neural plate. This phenotype can be rescued by injection of wild-type human SF3B4 mRNA but not by mRNAs carrying mutations that cause Nager syndrome. At the tailbud stage, morphant embryos had decreased sox10 and tfap2a expression in the pharyngeal arches, indicative of a reduced number of neural crest cells. Later in development, Sf3b4-depleted tadpoles exhibited hypoplasia of neural crest-derived craniofacial cartilages, phenocopying aspects of the craniofacial skeletal defects seen in Nager syndrome patients. With this animal model we are now poised to gain important insights into the etiology and pathogenesis of Nager type MFD, and to identify the molecular targets of Sf3b4. PMID:26874011
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Ramasamy, Srinivas; Wang, Hui; Quach, Helen Ngoc Bao; Sampath, Karuna
2006-04-15
In sexually reproducing organisms, primordial germ cells (PGCs) give rise to the cells of the germ line, the gametes. In many animals, PGCs are set apart from somatic cells early during embryogenesis. Work in Drosophila, C. elegans, Xenopus, and zebrafish has shown that maternally provided localized cytoplasmic determinants specify the germ line in these organisms (Raz, E., 2003. Primordial germ-cell development: the zebrafish perspective. Nat. Rev., Genet. 4, 690--700; Santos, A.C., Lehmann, R., 2004. Germ cell specification and migration in Drosophila and beyond. Curr. Biol. 14, R578-R589). The Drosophila RNA-binding protein, Staufen is required for germ cell formation, and mutations in stau result in a maternal effect grandchild-less phenotype (Schupbach,T., Weischaus, E., 1989. Female sterile mutations on the second chromosome of Drosophila melanogaster:1. Maternal effect mutations. Genetics 121, 101-17). Here we describe the functions of two zebrafish Staufen-related proteins, Stau1 and Stau2. When Stau1 or Stau2 functions are compromised in embryos by injecting antisense morpholino modified oligonucleotides or dominant-negative Stau peptides, germ layer patterning is not affected. However, expression of the PGC marker vasa is not maintained. Furthermore, expression of a green fluorescent protein (GFP):nanos 3'UTR fusion protein in germ cells shows that PGC migration is aberrant, and the mis-migrating PGCs do not survive in Stau-compromised embryos. Stau2 is also required for survival of neurons in the central nervous system (CNS). These phenotypes are rescued by co-injection of Drosophila stau mRNA. Thus, staufen has an evolutionarily conserved function in germ cells. In addition, we have identified a function for Stau proteins in PGC migration.
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Xu, Jin; Lu, Zhigang; Xu, Mingming; Pan, Ling; Deng, Yi; Xie, Xiaohu; Liu, Huifen; Ding, Shixiong; Hurd, Yasmin L.; Pasternak, Gavril W.; Klein, Robert J.; Cartegni, Luca
2014-01-01
Single nucleotide polymorphisms (SNPs) in the OPRM1 gene have been associated with vulnerability to opioid dependence. The current study identifies an association of an intronic SNP (rs9479757) with the severity of heroin addiction among Han-Chinese male heroin addicts. Individual SNP analysis and haplotype-based analysis with additional SNPs in the OPRM1 locus showed that mild heroin addiction was associated with the AG genotype, whereas severe heroin addiction was associated with the GG genotype. In vitro studies such as electrophoretic mobility shift assay, minigene, siRNA, and antisense morpholino oligonucleotide studies have identified heterogeneous nuclear ribonucleoprotein H (hnRNPH) as the major binding partner for the G-containing SNP site. The G-to-A transition weakens hnRNPH binding and facilitates exon 2 skipping, leading to altered expressions of OPRM1 splice-variant mRNAs and hMOR-1 proteins. Similar changes in splicing and hMOR-1 proteins were observed in human postmortem prefrontal cortex with the AG genotype of this SNP when compared with the GG genotype. Interestingly, the altered splicing led to an increase in hMOR-1 protein levels despite decreased hMOR-1 mRNA levels, which is likely contributed by a concurrent increase in single transmembrane domain variants that have a chaperone-like function on MOR-1 protein stability. Our studies delineate the role of this SNP as a modifier of OPRM1 alternative splicing via hnRNPH interactions, and suggest a functional link between an SNP-containing splicing modifier and the severity of heroin addiction. PMID:25122903
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Rösel-Hillgärtner, Tanja Dorothe; Hung, Lee-Hsueh; Khrameeva, Ekaterina; Le Querrec, Patrick; Gelfand, Mikhail S.; Bindereif, Albrecht
2013-01-01
The U1 small nuclear ribonucleoprotein (snRNP)-specific U1C protein participates in 5′ splice site recognition and regulation of pre-mRNA splicing. Based on an RNA-Seq analysis in HeLa cells after U1C knockdown, we found a conserved, intra-U1 snRNP cross-regulation that links U1C and U1-70K expression through alternative splicing and U1 snRNP assembly. To investigate the underlying regulatory mechanism, we combined mutational minigene analysis, in vivo splice-site blocking by antisense morpholinos, and in vitro binding experiments. Alternative splicing of U1-70K pre-mRNA creates the normal (exons 7–8) and a non-productive mRNA isoform, whose balance is determined by U1C protein levels. The non-productive isoform is generated through a U1C-dependent alternative 3′ splice site, which requires an adjacent cluster of regulatory 5′ splice sites and binding of intact U1 snRNPs. As a result of nonsense-mediated decay (NMD) of the non-productive isoform, U1-70K mRNA and protein levels are down-regulated, and U1C incorporation into the U1 snRNP is impaired. U1-70K/U1C-deficient particles are assembled, shifting the alternative splicing balance back towards productive U1-70K splicing, and restoring assembly of intact U1 snRNPs. Taken together, we established a novel feedback regulation that controls U1-70K/U1C homeostasis and ensures correct U1 snRNP assembly and function. PMID:24146627
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Small GTPase R-Ras participates in neural tube formation in zebrafish embryonic spinal cord.
Ohata, Shinya; Uga, Hideko; Okamoto, Hitoshi; Katada, Toshiaki
2018-06-27
Ras related (R-Ras), a small GTPase, is involved in the maintenance of apico-basal polarity in neuroepithelial cells of the zebrafish hindbrain, axonal collapse in cultured murine hippocampal neurons, and maturation of blood vessels in adult mice. However, the role of R-Ras in neural tube formation remains unknown. Using antisense morpholino oligonucleotides (AMOs), we found that in the spinal cord of zebrafish embryos, the lumen was formed bilaterally in rras morphants, whereas it was formed at the midline in control embryos. As AMO can cause off-target effects, we generated rras mutant zebrafish lines using CRISPR/Cas9 technology. Although these rras mutant embryos did not have a bilateral lumen in the spinal cord, the following findings suggest that the phenotype is unlikely due to an off-target effect of rras AMO: 1) The rras morphant phenotype was rescued by an injection of AMO-resistant rras mRNA, and 2) a bilaterally segregated spinal cord was not observed in rras mutant embryos injected with rras AMO. The results suggest that the function of other ras family genes may be redundant in rras mutants. Previous research reported a bilaterally formed lumen in the spinal cord of zebrafish embryos with a mutation in a planar cell polarity (PCP) gene, van gogh-like 2 (vangl2). In the present study, in cultured cells, R-Ras was co-immunoprecipitated with Vangl2 but not with another PCP regulator, Pricke1. Interestingly, the interaction between R-Ras and Vangl2 was stronger in guanine-nucleotide free point mutants of R-Ras than in wild-type or constitutively active (GTP-bound) forms of R-Ras. R-Ras may regulate neural tube formation in cooperation with Vangl2 in the developing zebrafish spinal cord. Copyright © 2018 Elsevier Inc. All rights reserved.
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Fgf signaling is required for zebrafish tooth development.
Jackman, William R; Draper, Bruce W; Stock, David W
2004-10-01
We have investigated fibroblast growth factor (FGF) signaling during the development of the zebrafish pharyngeal dentition with the goal of uncovering novel roles for FGFs in tooth development as well as phylogenetic and topographic diversity in the tooth developmental pathway. We found that the tooth-related expression of several zebrafish genes is similar to that of their mouse orthologs, including both epithelial and mesenchymal markers. Additionally, significant differences in gene expression between zebrafish and mouse teeth are indicated by the apparent lack of fgf8 and pax9 expression in zebrafish tooth germs. FGF receptor inhibition with SU5402 at 32 h blocked dental epithelial morphogenesis and tooth mineralization. While the pharyngeal epithelium remained intact as judged by normal pitx2 expression, not only was the mesenchymal expression of lhx6 and lhx7 eliminated as expected from mouse studies, but the epithelial expression of dlx2a, dlx2b, fgf3, and fgf4 was as well. This latter result provides novel evidence that the dental epithelium is a target of FGF signaling. However, the failure of SU5402 to block localized expression of pitx2 suggests that the earliest steps of tooth initiation are FGF-independent. Investigations of specific FGF ligands with morpholino antisense oligonucleotides revealed only a mild tooth shape phenotype following fgf4 knockdown, while fgf8 inhibition revealed only a subtle down-regulation of dental dlx2b expression with no apparent effect on tooth morphology. Our results suggest redundant FGF signals target the dental epithelium and together are required for dental morphogenesis. Further work will be required to elucidate the nature of these signals, particularly with respect to their origins and whether they act through the mesenchyme.
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Catena, Raul; Muniz-Medina, Vanessa; Moralejo, Beatriz; Javierre, Biola; Best, Carolyn J M; Emmert-Buck, Michael R; Green, Jeffrey E; Baker, Carl C; Calvo, Alfonso
2007-05-15
Vascular endothelial growth factor (VEGF) is a proangiogenic factor upregulated in many tumors. The alternative splicing of VEGF mRNA renders 3 major isoforms of 121, 165 and 189 amino-acids in humans (1 less amino-acid for each mouse VEGF isoform). We have designed isoform specific real time QRT-PCR assays to quantitate VEGF transcripts in mouse and human normal and malignant prostates. In the human normal prostate, VEGF(165) was the predominant isoform (62.8% +/- 5.2%), followed by VEGF(121) (22.5% +/- 6.3%) and VEGF(189) (p < 0.001) (14.6% +/- 2.1%). Prostate tumors showed a significant increase in the percentage of VEGF(121) and decreases in VEGF(165) (p < 0.01) and VEGF(189) (p < 0.05). However, the amount of total VEGF mRNA was similar between normal and malignant prostates. VEGF(164) was the transcript with the highest expression in the mouse normal prostate. Unlike human prostate cancer, tumors from TRAMP mice demonstrated a significant increase in total VEGF mRNA levels and in each of the VEGF isoforms, without changes in the relative isoform ratios. Morpholino phosphorodiamide antisense oligonucleotide technology was used to increase the relative amount of VEGF(121) while proportionally decreasing VEGF(165) and VEGF(189) levels in human prostate cell lines, through the modification of alternative splicing, without changing transcription levels and total amount of VEGF. The increase in the VEGF(121)/VEGF(165-189) ratio in PC3 cells resulted in a dramatic increase in prostate tumor angiogenesis in vivo. Our results underscore the importance of VEGF(121) in human prostate carcinoma and demonstrate that the relative expression of the different VEGF isoforms has an impact on prostate carcinogenesis. (c) 2007 Wiley-Liss, Inc.
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Horn, Eberhard R; El-Yamany, Nabil A; Gradl, Dietmar
2013-02-15
Development of the amphibian vestibular organ is regulated by molecular and neuronal mechanisms and by environmental input. The molecular component includes inductive signals derived from neural tissue of the hindbrain and from the surrounding mesoderm. The integrity of hindbrain patterning, on the other hand, depends on instructive signals from the isthmus organizer of the midbrain, including the transcription factor XTcf-4. If the development of the vestibular system depends on the integrity of the isthmus as the organizing centre, suppression of isthmus maintenance should modify vestibular morphology and function. We tested this hypothesis by downregulation of the transcription factor XTcf-4. 10 pmol l(-1) XTcf-4-specific antisense morpholino oligonucleotide was injected in one blastomere of two-cell-stage embryos of Xenopus laevis. For reconstitution experiments, 500 pg mRNA of the repressing XTcf-4A isoform or the activating XTcf-4C isoform were co-injected. Overexpression experiments were included using the same isoforms. Otoconia formation and vestibular controlled behaviour such as the roll-induced vestibuloocular reflex (rVOR) and swimming were recorded two weeks later. In 50% of tadpoles, downregulation of XTcf-4 induced (1) a depression of otoconia formation accompanied by a reduction of the rVOR, (2) abnormal tail development and (3) loop swimming behaviour. (4) All effects were rescued by co-injection of XTcf-4C but not, or only partially, by XTcf-4A. (5) Overexpression of XTcf-4A caused similar morphological and rVOR modifications as XTcf-4 depletion, while overexpression of XTcf-4C had no effect. Because XTcf-4C has been described as an essential factor for isthmus development, we postulate that the isthmus is strongly involved in vestibular development.
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Chiou, Ming-Jyun; Chao, Tsung-Tai; Wu, Jen-Leih; Kuo, Ching-Ming; Chen, Jyh-Yih
2006-10-20
During mouse embryogenesis, CTGF/CCN2 is expressed in zones containing hypertrophic chondroctyes and calcifying cartilage such as long bones, ribs, vertebral column, and phalanges. But in fish, its expression is yet unclear. Development of the vertebrae is morphologically similar among vertebrates, indicating that the underlying mechanism regulating the process is highly conserved during evolution. Analysis of 3.2kb of the CTGF/CCN2 proximal promoter sequence revealed a consensus TATAA box, putative AP1, Brn-2, CdxA, C/EBP alpha, C/EBP beta, C-Ets-, delta E, HFH-2, and HSF2 binding sites. Transient expression experiments with a 5'-deletion revealed at least 4 regulatory regions in the zebrafish CTGF/CCN2 gene, 2 with a stimulatory effect on transcription and 2 with an apparent inhibitory effect after IGF-I treatment in the ZFL cell line. To study the promoter-specific expression, we constructed a series of CTGF/CCN2 (3.0-, 2.5-, 2.0-, 1.5-, 1.0-, and 0.4-kb) promoter-driven green fluorescent protein (GFP) fragments encoding the GFP cDNA transgene which was microinjected into zebrafish embryos. Morphological studies of transgenic zebrafish indicated that the CTGF/CCN2 promoter-driven GFP transcripts appeared in the notochord. Targeted knockdown of the CTGF/CCN2 gene by two antisense morpholino oligonucleotides resulted in disruptions to notochord development. From a comparative point of view, this study of the CTGF/CCN2 gene in zebrafish may correlate well with those previously published on the mouse. These molecular results suggest that CTGF/CCN2 plays an important role in notochord development and is required for general embryonic development.
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Rex, Maria; Hilton, Emma; Old, Robert
2002-03-01
We have investigated the induction of the six Xenopus nodal-related genes, Xnr1-Xnr6, by maternal determinants. The beta-catenin pathway was modelled by stimulation using Xwnt8, activin-like signalling was modelled by activin, and VegT action was studied by overexpression in animal cap explants. Combinations of factors were examined, and previously unrecognised interactions were revealed in animal caps and whole embryos. For the induction of Xnr5 and Xnr6 in whole embryos, using a beta-catenin antisense morpholino oligonucleotide or a dominant negative XTcf3, we have demonstrated an absolute permissive requirement for the beta-catenin/Tcf pathway, in addition to the requirement for VegT action. In animal caps Xnr5 and Xnr6 are induced in response to VegT overexpression, and this induction is dependent upon the concomitant activation of the beta-catenin pathway that VegT initiates in animal caps. For the induction of Xnr3, VegT interacts negatively so as to inhibit the induction otherwise observed with wnt-signalling alone. The negative effect of VegT is not the result of a general inhibition of wnt-signalling, and does not result from an inhibition of wnt-induced siamois expression. A 294 bp proximal promoter fragment of the Xnr3 gene is sufficient to mediate the negative effect of VegT. Further experiments, employing cycloheximide to examine the dependence of Xnr gene expression upon proteins translated after the mid-blastula stage, demonstrated that Xnrs 4, 5 and 6 are 'primary' Xnr genes whose expression in the late blastula is solely dependent upon factors present before the mid-blastula stage.
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Hirata, Hiromi; Nanda, Indrajit; van Riesen, Anne; McMichael, Gai; Hu, Hao; Hambrock, Melanie; Papon, Marie-Amélie; Fischer, Ute; Marouillat, Sylviane; Ding, Can; Alirol, Servane; Bienek, Melanie; Preisler-Adams, Sabine; Grimme, Astrid; Seelow, Dominik; Webster, Richard; Haan, Eric; MacLennan, Alastair; Stenzel, Werner; Yap, Tzu Ying; Gardner, Alison; Nguyen, Lam Son; Shaw, Marie; Lebrun, Nicolas; Haas, Stefan A.; Kress, Wolfram; Haaf, Thomas; Schellenberger, Elke; Chelly, Jamel; Viot, Géraldine; Shaffer, Lisa G.; Rosenfeld, Jill A.; Kramer, Nancy; Falk, Rena; El-Khechen, Dima; Escobar, Luis F.; Hennekam, Raoul; Wieacker, Peter; Hübner, Christoph; Ropers, Hans-Hilger; Gecz, Jozef; Schuelke, Markus; Laumonnier, Frédéric; Kalscheuer, Vera M.
2013-01-01
Arthrogryposis multiplex congenita (AMC) is caused by heterogeneous pathologies leading to multiple antenatal joint contractures through fetal akinesia. Understanding the pathophysiology of this disorder is important for clinical care of the affected individuals and genetic counseling of the families. We thus aimed to establish the genetic basis of an AMC subtype that is associated with multiple dysmorphic features and intellectual disability (ID). We used haplotype analysis, next-generation sequencing, array comparative genomic hybridization, and chromosome breakpoint mapping to identify the pathogenic mutations in families and simplex cases. Suspected disease variants were verified by cosegregation analysis. We identified disease-causing mutations in the zinc-finger gene ZC4H2 in four families affected by X-linked AMC plus ID and one family affected by cerebral palsy. Several heterozygous females were also affected, but to a lesser degree. Furthermore, we found two ZC4H2 deletions and one rearrangement in two female and one male unrelated simplex cases, respectively. In mouse primary hippocampal neurons, transiently produced ZC4H2 localized to the postsynaptic compartment of excitatory synapses, and the altered protein influenced dendritic spine density. In zebrafish, antisense-morpholino-mediated zc4h2 knockdown caused abnormal swimming and impaired α-motoneuron development. All missense mutations identified herein failed to rescue the swimming defect of zebrafish morphants. We conclude that ZC4H2 point mutations, rearrangements, and small deletions cause a clinically variable broad-spectrum neurodevelopmental disorder of the central and peripheral nervous systems in both familial and simplex cases of both sexes. Our results highlight the importance of ZC4H2 for genetic testing of individuals presenting with ID plus muscle weakness and minor or major forms of AMC. PMID:23623388
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Nuzzo, F; Bulato, C; Nielsen, B I; Lee, K; Wielders, S J; Simioni, P; Key, N S; Castoldi, E
2015-03-01
Coagulation factor V (FV) deficiency is a rare autosomal recessive bleeding disorder. We investigated a patient with severe FV deficiency (FV:C < 3%) and moderate bleeding symptoms. Thrombin generation experiments showed residual FV expression in the patient's plasma, which was quantified as 0.7 ± 0.3% by a sensitive prothrombinase-based assay. F5 gene sequencing identified a novel missense mutation in exon 4 (c.578G>C, p.Cys193Ser), predicting the abolition of a conserved disulphide bridge, and an apparently synonymous variant in exon 8 (c.1281C>G). The observation that half of the patient's F5 mRNA lacked the last 18 nucleotides of exon 8 prompted us to re-evaluate the c.1281C>G variant for its possible effects on splicing. Bioinformatics sequence analysis predicted that this transversion would activate a cryptic donor splice site and abolish an exonic splicing enhancer. Characterization in a F5 minigene model confirmed that the c.1281C>G variant was responsible for the patient's splicing defect, which could be partially corrected by a mutation-specific morpholino antisense oligonucleotide. The aberrantly spliced F5 mRNA, whose stability was similar to that of the normal mRNA, encoded a putative FV mutant lacking amino acids 427-432. Expression in COS-1 cells indicated that the mutant protein is poorly secreted and not functional. In conclusion, the c.1281C>G mutation, which was predicted to be translationally silent and hence neutral, causes FV deficiency by impairing pre-mRNA splicing. This finding underscores the importance of cDNA analysis for the correct assessment of exonic mutations. © 2014 John Wiley & Sons Ltd.
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Sema4d is required for the development of the hindbrain boundary and skeletal muscle in zebrafish
DOE Office of Scientific and Technical Information (OSTI.GOV)
Yang, Jie; Zeng, Zhen; Wei, Juncheng
2013-04-05
Highlights: ► Sema4d was expressed at all developmental stages of zebrafish. ► Knockdown of sema4d in embryos resulted in defects in the hindbrain and the trunk structure. ► Knockdown of sema4d in embryos upregulated the expression of three hindbrain rhombomere markers. ► Knockdown of sema4d in embryos increased the expression of myogenic regulatory factors. ► Knockdown of sema4d in embryos resulted in an obvious increase of cell apoptosis. -- Abstract: Semaphorin4d (SEMA4D), also known as CD100, an oligodendrocyte secreted R-Ras GTPase-activating protein (GAP), affecting axonal growth is involved in a range of processes including cell adhesion, motility, angiogenesis, immune responsesmore » and tumour progression. However, its actual physiological mechanisms and its role in development remain unclear. This study has focused on the role of sema4d in the development and expression patterns in zebrafish embryos and the effect of its suppression on development using sema4d-specific antisense morpholino-oligonucleotides. In this study the knockdown of sema4d, expressed at all developmental stages, lead to defects in the hindbrain and trunk structure of zebrafish embryos. In addition, these phenotypes appeared to be associated with the abnormal expression of three hindbrain rhombomere boundary markers, wnt1, epha4a and foxb1.2, and two myogenic regulatory factors, myod and myog. Further, a notable increase of cell apoptosis appeared in the sema4d knockdown embryos, while no obvious reduction in cell proliferation was observed. Collectively, these data suggest that sema4d plays an important role in the development of the hindbrain and skeletal muscle.« less
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Hedgehog signaling is required at multiple stages of zebrafish tooth development.
Jackman, William R; Yoo, James J; Stock, David W
2010-11-30
The accessibility of the developing zebrafish pharyngeal dentition makes it an advantageous system in which to study many aspects of tooth development from early initiation to late morphogenesis. In mammals, hedgehog signaling is known to be essential for multiple stages of odontogenesis; however, potential roles for the pathway during initiation of tooth development or in later morphogenesis are incompletely understood. We have identified mRNA expression of the hedgehog ligands shha and the receptors ptc1 and ptc2 during zebrafish pharyngeal tooth development. We looked for, but did not detect, tooth germ expression of the other known zebrafish hedgehog ligands shhb, dhh, ihha, or ihhb, suggesting that as in mammals, only Shh participates in zebrafish tooth development. Supporting this idea, we found that morphological and gene expression evidence of tooth initiation is eliminated in shha mutant embryos, and that morpholino antisense oligonucleotide knockdown of shha, but not shhb, function prevents mature tooth formation. Hedgehog pathway inhibition with the antagonist compound cyclopamine affected tooth formation at each stage in which we applied it: arresting development at early stages and disrupting mature tooth morphology when applied later. These results suggest that hedgehog signaling is required continuously during odontogenesis. In contrast, over-expression of shha had no effect on the developing dentition, possibly because shha is normally extensively expressed in the zebrafish pharyngeal region. We have identified previously unknown requirements for hedgehog signaling for early tooth initiation and later morphogenesis. The similarity of our results with data from mouse and other vertebrates suggests that despite gene duplication and changes in the location of where teeth form, the roles of hedgehog signaling in tooth development have been largely conserved during evolution.
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Cyp1b1 Regulates Ocular Fissure Closure Through a Retinoic Acid–Independent Pathway
Williams, Antionette L.; Eason, Jessica; Chawla, Bahaar; Bohnsack, Brenda L.
2017-01-01
Purpose Mutations in the CYP1B1 gene are the most commonly identified genetic causes of primary infantile-onset glaucoma. Despite this disease association, the role of CYP1B1 in eye development and its in vivo substrate remain unknown. In the present study, we used zebrafish to elucidate the mechanism by which cyp1b1 regulates eye development. Methods Zebrafish eye and neural crest development were analyzed using live imaging of transgenic zebrafish embryos, in situ hybridization, immunostaining, TUNEL assay, and methylacrylate sections. Cyp1b1 and retinoic acid (RA) levels were genetically (morpholino oligonucleotide antisense and mRNA) and pharmacologically manipulated to examine gene function. Results Using zebrafish, we observed that cyp1b1 was expressed in a specific spatiotemporal pattern in the ocular fissures of the developing zebrafish retina and regulated fissure patency. Decreased Cyp1b1 resulted in the premature breakdown of laminin in the ventral fissure and altered subsequent neural crest migration into the anterior segment. In contrast, cyp1b1 overexpression inhibited cell survival in the ventral ocular fissure and prevented fissure closure via an RA-independent pathway. Cyp1b1 overexpression also inhibited the ocular expression of vsx2, pax6a, and pax6b and increased the extraocular expression of shha. Importantly, embryos injected with human wild-type but not mutant CYP1B1 mRNA also showed colobomas, demonstrating the evolutionary and functional conservation of gene function between species. Conclusions Cyp1b1 regulation of ocular fissure closure indirectly affects neural crest migration and development through an RA-independent pathway. These studies provide insight into the role of Cyp1b1 in eye development and further elucidate the pathogenesis of primary infantile-onset glaucoma. PMID:28192799
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Rizzo, Francesca; Coffman, James A; Arnone, Maria Ina
2016-08-01
Elk proteins are Ets family transcription factors that regulate cell proliferation, survival, and differentiation in response to ERK (extracellular-signal regulated kinase)-mediated phosphorylation. Here we report the embryonic expression and function of Sp-Elk, the single Elk gene of the sea urchin Strongylocentrotus purpuratus. Sp-Elk is zygotically expressed throughout the embryo beginning at late cleavage stage, with peak expression occurring at blastula stage. Morpholino antisense-mediated knockdown of Sp-Elk causes blastula-stage developmental arrest and embryo disintegration due to apoptosis, a phenotype that is rescued by wild-type Elk mRNA. Development is also rescued by Elk mRNA encoding a serine to aspartic acid substitution (S402D) that mimics ERK-mediated phosphorylation of a conserved site that enhances DNA binding, but not by Elk mRNA encoding an alanine substitution at the same site (S402A). This demonstrates both that the apoptotic phenotype of the morphants is specifically caused by Elk depletion, and that phosphorylation of serine 402 of Sp-Elk is critical for its anti-apoptotic function. Knockdown of Sp-Elk results in under-expression of several regulatory genes involved in cell fate specification, cell cycle control, and survival signaling, including the transcriptional regulator Sp-Runt-1 and its target Sp-PKC1, both of which were shown previously to be required for cell survival during embryogenesis. Both Sp-Runt-1 and Sp-PKC1 have sequences upstream of their transcription start sites that specifically bind Sp-Elk. These results indicate that Sp-Elk is the signal-dependent activator of a feed-forward gene regulatory circuit, consisting also of Sp-Runt-1 and Sp-PKC1, which actively suppresses apoptosis in the early embryo. Copyright © 2016 Elsevier Inc. All rights reserved.
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Knierim, Ellen; Hirata, Hiromi; Wolf, Nicole I.; Morales-Gonzalez, Susanne; Schottmann, Gudrun; Tanaka, Yu; Rudnik-Schöneborn, Sabine; Orgeur, Mickael; Zerres, Klaus; Vogt, Stefanie; van Riesen, Anne; Gill, Esther; Seifert, Franziska; Zwirner, Angelika; Kirschner, Janbernd; Goebel, Hans Hilmar; Hübner, Christoph; Stricker, Sigmar; Meierhofer, David; Stenzel, Werner; Schuelke, Markus
2016-01-01
Transcriptional signal cointegrators associate with transcription factors or nuclear receptors and coregulate tissue-specific gene transcription. We report on recessive loss-of-function mutations in two genes (TRIP4 and ASCC1) that encode subunits of the nuclear activating signal cointegrator 1 (ASC-1) complex. We used autozygosity mapping and whole-exome sequencing to search for pathogenic mutations in four families. Affected individuals presented with prenatal-onset spinal muscular atrophy (SMA), multiple congenital contractures (arthrogryposis multiplex congenita), respiratory distress, and congenital bone fractures. We identified homozygous and compound-heterozygous nonsense and frameshift TRIP4 and ASCC1 mutations that led to a truncation or the entire absence of the respective proteins and cosegregated with the disease phenotype. Trip4 and Ascc1 have identical expression patterns in 17.5-day-old mouse embryos with high expression levels in the spinal cord, brain, paraspinal ganglia, thyroid, and submandibular glands. Antisense morpholino-mediated knockdown of either trip4 or ascc1 in zebrafish disrupted the highly patterned and coordinated process of α-motoneuron outgrowth and formation of myotomes and neuromuscular junctions and led to a swimming defect in the larvae. Immunoprecipitation of the ASC-1 complex consistently copurified cysteine and glycine rich protein 1 (CSRP1), a transcriptional cofactor, which is known to be involved in spinal cord regeneration upon injury in adult zebrafish. ASCC1 mutant fibroblasts downregulated genes associated with neurogenesis, neuronal migration, and pathfinding (SERPINF1, DAB1, SEMA3D, SEMA3A), as well as with bone development (TNFRSF11B, RASSF2, STC1). Our findings indicate that the dysfunction of a transcriptional coactivator complex can result in a clinical syndrome affecting the neuromuscular system. PMID:26924529
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Coate, Thomas M.; Swanson, Tracy L.; Copenhaver, Philip F.
2011-01-01
Reverse signaling via GPI-linked Ephrins may help control cell proliferation and outgrowth within the nervous system, but the mechanisms underlying this process remain poorly understood. In the embryonic enteric nervous system (ENS) of the moth Manduca sexta, migratory neurons forming the enteric plexus (EP cells) express a single Ephrin ligand (GPI-linked MsEphrin), while adjacent midline cells that are inhibitory to migration express the cognate receptor (MsEph). Knocking down MsEph receptor expression in cultured embryos with antisense morpholino oligonucleotides allowed the EP cells to cross the midline inappropriately, consistent with the model that reverse signaling via MsEphrin mediates a repulsive response in the ENS. Src family kinases have been implicated in reverse signaling by type-A Ephrins in other contexts, and MsEphrin colocalizes with activated forms of endogenous Src in the leading processes of the EP cells. Pharmacological inhibition of Src within the developing ENS induced aberrant midline crossovers, similar to the effect of blocking MsEphrin reverse signaling. Hyperstimulating MsEphrin reverse signaling with MsEph-Fc fusion proteins induced the rapid activation of endogenous Src specifically within the EP cells, as assayed by Western blots of single embryonic gut explants and by whole-mount immunostaining of cultured embryos. In longer cultures, treatment with MsEph-Fc caused a global inhibition of EP cell migration and outgrowth, an effect that was prevented by inhibiting Src activation. These results support the model that MsEphrin reverse signaling induces the Src-dependent retraction of EP cell processes away from the enteric midline, thereby helping to confine the neurons to their appropriate pathways. PMID:19295147
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Nakashima, N; Tamura, T
2013-06-01
Here, we report on the construction of doxycycline (tetracycline analogue)-inducible vectors that express antisense RNAs in Escherichia coli. Using these vectors, the expression of genes of interest can be silenced conditionally. The expression of antisense RNAs from the vectors was more tightly regulated than the previously constructed isopropyl-β-D-galactopyranoside-inducible vectors. Furthermore, expression levels of antisense RNAs were enhanced by combining the doxycycline-inducible promoter with the T7 promoter-T7 RNA polymerase system; the T7 RNA polymerase gene, under control of the doxycycline-inducible promoter, was integrated into the lacZ locus of the genome without leaving any antibiotic marker. These vectors are useful for investigating gene functions or altering cell phenotypes for biotechnological and industrial applications. A gene silencing method using antisense RNAs in Escherichia coli is described, which facilitates the investigation of bacterial gene function. In particular, the method is suitable for comprehensive analyses or phenotypic analyses of genes essential for growth. Here, we describe expansion of vector variations for expressing antisense RNAs, allowing choice of a vector appropriate for the target genes or experimental purpose. © 2013 The Society for Applied Microbiology.
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Characterization of Antisense Transformed Plants Deficient in the Tobacco Anionic Peroxidase.
Lagrimini, L. M.; Gingas, V.; Finger, F.; Rothstein, S.; Liu, TTY.
1997-01-01
On the basis of the biological compounds that they metabolize, plant peroxidases have long been implicated in plant growth, cell wall biogenesis, lignification, and host defenses. Transgenic tobacco (Nicotiana tabacum L.) plants that underexpress anionic peroxidase were generated using antisense RNA. The antisense RNA was found to be specific for the anionic isoenzyme and highly effective, reducing endogenous transcript levels and total peroxidase activity by as much as 1600-fold. Antisense-transformed plants appeared normal at initial observation; however, growth studies showed that plants with reduced peroxidase activity grow taller and flower sooner than control plants. In contrast, previously transformed plants overproducing anionic peroxidase were shorter and flowered later than controls. Axillary buds were more developed in antisense-transformed plants and less developed in plants overproducing this enzyme. It was found that the lignin content in leaf, stem, and root was unchanged in antisense-transformed plants, which does not support a role for anionic peroxidase in the lignification of secondary xylem vessels. However, studies of wounded tissue show some reduction in wound-induced deposition of lignin-like polymers. The data support a possible role for tobacco anionic peroxidase in host defenses but not without a reduction in growth potential. PMID:12223765
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Characterization of Antisense Transformed Plants Deficient in the Tobacco Anionic Peroxidase.
Lagrimini, L. M.; Gingas, V.; Finger, F.; Rothstein, S.; Liu, TTY.
1997-08-01
On the basis of the biological compounds that they metabolize, plant peroxidases have long been implicated in plant growth, cell wall biogenesis, lignification, and host defenses. Transgenic tobacco (Nicotiana tabacum L.) plants that underexpress anionic peroxidase were generated using antisense RNA. The antisense RNA was found to be specific for the anionic isoenzyme and highly effective, reducing endogenous transcript levels and total peroxidase activity by as much as 1600-fold. Antisense-transformed plants appeared normal at initial observation; however, growth studies showed that plants with reduced peroxidase activity grow taller and flower sooner than control plants. In contrast, previously transformed plants overproducing anionic peroxidase were shorter and flowered later than controls. Axillary buds were more developed in antisense-transformed plants and less developed in plants overproducing this enzyme. It was found that the lignin content in leaf, stem, and root was unchanged in antisense-transformed plants, which does not support a role for anionic peroxidase in the lignification of secondary xylem vessels. However, studies of wounded tissue show some reduction in wound-induced deposition of lignin-like polymers. The data support a possible role for tobacco anionic peroxidase in host defenses but not without a reduction in growth potential.
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Leonetti, C; Biroccio, A; Benassi, B; Stringaro, A; Stoppacciaro, A; Semple, S C; Zupi, G
2001-06-01
Phosphorothioate c-myc antisense oligodeoxynucleotides [S]ODNs (free INX-6295) were encapsulated in a new liposome formulation and the antitumor activity was compared to the unencapsulated antisense in a human melanoma xenograft. The systemic administration of INX-6295 encapsulated in stabilized antisense lipid particles (SALP INX-6295) improved plasma AUC (area under the plasma concentration-time curve) and initial half-life of free INX-6295, resulting in a significant enhancement in tumor accumulation and improvement in tumor distribution of antisense oligodeoxynucleotides. Animals treated with SALP INX-6295 exhibited a prolonged reduction of c-myc expression, reduced tumor growth and increased mice survival. When administered in combination with cisplatin (DDP), SALP INX-6295 produced a complete tumor regression in approximately 30% of treated mice, which persisted for at least 60 days following the first cycle of treatment. Finally, the median survival of mice treated with DDP/SALP INX-6295 increased by 105% compared to 84% for animals treated with the combination DDP/free INX-6295. These data indicate that the biological activity and the therapeutic efficacy of c-myc antisense therapy may be improved when these agents are administered in lipid-based delivery systems.
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Optimization of gefitinib analogues with potent anticancer activity.
Yin, Kai-Hao; Hsieh, Yi-Han; Sulake, Rohidas S; Wang, Su-Pei; Chao, Jui-I; Chen, Chinpiao
2014-11-15
The interactions of gefitinib (Iressa) in EGFR are hydrogen bonding and van der Waals forces through quinazoline and aniline rings. However the morpholino group of gefitinib is poorly ordered due to its weak electron density. A series of novel piperazino analogues of gefitinib where morpholino group substituted with various piperazino groups were designed and synthesized. Most of them indicated significant anti-cancer activities against human cancer cell lines. In particular, compounds 52-54 showed excellent potency against cancer cells. Convergent synthetic approach has been developed for the synthesis of gefitinib intermediate which can lead to gefitinib as well as numerous analogues. Copyright © 2014 Elsevier Ltd. All rights reserved.
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Simultaneous assessment of glomerular filtration and barrier function in live zebrafish
Kotb, Ahmed M.; Müller, Tobias; Xie, Jing; Anand-Apte, Bela; Endlich, Nicole
2014-01-01
The zebrafish pronephros is a well-established model to study glomerular development, structure, and function. A few methods have been described to evaluate glomerular barrier function in zebrafish larvae so far. However, there is a need to assess glomerular filtration as well. In the present study, we extended the available methods by simultaneously measuring the intravascular clearances of Alexa fluor 647-conjugated 10-kDa dextran and FITC-conjugated 500-kDa dextran as indicators of glomerular filtration and barrier function, respectively. After intravascular injection of the dextrans, mean fluorescence intensities of both dextrans were measured in the cardinal vein of living zebrafish (4 days postfertilization) by confocal microscopy over time. We demonstrated that injected 10-kDa dextran was rapidly cleared from the circulation, became visible in the lumen of the pronephric tubule, quickly accumulated in tubular cells, and was detectably excreted at the cloaca. In contrast, 500-kDa dextran could not be visualized in the tubule at any time point. To check whether alterations in glomerular function can be quantified by our method, we injected morpholino oligonucleotides (MOs) against zebrafish nonmuscle myosin heavy chain IIA (zMyh9) or apolipoprotein L1 (zApol1). While glomerular filtration was reduced in zebrafish nonmuscle myosin heavy chain IIA MO-injected larvae, glomerular barrier function remained intact. In contrast, in zebrafish apolipoprotein L1 MO-injected larvae, glomerular barrier function was compromised as 500-kDa dextran disappeared from the circulation and became visible in tubular cells. In summary, we present a novel method that allows to simultaneously assess glomerular filtration and barrier function in live zebrafish. PMID:25298528
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Chen, Huei-Mei; Rosebrock, Adam P.; Khan, Sohail R.; Futcher, Bruce; Leatherwood, Janet K.
2012-01-01
In S. pombe, about 5% of genes are meiosis-specific and accumulate little or no mRNA during vegetative growth. Here we use Affymetrix tiling arrays to characterize transcripts in vegetative and meiotic cells. In vegetative cells, many meiotic genes, especially those induced in mid-meiosis, have abundant antisense transcripts. Disruption of the antisense transcription of three of these mid-meiotic genes allowed vegetative sense transcription. These results suggest that antisense transcription represses sense transcription of meiotic genes in vegetative cells. Although the mechanism(s) of antisense mediated transcription repression need to be further explored, our data indicates that RNAi machinery is not required for repression. Previously, we and others used non-strand specific methods to study splicing regulation of meiotic genes and concluded that 28 mid-meiotic genes are spliced only in meiosis. We now demonstrate that the “unspliced” signal in vegetative cells comes from the antisense RNA, not from unspliced sense RNA, and we argue against the idea that splicing regulates these mid-meiotic genes. Most of these mid-meiotic genes are induced in mid-meiosis by the forkhead transcription factor Mei4. Interestingly, deletion of a different forkhead transcription factor, Fkh2, allows low levels of sense expression of some mid-meiotic genes in vegetative cells. We propose that vegetative expression of mid-meiotic genes is repressed at least two independent ways: antisense transcription and Fkh2 repression. PMID:22238674
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Antisense imaging of gene expression in the brain in vivo
NASA Astrophysics Data System (ADS)
Shi, Ningya; Boado, Ruben J.; Pardridge, William M.
2000-12-01
Antisense radiopharmaceuticals could be used to image gene expression in the brain in vivo, should these polar molecules be made transportable through the blood-brain barrier. The present studies describe an antisense imaging agent comprised of an iodinated peptide nucleic acid (PNA) conjugated to a monoclonal antibody to the rat transferrin receptor by using avidin-biotin technology. The PNA was a 16-mer antisense to the sequence around the methionine initiation codon of the luciferase mRNA. C6 rat glioma cells were permanently transfected with a luciferase expression plasmid, and C6 experimental brain tumors were developed in adult rats. The expression of the luciferase transgene in the tumors in vivo was confirmed by measurement of luciferase enzyme activity in the tumor extract. The [125I]PNA conjugate was injected intravenously in anesthetized animals with brain tumors and killed 2 h later for frozen sectioning of brain and film autoradiography. No image of the luciferase gene expression was obtained after the administration of either the unconjugated antiluciferase PNA or a PNA conjugate that was antisense to the mRNA of a viral transcript. In contrast, tumors were imaged in all rats administered the [125I]PNA that was antisense to the luciferase sequence and was conjugated to the targeting antibody. In conclusion, these studies demonstrate gene expression in the brain in vivo can be imaged with antisense radiopharmaceuticals that are conjugated to a brain drug-targeting system.
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NASA Technical Reports Server (NTRS)
Murashov, A. K.; Wolgemuth, D. J.
1996-01-01
We have examined the spatial pattern of expression of a member of the hsp70 gene family, hsp70.2, in the mouse central nervous system. Surprisingly, RNA blot analysis and in situ hybridization revealed abundant expression of an 'antisense' hsp70.2 transcript in several areas of adult mouse brain. Two different transcripts recognized by sense and antisense riboprobes for the hsp70.2 gene were expressed in distinct and only partially overlapping neuronal populations. RNA blot analysis revealed low levels of the 2.7 kb transcript of hsp70.2 in several areas of the brain, with highest signal in the hippocampus. Abundant expression of a slightly larger (approximately 2.8 kb) 'antisense' transcript was detected in several brain regions, notably in the brainstem, cerebellum, mesencephalic tectum, thalamus, cortex, and hippocampus. In situ hybridization revealed that the sense and antisense transcripts were both predominantly neuronal and localized to the same cell types in the granular layer of the cerebellum, trapezoid nucleus of the superior olivary complex, locus coeruleus and hippocampus. The hsp70.2 antisense transcripts were particularly abundant in the frontal cortex, dentate gyrus, subthalamic nucleus, zona incerta, superior and inferior colliculi, central gray, brainstem, and cerebellar Purkinje cells. Our findings have revealed a distinct cellular and spatial localization of both sense and antisense transcripts, demonstrating a new level of complexity in the function of the heat shock genes.
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2007-02-01
antisense RNA for suppressing gene expression in nematode worms (Caenorhabditis elegans) 2. This was followed by the introduction of dsRNA into worms...When single-stranded antisense RNA and double stranded RNA was introduced into worms, they found that dsRNA was more effective than either strand...RISC ( RNA -induced silencing complex), which contains helicase activity that unwinds the two strands 3 of RNA molecules, allowing the antisense
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Miller, Andrew D
2015-02-01
A sense peptide can be defined as a peptide whose sequence is coded by the nucleotide sequence (read 5' → 3') of the sense (positive) strand of DNA. Conversely, an antisense (complementary) peptide is coded by the corresponding nucleotide sequence (read 5' → 3') of the antisense (negative) strand of DNA. Research has been accumulating steadily to suggest that sense peptides are capable of specific interactions with their corresponding antisense peptides. Unfortunately, although more and more examples of specific sense-antisense peptide interactions are emerging, the very idea of such interactions does not conform to standard biology dogma and so there remains a sizeable challenge to lift this concept from being perceived as a peripheral phenomenon if not worse, into becoming part of the scientific mainstream. Specific interactions have now been exploited for the inhibition of number of widely different protein-protein and protein-receptor interactions in vitro and in vivo. Further, antisense peptides have also been used to induce the production of antibodies targeted to specific receptors or else the production of anti-idiotypic antibodies targeted against auto-antibodies. Such illustrations of utility would seem to suggest that observed sense-antisense peptide interactions are not just the consequence of a sequence of coincidental 'lucky-hits'. Indeed, at the very least, one might conclude that sense-antisense peptide interactions represent a potentially new and different source of leads for drug discovery. But could there be more to come from studies in this area? Studies on the potential mechanism of sense-antisense peptide interactions suggest that interactions may be driven by amino acid residue interactions specified from the genetic code. If so, such specified amino acid residue interactions could form the basis for an even wider amino acid residue interaction code (proteomic code) that links gene sequences to actual protein structure and function, even entire genomes to entire proteomes. The possibility that such a proteomic code should exist is discussed. So too the potential implications for biology and pharmaceutical science are also discussed were such a code to exist.
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Chemistry, mechanism and clinical status of antisense oligonucleotides and duplex RNAs
Shen, Xiulong; Corey, David R
2018-01-01
Abstract RNA plays a central role in the expression of all genes. Because any sequence within RNA can be recognized by complementary base pairing, synthetic oligonucleotides and oligonucleotide mimics offer a general strategy for controlling processes that affect disease. The two primary antisense approaches for regulating expression through recognition of cellular RNAs are single-stranded antisense oligonucleotides and duplex RNAs. This review will discuss the chemical modifications and molecular mechanisms that make synthetic nucleic acid drugs possible. Lessons learned from recent clinical trials will be summarized. Ongoing clinical trials are likely to decisively test the adequacy of our current generation of antisense nucleic acid technologies and highlight areas where more basic research is needed. PMID:29240946
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Wu, Li; Wang, Yuan; Wu, Junzhou; Lv, Cong; Wang, Jie; Tang, Xinjing
2013-01-01
We synthesized three 20mer caged circular antisense oligodeoxynucleotides (R20, R20B2 and R20B4) with a photocleavable linker and an amide bond linker between two 10mer oligodeoxynucleotides. With these caged circular antisense oligodeoxynucleotides, RNA-binding affinity and its digestion by ribonuclease H were readily photomodulated. RNA cleavage rates were upregulated ∼43-, 25- and 15-fold for R20, R20B2 and R20B4, respectively, upon light activation in vitro. R20B2 and R20B4 with 2- or 4-nt gaps in the target RNA lost their ability to bind the target RNA even though a small amount of RNA digestion was still observed. The loss of binding ability indicated promising gene photoregulation through a non-enzymatic strategy. To test this strategy, three caged circular antisense oligonucleotides (PS1, PS2 and PS3) with 2′-OMe RNA and phosphorothioate modifications were synthesized to target GFP expression. Upon light activation, photomodulation of target hybridization and GFP expression in cells was successfully achieved with PS1, PS2 and PS3. These caged circular antisense oligonucleotides show promising applications of photomodulating gene expression through both ribonuclease H and non-enzyme involved antisense strategies. PMID:23104375
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Li, Yin-Ji; Kukita, Akiko; Kyumoto-Nakamura, Yukari; Kukita, Toshio
2016-09-01
Wilms' tumor 1 (WT1), a zinc-finger transcription regulator of the early growth response family, identified as the product of a tumor suppressor gene of Wilms' tumors, bears potential ability to induce macrophage differentiation in blood cell differentiation. Herein, we examined the involvement of WT1 in the regulation of osteoclastogenesis. We detected a high level of WT1 protein expression in osteoclast precursors; however, WT1 expression was markedly suppressed during osteoclastogenesis. We examined expression of WT1 transcripts in bone tissue by RNA in situ hybridization. We found a high level of antisense transcripts in osteoclasts actively resorbing bone in mandible of newborn rats. Expression of antisense WT1 RNA in mandible was also confirmed by Northern blot analysis and strand-specific RT-PCR. Overexpression of antisense WT1 RNA in RAW-D cells, an osteoclast precursor cell line, resulted in a marked enhancement of osteoclastogenesis, suggesting that antisense WT1 RNA functions to suppress expression of WT1 protein in osteoclastogenesis. High level expression of antisense WT1 RNA may contribute to commitment to osteoclastogenesis, and may allow osteoclasts to maintain or stabilize their differentiation state. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.
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Posé, Sara; Kirby, Andrew R; Paniagua, Candelas; Waldron, Keith W; Morris, Victor J; Quesada, Miguel A; Mercado, José A
2015-11-05
To ascertain the role of pectin disassembly in fruit softening, chelated- (CSP) and sodium carbonate-soluble (SSP) pectins from plants with a pectate lyase, FaplC, or a polygalacturonase, FaPG1, downregulated by antisense transformation were characterized at the nanostructural level. Fruits from transgenic plants were firmer than the control, although FaPG1 suppression had a greater effect on firmness. Size exclusion chromatography showed that the average molecular masses of both transgenic pectins were higher than that of the control. Atomic force microscopy analysis of pectins confirmed the higher degree of polymerization as result of pectinase silencing. The mean length values for CSP chains increased from 84 nm in the control to 95.5 and 101 nm, in antisense FaplC and antisense FaPG1 samples, respectively. Similarly, SSP polyuronides were longer in transgenic fruits (61, 67.5 and 71 nm, in the control, antisense FaplC and antisense FaPG1 samples, respectively). Transgenic pectins showed a more complex structure, with a higher percentage of branched chains than the control, especially in the case of FaPG1 silenced fruits. Supramolecular pectin aggregates, supposedly formed by homogalacturonan and rhamnogalacturonan I, were more frequently observed in antisense FaPG1 samples. The larger modifications in the nanostructure of pectins in FaPG1 silenced fruits when compared with antisense pectate lyase plants correlate with the higher impact of polygalacturonase silencing on reducing strawberry fruit softening. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Inhibition of B cell proliferation by antisense DNA to both alpha and beta forms of Fc epsilon R II.
Bhatti, L; Behle, K; Stevens, R H
1992-10-01
Epstein-Barr Virus (EBV) infection activates B lymphocyte proliferation through partially understood mechanisms, resulting in phenotypic changes, including the appearance of new antigens. One such antigen is Fc epsilon R II/CD-23 which may be relevant for B cell proliferation. We have used anti-sense oligonucleotides to study the importance of the two forms of this molecule for proliferation in the EBV-transformed, Fc epsilon R II +ve lymphoblastoid B cell line, RPMI 8866. Anti-sense oligodeoxynucleotides were generated to the two forms of Fc epsilon R II; Fc epsilon R IIa (alpha) and IIb (beta) which differ only in their intracytoplasmic domains. Addition of increasing concentrations of anti-sense oligonucleotides, ranging from 1 to 30 microM, significantly decreased cellular proliferation as measured by the incorporation of [3H]thymidine (inhibition range 8-88%). Optimum inhibition of cellular proliferation was apparent at 15 microM concentration of both anti-sense Fc epsilon R IIa and IIb (Fc epsilon R IIa, mean +/- SE = 75 +/- 7% inhibition, p less than 0.001; Fc epsilon R IIb, mean +/- SE = 71 +/- 7% inhibition, p less than 0.001). Anti-sense oligonucleotides complementary to the common part of Fc epsilon R II resulted in a similar inhibition of proliferation. Sense oligonucleotides did not induce significant inhibition. Preincubation of sense and anti-sense oligonucleotides resulted in an abrogation of proliferation inhibition. Moreover, none of these oligonucleotides had any effect on a Fc epsilon R II -ve cell line. Incubation with both anti-sense IIa and IIb resulted in additive, but not synergistic inhibition of proliferation. Addition of soluble Fc epsilon R II did not reverse inhibition of proliferation, suggesting that membrane-bound or intracellular rather than soluble Fc epsilon R II was important for the induced proliferation. Analysis of cell surface expression for Fc epsilon II indicated that while there was a pronounced effect on cell number following incubation with anti-sense oligonucleotides, surface expression of Fc epsilon R II was consistent as measured over different time points. PCR analysis revealed that while most cells expressed either the alpha or the beta form of Fc epsilon R II, EBV-transformed cell lines, particularly RPMI 8866, were found to express both alpha and beta forms simultaneously. This may constitute a mechanism whereby EBV infection confers an immortal state to the cell, resulting in its uncontrolled proliferation. Cell lines expressing only one receptor form, either alpha or beta, were unaffected after incubation with anti-sense oligonucleotides.(ABSTRACT TRUNCATED AT 400 WORDS)
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Activation of Antitumorigenic Stat3beta in Breast Cancer by Splicing Redirection
2013-07-01
4175) model system REPORTABLE OUTCOMES 1. Lee Spraggon and Luca Cartegni; Antisense Modulation of RNA Processing as a Therapeutic Approach in...modulation. Proc Natl Acad Sci U S A 108: 17779-17784. 26. Spraggon L, Cartegni L (2013) Antisense modulation of RNA processing as a therapeutic...pre-print copy 1 Antisense Modulation of RNA Processing as a Therapeutic Approach in Cancer Therapy Lee Spraggon and Luca Cartegni Molecular
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Inhibition of Human Immunodeficiency Virus Replication by Antisense Oligodeoxynucleotides
NASA Astrophysics Data System (ADS)
Goodchild, John; Agrawal, Sudhir; Civeira, Maria P.; Sarin, Prem S.; Sun, Daisy; Zamecnik, Paul C.
1988-08-01
Twenty different target sites within human immunodeficiency virus (HIV) RNA were selected for studies of inhibition of HIV replication by antisense oligonucleotides. Target sites were selected based on their potential capacity to block recognition functions during viral replication. Antisense oligomers complementary to sites within or near the sequence repeated at the ends of retrovirus RNA (R region) and to certain splice sites were most effective. The effect of antisense oligomer length on inhibiting virus replication was also investigated, and preliminary toxicity studies in mice show that these compounds are toxic only at high levels. The results indicate potential usefulness for these oligomers in the treatment of patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex either alone or in combination with other drugs.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Shi, Yanan; Liu, Xiaochun; Zhu, Pei
Highlights: •The Gper expression was detected in the developing brain of zebrafish. •Gper morpholino knockdown induced apoptosis of brain cells. •Gper morpholino knockdown reduced expression in neuron markers. •Zebrafish Gper may be involved in neuronal development. -- Abstract: G-protein-coupled estrogen receptor 1 (Gper, formerly known as GPR30) is found to be a trophic and protective factor in mediating action of estrogen in adult brain, while its role in developing brain remains to be elucidated. Here we present the expression pattern of Gper and its functions during embryogenesis in zebrafish. Both the mRNA and protein of Gper were detected throughout embryogenesis.more » Whole mount in situ hybridization (WISH) revealed a wide distribution of gper mRNAs in various regions of the developing brain. Gper knockdown by specific morpholinos resulted in growth retardation in embryos and morphological defects in the developing brain. In addition, induced apoptosis, decreased proliferation of the brain cells and maldevelopment of sensory and motor neurons were also found in the morphants. Our results provide novel insights into Gper functions in the developing brain, revealing that Gper can maintain the survival of the brain cells, and formation and/or differentiation of the sensory and motor neurons.« less
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A Morpholino-based screen to identify novel genes involved in craniofacial morphogenesis
Melvin, Vida Senkus; Feng, Weiguo; Hernandez-Lagunas, Laura; Artinger, Kristin Bruk; Williams, Trevor
2014-01-01
BACKGROUND The regulatory mechanisms underpinning facial development are conserved between diverse species. Therefore, results from model systems provide insight into the genetic causes of human craniofacial defects. Previously, we generated a comprehensive dataset examining gene expression during development and fusion of the mouse facial prominences. Here, we used this resource to identify genes that have dynamic expression patterns in the facial prominences, but for which only limited information exists concerning developmental function. RESULTS This set of ~80 genes was used for a high throughput functional analysis in the zebrafish system using Morpholino gene knockdown technology. This screen revealed three classes of cranial cartilage phenotypes depending upon whether knockdown of the gene affected the neurocranium, viscerocranium, or both. The targeted genes that produced consistent phenotypes encoded proteins linked to transcription (meis1, meis2a, tshz2, vgll4l), signaling (pkdcc, vlk, macc1, wu:fb16h09), and extracellular matrix function (smoc2). The majority of these phenotypes were not altered by reduction of p53 levels, demonstrating that both p53 dependent and independent mechanisms were involved in the craniofacial abnormalities. CONCLUSIONS This Morpholino-based screen highlights new genes involved in development of the zebrafish craniofacial skeleton with wider relevance to formation of the face in other species, particularly mouse and human. PMID:23559552
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Tabler, M; Homann, M; Tzortzakaki, S; Sczakiel, G
1994-01-01
Trans-cleaving hammerhead ribozymes with long target-specific antisense sequences flanking the catalytic domain share some features with conventional antisense RNA and are therefore termed 'catalytic antisense RNAs'. Sequences 5' to the catalytic domain form helix I and sequences 3' to it form helix III when complexed with the target RNA. A catalytic antisense RNA of more than 400 nucleotides, and specific for the human immunodeficiency virus type 1 (HIV-1), was systematically truncated within the arm that constituted originally a helix I of 128 base pairs. The resulting ribozymes formed helices I of 13, 8, 5, 3, 2, 1 and 0 nucleotides, respectively, and a helix III of about 280 nucleotides. When their in vitro cleavage activity was compared with the original catalytic antisense RNA, it was found that a helix I of as little as three nucleotides was sufficient for full endonucleolytic activity. The catalytically active constructs inhibited HIV-1 replication about four-fold more effectively than the inactive ones when tested in human cells. A conventional hammerhead ribozyme having helices of just 8 nucleotides on either side failed to cleave the target RNA in vitro when tested under the conditions for catalytic antisense RNA. Cleavage activity could only be detected after heat-treatment of the ribozyme substrate mixture which indicates that hammerhead ribozymes with short arms do not associate as efficiently to the target RNA as catalytic antisense RNA. The requirement of just a three-nucleotide helix I allows simple PCR-based generation strategies for asymmetric hammerhead ribozymes. Advantages of an asymmetric design will be discussed. Images PMID:7937118
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Down-regulation of the IbEXP1 gene enhanced storage root development in sweetpotato
Bae, Jung Myung
2013-01-01
The role of an expansin gene (IbEXP1) in the formation of the storage root (SR) was investigated by expression pattern analysis and characterization of IbEXP1-antisense sweetpotato (Ipomoea batatas cv. Yulmi) plants in an attempt to elucidate the molecular mechanism underlying SR development in sweetpotato. The transcript level of IbEXP1 was high in the fibrous root (FR) and petiole at the FR stage, but decreased significantly at the young storage root (YSR) stage. IbEXP1-antisense plants cultured in vitro produced FRs which were both thicker and shorter than those of wild-type (WT) plants. Elongation growth of the epidermal cells was significantly reduced, and metaxylem and cambium cell proliferation was markedly enhanced in the FRs of IbEXP1-antisense plants, resulting in an earlier thickening growth in these plants relative to WT plants. There was a marked reduction in the lignification of the central stele of the FRs of the IbEXP1-antisense plants, suggesting that the FRs of the mutant plants possessed a higher potential than those of WT plants to develop into SRs. IbEXP1-antisense plants cultured in soil produced a larger number of SRs and, consequently, total SR weight per IbEXP1-antisense plant was greater than that per WT plant. These results demonstrate that SR development was accelerated in IbEXP1-antisense plants and suggest that IbEXP1 plays a negative role in the formation of SR by suppressing the proliferation of metaxylem and cambium cells to inhibit the initial thickening growth of SRs. IbEXP1 is the first sweetpotato gene whose role in SR development has been directly identified in soil-grown transgenic sweetpotato plants. PMID:22945944
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Jansson, Christer; Sun, Chuanxin; Ghebramedhin, Haile
Antisense oligodeoxynucleotide (ODN) inhibition emerges as an effective means for probing gene function in plant cells. Employing this method we have established the importance of the SUSIBA2 transcription factor for regulation of starch synthesis in barley endosperm, and arrived at a model for the role of the SUSIBAs in sugar signaling and source-sink commutation during cereal endosperm development. In this addendum we provide additional data demonstrating the suitability of the antisense ODN technology in studies on starch branching enzyme activities in barley leaves. We also comment on the mechanism for ODN uptake in plant cells. Antisense ODNs are short (12-25more » nt-long) stretches of single-stranded ODNs that hybridize to the cognate mRNA in a sequence-specific manner, thereby inhibiting gene expression. They are naturally occurring in both prokaryotes and eukaryotes where they partake in gene regulation and defense against viral infection. The mechanisms for antisense ODN inhibition are not fully understood but it is generally considered that the ODN either sterically interferes with translation or promotes transcript degradation by RNase H activation. The earliest indication of the usefulness of antisense ODN technology for the purposes of molecular biology and medical therapy was the demonstration in 1978 that synthetic ODNs complementary to Raos sarcoma virus could inhibit virus replication in tissue cultures of chick embryo fibroblasts. Since then the antisense ODN technology has been widely used in animal sciences and as an important emerging therapeutic approach in clinical medicine. However, antisense ODN inhibition has been an under-exploited strategy for plant tissues, although the prospects for plant cells in suspension cultures to take up single-stranded ODNs was reported over a decade ago. In 2001, two reports from Malho and coworker demonstrated the use of cationic-complexed antisense ODNs to suppress expression of genes encoding pollen-signaling proteins in pollen tubes from the lilly Agapanthus umbellatus. For the uptake of DNA pollen tubes represent a unique system since the growing tip is surrounded by a loose matrix of hemicellulose and pectins, exposing the plasma membrane7 and the first uptake of ODNs by pollen tubes was reported as early as 1994. A breakthrough in the employment of antisense ODN inhibition as a powerful approach in plant biology was recently presented through our work on intact barley leaves. As was illustrated by confocal microscopy and fluorescently labeled ODNs, naked ODNs were taken up through the leaf petiole and efficiently imported into the plant cell and the nucleus. The work portrayed in that study demonstrate the applicability of antisense ODN inhibition in plant biology, e.g. as a rapid antecedent to time-consuming transgenic studies, and that it operates through RNase H degradation. We employed the antisense ODN strategy to demonstrate the importance of the SUSIBA2 transcription factor in regulation of starch synthesis, and to depict a possible mechanism for sugar signaling in plants and how it might confer endosperm-specific gene expression during seed development. We also described the employment of the antisense ODN strategy for studies on in vitro spike cultures of barley. Here we present further evidence as to the value of the antisense ODN approach in plant biology by following the effects on starch branching enzyme (SBE) accumulation in barley leaves after suppression of individual SBE genes. In agreement with transcript analyses of SBE expression in barley leaves, a zymogram assay (Fig. 1) revealed that sucrose treatment of barley leaves increased the number of SBE activity bands as compared to sorbitol treatment. In the presence of antisense SBEI or SBEIIA ODNs, zymograms of sucrose-treated leaves displayed only a subset of these activities with bands in the top portion of the zymogram gel missing or diminished. With antisense SBEIIB ODN, all activity bands in the top portion of the gel as well as the lowest band were absent. Based on these data we provide a tentative annotation for the various SBE activity bands. In animal experiments, naked ODNs are usually not taken up by the cells since both the ODNs and the outside of the plasma membrane carry a net negative charge. Thus the uptake of naked ODNs into barley leaf cells was surprising and called for an explanation. As demonstrated in our subsequent paper, the answer seems to be that the ODNs slip into the cells through sugar translocators as they are activated in the presence of the appropriate sugar (Fig. 2). Whether it is the structural resemblance between the sugar (deoxyribose) backbone of the ODNs and the transported sugars that allows for the ODNs to be transferred, or if other mechanisms are involved, remains to be elucidated.« less
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Psf2 plays important roles in normal eye development in Xenopus laevis
Walter, Brian E.; Perry, Kimberly J.; Fukui, Lisa; Malloch, Erica L.; Wever, Jason
2008-01-01
Purpose Psf2 (partner of Sld5 2) represents a member of the GINS (go, ichi, ni, san) heterotetramer [1] and functions in DNA replication as a “sliding clamp.” Previous in situ hybridization analyses revealed that Psf2 is expressed during embryonic development in a tissue-specific manner, including the optic cup (retina) and the lens [2]. This article provides an analysis of Psf2 function during eye development in Xenopus laevis. Methods A morpholino targeted to Psf2 mRNA was designed to knockdown Psf2 translation and was injected into specific embryonic cells during early cleavage stages in the frog, Xenopus laevis. Injected embryos were assayed for specific defects in morphology, cell proliferation, and apoptosis. Synthetic Psf2 RNA was also co-injected with the morpholino to rescue morpholino-mediated developmental defects. It is well known that reciprocal inductive interactions control the development of the optic cup and lens. Therefore, control- and morpholino-injected embryos were used for reciprocal transplantation experiments to distinguish the intrinsic role of Psf2 in the development of the optic cup (retina) versus the lens. Results Morpholino-mediated knockdown of Psf2 expression resulted in dosage-dependent phenotypes, which included microphthalmia, incomplete closure of the ventral retinal fissure, and retinal and lens dysgenesis. Defects were also observed in other embryonic tissues that normally express Psf2 including the pharyngeal arches and the otic vesicle, although other tissues that express Psf2 were not found to be grossly defective. Eye defects could be rescued by co-injection of synthetic Psf2 RNA. Examination of cell proliferation via an antibody against phospho-histone H3 S10P revealed no significant differences in the retina and lens following Psf2 knockdown. However, there was a significant increase in the level of apoptosis in retinal as well as forebrain tissues, as revealed by TUNEL (terminal deoxynucleotide transferase dUTP nick end labeling) assay. Conclusions The results demonstrate intrinsic roles for Psf2 in both retinal and to a lesser extent, lens tissues. Observed lens defects can mainly be attributed to deficiencies in retinal development and consequently the late phase of lens induction, which involves instructive cues from the optic cup. Developmental defects were not observed in all tissues that express Psf2, which could be related to differences in the translation of Psf2 or redundant effects of related factors such as proliferating cell nuclear antigen (PCNA). PMID:18509549
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Sun, Wei-Hong; Wang, Yong; He, Hua-Gang; Li, Xue; Song, Wan; Du, Bin; Meng, Qing-Wei
2013-07-01
Ascorbate peroxidases are directly involved in reactive oxygen species (ROS) scavenging by reducing hydrogen peroxide to water. The tomato thylakoid-bound ascorbate peroxidase gene (StAPX) was introduced into tobacco. RNA gel blot analysis confirmed that StAPX in tomato leaves was induced by methylviologen-mediated oxidative stress. The sense transgenic seedlings exhibited higher tAPX activity than that of the wild type (WT) plants under oxidative stress conditions, while the antisense seedlings exhibited lower tAPX activity. Lower APX activities of antisense transgenic seedlings caused higher malondialdehyde contents and relative electrical conductivity. The sense transgenic seedlings with higher tAPX activity maintained higher chlorophyll content and showed the importance of tAPX in maintaining the optimal chloroplast development under methylviologen stress conditions, whereas the antisense lines maintained lower chlorophyll content than WT seedlings. Results indicated that the over-expression of StAPX enhanced tolerance to methylviologen-mediated oxidative stress in sense transgenic tobacco early seedlings, whereas the suppression of StAPX in antisense transgenic seedlings showed high sensitivity to oxidative stress.
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Bannai, M; Ichikawa, M; Nishihara, M; Takahashi, M
1998-02-16
In the ventromedial hypothalamus (VMH), gamma-aminobutyric acid (GABA) plays a role in regulating feeding and running behaviors. The GABA synthetic enzyme, glutamic acid decarboxylase (GAD), consists of two isozymes, GAD65 and GAD67. In the present study, the phosphorothioated antisense oligodeoxynucleotides (ODNs) of each GAD isozyme were injected bilaterally into the VMH of male rats, and food intake, body weight and locomotor activity were monitored. ODNs were incorporated in the water-absorbent polymer (WAP, 0.2 nmol/microliter) so that ODNs were retained at the injection site. Each antisense ODN of GAD65 or GAD67 tended to reduce food intake on day 1 (day of injection=day 0) though not significantly. An injection combining both antisense ODNs significantly decreased food intake only on day 1, but body weight remained significantly lower than the control for 5 days. This suppression of body weight gain could be attributed to a significant increase in locomotor activity between days 3 and 5. Individual treatment with either ODNs did not change locomotor activity. The increase in daily locomotor activity in the group receiving the combined antisense ODNs occurred mainly during the light phase. Neither vehicle (WAP) nor control ODN affected food intake, body weight and locomotor activity. Histological studies indicated that antisense ODN distributed within 800 micron from the edge of the area where WAP was located 24 h after the injection gradually disappeared within days, but still remained within 300 micron m distance even 7 days after the injection. Antisense ODN was effectively incorporated by all the cell types examined, i.e., neurons, astrocytes and microglias. Further, HPLC analysis revealed that antisense ODNs of GAD isozymes, either alone or combined, decreased the content of GABA by 50% in VMH 24 h after the injection. These results indicate that suppression of GABA synthesis by either of the GAD isozymes is synergistically involved in suppressing food intake and enhancing locomotor activity in rat VMH. Copyright 1998 Elsevier Science B.V.
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Flowering time control: another window to the connection between antisense RNA and chromatin.
Ietswaart, Robert; Wu, Zhe; Dean, Caroline
2012-09-01
A high proportion of all eukaryotic genes express antisense RNA (asRNA), which accumulates to varying degrees at different loci. Whether there is a general function for asRNA is unknown, but its widespread occurrence and frequent regulation by stress suggest an important role. The best-characterized plant gene exhibiting a complex antisense transcript pattern is the Arabidopsis floral regulator FLOWERING LOCUS C (FLC). Changes occur in the accumulation, splicing, and polyadenylation of this antisense transcript, termed COOLAIR, in different environments and genotypes. These changes are associated with altered chromatin regulation and differential FLC expression, provoking mechanistic comparisons with many well-studied loci in yeast and mammals. Detailed analysis of these specific examples may shed light on the complex interplay between asRNA and chromatin modifications in different genomes. Copyright © 2012 Elsevier Ltd. All rights reserved.
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Laitala, T; Väänänen, H K
1994-01-01
The bone resorbing cells, osteoclasts, express high levels of carbonic anhydrase II (CA II) and vacuolar H(+)-ATPase (V-ATPase) during bone resorption. We have used antisense RNA and DNA molecules targeted against CA II, and against 16- and 60-kD subunits of vacuolar H(+)-ATPase (V-ATPase), to block the expression of these proteins in vitro. Osteoclastic bone resorption was studied in two in vitro culture systems: release of 45Calcium from prelabeled newborn mouse calvaria cultures, and resorption pit assays performed with rat osteoclasts cultured on bovine bone slices. Both antisense RNA and DNA against CA II and the V-ATPase were used to compare their specificities as regards inhibiting bone resorption in vitro. The antisense molecules inhibited the synthesis of these proteins by decreasing the amounts of mRNA in the cells in a highly specific manner. In osteoclast cultures treated with the 16-kD V-ATPase antisense RNA, acidification of an unknown population of intracellular vesicles was highly stimulated. The acidification of these vesicles was not sensitive to amiloride or bafilomycin A1. This suggests the existence of a back-up system for acidification of intracellular vesicles, when the expression of the V-ATPase is blocked. Our results further indicate that blocking the expression of CA II and V-ATPase with antisense RNA or DNA leads to decreased bone resorption. Images PMID:8200964
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Krieg, A M; Tonkinson, J; Matson, S; Zhao, Q; Saxon, M; Zhang, L M; Bhanja, U; Yakubov, L; Stein, C A
1993-02-01
Phosphodiester oligodeoxynucleotides bearing a 5' cholesteryl (chol) modification bind to low density lipoprotein (LDL), apparently by partitioning the chol-modified oligonucleotides into the lipid layer. Both HL60 cells and primary mouse spleen T and B cells incubated with fluorescently labeled chol-modified oligonucleotide showed substantially increased cellular association by flow cytometry and increased internalization by confocal microscopy compared to an identical molecule not bearing the chol group. Cellular internalization of chol-modified oligonucleotide occurred at least partially through the LDL receptor; it was increased in mouse spleen cells by cell culture in lipoprotein-deficient medium and/or lovastatin, and it was decreased by culture in high serum medium. To determine whether chol-modified oligonucleotides are more potent antisense agents, we titered antisense unmodified phosphodiester and chol-modified oligonucleotides targeted against a mouse immunosuppressive protein. Murine spleen cells cultured with 20 microM phosphodiester antisense oligonucleotides had a 2-fold increase in RNA synthesis, indicating the expected lymphocyte activation. Antisense chol-modified oligonucleotides showed an 8-fold increase in relative potency: they caused a 2-fold increase in RNA synthesis at just 2.5 microM. The increased efficacy was blocked by heparin and was further increased by cell culture in 1% (vs. 10%) fetal bovine serum, suggesting that the effect may, at least in part, be mediated via the LDL receptor. Antisense chol-modified oligonucleotides are sequence specific and have increased potency as compared to unmodified oligonucleotides.
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Dworkin, Sebastian; Simkin, Johanna; Darido, Charbel; Partridge, Darren D; Georgy, Smitha R; Caddy, Jacinta; Wilanowski, Tomasz; Lieschke, Graham J; Doggett, Karen; Heath, Joan K; Jane, Stephen M
2014-08-01
Craniofacial development is a highly conserved process that requires complex interactions between neural crest cells (NCCs) and pharyngeal tissues derived from all three germ layers. Signals emanating from the pharyngeal endoderm drive differentiation of NCCs into craniofacial cartilage, and disruption of this process underpins several human craniofacial defects (CFD). Here, we demonstrate that morpholino (MO)-mediated knockdown in zebrafish of the highly conserved transcription factor grainyhead-like 3 (grhl3), which is selectively expressed in the pharyngeal endoderm, leads to severe hypoplasia of the lower jaw cartilages. Phylogenetic analysis of conserved grhl-binding sites in gene regulatory regions identified endothelin-1 (edn1) as a putative direct grhl3 target gene, and this was confirmed by chromatin precipitation (ChIP) assays in zebrafish embryos. Injection of sub-phenotypic concentrations of MOs targeting both grhl3 and edn1 induced jaw abnormalities, and injection of edn1 mRNA into grhl3-morphants rescued both pharyngeal expression of the downstream effectors of edn1, and jaw cartilage formation. This study sheds new light on the role of endodermal endothelin-1 in vertebrate jaw development, and highlights potential new genetic defects that could underpin human CFD. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.
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Nomani, Alireza; Haririan, Ismaeil; Rahimnia, Ramin; Fouladdel, Shamileh; Gazori, Tarane; Dinarvand, Rassoul; Omidi, Yadollah; Azizi, Ebrahim
2010-01-01
To gain a deeper understanding of the physicochemical phenomenon of self-assembled nanoparticles of different generations and ratios of poly (amidoamine) dendrimer (PAMAM) dendrimer and a short-stranded DNA (antisense oligonucleotide), multiple methods were used to characterize these nanoparticles including photon correlation spectroscopy (PCS); zeta potential measurement; and atomic force microscopy (AFM). PCS and AFM results revealed that, in contrast to larger molecules of DNA, smaller molecules produce more heterodisperse and large nanoparticles when they are condensed with a cationic dendrimer. AFM images also showed that such nanoparticles were spherical. The stability of the antisense content of the nanoparticles was investigated over different charge ratios using polyacrylamide gel electrophoresis. It was clear from such analyses that much more than charge neutrality point was required to obtain stable nanoparticles. For cell uptake, self-assembled nanoparticles were prepared with PAMAM G5 and 5’-FITC labeled antisense and the uptake experiment was carried out in T47D cell culture. This investigation also shows that the cytotoxicity of the nanoparticles was dependent upon the generation and charge ratio of the PAMAM dendrimer, and the antisense concentration had no significant effect on the cytotoxicity. PMID:20517481
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Antisense apolipoprotein B therapy: where do we stand?
Akdim, Fatima; Stroes, Erik S G; Kastelein, John J P
2007-08-01
Antisense oligonucleotides are novel therapeutic agents that reduce the number of specific mRNAs available for translation of the encoded protein. ISIS 301012 is an antisense oligonucleotide developed to reduce the hepatic synthesis of apolipoprotein B-100. Apolipoprotein B-100 is made in the liver, and antisense oligonucleotides preferentially distribute to that organ, so antisense apolipoprotein B-100 may have potential as an efficacious lipid-lowering agent. Recently, in healthy volunteers and in mild dyslipidaemic patients, this strategy as monotherapy or in conjunction with statins has shown unparalleled efficacy in reducing apolipoprotein B-100 and LDL-cholesterol. Tolerance for this novel therapy is encouraging and safety concerns currently only relate to mild injection-site reactions and rare liver-function test abnormalities. It should be noted, however, that these safety results were obtained in relatively few individuals. ISIS 301012 has initially shown promising results in experimental animal models, and in clinical trials in humans. Besides the effect of reducing apolipoprotein B-100 and LDL-cholesterol, this compound also significantly lowers plasma triglycerides. Safety concerns related to the drug include increased liver-function tests. To date no evidence of hepatic steatosis has been reported. Nonetheless, clinical trials of longer duration are required to demonstrate further safety.
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RNase III-Binding-mRNAs Revealed Novel Complementary Transcripts in Streptomyces
Šetinová, Dita; Šmídová, Klára; Pohl, Pavel; Musić, Inesa; Bobek, Jan
2018-01-01
cis-Antisense RNAs (asRNAs) provide very simple and effective gene expression control due to the perfect complementarity between regulated and regulatory transcripts. In Streptomyces, the antibiotic-producing clade, the antisense control system is not yet understood, although it might direct the organism's complex development. Initial studies in Streptomyces have found a number of asRNAs. Apart from this, hundreds of mRNAs have been shown to bind RNase III, the double strand-specific endoribonuclease. In this study, we tested 17 mRNAs that have been previously co-precipitated with RNase III for antisense expression. Our RACE mapping showed that all of these mRNAs possess cognate asRNA. Additional tests for antisense expression uncovered as-adpA, as-rnc, as3983, as-sigB, as-sigH, and as-sigR RNAs. Northern blots detected the expression profiles of 18 novel transcripts. Noteworthy, we also found that only a minority of asRNAs respond to the absence of RNase III enzyme by increasing their cellular levels. Our findings suggest that antisense expression is widespread in Streptomyces, including genes of such important developmental regulators, as AdpA, RNase III, and sigma factors. PMID:29379487
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RNase III-Binding-mRNAs Revealed Novel Complementary Transcripts in Streptomyces.
Šetinová, Dita; Šmídová, Klára; Pohl, Pavel; Musić, Inesa; Bobek, Jan
2017-01-01
cis -Antisense RNAs (asRNAs) provide very simple and effective gene expression control due to the perfect complementarity between regulated and regulatory transcripts. In Streptomyces , the antibiotic-producing clade, the antisense control system is not yet understood, although it might direct the organism's complex development. Initial studies in Streptomyces have found a number of asRNAs. Apart from this, hundreds of mRNAs have been shown to bind RNase III, the double strand-specific endoribonuclease. In this study, we tested 17 mRNAs that have been previously co-precipitated with RNase III for antisense expression. Our RACE mapping showed that all of these mRNAs possess cognate asRNA. Additional tests for antisense expression uncovered as-adpA, as-rnc, as3983, as-sigB, as-sigH , and as-sigR RNAs. Northern blots detected the expression profiles of 18 novel transcripts. Noteworthy, we also found that only a minority of asRNAs respond to the absence of RNase III enzyme by increasing their cellular levels. Our findings suggest that antisense expression is widespread in Streptomyces , including genes of such important developmental regulators, as AdpA, RNase III, and sigma factors.
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ERIC Educational Resources Information Center
Weiss, Rick
1991-01-01
Discussed is a designer-drug technology called antisense which blocks messenger RNA's ability to carry information to protein producing sites in the cell. The applications of this drug to AIDS research, cancer therapy, and other diseases are discussed. (KR)
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Lavorgna, Giovanni; Triunfo, Riccardo; Santoni, Federico; Orfanelli, Ugo; Noci, Sara; Bulfone, Alessandro; Zanetti, Gianluigi; Casari, Giorgio
2005-07-01
An increasing number of eukaryotic and prokaryotic genes are being found to have natural antisense transcripts (NATs). There is also growing evidence to suggest that antisense transcription could play a key role in many human diseases. Consequently, there have been several recent attempts to set up computational procedures aimed at identifying novel NATs. Our group has developed the AntiHunter program for the identification of expressed sequence tag (EST) antisense transcripts from BLAST output. In order to perform an analysis, the program requires a genomic sequence plus an associated list of transcript names and coordinates of the genomic region. After masking the repeated regions, the program carries out a BLASTN search of this sequence in the selected EST database, reporting via email the EST entries that reveal an antisense transcript according to the user-supplied list. Here, we present the newly developed version 2.0 of the AntiHunter tool. Several improvements have been added to this version of the program in order to increase its ability to detect a larger number of antisense ESTs. As a result, AntiHunter can now detect, on average, >45% more antisense ESTs with little or no increase in the percentage of the false positives. We also raised the maximum query size to 3 Mb (previously 1 Mb). Moreover, we found that a reasonable trade-off between the program search sensitivity and the maximum allowed size of the input-query sequence could be obtained by querying the database with the MEGABLAST program, rather than by using the BLAST one. We now offer this new opportunity to users, i.e. if choosing the MEGABLAST option, users can input a query sequence up to 30 Mb long, thus considerably improving the possibility to analyze longer query regions. The AntiHunter tool is freely available at http://bioinfo.crs4.it/AH2.0.
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Azizi, Ebrahim; Namazi, Alireza; Haririan, Ismaeil; Fouladdel, Shamileh; Khoshayand, Mohammad R; Shotorbani, Parisa Y; Nomani, Alireza; Gazori, Taraneh
2010-01-01
Chitosan/alginate nanoparticles which had been optimized in our previous study using two different N/P ratios were chosen and their ability to release epidermal growth factor receptor (EGFR) antisense was investigated. In addition, the stability of these nanoparticles in aqueous medium and after freeze-drying was investigated. In the case of both N/P ratios (5, 25), nanoparticles started releasing EGFR antisense as soon as they were exposed to the medium and the release lasted for approximately 50 hours. Nanoparticle size, shape, zeta potential, and release profile did not show any significant change after the freeze-drying process (followed by reswelling). The nanoparticles were reswellable again after freeze-drying in phosphate buffer with a pH of 7.4 over a period of six hours. Agarose gel electrophoresis of the nanoparticles with the two different N/P ratios showed that these nanoparticles could protect EGFR antisense molecules for six hours. PMID:20957167
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Repair of Thalassemic Human β -globin mRNA in Mammalian Cells by Antisense Oligonucleotides
NASA Astrophysics Data System (ADS)
Sierakowska, Halina; Sambade, Maria J.; Agrawal, Sudhir; Kole, Ryszard
1996-11-01
In one form of β -thalassemia, a genetic blood disorder, a mutation in intron 2 of the β -globin gene (IVS2-654) causes aberrant splicing of β -globin pre-mRNA and, consequently, β -globin deficiency. Treatment of mammalian cells stably expressing the IVS2-654 human β -globin gene with antisense oligonucleotides targeted at the aberrant splice sites restored correct splicing in a dose-dependent fashion, generating correct human β -globin mRNA and polypeptide. Both products persisted for up to 72 hr posttreatment. The oligonucleotides modified splicing by a true antisense mechanism without overt unspecific effects on cell growth and splicing of other pre-mRNAs. This novel approach in which antisense oligonucleotides are used to restore rather than to down-regulate the activity of the target gene is applicable to other splicing mutants and is of potential clinical interest.
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Niessen, Neville-Andrew; Balthazart, Jacques; Ball, Gregory F.; Charlier, Thierry D.
2013-01-01
Environmental stimulation results in an increased expression of transcription factors called immediate early genes (IEG) in specific neuronal populations. In male Japanese quail, copulation with a female increases the expression of the IEGs zenk and c-fos in the medial preoptic nucleus (POM), a key nucleus controlling male sexual behavior. The functional significance of this increased IEG expression that follows performance of copulatory behavior is unknown. We addressed this question by repeatedly quantifying the performance of appetitive (learned social proximity response) and consummatory (actual copulation) sexual behavior in castrated, testosterone-treated males that received daily intracerebroventricular injection of an antisense oligodeoxynucleotide targeting c-fos or control vehicle. Daily antisense injections significantly inhibited expression of copulatory behavior as well as acquisition of the learned social proximity response. A strong reduction of the proximity response was still observed in antisense-treated birds that copulated with a female, ruling out the indirect effect of the absence of interactions with females on the learning process. After a two-day interruption of behavioral testing but not of antisense injections, birds were submitted to a final copulatory test that confirmed the behavioral inhibition in antisense-injected birds. Brains were collected 90 min after the behavioral testing for quantification of c-fos immunoreactive cells. A significant reduction of the number of c-fos-positive cells in POM but not in other brain regions was observed following antisense injection. Together, data suggest that c-fos expression in POM modulates copulatory behavior and sexual learning in male quail. PMID:23895306
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Down-Regulating α-Galactosidase Enhances Freezing Tolerance in Transgenic Petunia1
Pennycooke, Joyce C.; Jones, Michelle L.; Stushnoff, Cecil
2003-01-01
α-Galactosidase (α-Gal; EC 3.2.1.22) is involved in many aspects of plant metabolism, including hydrolysis of the α-1,6 linkage of raffinose oligosaccharides during deacclimation. To examine the relationship between endogenous sugars and freezing stress, the expression of α-Gal was modified in transgenic petunia (Petunia × hybrida cv Mitchell). The tomato (Lycopersicon esculentum) Lea-Gal gene under the control of the Figwort Mosaic Virus promoter was introduced into petunia in the sense and antisense orientations using Agrobacterium tumefaciens-mediated transformation. RNA gel blots confirmed that α-Gal transcripts were reduced in antisense lines compared with wild type, whereas sense plants had increased accumulation of α-Gal mRNAs. α-Gal activity followed a similar trend, with reduced activity in antisense lines and increased activity in all sense lines evaluated. Raffinose content of nonacclimated antisense plants increased 12- to 22-fold compared with wild type, and 22- to 53-fold after cold acclimation. Based upon electrolyte leakage tests, freezing tolerance of the antisense lines increased from –4°C for cold-acclimated wild-type plants to –8°C for the most tolerant antisense line. Down-regulating α-Gal in petunia results in an increase in freezing tolerance at the whole-plant level in nonacclimated and cold-acclimated plants, whereas overexpression of the α-Gal gene caused a decrease in endogenous raffinose and impaired freezing tolerance. These results suggest that engineering raffinose metabolism by transformation with α-Gal provides an additional method for improving the freezing tolerance of plants. PMID:14500789
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Margaritis, Thanasis; Oreal, Vincent; Brabers, Nathalie; Maestroni, Laetitia; Vitaliano-Prunier, Adeline; Benschop, Joris J.; van Hooff, Sander; van Leenen, Dik
2012-01-01
Histone H3 di- and trimethylation on lysine 4 are major chromatin marks that correlate with active transcription. The influence of these modifications on transcription itself is, however, poorly understood. We have investigated the roles of H3K4 methylation in Saccharomyces cerevisiae by determining genome-wide expression-profiles of mutants in the Set1 complex, COMPASS, that lays down these marks. Loss of H3K4 trimethylation has virtually no effect on steady-state or dynamically-changing mRNA levels. Combined loss of H3K4 tri- and dimethylation results in steady-state mRNA upregulation and delays in the repression kinetics of specific groups of genes. COMPASS-repressed genes have distinct H3K4 methylation patterns, with enrichment of H3K4me3 at the 3′-end, indicating that repression is coupled to 3′-end antisense transcription. Further analyses reveal that repression is mediated by H3K4me3-dependent 3′-end antisense transcription in two ways. For a small group of genes including PHO84, repression is mediated by a previously reported trans-effect that requires the antisense transcript itself. For the majority of COMPASS-repressed genes, however, it is the process of 3′-end antisense transcription itself that is the important factor for repression. Strand-specific qPCR analyses of various mutants indicate that this more prevalent mechanism of COMPASS-mediated repression requires H3K4me3-dependent 3′-end antisense transcription to lay down H3K4me2, which seems to serve as the actual repressive mark. Removal of the 3′-end antisense promoter also results in derepression of sense transcription and renders sense transcription insensitive to the additional loss of SET1. The derepression observed in COMPASS mutants is mimicked by reduction of global histone H3 and H4 levels, suggesting that the H3K4me2 repressive effect is linked to establishment of a repressive chromatin structure. These results indicate that in S. cerevisiae, the non-redundant role of H3K4 methylation by Set1 is repression, achieved through promotion of 3′-end antisense transcription to achieve specific rather than global effects through two distinct mechanisms. PMID:23028359
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NASA Technical Reports Server (NTRS)
Murashov, A. K.; Wolgemuth, D. J.
1996-01-01
The expression of hsp70.2, an hsp70 gene family member, originally characterized by its high levels of expression in germ cells in the adult mouse testis, was detected in several other reproductive tissues, including epididymis, prostate, and seminal vesicles, as well as in extraembryonic tissues of mid-gestation fetuses. In addition, hybridization with RNA probes transcribed in the sense orientation surprisingly indicated the presence of slightly larger "antisense" transcripts in several tissues. The levels of antisense transcripts varied among the tissues, with the highest signal detected in the prostate and no signal being detectable in the testis. Consistent with these results, in situ hybridization analysis clearly localized the sense-orientation transcripts to pachytene spermatocytes, while no antisense-orientation transcripts were observed in adjacent sections of the same tubules. Our findings have thus shown that although hsp70.2 was expressed abundantly and in a highly stage-specific manner in the male germ line, it was also expressed in other murine tissues. Furthermore, we have made the surprising observation of antisense transcription of the hsp70.2 gene in several mouse tissues, revealing another level of complexity in the regulation and function of heat shock proteins.
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Yessine, Marie-Andrée; Meier, Christian; Petereit, Hans-Ulrich; Leroux, Jean-Christophe
2006-05-01
The delivery of active biomacromolecules to the cytoplasm is a major challenge as it is generally hindered by the endosomal/lysosomal barrier. Synthetic titratable polyanions can overcome this barrier by destabilizing membrane bilayers at pH values typically found in endosomes. This study investigates how anionic polyelectrolytes can enhance the cytoplasmic delivery of an antisense oligonucleotide (ODN). Novel methacrylic acid (MAA) copolymers were examined for their pH-sensitive properties and ability to destabilize cell membranes in a pH-dependent manner. Ternary complex formulations prepared with the ODN, a cationic lipid and a MAA copolymer were systematically characterized with respect to their size, zeta potential, antisense activity, cytotoxicity and cellular uptake using the A549 human lung carcinoma cell line. The MAA copolymer substantially increased the activity of the antisense ODN in inhibiting the expression of protein kinase C-alpha. Uptake, cytotoxicity and antisense activity were strongly dependent on copolymer concentration. Metabolic inhibitors demonstrated that endocytosis was the major internalization pathway of the complexes, and that endosomal acidification was essential for ODN activity. Confocal microscopy analysis of cells incubated with fluorescently-labeled complexes revealed selective delivery of the ODN, but not of the copolymer, to the cytoplasm/nucleus. This study provides new insight into the mechanisms of intracellular delivery of macromolecular drugs, using synthetic anionic polyelectrolytes.
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Caruso, Gerardo; Caffo, Mariella; Raudino, Giuseppe; Alafaci, Concetta; Salpietro, Francesco M; Tomasello, Francesco
2010-01-01
Despite the intensive recent research in cancer therapy, the prognosis in patients affected by high-grade gliomas is still very unfavorable. The efficacy of classical anti-cancer strategies is seriously limited by lack of specific therapies against malignant cells. The extracellular matrix plays a pivotal role in processes such as differentiation, apoptosis, and migration in both the normal and the pathologic nervous system. Glial tumors seem to be able to create a favorable environment for the invasion of glioma cells in cerebral parenchyma when they combine with the extracellular matrix via cell surface receptors. Glioma cells synthesize matrix proteins, such as tenascin, laminin, fibronectin that facilitate the tumor cell's motility. New treatments have shown to hit the acting molecules in the tumor growth and to increase the efficacy and minimize the toxicity. Antisense oligonucleotides are synthetic stretches of DNA which hybridize with specific mRNA strands. The specificity of hybridization makes antisense method an interesting strategy to selectively modulate the expression of genes involved in tumorigenesis. In this review we will focus on the mechanisms of action of antisense oligonucleotides and report clinical and experimental studies on the treatment of high-grade gliomas. We will also report the patents of preclinical and/or clinical studies that adopt the antisense oligonucleotide therapy list in cerebral gliomas.
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Antisense Transcription Is Pervasive but Rarely Conserved in Enteric Bacteria
Raghavan, Rahul; Sloan, Daniel B.; Ochman, Howard
2012-01-01
ABSTRACT Noncoding RNAs, including antisense RNAs (asRNAs) that originate from the complementary strand of protein-coding genes, are involved in the regulation of gene expression in all domains of life. Recent application of deep-sequencing technologies has revealed that the transcription of asRNAs occurs genome-wide in bacteria. Although the role of the vast majority of asRNAs remains unknown, it is often assumed that their presence implies important regulatory functions, similar to those of other noncoding RNAs. Alternatively, many antisense transcripts may be produced by chance transcription events from promoter-like sequences that result from the degenerate nature of bacterial transcription factor binding sites. To investigate the biological relevance of antisense transcripts, we compared genome-wide patterns of asRNA expression in closely related enteric bacteria, Escherichia coli and Salmonella enterica serovar Typhimurium, by performing strand-specific transcriptome sequencing. Although antisense transcripts are abundant in both species, less than 3% of asRNAs are expressed at high levels in both species, and only about 14% appear to be conserved among species. And unlike the promoters of protein-coding genes, asRNA promoters show no evidence of sequence conservation between, or even within, species. Our findings suggest that many or even most bacterial asRNAs are nonadaptive by-products of the cell’s transcription machinery. PMID:22872780
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Polakowski, Nicholas; Hoang, Kimson
2016-01-01
ABSTRACT Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus, and, as such, its genome becomes chromosomally integrated following infection. The resulting provirus contains identical 5′ and 3′ peripheral long terminal repeats (LTRs) containing bidirectional promoters. Antisense transcription from the 3′ LTR regulates expression of a single gene, hbz, while sense transcription from the 5′ LTR controls expression of all other viral genes, including tax. Both the HBZ and Tax proteins are implicated in the development of adult T-cell leukemia (ATL), a T-cell malignancy caused by HTLV-1 infection. However, these proteins appear to harbor opposing molecular functions, indicating that they may act independently and at different time points prior to leukemogenesis. Here, we used bidirectional reporter constructs to test whether transcriptional interference serves as a mechanism that inhibits simultaneous expression of Tax and HBZ. We found that sense transcription did not interfere with antisense transcription from the 3′ LTR and vice versa, even with strong transcription emanating from the opposing direction. Therefore, bidirectional transcription across the provirus might not restrict hbz or tax expression. Single-cell analyses revealed that antisense transcription predominates in the absence of Tax, which transactivates viral sense transcription. Interestingly, a population of Tax-expressing cells exhibited antisense but not activated sense transcription. Consistent with the ability of Tax to induce cell cycle arrest, this population was arrested in G0/G1 phase. These results imply that cell cycle arrest inhibits Tax-mediated activation of sense transcription without affecting antisense transcription, which may be important for long-term viral latency. IMPORTANCE The chromosomally integrated form of the retrovirus human T-cell leukemia virus type 1 (HTLV-1) contains identical DNA sequences, known as long terminal repeats (LTRs), at its 5′ and 3′ ends. The LTRs modulate transcription in both forward (sense) and reverse (antisense) directions. We found that sense transcription from the 5′ LTR does not interfere with antisense transcription from the 3′ LTR, allowing viral genes encoded on opposite DNA strands to be simultaneously transcribed. Two such genes are tax and hbz, and while they are thought to function at different times during the course of infection to promote leukemogenesis of infected T cells, our results indicate that they can be simultaneously transcribed. We also found that the ability of Tax to induce cell cycle arrest inhibits its fundamental function of activating viral sense transcription but does not affect antisense transcription. This regulatory mechanism may be important for long-term HTLV-1 infection. PMID:26792732
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Vedula, Vijay; Lee, Juhyun; Xu, Hao; Hsiai, Tzung K.
2017-01-01
Blood flow and mechanical forces in the ventricle are implicated in cardiac development and trabeculation. However, the mechanisms of mechanotransduction remain elusive. This is due in part to the challenges associated with accurately quantifying mechanical forces in the developing heart. We present a novel computational framework to simulate cardiac hemodynamics in developing zebrafish embryos by coupling 4-D light sheet imaging with a stabilized finite element flow solver, and extract time-dependent mechanical stimuli data. We employ deformable image registration methods to segment the motion of the ventricle from high resolution 4-D light sheet image data. This results in a robust and efficient workflow, as segmentation need only be performed at one cardiac phase, while wall position in the other cardiac phases is found by image registration. Ventricular hemodynamics are then quantified by numerically solving the Navier-Stokes equations in the moving wall domain with our validated flow solver. We demonstrate the applicability of the workflow in wild type zebrafish and three treated fish types that disrupt trabeculation: (a) chemical treatment using AG1478, an ErbB2 signaling inhibitor that inhibits proliferation and differentiation of cardiac trabeculation; (b) injection of gata1a morpholino oligomer (gata1aMO) suppressing hematopoiesis and resulting in attenuated trabeculation; (c) weak-atriumm58 mutant (wea) with inhibited atrial contraction leading to a highly undeveloped ventricle and poor cardiac function. Our simulations reveal elevated wall shear stress (WSS) in wild type and AG1478 compared to gata1aMO and wea. High oscillatory shear index (OSI) in the grooves between trabeculae, compared to lower values on the ridges, in the wild type suggest oscillatory forces as a possible regulatory mechanism of cardiac trabeculation development. The framework has broad applicability for future cardiac developmental studies focused on quantitatively investigating the role of hemodynamic forces and mechanotransduction during morphogenesis. PMID:29084212
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NASA Technical Reports Server (NTRS)
Duncan, R. L.; Kizer, N.; Barry, E. L.; Friedman, P. A.; Hruska, K. A.
1996-01-01
By patch-clamp analysis, we have shown that chronic, intermittent mechanical strain (CMS) increases the activity of stretch-activated cation channels of osteoblast-like UMR-106.01 cells. CMS also produces a swelling-activated whole-cell conductance (Gm) regulated by varying strain levels. We questioned whether the swelling-activated conductance was produced by stretch-activated cation channel activity. We have identified a gene involved in the increase in conductance by using antisense oligodeoxynucleotides (ODN) derived from the alpha 1-subunit genes of calcium channels found in UMR-106.01 cells (alpha1S, alpha1C, and alpha1D). We demonstrate that alpha 1C antisense ODNs abolish the increase in Gm in response to hypotonic swelling following CMS. Antisense ODNs to alpha1S and alpha1D, sense ODNs to alpha1C, and sham permeabilization had no effect on the conductance increase. In addition, during cell-attached patch-clamp studies, antisense ODNs to alpha1c completely blocked the swelling-activated and stretch-activated nonselective cation channel response to strain. Antisense ODNs to alpha1S treatment produced no effect on either swelling-activated or stretch-activated cation channel activity. There were differences in the stretch-activated and swelling-activated cation channel activity, but whether they represent different channels could not be determined from our data. Our data indicate that the alpha1C gene product is involved in the Gm and the activation of the swelling-activated cation channels induced by CMS. The possibility that swelling-activated cation channel genes are members of the calcium channel superfamily exists, but if alpha1c is not the swelling-activated cation channel itself, then its expression is required for induction of swelling-activated cation channel activity by CMS.
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PETERS, NICK T.; ROHRBACH, JUSTIN A.; ZALEWSKI, BRIAN A.; BYRKETT, COLLEEN M.; VAUGHN, JACK C.
2003-01-01
We have previously described an example of extensively A-to-G edited cDNA derived from adult heads of the fruitfly Drosophila melanogaster. In that study, the source of the predicted antisense RNA pairing strand for template recognition by dADAR editase was not identified, and the biological significance of the observed hyperediting was not known. Here, we address each of these questions. 4f-rnp and sas-10 are closely adjacent X-linked genes located on opposite DNA strands that produce convergent transcripts. We show that developmentally regulated antisense sas-10 readthrough mRNA arises by activation of an upstream promoter P2 during the late embryo stage of fly development. The sas-10 readthrough transcripts pair with 4f-rnp mRNA to form double-stranded molecules, as indicated by A-to-G editing observed in both RNA strands. It would be predicted that perfect RNA duplexes would be targeted for modification/degradation by enzyme pathways that recognize double-stranded RNAs, leading to decline in 4f-rnp mRNA levels, and this is what we observe. The observation using quantitative RT-PCR that sas-10 readthrough and 4f-rnp transcript levels are inversely related suggests a role for the antisense RNA in posttranscriptional regulation of 4f-rnp gene expression during development. Potential molecular mechanisms that could lead to this result are discussed, one of which is targeted transcript degradation via the RNAi pathway. Insofar as the dADAR editase and RNAi pathways are known to be constitutive in this system, it is likely that control of antisense RNA transcription is the rate-limiting factor. The results provide insight into roles of naturally occurring antisense RNAs in regulation of eukaryotic gene expression. PMID:12756328
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Goh, Shan; Loeffler, Anette; Lloyd, David H; Nair, Sean P; Good, Liam
2015-11-11
Antibiotic resistance genes can be targeted by antisense agents, which can reduce their expression and thus restore cellular susceptibility to existing antibiotics. Antisense inhibitors can be gene and pathogen specific, or designed to inhibit a group of bacteria having conserved sequences within resistance genes. Here, we aimed to develop antisense peptide nucleic acids (PNAs) that could be used to effectively restore susceptibility to β-lactams in methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP). Antisense PNAs specific for conserved regions of the mobilisable gene mecA, and the growth essential gene, ftsZ, were designed. Clinical MRSA and MRSP strains of high oxacillin resistance were treated with PNAs and assayed for reduction in colony forming units on oxacillin plates, reduction in target gene mRNA levels, and cell size. Anti-mecA PNA at 7.5 and 2.5 μM reduced mecA mRNA in MRSA and MRSP (p < 0.05). At these PNA concentrations, 66 % of MRSA and 92 % of MRSP cells were killed by oxacillin (p < 0.01). Anti-ftsZ PNA at 7.5 and 2.5 μM reduced ftsZ mRNA in MRSA and MRSP, respectively (p ≤ 0.05). At these PNA concentrations, 86 % of MRSA cells and 95 % of MRSP cells were killed by oxacillin (p < 0.05). Anti-ftsZ PNAs resulted in swelling of bacterial cells. Scrambled PNA controls did not affect MRSA but sensitized MRSP moderately to oxacillin without affecting mRNA levels. The antisense PNAs effects observed provide in vitro proof of concept that this approach can be used to reverse β-lactam resistance in staphylococci. Further studies are warranted as clinical treatment alternatives are needed.
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Edwards, Mary E.; Choo, Tze-Siang; Dickson, Cathryn A.; Scott, Catherine; Gidley, Michael J.; Reid, J.S. Grant
2004-01-01
Galactomannan biosynthesis in legume seed endosperms involves two Golgi membrane-bound glycosyltransferases, mannan synthase and galactomannan galactosyltransferase (GMGT). GMGT specificity is an important factor regulating the distribution and amount of (1→6)-α-galactose (Gal) substitution of the (1→4)-β-linked mannan backbone. The model legume Lotus japonicus is shown now to have endospermic seeds with endosperm cell walls that contain a high-Gal galactomannan (mannose [Man]/Gal = 1.2-1.3). Galactomannan biosynthesis in developing L. japonicus endosperms has been mapped, and a cDNA encoding a functional GMGT has been obtained from L. japonicus endosperms during galactomannan deposition. L. japonicus has been transformed with sense, antisense, and sense/antisense (“hairpin loop”) constructs of the GMGT cDNA. Some of the sense, antisense, and sense/antisense transgenic lines exhibited galactomannans with altered (higher) Man/Gal values in their (T1 generation) seeds, at frequencies that were consistent with posttranscriptional silencing of GMGT. For T1 generation individuals, transgene inheritance was correlated with galactomannan composition and amount in the endosperm. All the azygous individuals had unchanged galactomannans, whereas those that had inherited a GMGT transgene exhibited a range of Man/Gal values, up to about 6 in some lines. For Man/Gal values up to 4, the results were consistent with lowered Gal substitution of a constant amount of mannan backbone. Further lowering of Gal substitution was accompanied by a slight decrease in the amount of mannan backbone. Microsomal membranes prepared from the developing T2 generation endosperms of transgenic lines showed reduced GMGT activity relative to mannan synthase. The results demonstrate structural modification of a plant cell wall polysaccharide by designed regulation of a Golgi-bound glycosyltransferase. PMID:14988472
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Pinçon, Gaelle; Chabannes, Matthieu; Lapierre, Catherine; Pollet, Brigitte; Ruel, Katia; Joseleau, Jean-Paul; Boudet, Alain M.; Legrand, Michel
2001-01-01
Inhibition of specific lignin biosynthetic steps by antisense strategy has previously been shown to alter lignin content and/or structure. In this work, homozygous tobacco (Nicotiana tabacum) lines transformed with cinnamoyl-coenzyme A reductase (CCR) or caffeic acid/5-hydroxy ferulic acid-O-methyltransferase I (COMT I) antisense sequences have been crossed and enzyme activities, lignin synthesis, and cell wall structure of the progeny have been analyzed. In single transformed parents, CCR inhibition did not affect COMT I expression, whereas marked increases in CCR activity were observed in COMT I antisense plants, suggesting potential cross talk between some genes of the pathway. In the progeny, both CCR and COMT I activities were shown to be markedly decreased due to the simultaneous repression of the two genes. In these double transformants, the lignin profiles were dependent on the relative extent of down-regulation of each individual enzyme. For the siblings issued from a strongly repressed antisense CCR parent, the lignin patterns mimicked the patterns obtained in single transformants with a reduced CCR activity. In contrast, the specific lignin profile of COMT I repression could not be detected in double transformed siblings. By transmission electron microscopy some cell wall loosening was detected in the antisense CCR parent but not in the antisense COMT I parent. In double transformants, immunolabeling of non-condensed guaiacyl-syringyl units was weaker and revealed changes in epitope distribution that specifically affected vessels. Our results more widely highlight the impact of culture conditions on phenotypes and gene expression of transformed plants. PMID:11351078
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NASA Astrophysics Data System (ADS)
Bredikhin, Alexander A.; Zakharychev, Dmitry V.; Fayzullin, Robert R.; Bredikhina, Zemfira A.; Gubaidullin, Aidar T.
2015-05-01
Solid state properties of 3-(4-N-morpholino-1,2,5-thiadiazol-3-yloxy)-propane-1,2-diol 3, the synthetic precursor of popular drug timolol, have been investigated. The original solubility test, the data of X-ray diffraction and DSC methods indicate that the compound is prone to spontaneous resolution. Diol 3 crystallizing from both enantiopure or racemic feed material forms "guaifenesin-like" crystal packing in which the classic H-bonded bilayers, framed in both sides by hydrophobic molecular fragments, act as the basic supramolecular motif. The main chain conformation of the molecules in the crystals of diol 3 differs from that in the guaifenesin crystals, and this fact changes the absolute configuration of spiral columns formed by intermolecular hydrogen bonds in crystals of 3 as compared with guaifenesin crystals.
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CTCF knockout reveals an essential role for this protein during the zebrafish development.
Carmona-Aldana, Francisco; Zampedri, Cecilia; Suaste-Olmos, Fernando; Murillo-de-Ozores, Adrián; Guerrero, Georgina; Arzate-Mejía, Rodrigo; Maldonado, Ernesto; Navarro, Rosa; Chimal-Monroy, Jesús; Recillas-Targa, Félix
2018-05-01
Chromatin regulation and organization are essential processes that regulate gene activity. The CCCTC-binding factor (CTCF) is a protein with different and important molecular functions related with chromatin dynamics. It is conserved since invertebrates to vertebrates, posing it as a factor with an important role in the physiology. In this work, we aimed to understand the distribution and functional relevance of CTCF during the embryonic development of the zebrafish (Danio rerio). We generated a zebrafish specific anti-Ctcf antibody, and found this protein to be ubiquitous, through different stages and tissues. We used the CRISPR-Cas9 system to induce molecular alterations in the locus. This resulted in early lethality. We delayed the lethality performing knockdown morpholino experiments, and found an aberrant embryo morphology involving malformations in structures through all the length of the embryo. These phenotypes were rescued with human CTCF mRNA injections, showing the specificity of the morpholinos and a partial functional conservation between the fish and the human proteins. Lastly, we found that the pro-apoptotic genes p53 and bbc3/PUMA are deregulated in the ctcf morpholino-injected embryos. In conclusion, CTCF is a ubiquitous factor during the zebrafish development, which regulates the correct formation of different structures of the embryo, and its deregulation impacts on essential cell survival genes. Overall, this work provides a basis to look for the particular functions of CTCF in the different developing tissues and organs of the zebrafish. Copyright © 2018. Published by Elsevier B.V.
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Strategies to introduce resistance to viroids (Book Chapter)
USDA-ARS?s Scientific Manuscript database
Little or no naturally occurring durable resistance to viroids has been found in most viroid host species; therefore efforts to engineer viroid resistance in these plant hosts have been made. These efforts include strategies that incorporate viroid-specific antisense RNAs, sense and antisense viroid...
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Bcl-2 antisense therapy in B-cell malignant proliferative disorders.
Chanan-Khan, Asher; Czuczman, Myron S
2004-08-01
Overexpression of Bcl-2 oncogene has been clinically associated with an aggressive clinical course, chemotherapy and radiotherapy resistance, and poor survival in patients with malignant B-cell disorders. Patients with relapsed or refractory chronic lymphocytic leukemia, multiple myeloma, or non-Hodgkin's lymphoma have limited therapeutic options. Preclinical and early clinical data have shown that Bcl-2 oncoprotein can be decreased by Bcl-2 antisense therapy. Also, downregulation of Bcl-2 protein can result in reversal of chemotherapy resistance and improved antitumor activity of biologic agents. Various clinical trials are evaluating the role of targeting Bcl-2 as a mechanism to enhance the antitumor potential of chemotherapy and immunotherapy. Early results from these clinical studies are encouraging and confirm the proof of principle for antisense therapy. As current data mature, these trials will hopefully validate preliminary results and establish Bcl-2 antisense as an important addition to the current armamentarium used in the treatment of patients with B-cell neoplasms.
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A long noncoding RNA contributes to neuropathic pain by silencing Kcna2 in primary afferent neurons
Zhao, Xiuli; Tang, Zongxiang; Zhang, Hongkang; Atianjoh, Fidelis E.; Zhao, Jian-Yuan; Liang, Lingli; Wang, Wei; Guan, Xiaowei; Kao, Sheng-Chin; Tiwari, Vinod; Gao, Yong-Jing; Hoffman, Paul N.; Cui, Hengmi; Li, Min; Dong, Xinzhong; Tao, Yuan-Xiang
2013-01-01
Neuropathic pain is a refractory disease characterized by maladaptive changes in gene transcription and translation within the sensory pathway. Long noncoding RNAs (lncRNAs) are emerging as new players in gene regulation, but how lncRNAs operate in the development of neuropathic pain is unclear. Here we identify a conserved lncRNA for Kcna2 (named Kcna2 antisense RNA) in first-order sensory neurons of rat dorsal root ganglion (DRG). Peripheral nerve injury increases Kcna2 antisense RNA expression in injured DRG through activation of myeloid zinc finger protein 1, a transcription factor that binds to Kcna2 antisense RNA gene promoter. Mimicking this increase downregulates Kcna2, reduces total Kv current, increases excitability in DRG neurons, and produces neuropathic pain symptoms. Blocking this increase reverses nerve injury-induced downregulation of DRG Kcna2 and attenuates development and maintenance of neuropathic pain. These findings suggest native Kcna2 antisense RNA as a new therapeutic target for the treatment of neuropathic pain. PMID:23792947
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Jaeger, Laura B; Dohgu, Shinya; Hwang, Mark C; Farr, Susan A; Murphy, M Paul; Fleegal-DeMotta, Melissa A; Lynch, Jessica L; Robinson, Sandra M; Niehoff, Michael L; Johnson, Steven N; Kumar, Vijaya B; Banks, William A
2009-01-01
Decreased clearance is the main reason amyloid-beta protein (Abeta) is increased in the brains of patients with Alzheimer's disease (AD). The neurovascular hypothesis states that this decreased clearance is caused by impairment of low density lipoprotein receptor related protein-1 (LRP-1), the major brain-to-blood transporter of Abeta at the blood-brain barrier (BBB). As deletion of the LRP-1 gene is a lethal mutation, we tested the neurovascular hypothesis by developing a cocktail of phosphorothioate antisenses directed against LRP-1 mRNA. We found these antisenses in comparison to random antisense selectively decreased LRP-1 expression, reduced BBB clearance of Abeta42, increased brain levels of Abeta42, and impaired learning ability and recognition memory in mice. These results support dysfunction of LRP-1 at the BBB as a mechanism by which brain levels of Abeta could increase and AD would be promoted.
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Crooke, Rosanne M; Graham, Mark J
2013-01-01
Antisense oligonucleotides (ASOs) are a new class of specific therapeutic agents that alter the intermediary metabolism of mRNA, resulting in the suppression of disease-associated gene products. ASOs exert their pharmacological effects after hybridizing, via Watson-Crick base pairing, to a specific target RNA. If appropriately designed, this event results in the recruitment of RNase H, the degradation of targeted mRNA or pre-mRNA, and subsequent inhibition of the synthesis of a specific protein. A key advantage of the technology is the ability to selectively inhibit targets that cannot be modulated by traditional therapeutics such as structural proteins, transcription factors, and, of topical interest, lipoproteins. In this chapter, we will first provide an overview of antisense technology, then more specifically describe the status of lipoprotein-related genes that have been studied using the antisense platform, and finally, outline the general methodology required to design and evaluate the in vitro and in vivo efficacy of those drugs.
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Ramazeilles, C; Mishra, R K; Moreau, S; Pascolo, E; Toulmé, J J
1994-08-16
We targeted the mini-exon sequence, present at the 5' end of every mRNA of the protozoan parasite Leishmania amazonensis, by phosphorothioate oligonucleotides. A complementary 16-mer (16PS) was able to kill amastigotes--the intracellular stage of the parasite--in murine macrophages in culture. After 24 hr of incubation with 10 microM 16PS, about 30% infected macrophages were cured. The oligomer 16PS acted through antisense hybridization in a sequence-dependent way; no effect on parasites was observed with noncomplementary phosphorothioate oligonucleotides. The antisense oligonucleotide 16PS was a selective killer of the protozoans without any detrimental effect to the host macrophage. Using 16PS linked to a palmitate chain, which enabled it to complex with low density lipoproteins, improved the leishmanicidal efficiency on intracellular amastigotes, probably due to increased endocytosis. Phosphorothioate oligonucleotides complementary to the intron part of the mini-exon pre-RNA were also effective, suggesting that antisense oligomers could prevent trans-splicing in these parasites.
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Islam, N; Poitras, L; Gagnon, F; Moss, T
1996-10-17
The structure and temporal expression of two Xenopus cDNAs encoding the beta subunit of pyruvate dehydrogenase (XPdhE1 beta) have been determined. XPdhE1 beta was 88% homologous to mature human PdhE1 beta, but the putative N-terminal mitochondrial signal peptide was poorly conserved. Zygotic expression of XPdhE1 beta mRNA was detected at neural tube closure and increased until stage 40. RT-PCR cloning identified a short homology to a protein kinase open reading frame within the 3' non-coding sequence of the XPdhE1 beta cDNAs. This homology, which occurred on the antisense cDNA strand, was shown by strand specific RT-PCR to be transcribed in vivo as part of an antisense RNA. Northern analysis showed that this RNA formed part of an abundant and heterogeneous population of antisense and sense poly(A)-RNAs transcribed from the XPdhE1 beta loci and coordinately regulated with message production.
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Streets, Andrew J; Moon, David J; Kane, Michelle E; Obara, Tomoko; Ong, Albert CM
2008-01-01
PKD2 is mutated in 15% of patients with autosomal dominant polycystic kidney disease (ADPKD). Polycystin-2 (PC2), the PKD2 protein, is a nonselective Ca2+-permeable cation channel which may function at the cell surface and ER. Nevertheless, the factors that regulate the dynamic translocation of PC2 between the ER and other compartments are not well understood. Constitutive phosphorylation of PC2 at a single C-terminal site (Ser812) has been previously reported. Since we were unable to abolish phospholabelling of PC2 in HEK293 cells by site-directed mutagenesis of Ser812 or all 5 predicted phosphorylation sites in the C-terminus, we hypothesised that PC2 could also be phosphorylated at the N-terminus. In this paper, we report the identification of a new phosphorylation site for PC2 within its N-terminal domain (Ser76) and demonstrate that this residue is phosphorylated by glycogen synthase kinase 3 (GSK-3). The consensus recognition sequence for GSK-3 (Ser76/Ser80) is evolutionarily conserved down to lower vertebrates. In the presence of specific GSK-3 inhibitors, the lateral plasma membrane pool of endogenous PC2 redistributes into an intracellular compartment in MDCK cells without a change in primary cilia localization. Finally, co-injection of wild-type but not a S76A/S80A mutant PKD2 capped mRNA could rescue the cystic phenotype induced by an antisense morpholino oligonucleotide to pkd2 in zebrafish pronephric kidney. We conclude that surface localization of PC2 is regulated by phosphorylation at a unique GSK-3 site in its N-terminal domain in vivo and in vitro. This site is functionally significant for the maintenance of normal glomerular and tubular morphology. PMID:16551655
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Müller, Miriam; Persson, Anja Bondke; Krueger, Katharina; Kirschner, Karin M; Scholz, Holger
2017-07-01
Insulin-like growth factor (IGF) binding proteins (IGFBPs) constitute a family of six secreted proteins that regulate the signaling of insulin-like growth factors (IGFs). IGFBP5 is the most conserved family member in vertebrates and the major IGF binding protein in bone. IGFBP5 is required for normal development of the musculoskeletal system, and various types of cancer frequently express high levels of IGFP5. Here we identify the gene encoding IGFBP5 as a novel downstream target of the Wilms tumor protein WT1. IGFBP5 and WT1 are expressed in an overlapping pattern in the condensing metanephric mesenchyme of embryonic murine kidneys. Down-regulation of WT1 by transfection with antisense vivo-morpholino significantly decreased Igfbp5 transcripts in murine embryonic kidney explants. Likewise, silencing of Wt1 in a mouse mesonephros-derived cell line reduced Igfbp5 mRNA levels by approximately 80%. Conversely, induction of the WT1(-KTS) isoform, whose role as transcriptional regulator has been firmly established, significantly increased IGFBP5 mRNA and protein levels in osteosarcoma cells. IGFBP5 expression was not significantly changed by WT1(+KTS) protein, which exhibits lower DNA binding affinity than the WT1(-KTS) isoform and has a presumed role in post-transcriptional gene regulation. Luciferase reporter constructs harboring 0.8 and 1.6 kilobases of the murine Igfbp5 promoter, respectively, were stimulated approximately 5-fold by co-transfection of WT1(-KTS). The WT1(+KTS) variant had no significant effect on IGFBP5 promoter activity. Binding of WT1(-KTS), but not of WT1(+KTS) protein, to the IGFBP5 promoter in human osteosarcoma cells was proven by chromatin immunoprecipitation (ChIP) and confirmed by electrophoretic mobility shift assay. These findings demonstrate that WT1 activates transcription of the IGFBP5 gene with possible implications for kidney development and bone (patho)physiology. Copyright © 2017 Elsevier B.V. All rights reserved.
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Schmidt, Jennifer; Schuff, Maximilian; Olsson, Lennart
2011-01-01
The origin of morphological novelties is a controversial topic in evolutionary developmental biology. The heads of anuran larvae have several unique structures, including the supra- and infrarostral cartilages, the specialised structure of the gill basket (used for filtration), and novel cranial muscle arrangements. FoxN3, a member of the forkhead/winged helix family of transcription factors, has been implicated as important for normal craniofacial development in the pipid anuran Xenopus laevis. We have investigated the effects of functional knockdown of FoxN3 (using antisense oligonucleotide morpholino) on the development of the larval head skeleton and the associated cranial muscles in X. laevis. Our data complement earlier studies and provide a more complete account of the requirement of FoxN3 in chondrocranium development. In addition, we analyse the effects of FoxN3 knockdown on cranial muscle development. We show that FoxN3 knockdown primarily affects the novel skeletal structures unique to anuran larvae, i.e. the rostralia or the fine structure of the gill apparatus. The articulation between the infrarostral and Meckel's cartilage is malformed and the filigreed processes of the gill basket do not develop. Because these features do not develop after FoxN3 knockdown, the head morphology resembles that in the less specialised larvae of salamanders. Furthermore, the development of all cartilages derived from the neural crest is delayed and cranial muscle fibre development incomplete. The cartilage precursors initially condense in their proper position but later differentiate incompletely; several visceral arch muscles start to differentiate at their origin but fail to extend toward their insertion. Our findings indicate that FoxN3 is essential for the development of novel cartilages such as the infrarostral and other cranial tissues derived from the neural crest and, indirectly, also for muscle morphogenesis. PMID:21050205
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Schumacher, Jennifer A; Hashiguchi, Megumi; Nguyen, Vu H; Mullins, Mary C
2011-01-01
The specification of the neural crest progenitor cell (NCPC) population in the early vertebrate embryo requires an elaborate network of signaling pathways, one of which is the Bone Morphogenetic Protein (BMP) pathway. Based on alterations in neural crest gene expression in zebrafish BMP pathway component mutants, we previously proposed a model in which the gastrula BMP morphogen gradient establishes an intermediate level of BMP activity establishing the future NCPC domain. Here, we tested this model and show that an intermediate level of BMP signaling acts directly to specify the NCPC. We quantified the effects of reducing BMP signaling on the number of neural crest cells and show that neural crest cells are significantly increased when BMP signaling is reduced and that this increase is not due to an increase in cell proliferation. In contrast, when BMP signaling is eliminated, NCPC fail to be specified. We modulated BMP signaling levels in BMP pathway mutants with expanded or no NCPCs to demonstrate that an intermediate level of BMP signaling specifies the NCPC. We further investigated the ability of Smad5 to act in a graded fashion by injecting smad5 antisense morpholinos and show that increasing doses first expand the NCPCs and then cause a loss of NCPCs, consistent with Smad5 acting directly in neural crest progenitor specification. Using Western blot analysis, we show that P-Smad5 levels are dose-dependently reduced in smad5 morphants, consistent with an intermediate level of BMP signaling acting through Smad5 to specify the neural crest progenitors. Finally, we performed chimeric analysis to demonstrate for the first time that BMP signal reception is required directly by NCPCs for their specification. Together these results add substantial evidence to a model in which graded BMP signaling acts as a morphogen to pattern the ectoderm, with an intermediate level acting in neural crest specification.
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Chen, Wen-Chuan; Kou, Fong-Ji; Lu, Jeng-Wei; Wang, Horng-Dar; Huang, Chou-Long; Yuh, Chiou-Hwa
2014-01-01
The WNK1 (WNK lysine deficient protein kinase 1) protein is a serine/threonine protein kinase with emerging roles in cancer. WNK1 causes hypertension and hyperkalemia when overexpressed and cardiovascular defects when ablated in mice. In this study, the role of Wnk1 in angiogenesis was explored using the zebrafish model. There are two zebrafish wnk1 isoforms, wnk1a and wnk1b, and both contain all the functional domains found in the human WNK1 protein. Both isoforms are expressed in the embryo at the initiation of angiogenesis and in the posterior cardinal vein (PCV), similar to fms-related tyrosine kinase 4 (flt4). Using morpholino antisense oligonucleotides against wnk1a and wnk1b, we observed that wnk1 morphants have defects in angiogenesis in the head and trunk, similar to flk1/vegfr2 morphants. Furthermore, both wnk1a and wnk1b mRNA can partially rescue the defects in vascular formation caused by flk1/vegfr2 knockdown. Mutation of the kinase domain or the Akt/PI3K phosphorylation site within wnk1 destroys this rescue capability. The rescue experiments provide evidence that wnk1 is a downstream target for Vegfr2 (vascular endothelial growth factor receptor-2) and Akt/PI3K signaling and thereby affects angiogenesis in zebrafish embryos. Furthermore, we found that knockdown of vascular endothelial growth factor receptor-2 (flk1/vegfr2) or vascular endothelial growth factor receptor-3 (flt4/vegfr3) results in a decrease in wnk1a expression, as assessed by in situ hybridization and q-RT-PCR analysis. Thus, the Vegf/Vegfr signaling pathway controls angiogenesis in zebrafish via Akt kinase-mediated phosphorylation and activation of Wnk1 as well as transcriptional regulation of wnk1 expression. PMID:25171174
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Gebruers, Evelien; Cordero-Maldonado, María Lorena; Gray, Alexander I.; Clements, Carol; Harvey, Alan L.; Edrada-Ebel, Ruangelie; de Witte, Peter A. M.; Crawford, Alexander D.; Esguerra, Camila V.
2013-01-01
Zebrafish have recently emerged as an attractive model for the in vivo bioassay-guided isolation and characterization of pharmacologically active small molecules of natural origin. We carried out a zebrafish-based phenotypic screen of over 3000 plant-derived secondary metabolite extracts with the goal of identifying novel small-molecule modulators of the BMP and Wnt signaling pathways. One of the bioactive plant extracts identified in this screen – Jasminum gilgianum, an Oleaceae species native to Papua New Guinea – induced ectopic tails during zebrafish embryonic development. As ectopic tail formation occurs when BMP or non-canonical Wnt signaling is inhibited during the tail protrusion process, we suspected a constituent of this extract to act as a modulator of these pathways. A bioassay-guided isolation was carried out on the basis of this zebrafish phenotype, identifying para-coumaric acid methyl ester (pCAME) as the active compound. We then performed an in-depth phenotypic analysis of pCAME-treated zebrafish embryos, including a tissue-specific marker analysis of the secondary tails. We found pCAME to synergize with the BMP-inhibitors dorsomorphin and LDN-193189 in inducing ectopic tails, and causing convergence-extension defects in compound-treated embryos. These results indicate that pCAME may interfere with non-canonical Wnt signaling. Inhibition of Jnk, a downstream target of Wnt/PCP signaling (via morpholino antisense knockdown and pharmacological inhibition with the kinase inhibitor SP600125) phenocopied pCAME-treated embryos. However, immunoblotting experiments revealed pCAME to not directly inhibit Jnk-mediated phosphorylation of c-Jun, suggesting additional targets of SP600125, and/or other pathways, as possibly being involved in the ectopic tail formation activity of pCAME. Further investigation of pCAME’s mechanism of action will help determine this compound’s pharmacological utility. PMID:24349481
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Noda, Takeshi
2011-12-01
I isolated a Ciona intestinalis homolog of p53, Ci-p53/p73-a, in a microarray screen of rapidly degraded maternal mRNA by comparing the transcriptomes of unfertilized eggs and 32-cell stage embryos. Higher expression of the gene in eggs and lower expression in later embryonic stages were confirmed by whole-mount in situ hybridization (WISH) and quantitative reverse transcription-PCR (qRT-PCR); expression was ubiquitous in eggs and early embryos. Knockdown of Ci-p53/p73-a by injection of antisense morpholino oligonucleotides (MOs) severely perturbed gastrulation cell movements and expression of notochord marker genes. A key regulator of notochord differentiation in Ciona embryos is Brachyury (Ci-Bra), which is directly activated by a zic-like gene (Ci-ZicL). The expression of Ci-ZicL and Ci-Bra in A-line notochord precursors was downregulated in Ci-p53/p73-a knockdown embryos. Maternal expression of Ci-p53/p73-b, a homolog of Ci-p53/p73-a, was also detected. In Ci-p53/p73-b knockdown embryos, gastrulation cell movements, expression of Ci-ZicL and Ci-Bra in A-line notochord precursors, and expression of notochord marker gene at later stages were perturbed. The upstream region of Ci-ZicL contains putative p53-binding sites. Cis-regulatory analysis of Ci-ZicL showed that these sites are involved in expression of Ci-ZicL in A-line notochord precursors at the 32-cell and early gastrula stages. These results suggest that p53 genes are maternal factors that play a crucial role in A-line notochord differentiation in C. intestinalis embryos by regulating Ci-ZicL expression. Copyright © 2011 Elsevier Inc. All rights reserved.
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Two novel COLVI long chains in zebrafish that are essential for muscle development.
Ramanoudjame, Laetitia; Rocancourt, Claire; Lainé, Jeanne; Klein, Arnaud; Joassard, Lucette; Gartioux, Corine; Fleury, Marjory; Lyphout, Laura; Kabashi, Edor; Ciura, Sorana; Cousin, Xavier; Allamand, Valérie
2015-12-01
Collagen VI (COLVI), a protein ubiquitously expressed in connective tissues, is crucial for structural integrity, cellular adhesion, migration and survival. Six different genes are recognized in mammalians, encoding six COLVI-chains that assemble as two 'short' (α1, α2) and one 'long' chain (theoretically any one of α3-6). In humans, defects in the most widely expressed heterotrimer (α123), due to mutations in the COL6A1-3 genes, cause a heterogeneous group of neuromuscular disorders, collectively termed COLVI-related muscle disorders. Little is known about the function(s) of the recently described α4-6 chains and no mutations have been detected yet. In this study, we characterized two novel COLVI long chains in zebrafish that are most homologous to the mammalian α4 chain; therefore, we named the corresponding genes col6a4a and col6a4b. These orthologues represent ancestors of the mammalian Col6a4-6 genes. By in situ hybridization and RT-qPCR, we unveiled a distinctive expression kinetics for col6a4b, compared with the other col6a genes. Using morpholino antisense oligonucleotides targeting col6a4a, col6a4b and col6a2, we modelled partial and complete COLVI deficiency, respectively. All morphant embryos presented altered muscle structure and impaired motility. While apoptosis was not drastically increased, autophagy induction was defective in all morphants. Furthermore, motoneuron axon growth was abnormal in these morphants. Importantly, some phenotypical differences emerged between col6a4a and col6a4b morphants, suggesting only partial functional redundancy. Overall, our results further confirm the importance of COLVI in zebrafish muscle development and may provide important clues for potential human phenotypes associated with deficiency of the recently described COLVI-chains. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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Ciura, Sorana; Lattante, Serena; Le Ber, Isabelle; Latouche, Morwena; Tostivint, Hervé; Brice, Alexis; Kabashi, Edor
2013-08-01
To define the role that repeat expansions of a GGGGCC hexanucleotide sequence of the C9orf72 gene play in the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). A genetic model for ALS was developed to determine whether loss of function of the zebrafish orthologue of C9orf72 (zC9orf72) leads to abnormalities in neuronal development. C9orf72 mRNA levels were quantified in brain and lymphoblasts derived from FTLD and ALS/FTLD patients and in zebrafish. Knockdown of the zC9orf72 was performed using 2 specific antisense morpholino oligonucleotides to block transcription. Quantifications of spontaneous swimming and tactile escape response, as well as measurements of axonal projections from the spinal cord, were performed. Significantly decreased expression of C9orf72 transcripts in brain and lymphoblasts was found in sporadic FTLD and ALS/FTLD patients with normal-size or expanded hexanucleotide repeats. The zC9orf72 is selectively expressed in the developing nervous system at developmental stages. Loss of function of the zC9orf72 transcripts causes both behavioral and cellular deficits related to locomotion without major morphological abnormalities. These deficits were rescued upon overexpression of human C9orf72 mRNA transcripts. Our results indicate C9orf72 haploinsufficiency could be a contributing factor in the spectrum of ALS/FTLD neurodegenerative disorders. Loss of function of the zebrafish orthologue of zC9orf72 expression in zebrafish is associated with axonal degeneration of motor neurons that can be rescued by expressing human C9orf72 mRNA, highlighting the specificity of the induced phenotype. These results reveal a pathogenic consequence of decreased C9orf72 levels, supporting a loss of function mechanism of disease. © 2013 American Neurological Association.
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Wu, Mary Y.; Ramel, Marie-Christine; Howell, Michael; Hill, Caroline S.
2011-01-01
Bone morphogenetic protein (BMP) gradients provide positional information to direct cell fate specification, such as patterning of the vertebrate ectoderm into neural, neural crest, and epidermal tissues, with precise borders segregating these domains. However, little is known about how BMP activity is regulated spatially and temporally during vertebrate development to contribute to embryonic patterning, and more specifically to neural crest formation. Through a large-scale in vivo functional screen in Xenopus for neural crest fate, we identified an essential regulator of BMP activity, SNW1. SNW1 is a nuclear protein known to regulate gene expression. Using antisense morpholinos to deplete SNW1 protein in both Xenopus and zebrafish embryos, we demonstrate that dorsally expressed SNW1 is required for neural crest specification, and this is independent of mesoderm formation and gastrulation morphogenetic movements. By exploiting a combination of immunostaining for phosphorylated Smad1 in Xenopus embryos and a BMP-dependent reporter transgenic zebrafish line, we show that SNW1 regulates a specific domain of BMP activity in the dorsal ectoderm at the neural plate border at post-gastrula stages. We use double in situ hybridizations and immunofluorescence to show how this domain of BMP activity is spatially positioned relative to the neural crest domain and that of SNW1 expression. Further in vivo and in vitro assays using cell culture and tissue explants allow us to conclude that SNW1 acts upstream of the BMP receptors. Finally, we show that the requirement of SNW1 for neural crest specification is through its ability to regulate BMP activity, as we demonstrate that targeted overexpression of BMP to the neural plate border is sufficient to restore neural crest formation in Xenopus SNW1 morphants. We conclude that through its ability to regulate a specific domain of BMP activity in the vertebrate embryo, SNW1 is a critical regulator of neural plate border formation and thus neural crest specification. PMID:21358802
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Ribeiro, Carla M. S.; Bird, Steve; Raes, Geert; Ghassabeh, Gholamreza H.; Schijns, Virgil E. J. C.; Pontes, Maria J. S. L.; Savelkoul, Huub F. J.; Wiegertjes, Geert F.
2011-01-01
Background The innate immune system relies upon a wide range of germ-line encoded receptors including a large number of immunoglobulin superfamily (IgSF) receptors. Different Ig-like immune receptor families have been reported in mammals, birds, amphibians and fish. Most innate immune receptors of the IgSF are type I transmembrane proteins containing one or more extracellular Ig-like domains and their regulation of effector functions is mediated intracellularly by distinct stimulatory or inhibitory pathways. Methodology/Principal Findings Carp SITR was found in a substracted cDNA repertoire from carp macrophages, enriched for genes up-regulated in response to the protozoan parasite Trypanoplasma borreli. Carp SITR is a type I protein with two extracellular Ig domains in a unique organisation of a N-proximal V/C2 (or I-) type and a C-proximal V-type Ig domain, devoid of a transmembrane domain or any intracytoplasmic signalling motif. The carp SITR C-proximal V-type Ig domain, in particular, has a close sequence similarity and conserved structural characteristics to the mammalian CD300 molecules. By generating an anti-SITR antibody we could show that SITR protein expression was restricted to cells of the myeloid lineage. Carp SITR is abundantly expressed in macrophages and is secreted upon in vitro stimulation with the protozoan parasite T. borreli. Secretion of SITR protein during in vivo T. borreli infection suggests a role for this IgSF receptor in the host response to this protozoan parasite. Overexpression of carp SITR in mouse macrophages and knock-down of SITR protein expression in carp macrophages, using morpholino antisense technology, provided evidence for the involvement of carp SITR in the parasite-induced NO production. Conclusion/Significance We report the structural and functional characterization of a novel soluble immune-type receptor (SITR) in a teleost fish and propose a role for carp SITR in the NO-mediated response to a protozoan parasite. PMID:21305002
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Antisense oligonucleotide technologies in drug discovery.
Aboul-Fadl, Tarek
2006-09-01
The principle of antisense oligonucleotide (AS-OD) technologies is based on the specific inhibition of unwanted gene expression by blocking mRNA activity. It has long appeared to be an ideal strategy to leverage new genomic knowledge for drug discovery and development. In recent years, AS-OD technologies have been widely used as potent and promising tools for this purpose. There is a rapid increase in the number of antisense molecules progressing in clinical trials. AS-OD technologies provide a simple and efficient approach for drug discovery and development and are expected to become a reality in the near future. This editorial describes the established and emerging AS-OD technologies in drug discovery.
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Matveeva, O. V.; Tsodikov, A. D.; Giddings, M.; Freier, S. M.; Wyatt, J. R.; Spiridonov, A. N.; Shabalina, S. A.; Gesteland, R. F.; Atkins, J. F.
2000-01-01
Design of antisense oligonucleotides targeting any mRNA can be much more efficient when several activity-enhancing motifs are included and activity-decreasing motifs are avoided. This conclusion was made after statistical analysis of data collected from >1000 experiments with phosphorothioate-modified oligonucleotides. Highly significant positive correlation between the presence of motifs CCAC, TCCC, ACTC, GCCA and CTCT in the oligonucleotide and its antisense efficiency was demonstrated. In addition, negative correlation was revealed for the motifs GGGG, ACTG, AAA and TAA. It was found that the likelihood of activity of an oligonucleotide against a desired mRNA target is sequence motif content dependent. PMID:10908347
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Prhavc, M.; Prakash, T.P.; Minasov, G.
Oligonucleotides with a novel, 2'-O-[2-[2-(N,N-dimethylamino)ethoxy]ethyl] (2'-O-DMAEOE) modification have been synthesized. This modification, a cationic analogue of the 2'-O-(2-methoxyethyl) (2'-O-MOE) modification, exhibits high binding affinity to target RNA (but not to DNA) and exceptional resistance to nuclease degradation. Analysis of the crystal structure of a self-complementary oligonucleotide containing a single 2'-O-DMAEOE modification explains the importance of charge factors and gauche effects on the observed antisense properties. 2'-O-DMAEOE modified oligonucleotides are ideal candidates for antisense drugs.
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Natural antisense transcripts associated with salinity response in alfalfa
USDA-ARS?s Scientific Manuscript database
Natural antisense transcripts (NATs) are long non-coding RNAs (lncRNAs) complimentary to the messenger (sense) RNA (Wang et al. 2014). Many of them are involved in regulation of their own sense transcripts thus playing pivotal biological roles in all processes of organismal development and responses...
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Karimi, Mohammad Hossein; Ebadi, Padideh; Pourfathollah, Ali Akbar; Moazzeni, Mohammad; Soheili, Zahra Soheila; Samiee, Shahram
2010-12-01
In recent years, a new view of dendritic cells (DCs) as a main regulator of immunity to induce and maintain tolerance has been established. In vitro manipulation of their development and maturation is a topic of DC therapeutic application, which utilizes their inherent tolerogenicity. In this field, the therapeutic potential of antisense, siRNA, and blocking antibody are an interesting goal. In the present study, the efficiency of these three methods--siRNA, antisense, and blocking antibody--against CD40 molecule and its function in DCs and BCL1 cell line are compared. DCs were separated from mouse spleen and then cultured in vitro using Lipofectamine 2000 to deliver both silencers; the efficacy of transfection was estimated by flow cytometry. mRNA expression and protein synthesis were assessed by real time-PCR and flow cytometry, respectively. By Annexin V and propidium iodine staining, we could evaluate the viability of transfected cells. Knocking down the CD40 gene into separate groups of DCs by siRNA, antisense, and blocking antibody treated DCs can cause an increase in IL-4, decrease in IL-12, IFN-γ production, and allostimulation activity. Our results indicated that, in comparison to antisense and blocking antibody, siRNAs appear to be quantitatively more efficient in CD40 downregulation and their differences are significant.
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Chang, S H; Bugos, R C; Sun, W H; Yamamoto, H Y
2000-01-01
Violaxanthin de-epoxidase (VDE) catalyzes the de-epoxidation of violaxanthin to antheraxanthin and zeaxanthin in the xanthophyll cycle. Tobacco was transformed with an antisense VDE construct under control of the cauliflower mosaic virus 35S promoter to determine the effect of reduced levels of VDE on plant growth. Screening of 40 independent transformants revealed 18 antisense lines with reduced levels of VDE activity with two in particular (TAS32 and TAS39) having greater than 95% reduction in VDE activity. Northern analysis demonstrated that these transformants had greatly suppressed levels of VDE mRNA. De-epoxidation of violaxanthin was inhibited to such an extent that no zeaxanthin and only very low levels of antheraxanthin could be detected after exposure of leaves to high light (2000 mumol m(-2) s(-1) for 20 min) with no observable effect on levels of other carotenoids and chlorophyll. Non-photochemical quenching was greatly reduced in the antisense VDE tobacco, demonstrating that a significant level of the non-photochemical quenching in tobacco requires de-epoxidation of violaxanthin. Although the antisense plants demonstrated a greatly impaired de-epoxidation of violaxanthin, no effect on plant growth or photosynthetic rate was found when plants were grown at a photon flux density of 500 or 1000 mumol m(-2) s(-1) under controlled growth conditions as compared to wild-type tobacco.
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Sugano, M; Makino, N; Sawada, S; Otsuka, S; Watanabe, M; Okamoto, H; Kamada, M; Mizushima, A
1998-02-27
Cholesteryl ester transfer protein (CETP) is the enzyme that facilitates the transfer of cholesteryl ester from high density lipoprotein (HDL) to apolipoprotein B (apoB)-containing lipoproteins. However, the exact role of CETP in the development of atherosclerosis has not been determined. In the present study, we examined the effect of the suppression of increased plasma CETP by intravenous injection with antisense oligodeoxynucleotides (ODNs) against CETP targeted to the liver on the development of atherosclerosis in rabbits fed a cholesterol diet. The ODNs against rabbit CETP were coupled to asialoglycoprotein (ASOR) carrier molecules, which serve as an important method to regulate liver gene expression. Twenty-two male Japanese White rabbits were used in the experiment. Eighteen animals were fed a standard rabbit chow supplemented with 0.3% cholesterol throughout the experiment for 16 weeks. At 8 weeks, they were divided into three groups (six animals in each group), among which the plasma total and HDL cholesterol concentrations did not significantly change. The control group received nothing, the sense group were injected with the sense ODNs complex, and the antisense group were injected with the antisense ODNs complex, respectively, for subsequent 8 weeks. ASOR. poly(L-lysine) ODNs complex were injected via the ear veins twice a week. Four animals were fed a standard rabbit diet for 16 weeks. The total cholesterol concentrations and the CETP mass in the animals injected with antisense ODNs were all significantly decreased in 12 and 16 weeks compared with those injected with sense ODNs and the control animals. The HDL cholesterol concentrations measured by the precipitation assay did not significantly change among the groups fed a cholesterol diet, and triglyceride concentrations did not significantly change in the four groups. However, at the end of the study, when the HDL cholesterol concentrations were measured after the isolation by ultracentrifugation and a column chromotography, they were significantly higher in the animals injected with antisense ODNs than in the animals injected with sense ODNs and in the control animals. A reduction of CETP mRNA and an increase of LDL receptor mRNA in the liver were observed in the animals injected with antisense ODNs compared with those injected with sense ODNs and the control animals. Aortic cholesterol contents and the aortic percentage lesion to total surface area were significantly lower in the animals injected with antisense ODNs than in the animals injected with sense ODNs and in the control animals. These findings showed for the first time that suppression of increased plasma CETP by the injection with antisense ODNs against CETP coupled to ASOR carrier molecules targeted to the liver could thus inhibit the atherosclerosis possibly by decreasing the plasma LDL + very low density lipoprotein (VLDL) cholesterol in cholesterol-fed rabbits.
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Inositol-requiring enzyme 1α is required for gut development in Xenopus lavies embryos
Guo, Jing; Li, Xin-Xin; Feng, Jiao-Jiao; Yin, Chen-Yang; Wang, Xue-Jun; Wang, Ning; Yuan, Li
2013-01-01
AIM: To investigate the role of inositol-requiring enzyme 1α (IRE1α) in gut development of Xenopus lavies embryos. METHODS: Xenopus embryos were obtained with in vitro fertilization and cultured in 0.1 × MBSH. One and half nanogram of IRE1α, 1 ng of IRE1α-GR mRNA, 1 ng of IRE1αΔC-GR mRNA, and 50 ng of IRE1α morpholino oligonucleotide (MO) or XBP1(C)MO were injected into four blastomeres at 4-cell stage for scoring the phenotype and marker gene analysis. To rescue the effect of IRE1α MO, 1 ng of IRE1α-GR mRNA was co-injected with 50 ng of MO. For the activation of the GR-fusion proteins, dexamethasone was prepared as 5 mmol/L stock solutions in 100% ethanol and applied to the mRNA injected embryos at desired stages in a concentration of 10 μmol/L in 0.1 × MBSH. Embryos were kept in dexamethasone up to stage 41. Whole-mount in situ hybridization was used to determine specific gene expression, such as IRE1α, IRE1β, Xbra and Xsox17α. IRE1α protein expression during Xenopus embryogenesis was detected by Western blotting. RESULTS: In the whole-mount in situ hybridization analysis, xenopus IRE1α and IRE1β showed quite different expression pattern during tadpole stage. The relatively higher expression of IRE1α was observed in the pancreas, and significant transcription of IRE1β was found in the liver. IRE1α protein could be detected at all developmental stages analyzed, from stage 1 to stage 42. Gain-of-function assay showed that IRE1α mRNA injected embryos at tailbud stage were nearly normal and the expression of the pan-mesodermal marker gene Xbra and the endodermal gene Xsox17α at stage 10.5 was not significantly changed in embryos injected with IRE1α mRNA as compared to uninjected control embryos. And at tadpole stage, the embryos injected with IRE1α-GR mRNA did not display overt phenotype, such as gut-coiling defect. Loss-of-function assay demonstrated that the IRE1α MO injected embryos were morphologically normal before the tailbud stages. We did not observe a significant change of mesodermal and endodermal marker gene expression, while after stage 40, about 80% of the MO injected embryos exhibited dramatic gut defects in which the guts did not coil, but other structures outside the gastrointestinal tract were relatively normal. To test if the phenotypes were specifically caused by the knockdown of IRE1α, a rescue experiment was performed by co-injection of IRE1α-GR mRMA with IRE1α MO. The data obtained demonstrated that the gut coiling defect was rescued. The deletion mutant of IRE1α was constructed, consisting of the N-terminal part without the C-terminal kinase and RNase domains named IRE1αΔC, to investigate the functional domain of IRE1α. Injection of IRE1αΔC-GR mRNA caused similar morphological alterations with gut malformation by interfering with the function of endogenous xIRE1α. In order to investigate if IRE1α/XBP1 pathway was involved in gut development, 50 ng of XBP1 MO was injected and the results showed that knockdown of XBP1 resulted in similar morphological alterations with gut-coiling defect at tadpole stage. CONCLUSION: IRE1α is not required for germ layer formation but for gut development in Xenopus lavies and it may function via XBP1-dependent pathway. PMID:23345945
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Inositol-requiring enzyme 1α is required for gut development in Xenopus lavies embryos.
Guo, Jing; Li, Xin-Xin; Feng, Jiao-Jiao; Yin, Chen-Yang; Wang, Xue-Jun; Wang, Ning; Yuan, Li
2013-01-14
To investigate the role of inositol-requiring enzyme 1α (IRE1α) in gut development of Xenopus lavies embryos. Xenopus embryos were obtained with in vitro fertilization and cultured in 0.1 × MBSH. One and half nanogram of IRE1α, 1 ng of IRE1α-GR mRNA, 1 ng of IRE1αΔC-GR mRNA, and 50 ng of IRE1α morpholino oligonucleotide (MO) or XBP1(C)MO were injected into four blastomeres at 4-cell stage for scoring the phenotype and marker gene analysis. To rescue the effect of IRE1α MO, 1 ng of IRE1α-GR mRNA was co-injected with 50 ng of MO. For the activation of the GR-fusion proteins, dexamethasone was prepared as 5 mmol/L stock solutions in 100% ethanol and applied to the mRNA injected embryos at desired stages in a concentration of 10 μmol/L in 0.1 × MBSH. Embryos were kept in dexamethasone up to stage 41. Whole-mount in situ hybridization was used to determine specific gene expression, such as IRE1α, IRE1β, Xbra and Xsox17α. IRE1α protein expression during Xenopus embryogenesis was detected by Western blotting. In the whole-mount in situ hybridization analysis, xenopus IRE1α and IRE1β showed quite different expression pattern during tadpole stage. The relatively higher expression of IRE1α was observed in the pancreas, and significant transcription of IRE1β was found in the liver. IRE1α protein could be detected at all developmental stages analyzed, from stage 1 to stage 42. Gain-of-function assay showed that IRE1α mRNA injected embryos at tailbud stage were nearly normal and the expression of the pan-mesodermal marker gene Xbra and the endodermal gene Xsox17α at stage 10.5 was not significantly changed in embryos injected with IRE1α mRNA as compared to uninjected control embryos. And at tadpole stage, the embryos injected with IRE1α-GR mRNA did not display overt phenotype, such as gut-coiling defect. Loss-of-function assay demonstrated that the IRE1α MO injected embryos were morphologically normal before the tailbud stages. We did not observe a significant change of mesodermal and endodermal marker gene expression, while after stage 40, about 80% of the MO injected embryos exhibited dramatic gut defects in which the guts did not coil, but other structures outside the gastrointestinal tract were relatively normal. To test if the phenotypes were specifically caused by the knockdown of IRE1α, a rescue experiment was performed by co-injection of IRE1α-GR mRMA with IRE1α MO. The data obtained demonstrated that the gut coiling defect was rescued. The deletion mutant of IRE1α was constructed, consisting of the N-terminal part without the C-terminal kinase and RNase domains named IRE1αΔC, to investigate the functional domain of IRE1α. Injection of IRE1αΔC-GR mRNA caused similar morphological alterations with gut malformation by interfering with the function of endogenous xIRE1α. In order to investigate if IRE1α/XBP1 pathway was involved in gut development, 50 ng of XBP1 MO was injected and the results showed that knockdown of XBP1 resulted in similar morphological alterations with gut-coiling defect at tadpole stage. IRE1α is not required for germ layer formation but for gut development in Xenopus lavies and it may function via XBP1-dependent pathway.
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Bhattacharya, Dipankan; Marfo, Chris A; Li, Davis; Lane, Maura; Khokha, Mustafa K
2015-12-15
Congenital malformations are the major cause of infant mortality in the US and Europe. Due to rapid advances in human genomics, we can now efficiently identify sequence variants that may cause disease in these patients. However, establishing disease causality remains a challenge. Additionally, in the case of congenital heart disease, many of the identified candidate genes are either novel to embryonic development or have no known function. Therefore, there is a pressing need to develop inexpensive and efficient technologies to screen these candidate genes for disease phenocopy in model systems and to perform functional studies to uncover their role in development. For this purpose, we sought to test F0 CRISPR based gene editing as a loss of function strategy for disease phenocopy in the frog model organism, Xenopus tropicalis. We demonstrate that the CRISPR/Cas9 system can efficiently modify both alleles in the F0 generation within a few hours post fertilization, recapitulating even early disease phenotypes that are highly similar to knockdowns from morpholino oligos (MOs) in nearly all cases tested. We find that injecting Cas9 protein is dramatically more efficacious and less toxic than cas9 mRNA. We conclude that CRISPR based F0 gene modification in X. tropicalis is efficient and cost effective and readily recapitulates disease and MO phenotypes. Copyright © 2015 Elsevier Inc. All rights reserved.
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NASA Astrophysics Data System (ADS)
Zhang, Jie; Liu, Quan; Ren, Haojun; Zhang, Nanjie; Li, Pengfei; Yang, Kang
2017-11-01
The anionic ring-opening polymerization of propylene oxide (PO) was initiated with glycerol and catalyzed by three new synthetic phosphonium salts, tetrakis (pyrrolidino) phosphonium (Py4P1+), tetrakis (piperidino) phosphonium (Pi4P1+), tetrakis (morpholino) phosphonium (Mo4P1+), and the known tetrakis [cyclohexyl (methyl) amino] phosphonium (Cy4P1+) and tetrakis [tris (dimethylamino) phosphonoamino] phosphazene (P5+). The effects of substituents on the polymerization behavior, especially the molecular weight and its distribution, degree of unsaturation, and the sequential structures of poly (propylene oxide) (PPO) were investigated. The structures of these catalysts and PPOs were characterized by FT-IR, 1H and 13C NMR, and GPC. The results indicate that Cy4P1+, Py4P1+, and Pi4P1+ have lower optimum reaction temperatures at 90, 70, and 70 °C, respectively, and are better than traditional catalysts KOH and double metal cyanide. PPO samples with high molecular weight, narrow polydispersity, and high functionality were accessible when catalyzed with Cy4P1+, Pi4P1+, and P5+ at the optimum temperature. Notably, Pi4P1+ formed unimodal distribution PPO with 9000 g/mol, 2.93 of functionality, and 0.008 mmol/g degree of unsaturation. Majority segments of PPO from five catalysts adopted the stereoregular head-to-tail structure, exhibiting excellent regularity.
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Martin, Elodie; Yanicostas, Constantin; Rastetter, Agnès; Alavi Naini, Seyedeh Maryam; Maouedj, Alissia; Kabashi, Edor; Rivaud-Péchoux, Sophie; Brice, Alexis; Stevanin, Giovanni; Soussi-Yanicostas, Nadia
2012-12-01
Hereditary spastic paraplegias (HSPs) are rare neurological conditions caused by degeneration of the long axons of the cerebrospinal tracts, leading to locomotor impairment and additional neurological symptoms. There are more than 40 different causative genes, 24 of which have been identified, including SPG11 and SPG15 mutated in complex clinical forms. Since the vast majority of the causative mutations lead to loss of function of the corresponding proteins, we made use of morpholino-oligonucleotide (MO)-mediated gene knock-down to generate zebrafish models of both SPG11 and SPG15 and determine how invalidation of the causative genes (zspg11 and zspg15) during development might contribute to the disease. Micro-injection of MOs targeting each gene caused locomotor impairment and abnormal branching of spinal cord motor neurons at the neuromuscular junction. More severe phenotypes with abnormal tail developments were also seen. Moreover, partial depletion of both proteins at sub-phenotypic levels resulted in the same phenotypes, suggesting for the first time, in vivo, a genetic interaction between these genes. In conclusion, the zebrafish orthologues of the SPG11 and SPG15 genes are important for proper development of the axons of spinal motor neurons and likely act in a common pathway to promote their proper path finding towards the neuromuscular junction. Copyright © 2012 Elsevier Inc. All rights reserved.
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Synthesis and in Vitro and in Vivo Evaluation of Phosphoinositide-3-kinase Inhibitors.
Burger, Matthew T; Knapp, Mark; Wagman, Allan; Ni, Zhi-Jie; Hendrickson, Thomas; Atallah, Gordana; Zhang, Yanchen; Frazier, Kelly; Verhagen, Joelle; Pfister, Keith; Ng, Simon; Smith, Aaron; Bartulis, Sarah; Merrit, Hanne; Weismann, Marion; Xin, Xiaohua; Haznedar, Joshua; Voliva, Charles F; Iwanowicz, Ed; Pecchi, Sabina
2011-01-13
Phospoinositide-3-kinases (PI3K) are important oncology targets due to the deregulation of this signaling pathway in a wide variety of human cancers. A series of 2-morpholino, 4-substituted, 6-(3-hydroxyphenyl) pyrimidines have been reported as potent inhibitors of PI3Ks. Herein, we describe the structure-guided optimization of these pyrimidines with a focus on replacing the phenol moiety, while maintaining potent target inhibition and improving in vivo properties. A series of 2-morpholino, 4-substituted, 6-heterocyclic pyrimidines, which potently inhibit PI3K, were discovered. Within this series a compound, 17, was identified with suitable pharmacokinetic (PK) properties, which allowed for the establishment of a PI3K PK/pharmacodynamic-efficacy relationship as determined by in vivo inhibition of AKT(Ser473) phosphorylation and tumor growth inhibition in a mouse A2780 tumor xenograft model.
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Synthesis and in Vitro and in Vivo Evaluation of Phosphoinositide-3-kinase Inhibitors
2010-01-01
Phospoinositide-3-kinases (PI3K) are important oncology targets due to the deregulation of this signaling pathway in a wide variety of human cancers. A series of 2-morpholino, 4-substituted, 6-(3-hydroxyphenyl) pyrimidines have been reported as potent inhibitors of PI3Ks. Herein, we describe the structure-guided optimization of these pyrimidines with a focus on replacing the phenol moiety, while maintaining potent target inhibition and improving in vivo properties. A series of 2-morpholino, 4-substituted, 6-heterocyclic pyrimidines, which potently inhibit PI3K, were discovered. Within this series a compound, 17, was identified with suitable pharmacokinetic (PK) properties, which allowed for the establishment of a PI3K PK/pharmacodynamic−efficacy relationship as determined by in vivo inhibition of AKTSer473 phosphorylation and tumor growth inhibition in a mouse A2780 tumor xenograft model. PMID:24900252
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NASA Technical Reports Server (NTRS)
Bugbee, B. G.; Salisbury, F. B.
1985-01-01
All buffering agents used to stabilize pH in hydroponic research have disadvantages. Inorganic buffers are absorbed and may become phytotoxic. Solid carbonate salts temporarily mitigate decreasing pH but provide almost no protection against increasing pH, and they alter nutrient absorption. Exchange resins are more effective, but we find that they remove magnesium and manganese from solution. We have tested 2(N-Morpholino)ethanesulfonic acid (MES) as a buffering agent at concentrations of 1 and 10 mol m-3 (1 and 10 mM) with beans, corn, lettuce, tomatoes, and wheat. MES appears to be biologically inert and does not interact significantly with other solution ions. Relative growth rates among controls and MES treatments were nearly identical for each species during the trial period. The pH was stabilized by 1 mol m-3 MES. This buffer warrants further consideration in nutrient research.
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Venkatesan, Aranapakam M; Dehnhardt, Christoph M; Delos Santos, Efren; Chen, Zecheng; Dos Santos, Osvaldo; Ayral-Kaloustian, Semiramis; Khafizova, Gulnaz; Brooijmans, Natasja; Mallon, Robert; Hollander, Irwin; Feldberg, Larry; Lucas, Judy; Yu, Ker; Gibbons, James; Abraham, Robert T; Chaudhary, Inder; Mansour, Tarek S
2010-03-25
The PI3K/Akt signaling pathway is a key pathway in cell proliferation, growth, survival, protein synthesis, and glucose metabolism. It has been recognized recently that inhibiting this pathway might provide a viable therapy for cancer. A series of bis(morpholino-1,3,5-triazine) derivatives were prepared and optimized to provide the highly efficacious PI3K/mTOR inhibitor 1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea 26 (PKI-587). Compound 26 has shown excellent activity in vitro and in vivo, with antitumor efficacy in both subcutaneous and orthotopic xenograft tumor models when administered intravenously. The structure-activity relationships and the in vitro and in vivo activity of analogues in this series are described.
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USDA-ARS?s Scientific Manuscript database
Natural antisense transcripts (NATs) are transcripts of the opposite DNA strand to the sense-strand either at the same locus (cis-encoded) or a different locus (trans-encoded). They can affect gene expression at multiple stages including transcription, RNA processing and transport, and translation....
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Bcl-2 antisense therapy in B-cell malignancies.
Chanan-Khan, Asher
2005-07-01
Bcl-2 is an apoptosis regulating protein, overexpression of which is associated with chemotherapy resistant disease, aggressive clinical course, and poor survival in patients with B-cell lymphoproliferative disorders. Overexpression of Bcl-2 protein results in an aberrant intrinsic apoptotic pathway that confers a protective effect on malignant cells against a death signal (e.g., chemotherapy or radiotherapy). Downregulation of this oncoprotein, thus, represents a possible new way to target clinically aggressive disease. Preclinical studies have shown that this oncoprotein can be effectively decreased by Bcl-2 antisense in malignant lymphoid cells and can reverse chemotherapy resistance, as well as enhance the anti-apoptotic potential of both chemotherapeutic and biologic agents. Ongoing clinical trials are exploring the role of Bcl-2 downregulation with oblimersen (Bcl-2 antisense) in patients with non-Hodgkin's lymphoma, chronic lymphocytic leukemia and multiple myeloma. Early results from these studies are promising and support the proof of the principle. As these studies are completed and mature data emerges, the role of Bcl-2 antisense therapy in the treatment of B-cell malignancies will become clearer.
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Pierce, M L; Ruffner, D E
1998-01-01
Antisense-mediated gene inhibition uses short complementary DNA or RNA oligonucleotides to block expression of any mRNA of interest. A key parameter in the success or failure of an antisense therapy is the identification of a suitable target site on the chosen mRNA. Ultimately, the accessibility of the target to the antisense agent determines target suitability. Since accessibility is a function of many complex factors, it is currently beyond our ability to predict. Consequently, identification of the most effective target(s) requires examination of every site. Towards this goal, we describe a method to construct directed ribozyme libraries against any chosen mRNA. The library contains nearly equal amounts of ribozymes targeting every site on the chosen transcript and the library only contains ribozymes capable of binding to that transcript. Expression of the ribozyme library in cultured cells should allow identification of optimal target sites under natural conditions, subject to the complexities of a fully functional cell. Optimal target sites identified in this manner should be the most effective sites for therapeutic intervention. PMID:9801305
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Review on investigations of antisense oligonucleotides with the use of mass spectrometry.
Studzińska, Sylwia
2018-01-01
Antisense oligonucleotides have been investigated as potential drugs for years. They inhibit target gene or protein expression. The present review summarizes their modifications, modes of action, and applications of liquid chromatography coupled with mass spectrometry for qualitative and quantitative analysis of these compounds. The most recent reports on a given topic were given prominence, while some early studies were reviewed in order to provide a theoretical background. The present review covers the issues of using ion-exchange chromatography, ion-pair reversed-phase high performance liquid chromatography and hydrophilic interaction chromatography for the separation of antisense oligonucleotides. The application of mass spectrometry was described with regard to the ionization type used for the determination of these potential therapeutics. Moreover, the current approaches and applications of mass spectrometry for quantitative analysis of antisense oligonucleotides and their metabolites as well as their impurities during in vitro and in vivo studies were discussed. Finally, certain conclusions and perspectives on the determination of therapeutic oligonucleotides in various samples were briefly described. Copyright © 2017 Elsevier B.V. All rights reserved.
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Ohno, Satoshi; Yoshikawa, Katsunori; Shimizu, Hiroshi; Tamura, Tomohiro
2014-01-01
We describe here the construction of a series of 71 vectors to silence central carbon metabolism genes in Escherichia coli. The vectors inducibly express antisense RNAs called paired-terminus antisense RNAs, which have a higher silencing efficacy than ordinary antisense RNAs. By measuring mRNA amounts, measuring activities of target proteins, or observing specific phenotypes, it was confirmed that all the vectors were able to silence the expression of target genes efficiently. Using this vector set, each of the central carbon metabolism genes was silenced individually, and the accumulation of metabolites was investigated. We were able to obtain accurate information on ways to increase the production of pyruvate, an industrially valuable compound, from the silencing results. Furthermore, the experimental results of pyruvate accumulation were compared to in silico predictions, and both sets of results were consistent. Compared to the gene disruption approach, the silencing approach has an advantage in that any E. coli strain can be used and multiple gene silencing is easily possible in any combination. PMID:24212579
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Philipp, Katrin; Riedel, Frank; Germann, Günter; Hörmann, Karl; Sauerbier, Michael
2005-02-01
The pathology of chronic dermal ulcers is characterized by excessive proteolytic activity which degrades extracellular matrix. The transforming growth factor-beta (TGF-beta) has been identified as an important component of wound healing. Recent developments in molecular therapy offer exciting prospects for the modulation of wound healing, specifically those targeting TGF-beta. We investigated the effect of TGF-beta antisense oligonucleotides on the mRNA expression of matrix metalloproteinases in cultured human keratinocytes, fibroblasts and endothelial cells using multiplex RT-PCR. The treatment of keratinocytes and fibroblasts with TGF-beta antisense oligonucleotides resulted in a significant decrease of expression of mRNA of MMP-1 and MMP-9 compared to controls. Accordingly, a decreased expression of MMP-1 mRNA in endothelial cells was detectable. Other MMPs were not affected. Affecting all dermal wound-healing-related cell types, TGF-beta antisense oligonucleotide technology may be a potential therapeutic option for the inhibition of proteolytic tissue destruction in chronic wounds. Pharmaceutical intervention in this area ultimately may help clinicians to proactively intervene in an effort to prevent normal wounds from becoming chronic.
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Lnx2 ubiquitin ligase is essential for exocrine cell differentiation in the early zebrafish pancreas
Won, Minho; Ro, Hyunju; Dawid, Igor B.
2015-01-01
The gene encoding the E3 ubiquitin ligase Ligand of Numb protein-X (Lnx)2a is expressed in the ventral-anterior pancreatic bud of zebrafish embryos in addition to its expression in the brain. Knockdown of Lnx2a by using an exon 2/intron 2 splice morpholino resulted in specific inhibition of the differentiation of ventral bud derived exocrine cell types, with little effect on endocrine cell types. A frame shifting null mutation in lnx2a did not mimic this phenotype, but a mutation that removed the exon 2 splice donor site did. We found that Lnx2b functions in a redundant manner with its paralog Lnx2a. Inhibition of lnx2a exon 2/3 splicing causes exon 2 skipping and leads to the production of an N-truncated protein that acts as an interfering molecule. Thus, the phenotype characterized by inhibition of exocrine cell differentiation requires inactivation of both Lnx2a and Lnx2b. Human LNX1 is known to destabilize Numb, and we show that inhibition of Numb expression rescues the Lnx2a/b-deficient phenotype. Further, Lnx2a/b inhibition leads to a reduction in the number of Notch active cells in the pancreas. We suggest that Lnx2a/b function to fine tune the regulation of Notch through Numb in the differentiation of cell types in the early zebrafish pancreas. Further, the complex relationships among genotype, phenotype, and morpholino effect in this case may be instructive in the ongoing consideration of morpholino use. PMID:26392552
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Won, Minho; Ro, Hyunju; Dawid, Igor B
2015-10-06
The gene encoding the E3 ubiquitin ligase Ligand of Numb protein-X (Lnx)2a is expressed in the ventral-anterior pancreatic bud of zebrafish embryos in addition to its expression in the brain. Knockdown of Lnx2a by using an exon 2/intron 2 splice morpholino resulted in specific inhibition of the differentiation of ventral bud derived exocrine cell types, with little effect on endocrine cell types. A frame shifting null mutation in lnx2a did not mimic this phenotype, but a mutation that removed the exon 2 splice donor site did. We found that Lnx2b functions in a redundant manner with its paralog Lnx2a. Inhibition of lnx2a exon 2/3 splicing causes exon 2 skipping and leads to the production of an N-truncated protein that acts as an interfering molecule. Thus, the phenotype characterized by inhibition of exocrine cell differentiation requires inactivation of both Lnx2a and Lnx2b. Human LNX1 is known to destabilize Numb, and we show that inhibition of Numb expression rescues the Lnx2a/b-deficient phenotype. Further, Lnx2a/b inhibition leads to a reduction in the number of Notch active cells in the pancreas. We suggest that Lnx2a/b function to fine tune the regulation of Notch through Numb in the differentiation of cell types in the early zebrafish pancreas. Further, the complex relationships among genotype, phenotype, and morpholino effect in this case may be instructive in the ongoing consideration of morpholino use.
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Alper, O; De Santis, M L; Stromberg, K; Hacker, N F; Cho-Chung, Y S; Salomon, D S
2000-11-15
Over-expression of epidermal growth factor receptor (EGFR) in ovarian cancer has been well documented. Human NIH:OVCAR-8 ovarian carcinoma cells were transfected with an expression vector containing the anti-sense orientation of truncated human EGFR cDNA. EGFR anti-sense over-expression resulted in decreased EGFR protein and mRNA expression, cell proliferation and tumor formation in nude mice. In accordance with the reduced levels of EGFR in EGFR anti-sense-expressing cells, tyrosine phosphorylation of EGFR was decreased compared to untransfected parental cells treated with EGF. In EGFR anti-sense-transfected cells, expression of erbB-3, but not erbB-2, was increased. In addition, basal and heregulin-beta 1-stimulated tyrosine phosphorylation of erbB-3 was higher in EGFR anti-sense vector-transfected cells. A morphological alteration in EGFR anti-sense gene-expressing cells was correlated with a decrease in the expression of E-cadherin, alpha-catenin and, to a lesser extent, beta-catenin. Changes in the expression of these proteins were associated with a reduction in complex formation among E-cadherin, beta-catenin and alpha-catenin and between beta-catenin and EGFR in EGFR anti-sense-expressing cells compared to sense-transfected control cells. These results demonstrate that EGFR expression in ovarian carcinoma cells regulates expression of cell adhesion proteins that may enhance cell growth and invasiveness. Copyright 2000 Wiley-Liss, Inc.
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Tran, Chi Nhan; Giangrossi, Mara; Prosseda, Gianni; Brandi, Anna; Di Martino, Maria Letizia; Colonna, Bianca; Falconi, Maurizio
2011-10-01
The icsA gene of Shigella encodes a structural protein involved in colonization of the intestinal mucosa by bacteria. This gene is expressed upon invasion of the host and is controlled by a complex regulatory circuit involving the nucleoid protein H-NS, the AraC-like transcriptional activator VirF, and a 450 nt antisense RNA (RnaG) acting as transcriptional attenuator. We investigated on the interplay of these factors at the molecular level. DNase I footprints reveal that both H-NS and VirF bind to a region including the icsA and RnaG promoters. H-NS is shown to repress icsA transcription at 30°C but not at 37°C, suggesting a significant involvement of this protein in the temperature-regulated expression of icsA. We also demonstrate that VirF directly stimulates icsA transcription and is able to alleviate H-NS repression in vitro. According to these results, icsA expression is derepressed in hns- background and overexpressed when VirF is provided in trans. Moreover, we find that RnaG-mediated transcription attenuation depends on 80 nt at its 5'-end, a stretch carrying the antisense region. Bases engaged in the initial contact leading to sense-antisense pairing have been identified using synthetic RNA and DNA oligonucleotides designed to rebuild and mutagenize the two stem-loop motifs of the antisense region.
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Readman, John Benedict; Dickson, George; Coldham, Nick G
2017-06-01
The bacterial cell wall presents a barrier to the uptake of unmodified synthetic antisense oligonucleotides, such as peptide nucleic acids, and so is one of the greatest obstacles to the development of their use as therapeutic anti-bacterial agents. Cell-penetrating peptides have been covalently attached to antisense agents, to facilitate penetration of the bacterial cell wall and deliver their cargo into the cytoplasm. Although they are an effective vector for antisense oligonucleotides, they are not specific for bacterial cells and can exhibit growth inhibitory properties at higher doses. Using a bacterial cell growth assay in the presence of cefotaxime (CTX 16 mg/L), we have developed and evaluated a self-assembling non-toxic DNA tetrahedron nanoparticle vector incorporating a targeted anti-bla CTX-M-group 1 antisense peptide nucleic acid (PNA4) in its structure for penetration of the bacterial cell wall. A dose-dependent CTX potentiating effect was observed when PNA4 (0-40 μM) was incorporated into the structure of a DNA tetrahedron vector. The minimum inhibitory concentration (to CTX) of an Escherichia coli field isolate harboring a plasmid carrying bla CTX-M-3 was reduced from 35 to 16 mg/L in the presence of PNA4 carried by the DNA tetrahedron vector (40 μM), contrasting with no reduction in MIC in the presence of PNA4 alone. No growth inhibitory effects of the DNA tetrahedron vector alone were observed.
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Suppression of cell division by pKi-67 antisense-RNA and recombinant protein.
Duchrow, M; Schmidt, M H; Zingler, M; Anemüller, S; Bruch, H P; Broll, R
2001-01-01
The human antigen defined by the monoclonal antibody Ki-67 (pKi-67) is a human nuclear protein strongly associated with cell proliferation and found in all tissues studied. It is widely used as a marker of proliferating cells, yet its function is unknown. To investigate its function we suppressed pKi-67 expression by antisense RNA and overexpressed a partial structure of pKi-67 in HeLa cells. A BrdU-incorporation assay showed a significant decrease in DNA synthesis after antisense inhibition. Cell cycle analysis indicated a higher proportion of cells in G1 phase and a lower proportion of cells in S phase while the number of G(2)/M phase cells remained constant. Overexpression of a recombinant protein encoding three of the repetitive elements from exon 13 of pKi-67 had a similar effect to that obtained by antisense inhibition. The similarity of the effect of expressing 'Ki-67 repeats' and pKi-67 antisense RNA could be explained by a negative effect on the folding of the endogenous protein in the endoplasmatic reticulum. Furthermore excessive self-association of pKi-67 via the repeat structure could inhibit its nuclear transport, preventing it from getting to its presumptive site of action. We conclude that the Ki-67 protein has an important role in the regulation of the cell cycle, which is mediated in part by its repetitive elements. Copyright 2001 S. Karger AG, Basel
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Identification of targets of miRNA-221 and miRNA-222 in fulvestrant-resistant breast cancer
Liu, Pengfei; Sun, Manna; Jiang, Wenhua; Zhao, Jinkun; Liang, Chunyong; Zhang, Huilai
2016-01-01
The present study aimed to identify the differentially expressed genes (DEGs) regulated by microRNA (miRNA)-221 and miRNA-222 that are associated with the resistance of breast cancer to fulvestrant. The GSE19777 transcription profile was downloaded from the Gene Expression Omnibus database, and includes data from three samples of antisense miRNA-221-transfected fulvestrant-resistant MCF7-FR breast cancer cells, three samples of antisense miRNA-222-transfected fulvestrant-resistant MCF7-FR cells and three samples of control inhibitor (green fluorescent protein)-treated fulvestrant-resistant MCF7-FR cells. The linear models for microarray data package in R/Bioconductor was employed to screen for DEGs in the miRNA-transfected cells, and the pheatmap package in R was used to perform two-way clustering. Pathway enrichment was conducted using the Gene Set Enrichment Analysis tool. Furthermore, a miRNA-messenger (m) RNA regulatory network depicting interactions between miRNA-targeted upregulated DEGs was constructed and visualized using Cytoscape. In total, 492 and 404 DEGs were identified for the antisense miRNA-221-transfected MCF7-FR cells and the antisense miRNA-222-transfected MCF7-FR cells, respectively. Genes of the pentose phosphate pathway (PPP) were significantly enriched in the antisense miRNA-221-transfected MCF7-FR cells. In addition, components of the Wnt signaling pathway and cell adhesion molecules (CAMs) were significantly enriched in the antisense miRNA-222-transfected MCF7-FR cells. In the miRNA-mRNA regulatory network, miRNA-222 was demonstrated to target protocadherin 10 (PCDH10). The results of the present study suggested that the PPP and Wnt signaling pathways, as well as CAMs and PCDH10, may be associated with the resistance of breast cancer to fulvestrant. PMID:27895744
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Chen, Jian-jing; Raab-Traub, Nancy; Yao, Qing-yun; Zhang, Feng; Huang, Mei-ling; Kuang, Zhu-ji; Zhang, Xiao-shi; Ye, Yan-li; Gu, Li
2002-01-01
The latent membrane protein gene (LMP) of Epstein-Barr virus (EBV) was thought to play an important role in the carcinogenesis of nasopharyngeal carcinoma (NPC). In this study, the authors investigated the effects of antisense RNA (AsRNA) on LMP for down regulating at the target gene over expression in EBV transformed lymphoid cells, and set up an antisense system to inhibit LMP expression. Constructing the single strand antisense transcription system in vitro, the authors have got large amount of AsRNA. Designing and setting up an antisense tracing system in situ (ATSIS), the authors could observe the living particles of AsRNA/sense RNA duplex dimer. With time lapse phase-contrast microscopy, the agglutination phenotype on living cells was easily detected by MTT test, the inhibition rate on EBV transformed cells was calculated. LMP 1.9 fragment ligated into pGEM vector in reverse orientation have been constructed and produced a plentiful amount of AsLMPmRNA which could incorporated into both B95-8 and C1936 cell lines by endophagocytosis and formed the duplex dimer of As/Sense RNA. This particles have been visualized in situ when labelling 35S isotope by ATSIS. When AsLMPmRNA acted as agents for specific inhibition to LMP over expression, the transform phenotype of cell agglutination have been suppressed and MTT particle formatin was apparently reduced both two EBV tansformed cell lines. AsLMPmRNA targets at sense strand have a high effectiveness of down-regulation on EBV-LMP overexpression. This down regulating function of LMP and growth inhibition on transformed cell is demonstrated by the antisenes tracing system in situ (ATSIS). The results provide a clue to overcome the latent EBV infection in human bodies all living long time and to prevent it inducing NPC in high incidence area by antisense strategies.
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Wen, Ming; Li, Bibo; Ouyang, Yu; Luo, Yi; Li, Shaolin
2009-06-01
Molecular imaging of tumor antisense gene techniques have been applied to the study of magnetic resonance (MR) gene imaging associated with malignant tumors. In this study, we designed, synthesized, and tested a novel molecular probe, in which the antisense oligodeoxynucleotide (ASODN) was labeled with superparamagnetic iron oxide (SPIO), and its efficiency was examined by in vitro MR imaging after SK-Br-3 mammary carcinoma cell lines (oncocytes) transfection. The SPIO-labeled ASODN probe was prepared through SPIO conjugated to ASODN using a chemical cross linking method. Its morphology and size were detected by atomic force microscope, size distribution were detected by laser granulometer, the conjugating rate and biological activity were determined by high performance liquid chromatography, and the stability was determined by polyacrylamide gel electrophoresis. After that, the probes were transfected into the SK-Br-3 oncocytes, cellular iron uptake was analyzed qualitatively at light and electron microscopy and was quantified at atomic absorption spectrometry, and the signal change of the transfected cells was observed and measured using MR imaging. The morphology of the SPIO-labeled ASODN probe was mostly spherical with well-distributed scattering, and the diameters were between 25 and 40 nm (95%) by atomic force microscope and laser granulometer, the conjugating rate of the probe was 99%. Moreover, this probe kept its activity under physiological conditions and could conjugate with antisense oligodeoxynucleotide. In addition, light microscopy revealed an intracellular uptake of iron oxides in the cytosol and electron microscopic studies revealed a lysosomal deposition of iron oxides in the transfected SK-Br-3 oncocytes by antisense probes, some of them gathered stacks, and the iron content of the group of transfected SK-Br-3 oncocytes by antisense probe is significantly higher (18.37 +/- 0.42 pg) than other contrast groups, the MR imaging showed that transfected SK-Br-3 oncocytes by antisense probe had the lowest signal of all. The SPIO-labeled ASODN probe shows unique features including well-distributed spherical morphology, high conjugating rate and loading efficiency, and the signal intensity of SPIO-labeled ASODN-transfected SK-Br-3 oncocytes is reduced in MR imaging. These results indicate that the SPIO-labeled ASODN probe is potentially useful as a MR targeting contrast enhancing agent to specifically diagnose tumors which had over-expression of the c-erbB2 oncogene at an early stage.
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Expression of cathepsin S antisense transcripts by adenovirus in retinal pigment epithelial cells.
Rakoczy, P E; Lai, M C; Baines, M G; Spilsbury, K; Constable, I J
1998-10-01
To show the production of sense or antisense transcripts by recombinant adenoviruses, to investigate whether the transcripts produced were suitable for downregulating the expression of the targeted gene, cathepsin S (CatS), and to examine the effect of antisense transcript production on the biologic function of retinal pigment epithelial (RPE) cells, including the regulation of endogenous aspartic protease expression. Ad.MLP.CatSAS, Ad.RSV.CatSAS, and Ad.MLP.CatSS recombinant viruses were produced by homologous recombination. The recombinant viruses were tested by restriction enzyme digestion to confirm the orientation of the inserts. The expression of antisense transcripts was tested by northern blot analysis. Western blot analysis was used to study the regulation of the endogenous CatS protein in ARPE19 cells. The biologic effect of CatS downregulation in ARPE19 cells was tested by proliferation and phagocytosis assays, de novo cathepsin D (CatD) synthesis, and measurement of aspartic protease activity. After characterization of the recombinant adenovirus constructs, the production of antisense and sense CatS transcripts was shown in ARPE19 cells. The transcripts appeared at approximately 1.9 kb 48 hours after transduction, and the expression of the antisense transcripts was similar in constructs carrying either the MLP or the RSV promoter. Western blot analysis showed that ARPE19 cells transduced with Ad.MLP.CatSAS and Ad.RSV.CatSAS had no detectable CatS. In contrast, there was a strong signal appearing at 24 kDa in ARPE19 cells transduced with Ad.MLP.CatSS. ARPE19 cells were transduced to a high level. The transduction of ARPE19 cells with the recombinant adenoviruses did not affect the morphologic appearance of the cells, their proliferation, or their phagocytosing ability. However, ARPE19 cells transduced by Ad.MLP.CatSAS recombinant adenovirus showed a significant downregulation of de novo CatD synthesis and a twofold decrease in aspartic protease activity. Recombinant adenoviruses were shown to be suitable for producing antisense CatS transcripts to modulate endogenous CatS expression in RPE cells. It is proposed that CatS may play an important role, directly or indirectly, in the lysosomal digestion of outer segments through the regulation of other lysosomal enzyme activity, such as the expression of CatD.
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Antisense transcription is pervasive but rarely conserved in enteric bacteria.
Raghavan, Rahul; Sloan, Daniel B; Ochman, Howard
2012-01-01
Noncoding RNAs, including antisense RNAs (asRNAs) that originate from the complementary strand of protein-coding genes, are involved in the regulation of gene expression in all domains of life. Recent application of deep-sequencing technologies has revealed that the transcription of asRNAs occurs genome-wide in bacteria. Although the role of the vast majority of asRNAs remains unknown, it is often assumed that their presence implies important regulatory functions, similar to those of other noncoding RNAs. Alternatively, many antisense transcripts may be produced by chance transcription events from promoter-like sequences that result from the degenerate nature of bacterial transcription factor binding sites. To investigate the biological relevance of antisense transcripts, we compared genome-wide patterns of asRNA expression in closely related enteric bacteria, Escherichia coli and Salmonella enterica serovar Typhimurium, by performing strand-specific transcriptome sequencing. Although antisense transcripts are abundant in both species, less than 3% of asRNAs are expressed at high levels in both species, and only about 14% appear to be conserved among species. And unlike the promoters of protein-coding genes, asRNA promoters show no evidence of sequence conservation between, or even within, species. Our findings suggest that many or even most bacterial asRNAs are nonadaptive by-products of the cell's transcription machinery. IMPORTANCE Application of high-throughput methods has revealed the expression throughout bacterial genomes of transcripts encoded on the strand complementary to protein-coding genes. Because transcription is costly, it is usually assumed that these transcripts, termed antisense RNAs (asRNAs), serve some function; however, the role of most asRNAs is unclear, raising questions about their relevance in cellular processes. Because natural selection conserves functional elements, comparisons between related species provide a method for assessing functionality genome-wide. Applying such an approach, we assayed all transcripts in two closely related bacteria, Escherichia coli and Salmonella enterica serovar Typhimurium, and demonstrate that, although the levels of genome-wide antisense transcription are similarly high in both bacteria, only a small fraction of asRNAs are shared across species. Moreover, the promoters associated with asRNAs show no evidence of sequence conservation between, or even within, species. These findings indicate that despite the genome-wide transcription of asRNAs, many of these transcripts are likely nonfunctional.
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Role of TAF12 in the Increased VDR Activity in Paget’s Disease of Bone
2013-10-01
and 5’‐GCC AAA TGC AGT TTA AGC TCT GCT‐3’ (antisense). The gene‐specific primers for mouse b‐actin were 5’‐GGC CGT ACC ACT GGC ATC GTG ATG‐ 3...cycles. The gene‐specific primers for CYP24A1 mRNA were 5’‐CGG GTG GAC CAT TTA CAA CTC GG‐3’ (sense) and 5’‐CTC AAC AGG CTC ATT GTC TGT GG‐3’ (antisense...The gene specific designing primers for b‐actinwere 5’‐ GTG CGT GAC ATC AAA GAG‐3’ (sense) and 5’‐GCC ACA GGA TTC CAT ACC‐3’ (antisense). The
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Zebrafish – As an Integrative Model for Twenty-first Century Toxicity Testing
The zebrafish embryo is a useful small model for investigating vertebrate development because of its transparency, low cost, transgenic and morpholino capabilities, conservation of cell signaling, and concordance with mammalian developmental phenotypes. From these advantages, the...
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Divergent transcription is associated with promoters of transcriptional regulators
2013-01-01
Background Divergent transcription is a wide-spread phenomenon in mammals. For instance, short bidirectional transcripts are a hallmark of active promoters, while longer transcripts can be detected antisense from active genes in conditions where the RNA degradation machinery is inhibited. Moreover, many described long non-coding RNAs (lncRNAs) are transcribed antisense from coding gene promoters. However, the general significance of divergent lncRNA/mRNA gene pair transcription is still poorly understood. Here, we used strand-specific RNA-seq with high sequencing depth to thoroughly identify antisense transcripts from coding gene promoters in primary mouse tissues. Results We found that a substantial fraction of coding-gene promoters sustain divergent transcription of long non-coding RNA (lncRNA)/mRNA gene pairs. Strikingly, upstream antisense transcription is significantly associated with genes related to transcriptional regulation and development. Their promoters share several characteristics with those of transcriptional developmental genes, including very large CpG islands, high degree of conservation and epigenetic regulation in ES cells. In-depth analysis revealed a unique GC skew profile at these promoter regions, while the associated coding genes were found to have large first exons, two genomic features that might enforce bidirectional transcription. Finally, genes associated with antisense transcription harbor specific H3K79me2 epigenetic marking and RNA polymerase II enrichment profiles linked to an intensified rate of early transcriptional elongation. Conclusions We concluded that promoters of a class of transcription regulators are characterized by a specialized transcriptional control mechanism, which is directly coupled to relaxed bidirectional transcription. PMID:24365181
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Graham, Mark J; Lee, Richard G; Bell, Thomas A; Fu, Wuxia; Mullick, Adam E; Alexander, Veronica J; Singleton, Walter; Viney, Nick; Geary, Richard; Su, John; Baker, Brenda F; Burkey, Jennifer; Crooke, Stanley T; Crooke, Rosanne M
2013-05-24
Elevated plasma triglyceride levels have been recognized as a risk factor for the development of coronary heart disease. Apolipoprotein C-III (apoC-III) represents both an independent risk factor and a key regulatory factor of plasma triglyceride concentrations. Furthermore, elevated apoC-III levels have been associated with metabolic syndrome and type 2 diabetes mellitus. To date, no selective apoC-III therapeutic agent has been evaluated in the clinic. To test the hypothesis that selective inhibition of apoC-III with antisense drugs in preclinical models and in healthy volunteers would reduce plasma apoC-III and triglyceride levels. Rodent- and human-specific second-generation antisense oligonucleotides were identified and evaluated in preclinical models, including rats, mice, human apoC-III transgenic mice, and nonhuman primates. We demonstrated the selective reduction of both apoC-III and triglyceride in all preclinical pharmacological evaluations. We also showed that inhibition of apoC-III was well tolerated and not associated with increased liver triglyceride deposition or hepatotoxicity. A double-blind, placebo-controlled, phase I clinical study was performed in healthy subjects. Administration of the human apoC-III antisense drug resulted in dose-dependent reductions in plasma apoC-III, concomitant lowering of triglyceride levels, and produced no clinically meaningful signals in the safety evaluations. Antisense inhibition of apoC-III in preclinical models and in a phase I clinical trial with healthy subjects produced potent, selective reductions in plasma apoC-III and triglyceride, 2 known risk factors for cardiovascular disease. This compelling pharmacological profile supports further clinical investigations in hypertriglyceridemic subjects.
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Antisense Oligonucleotide Therapy for Patients with Advanced Cancer | Center for Cancer Research
Colorectal cancer (CRC) is the second leading cause of cancer-related death in the U.S. Improvements in therapy have increased the survival of patients with CRC from 10 months to two years, but for patients who stop responding to treatments, such as irinotecan, options for additional therapy are limited. Antisense oligonucleotides (ASOs) may offer advantages over traditional
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Anti-Angiogenic Action of Neutral Endopeptidase
2007-11-01
message levels of NEP in hypoxia treated PC cells. Messenger RNA levels of NEP decreased between 50-75% relative to normoxic controls with high...GAGCATC-3 (sense) and 5-ATATGAATTCTCAGCTCT- TAGCAGACATGGAAGAAAG-3 ( antisense ) for glutathione S-transferase (GST) fusion proteins and 5-ATGGCAGCCGG...GAGCATC-3 (sense) and 5-CCCCAAGCTTTTAGCTCT- TAGCAGACAT-3 ( antisense ) for maltose-binding protein fusion proteins, as previously described (13
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Kralovicova, Jana; Moreno, Pedro M D; Cross, Nicholas C P; Pêgo, Ana Paula; Vorechovsky, Igor
2016-12-01
ATM (ataxia-telangiectasia, mutated) is an important cancer susceptibility gene that encodes a key apical kinase in the DNA damage response pathway. ATM mutations in the germ line result in ataxia-telangiectasia (A-T), a rare genetic syndrome associated with hypersensitivity to double-strand DNA breaks and predisposition to lymphoid malignancies. ATM expression is limited by a tightly regulated nonsense-mediated RNA decay (NMD) switch exon (termed NSE) located in intron 28. In this study, we identify antisense oligonucleotides that modulate NSE inclusion in mature transcripts by systematically targeting the entire 3.1-kb-long intron. Their identification was assisted by a segmental deletion analysis of transposed elements, revealing NSE repression upon removal of a distant antisense Alu and NSE activation upon elimination of a long terminal repeat transposon MER51A. Efficient NSE repression was achieved by delivering optimized splice-switching oligonucleotides to embryonic and lymphoblastoid cells using chitosan-based nanoparticles. Together, these results provide a basis for possible sequence-specific radiosensitization of cancer cells, highlight the power of intronic antisense oligonucleotides to modify gene expression, and demonstrate transposon-mediated regulation of NSEs.
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Whitesell, L; Rosolen, A; Neckers, L M
1991-01-01
Neuroectodermal tumors of childhood provide a unique opportunity to examine the role of genes potentially regulating neuronal growth and differentiation because many cell lines derived from these tumors are composed of at least two distinct morphologic cell types. These types display variant phenotypic characteristics and spontaneously interconvert, or transdifferentiate, in vitro. The factors that regulate transdifferentiation are unknown. Application of antisense approaches to the transdifferentiation process has allowed us to explore the precise role that N-myc may play in regulating developing systems. We now report construction of an episomally replicating expression vector designed to generate RNA antisense to part of the human N-myc gene. Such a vector is able to specifically inhibit N-myc expression in cell lines carrying both normal and amplified N-myc alleles. Inhibition of N-myc expression blocks transdifferentiation in these lines, with accumulation of cells of an intermediate phenotype. A concomitant decrease in growth rate but not loss of tumorigenicity was observed in the N-myc nonamplified cell line CHP-100. Vector-generated antisense RNA should allow identification of genes specifically regulated by the proto-oncogene N-myc. Images PMID:1996098
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Falzarano, Maria Sofia; Passarelli, Chiara
2014-01-01
Antisense therapy is a powerful tool for inducing post-transcriptional modifications and thereby regulating target genes associated with disease. There are several classes of antisense oligonucleotides (AONs) with therapeutic use, such as double-stranded RNAs (interfering RNAs, utilized for gene silencing, and single-stranded AONs with various chemistries, which are useful for antisense targeting of micro-RNAs and mRNAs. In particular, the use of AONs for exon skipping, by targeting pre-mRNA, is proving to be a highly promising therapy for some genetic disorders like Duchenne muscular dystrophy and spinal muscular atrophy. However, AONs are unable to cross the plasma membrane unaided, and several other obstacles still remain to be overcome, in particular their instability due to their nuclease sensitivity and their lack of tissue specificity. Various drug delivery systems have been explored to improve the bioavailability of nucleic acids, and nanoparticles (NPs) have been suggested as potential vectors for DNA/RNA. This review describes the recent progress in AON conjugation with natural and synthetic delivery systems, and provides an overview of the efficacy of NP-AON complexes as an exon-skipping treatment for Duchenne muscular dystrophy. PMID:24506782
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Bernick, David L.; Dennis, Patrick P.; Lui, Lauren M.; Lowe, Todd M.
2012-01-01
A great diversity of small, non-coding RNA (ncRNA) molecules with roles in gene regulation and RNA processing have been intensely studied in eukaryotic and bacterial model organisms, yet our knowledge of possible parallel roles for small RNAs (sRNA) in archaea is limited. We employed RNA-seq to identify novel sRNA across multiple species of the hyperthermophilic genus Pyrobaculum, known for unusual RNA gene characteristics. By comparing transcriptional data collected in parallel among four species, we were able to identify conserved RNA genes fitting into known and novel families. Among our findings, we highlight three novel cis-antisense sRNAs encoded opposite to key regulatory (ferric uptake regulator), metabolic (triose-phosphate isomerase), and core transcriptional apparatus genes (transcription factor B). We also found a large increase in the number of conserved C/D box sRNA genes over what had been previously recognized; many of these genes are encoded antisense to protein coding genes. The conserved opposition to orthologous genes across the Pyrobaculum genus suggests similarities to other cis-antisense regulatory systems. Furthermore, the genus-specific nature of these sRNAs indicates they are relatively recent, stable adaptations. PMID:22783241
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Green, M M; LeBoeuf, R D; Churchill, P F
2000-01-01
Tetrahymena vorax (T. vorax) is an indigenous fresh water protozoan with the natural biological potential to maintain a specific aquatic microbial flora by ingesting and eliminating specific microorganism. To investigate the molecular mechanisms controlling Tetrahymena vorax (T. vorax) cellular differentiation from a small-mouth vegetative cell to a voracious large-mouth carnivore capable of ingesting prey ciliates and bacteria from aquatic environments, we use DNA subtraction and gene discovery techniques to identify and isolate T. vorax differentiation-specific genes. The physiological necessity for one newly discovered gene, SUBII-TG, was determined in vivo using an antisense oligonucleotide directed against the 5' SUBII-TG DNA sequence. The barriers to delivering antisense oligonucleotides to the cytoplasm of T. vorax were circumvented by employing a new but simple procedure of processing the oligonucleotide with the differentiation stimulus, stomatin. In these studies, the antisense oligonucleotide down-regulated SUBII-TG mRNA expression, and blocked differentiation and ingestion of prey ciliates. The ability to down-regulate SUBII-TG expression with the antisense oligonucleotide suggests that the molecular mechanisms controlling the natural biological activities of T. vorax can be manipulated to further study its cellular differentiation and potential as a biocontrol microorganism.
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Two classes of small antisense RNAs in fungal RNA silencing triggered by non-integrative transgenes
Nicolás, Francisco E.; Torres-Martínez, Santiago; Ruiz-Vázquez, Rosa M.
2003-01-01
Transformation of Mucor circinelloides with self-replicative plasmids containing a wild-type copy of the carotenogenic gene carB causes silencing of the carB function in 3% of transformants. Genomic analyses revealed a relationship between silenced phenotype and number of copies of plasmids. This phenotype results from a reduction of the steady-state levels of carB mRNA, a reduction that is not due to differences in the level of transcription, indicating that silencing is post-transcriptional. Small sense and antisense RNAs have been found to be associated with gene silencing in M.circinelloides. Two size classes of small antisense RNAs, differentially accumulated during the vegetative growth of silenced transformants, have been detected: a long 25-nucleotide RNA and a short 21-nucleotide RNA. Secondary sense and antisense RNAs corresponding to sequences of the endogenous gene downstream of the initial triggering molecule have also been detected, revealing the existence of spreading of RNA targeting in fungi. These findings, together with the self-replicative nature of the triggering molecules, make M.circinelloides a suitable organism for investigating some unresolved questions in RNA silencing. PMID:12881432
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Tian, Hongmei; Ma, Leyuan; Zhao, Cong
To unravel the roles of sucrose phosphate synthase (SPS) in muskmelon (Cucumis melo L.), we reduced its activity in transgenic muskmelon plants by an antisense approach. For this purpose, an 830 bp cDNA fragment of muskmelon sucrose phosphate synthase was expressed in antisense orientation behind the 35S promoter of the cauliflower mosaic virus. The phenotype of the antisense plants clearly differed from that of control plants. The transgenic plant leaves were markedly smaller, and the plant height and stem diameter were obviously shorter and thinner. Transmission electron microscope observation revealed that the membrane degradation of chloroplast happened in transgenic leavesmore » and the numbers of grana and grana lamella in the chloroplast were significantly less, suggesting that the slow growth and weaker phenotype of transgenic plants may be due to the damage of the chloroplast ultrastructure, which in turn results in the decrease of the net photosynthetic rate. The sucrose concentration and levels of sucrose phosphate synthase decreased in transgenic mature fruit, and the fruit size was smaller than the control fruit. Together, our results suggest that sucrose phosphate synthase may play an important role in regulating the muskmelon plant growth and fruit development.« less
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Pradeepkumar, Pushpangadan I; Cheruku, Pradeep; Plashkevych, Oleksandr; Acharya, Parag; Gohil, Suresh; Chattopadhyaya, Jyoti
2004-09-22
We have earlier reported the synthesis and antisense properties of the conformationally constrained oxetane-C and -T containing oligonucleotides, which have shown effective down-regulation of the proto-oncogene c-myb mRNA in the K562 human leukemia cells. Here we report on the straightforward syntheses of the oxetane-A and oxetane-G nucleosides as well as their incorporations into antisense oligonucleotides (AONs), and compare their structural and antisense properties with those of the T and C modified AONs (including the thermostability and RNase H recruitment capability of the AON/RNA hybrid duplex by Michaelis-Menten kinetic analyses, their resistance in the human serum, as well as in the presence of exo and endonucleases).
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Agarwala, Anandita; Jones, Peter; Nambi, Vijay
2015-01-01
Antisense oligonucleotide therapy is a promising approach for the treatment of a broad variety of medical conditions. It functions at the cellular level by interfering with RNA function, often leading to degradation of specifically targeted abnormal gene products implicated in the disease process. Mipomersen is a novel antisense oligonucleotide directed at apolipoprotein (apoB)-100, the primary apolipoprotein associated with low-density lipoprotein cholesterol (LDL-C), which has recently been approved for the treatment of familial hypercholesterolemia. A number of clinical studies have demonstrated its efficacy in lowering LDL-C and apoB levels in patients with elevated LDL-C despite maximal medical therapy using conventional lipid-lowering agents. This review outlines the risks and benefits of therapy and provides recommendations on the use of mipomersen.
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Martins, Diogo; Wei, Xi; Levicky, Rastislav; Song, Yong-Ak
2016-04-05
We describe a microfluidic concentration device to accelerate the surface hybridization reaction between DNA and morpholinos (MOs) for enhanced detection. The microfluidic concentrator comprises a single polydimethylsiloxane (PDMS) microchannel onto which an ion-selective layer of conductive polymer poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) ( PSS) was directly printed and then reversibly surface bonded onto a morpholino microarray for hybridization. Using this electrokinetic trapping concentrator, we could achieve a maximum concentration factor of ∼800 for DNA and a limit of detection of 10 nM within 15 min. In terms of the detection speed, it enabled faster hybridization by around 10-fold when compared to conventional diffusion-based hybridization. A significant advantage of our approach is that the fabrication of the microfluidic concentrator is completely decoupled from the microarray; by eliminating the need to deposit an ion-selective layer on the microarray surface prior to device integration, interfacing between both modules, the PDMS chip for electrokinetic concentration and the substrate for DNA sensing are easier and applicable to any microarray platform. Furthermore, this fabrication strategy facilitates a multiplexing of concentrators. We have demonstrated the proof-of-concept for multiplexing by building a device with 5 parallel concentrators connected to a single inlet/outlet and applying it to parallel concentration and hybridization. Such device yielded similar concentration and hybridization efficiency compared to that of a single-channel device without adding any complexity to the fabrication and setup. These results demonstrate that our concentrator concept can be applied to the development of a highly multiplexed concentrator-enhanced microarray detection system for either genetic analysis or other diagnostic assays.
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Echigoya, Yusuke; Nakamura, Akinori; Nagata, Tetsuya; Urasawa, Nobuyuki; Trieu, Nhu; Panesar, Dharminder; Kuraoka, Mutsuki; Moulton, Hong M.; Saito, Takashi; Aoki, Yoshitsugu; Iversen, Patrick; Sazani, Peter; Kole, Ryszard; Maruyama, Rika; Partridge, Terry; Takeda, Shin’ichi; Yokota, Toshifumi
2017-01-01
Duchenne muscular dystrophy (DMD) is a lethal genetic disorder caused by an absence of the dystrophin protein in bodywide muscles, including the heart. Cardiomyopathy is a leading cause of death in DMD. Exon skipping via synthetic phosphorodiamidate morpholino oligomers (PMOs) represents one of the most promising therapeutic options, yet PMOs have shown very little efficacy in cardiac muscle. To increase therapeutic potency in cardiac muscle, we tested a next-generation morpholino: arginine-rich, cell-penetrating peptide-conjugated PMOs (PPMOs) in the canine X-linked muscular dystrophy in Japan (CXMDJ) dog model of DMD. A PPMO cocktail designed to skip dystrophin exons 6 and 8 was injected intramuscularly, intracoronarily, or intravenously into CXMDJ dogs. Intravenous injections with PPMOs restored dystrophin expression in the myocardium and cardiac Purkinje fibers, as well as skeletal muscles. Vacuole degeneration of cardiac Purkinje fibers, as seen in DMD patients, was ameliorated in PPMO-treated dogs. Although symptoms and functions in skeletal muscle were not ameliorated by i.v. treatment, electrocardiogram abnormalities (increased Q-amplitude and Q/R ratio) were improved in CXMDJ dogs after intracoronary or i.v. administration. No obvious evidence of toxicity was found in blood tests throughout the monitoring period of one or four systemic treatments with the PPMO cocktail (12 mg/kg/injection). The present study reports the rescue of dystrophin expression and recovery of the conduction system in the heart of dystrophic dogs by PPMO-mediated multiexon skipping. We demonstrate that rescued dystrophin expression in the Purkinje fibers leads to the improvement/prevention of cardiac conduction abnormalities in the dystrophic heart. PMID:28373570
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Echigoya, Yusuke; Nakamura, Akinori; Nagata, Tetsuya; Urasawa, Nobuyuki; Lim, Kenji Rowel Q; Trieu, Nhu; Panesar, Dharminder; Kuraoka, Mutsuki; Moulton, Hong M; Saito, Takashi; Aoki, Yoshitsugu; Iversen, Patrick; Sazani, Peter; Kole, Ryszard; Maruyama, Rika; Partridge, Terry; Takeda, Shin'ichi; Yokota, Toshifumi
2017-04-18
Duchenne muscular dystrophy (DMD) is a lethal genetic disorder caused by an absence of the dystrophin protein in bodywide muscles, including the heart. Cardiomyopathy is a leading cause of death in DMD. Exon skipping via synthetic phosphorodiamidate morpholino oligomers (PMOs) represents one of the most promising therapeutic options, yet PMOs have shown very little efficacy in cardiac muscle. To increase therapeutic potency in cardiac muscle, we tested a next-generation morpholino: arginine-rich, cell-penetrating peptide-conjugated PMOs (PPMOs) in the canine X-linked muscular dystrophy in Japan (CXMD J ) dog model of DMD. A PPMO cocktail designed to skip dystrophin exons 6 and 8 was injected intramuscularly, intracoronarily, or intravenously into CXMD J dogs. Intravenous injections with PPMOs restored dystrophin expression in the myocardium and cardiac Purkinje fibers, as well as skeletal muscles. Vacuole degeneration of cardiac Purkinje fibers, as seen in DMD patients, was ameliorated in PPMO-treated dogs. Although symptoms and functions in skeletal muscle were not ameliorated by i.v. treatment, electrocardiogram abnormalities (increased Q-amplitude and Q/R ratio) were improved in CXMD J dogs after intracoronary or i.v. administration. No obvious evidence of toxicity was found in blood tests throughout the monitoring period of one or four systemic treatments with the PPMO cocktail (12 mg/kg/injection). The present study reports the rescue of dystrophin expression and recovery of the conduction system in the heart of dystrophic dogs by PPMO-mediated multiexon skipping. We demonstrate that rescued dystrophin expression in the Purkinje fibers leads to the improvement/prevention of cardiac conduction abnormalities in the dystrophic heart.
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Intravesical NGF Antisense Therapy Using Lipid Nanoparticle for Interstitial Cystitis
2016-12-01
bladder symptoms including urinary frequency and urgency. Previous studies have indicated that overexpression of nerve growth factor (NGF) is an... studies indicate overexpression of nerve growth factor (NGF) as a key factor in the symptom development of IC/BPS. NGF antisense oligonucleotides hold...Stability Testing Ex -vivo stress testing II-2. Research Accomplishment Description AIM 1 Regulatory approval for animal research ; Obtain
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Improving Breast Cancer Diagnosis by Antisense Targeting
2007-08-01
aminohexanoic acid linker (21st Century Biochemicals, Mar- lboro, MA). The biotinylated cholesterol was synthesized by reacting biotinyl-3,6...radiolabel was placed on the MORF. The model carriers were a tat and a polyarginine peptide and cholesterol . The 25 mer MORF was selected as a suitable test...the MORF/streptavidin/ cholesterol accumulations were lower but stil1 significant). Furthermore, accumulations of the antisense MORF/streptavidin
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Development of siRNA Technology to Prevent Scar Formation in Tendon Repair
2013-12-01
Anti-sense RNA technologies: Under normal conditions cells produce small interfering (si) RNAs that inhibit protein synthesis and stimulate...stimulation of fibroblast proliferation and migration, collagen and fibronectin synthesis , and altered tissue remodeling through regulation of MMPs...expression by an antisense oligonucleotide protects mice from fulminant hepatitis. Nat Biotechnol 2000;18:862-7. 7. Guha M, Xu ZG, Tung D, Lanting L
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Igondjo-Tchen, S; Pagès, N; Bac, P; Godeau, G; Durlach, J
2003-03-01
The medical management of Marfan Syndrome (MFS) mainly relies on early prevention of the aortic complications. Hemodynamic treatments try to diminish the forcefulness of cardiac contractions and to reduce blood pressure: for example long term administration of propranolol may significantly reduce the rate of increase in aortic ratio (aortic diameter/expected aortic diameter). Retardation of aortic dilatation may be most often observed by early treatment started when the baseline end-diastolic aortic root diameter is < 40 mm. It seems better to use beta-blockers without intrinsic sympathomimetic activity. Successful acceptance of beta-blockers may be limited by side-effects, but the efficiency of alternative hypotensive agents (calcium channel inhibitors, ACE inhibitors) is not yet validated. Gene therapy might constitute an etiologic specific treatment of MFS. FBN1-RZ1 hammerhead antisense ribozyme is able to suppress expression of the mutant FBN1 allele. The use of ribozymes as systemic therapeutic agents will depend on efficient delivery to its target, but the various proposed vectors raise yet unsolved problems. A hydrogel angioplasty balloon might be a possible vector for delivering an antisense ribozyme in the aortic wall specifically. Ribozymes--as deoxyribonucleotides--may be taken up by tissue upon local application. Further research should study ex vivo local application of antisense ribozyme on human aortic wall, before assessing in vivo efficiency and tolerance of this aortic local vectorisation. It is always necessary to maintain a balanced magnesium intake in patients with MFS. Firstly to prevent the multiple noxious effects of magnesium deficiency on cardiovascular targets. Secondly to ensure the best efficiency and the least toxicity of the hemodynamic drugs used as long term prophylactic treatment for cardiovascular complications and of the etiologic antisense magnesium-dependent gene therapy, in the future.
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Rutkauskaite, Edita; Volkmer, Dagmar; Shigeyama, Yukio; Schedel, Jörg; Pap, Geza; Müller-Ladner, Ulf; Meinecke, Ingmar; Alexander, Dorothea; Gay, Renate E; Drynda, Susanne; Neumann, Wolfram; Michel, Beat A; Aicher, Wilhelm K; Gay, Steffen; Pap, Thomas
2005-07-01
Membrane type 1 matrix metalloproteinase (MT1-MMP) is expressed prominently in rheumatoid arthritis synovial fibroblasts (RASFs), but the specific contribution of MT1-MMP to fibroblast-mediated destruction of articular cartilage is incompletely understood. This study used gene transfer of an antisense expression construct to assess the effects of MT1-MMP inhibition on the invasiveness of RASFs. Retroviral gene transfer of a pLXIN vector-based antisense RNA expression construct (MT1-MMPalphaS) to MT1-MMP was used to stably transduce RASFs. Levels of MT1-MMP RNA and protein were determined by quantitative polymerase chain reaction, Western blotting, and immunocytochemistry in MT1-MMPalphaS-transduced RASFs as well as in control cells, with monitoring for 60 days. The effects of MT1-MMPalphaS on the invasiveness of RASFs were analyzed in the SCID mouse co-implantation model of RA. MT1-MMPalphaS-transduced RASFs produced high levels of antisense RNA that exceeded endogenous levels of MT1-MMP messenger RNA by 15-fold and resulted in a down-regulation of MT1-MMP at the protein level. Inhibition of MT1-MMP production was maintained for 60 days and significantly reduced the invasiveness of RASFs in the SCID mouse model. Whereas prominent invasion into cartilage by non-transduced and mock-transduced RASFs was observed (mean invasion scores 3.0 and 3.1, respectively), MT1-MMPalphaS-transduced cells showed only moderate invasiveness (mean invasion score 1.8; P < 0.05). The data demonstrate that an antisense RNA expression construct against MT1-MMP can be generated and expressed in RASFs for at least 60 days. Inhibition of MT1-MMP significantly reduces the cartilage degradation by RASFs.
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Strand-specific transcriptome profiling with directly labeled RNA on genomic tiling microarrays
2011-01-01
Background With lower manufacturing cost, high spot density, and flexible probe design, genomic tiling microarrays are ideal for comprehensive transcriptome studies. Typically, transcriptome profiling using microarrays involves reverse transcription, which converts RNA to cDNA. The cDNA is then labeled and hybridized to the probes on the arrays, thus the RNA signals are detected indirectly. Reverse transcription is known to generate artifactual cDNA, in particular the synthesis of second-strand cDNA, leading to false discovery of antisense RNA. To address this issue, we have developed an effective method using RNA that is directly labeled, thus by-passing the cDNA generation. This paper describes this method and its application to the mapping of transcriptome profiles. Results RNA extracted from laboratory cultures of Porphyromonas gingivalis was fluorescently labeled with an alkylation reagent and hybridized directly to probes on genomic tiling microarrays specifically designed for this periodontal pathogen. The generated transcriptome profile was strand-specific and produced signals close to background level in most antisense regions of the genome. In contrast, high levels of signal were detected in the antisense regions when the hybridization was done with cDNA. Five antisense areas were tested with independent strand-specific RT-PCR and none to negligible amplification was detected, indicating that the strong antisense cDNA signals were experimental artifacts. Conclusions An efficient method was developed for mapping transcriptome profiles specific to both coding strands of a bacterial genome. This method chemically labels and uses extracted RNA directly in microarray hybridization. The generated transcriptome profile was free of cDNA artifactual signals. In addition, this method requires fewer processing steps and is potentially more sensitive in detecting small amount of RNA compared to conventional end-labeling methods due to the incorporation of more fluorescent molecules per RNA fragment. PMID:21235785
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Enzymatic and antisense effects of a specific anti-Ki-ras ribozyme in vitro and in cell culture.
Giannini, C D; Roth, W K; Piiper, A; Zeuzem, S
1999-01-01
Due to their mode of action, ribozymes show antisense effects in addition to their specific cleavage activity. In the present study we investigated whether a hammerhead ribozyme is capable of cleaving mutated Ki-ras mRNA in a pancreatic carcinoma cell line and whether antisense effects contribute to the activity of the ribozyme. A 2[prime]-O-allyl modified hammerhead ribozyme was designed to cleave specifically the mutated form of the Ki- ras mRNA (GUU motif in codon 12). The activity was monitored by RT-PCR on Ki- ras RNA expression by determination of the relative amount of wild type to mutant Ki-ras mRNA, by 5-bromo-2[prime]-deoxy-uridine incorporation on cell proliferation and by colony formation in soft agar on malignancy in the human pancreatic adenocarcinoma cell line CFPAC-1, which is heterozygous for the Ki-ras mutation. A catalytically inactive ribozyme was used as control to differentiate between antisense and cleavage activity and a ribozyme with random guide sequences as negative control. The catalytically active anti-Ki-ras ribozyme was at least 2-fold more potent in decreasing cellular Ki-ras mRNA levels, inhibiting cell proliferation and colony formation in soft agar than the catalytically inactive ribozyme. The catalytically active anti-Ki-ras ribozyme, but not the catalytically inactive or random ribozyme, increased the ratio of wild type to mutated Ki-ras mRNA in CFPAC-1 cells. In conclusion, both cleavage activity and antisense effects contribute to the activity of the catalytically active anti-Ki-ras hammerhead ribozyme. Specific ribozymes might be useful in the treatment of pancreatic carcinomas containing an oncogenic GTT mutation in codon 12 of the Ki-ras gene. PMID:10373591
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C-fos mediates antipsychotic-induced neurotensin gene expression in the rodent striatum.
Robertson, G S; Tetzlaff, W; Bedard, A; St-Jean, M; Wigle, N
1995-07-01
The ubiquitous inducibility of the immediate-early gene c-fos in the central nervous system has led to the search for downstream genes which are regulated by its product, Fos. Recent evidence suggests that c-fos induction by a single injection of the classical antipsychotic haloperidol may contribute to the subsequent increase in neurotensin gene expression in the rodent striatum. Consistent with this proposal, in the present study haloperidol-induced Fos-like immunoreactivity and neurotensin/neuromedin N messenger RNA were found to be expressed by the same population of striatal neurons. Moreover, inhibition of haloperidol-induced c-fos expression by intrastriatal injection of antisense phosphorothioate oligodeoxynucleotides complimentary either to bases 109-126 or 127-144 of c-fos attenuated the subsequent increase in neurotensin/neuromedin N messenger RNA. However, injection of a sense phosphorothioate oligodeoxynucleotide corresponding to bases 127-144 of c-fos did not reduce haloperidol-induced c-fos or neurotensin/neuromedin N expression. Furthermore, constitutive expression of Jun-like immunoreactivity in the striatum was not reduced by either the sense or antisense phosphorothioate oligodeoxynucleotides. Similarly, the sense and antisense phosphorothioate oligodeoxynucleotide failed to reduce proenkephalin messenger RNA, which is located in the same striatal neurons that express haloperidol-induced neurotensin/neuromedin N messenger RNA, which is located in the same striatal neurons that express haloperidol-induced neurotensin/neuromedin N messenger RNA. Lastly, haloperidol-induced increases in nerve growth factor I-A-, JunB- and FosB-like immunoreactivity and fosB messenger RNA were not decreased by intrastriatal injection of either the sense or antisense phosphorothioate oligodeoxynucleotides. These results indicate that the antisense phosphorothioate oligodeoxynucleotides attenuated haloperidol-induced neurotensin/neuromedin N expression by selectively reducing c-fos expression and emphasize the potential importance of immediate-early gene induction in the mechanism of action of this antipsychotic drug.
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Held, Michael A.; Penning, Bryan; Brandt, Amanda S.; Kessans, Sarah A.; Yong, Weidong; Scofield, Steven R.; Carpita, Nicholas C.
2008-01-01
Small-interfering RNAs (siRNAs) from natural cis-antisense pairs derived from the 3′-coding region of the barley (Hordeum vulgare) CesA6 cellulose synthase gene substantially increase in abundance during leaf elongation. Strand-specific RT-PCR confirmed the presence of an antisense transcript of HvCesA6 that extends ≥1230 bp from the 3′ end of the CesA-coding sequence. The increases in abundance of the CesA6 antisense transcript and the 21-nt and 24-nt siRNAs derived from the transcript are coincident with the down-regulation of primary wall CesAs, several Csl genes, and GT8 glycosyl transferase genes, and are correlated with the reduction in rates of cellulose and (1 → 3),(1 → 4)-β-D-glucan synthesis. Virus induced gene silencing using unique target sequences derived from HvCesA genes attenuated expression not only of the HvCesA6 gene, but also of numerous nontarget Csls and the distantly related GT8 genes and reduced the incorporation of D-14C-Glc into cellulose and into mixed-linkage (1 → 3),(1 → 4)-β-D-glucans of the developing leaves. Unique target sequences for CslF and CslH conversely silenced the same genes and lowered rates of cellulose and (1 → 3),(1 → 4)-β-D-glucan synthesis. Our results indicate that the expression of individual members of the CesA/Csl superfamily and glycosyl transferases share common regulatory control points, and siRNAs from natural cis-antisense pairs derived from the CesA/Csl superfamily could function in this global regulation of cell-wall synthesis. PMID:19075248
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Murphy, Jane; Hall, William W.; Ratner, Lee
The human T-cell leukaemia virus type 1 and type 2 (HTLV-1/HTLV-2) antisense proteins HBZ and APH-2 play key roles in the HTLV lifecycles and persistence in the host. Nuclear Factors Associated with double-stranded RNA (NFAR) proteins NF90/110 function in the lifecycles of several viruses and participate in host innate immunity against infection and oncogenesis. Using GST pulldown and co-immunoprecipitation assays we demonstrate specific novel interactions between HBZ/APH-2 and NF90/110 and characterised the protein domains involved. Moreover we show that NF90/110 significantly enhance Tax mediated LTR activation, an effect that was abolished by HBZ but enhanced by APH-2. Additionally we foundmore » that HBZ and APH-2 modulate the promoter activity of survivin and are capable of antagonising NF110-mediated survivin activation. Thus interactions between HTLV antisense proteins and the NFAR protein family have an overall positive impact on HTLV infection. Hence NFARs may represent potential therapeutic targets in HTLV infected cells. - Highlights: • This study demonstrates for the first time interactions between NF90/110 and the HTLV antisense proteins HBZ and APH-2. • We show that NF90/110 significantly enhance LTR activation by the HTLV Tax protein, an effect that is abolished by HBZ but enhanced by APH-2. • The study shows that even though the HTLV antisense proteins activate survivin expression they antagonize the ability of NF90/110 to do so. • Overall we found that NF90/110 positively regulate HTLV infection and as such might represent a therapeutic target in infected cells.« less
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Rakoczy, P E; Lai, M C; Watson, M; Seydel, U; Constable, I
1996-01-01
In this article, we describe the preliminary results of the development of an animal model that will enable us to study the effect of photoreceptor-derived debris accumulation on the normal function of the retina in vivo. An antisense oligonucleotide (Cat 5), saline, and two control oligonucleotides were injected into the vitreous of 7-week-old RCS-rdy+ rats. The uptake, distribution, and persistence of the antisense oligonucleotide in the retina was demonstrated by fluorescent confocal microscopy, and the stability of the oligonucleotide was shown by GeneScan analysis using a fluorescein-labeled derivative of Cat 5 (Cat 5F). The accumulation of photoreceptor-derived debris was monitored by the number of undigested phagosomes in the RPE layer by light microscopy. Following intravitreal injection of Cat 5F, penetration of the oligonucleotide was observed in the ganglion cell layer in 2 hours and in the photoreceptor and pigment epithelial layers 3 days later. However, at 7, 28, and 56 days postinjection, only the RPE layer had significant amounts of Cat 5F present. Using GeneScan analysis, it was demonstrated that the fluorescein-labeled oligonucleotide present in the RPE layer was not degraded and it retained its original 19-mer length. There was no statistically significant difference in the number of phagosomes found in the RPE layer of control uninjected, saline-injected, and two sense and two antisense oligonucleotides-injected animals at 7 and 28 days postinjection. In contrast, the number of phagosomes was significantly higher (p < 0.001) in the RPE layer of Cat 5 antisense oligonucleotide-injected animals at 7 and 28 days postinjection. This difference, however, disappeared by 56 days postinjection. The inner nuclear layers of the retina of control and experimental animals were not affected by the injections.
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Eisenhut, Marion; Georg, Jens; Klähn, Stephan; Sakurai, Isamu; Mustila, Henna; Zhang, Pengpeng; Hess, Wolfgang R.; Aro, Eva-Mari
2012-01-01
The functional relevance of natural cis-antisense transcripts is mostly unknown. Here we have characterized the association of three antisense RNAs and one intergenically encoded noncoding RNA with an operon that plays a crucial role in photoprotection of photosystem II under low carbon conditions in the cyanobacterium Synechocystis sp. PCC 6803. Cyanobacteria show strong gene expression dynamics in response to a shift of cells from high carbon to low levels of inorganic carbon (Ci), but the regulatory mechanisms are poorly understood. Among the most up-regulated genes in Synechocystis are flv4, sll0218, and flv2, which are organized in the flv4-2 operon. The flavodiiron proteins encoded by this operon open up an alternative electron transfer route, likely starting from the QB site in photosystem II, under photooxidative stress conditions. Our expression analysis of cells shifted from high carbon to low carbon demonstrated an inversely correlated transcript accumulation of the flv4-2 operon mRNA and one antisense RNA to flv4, designated as As1_flv4. Overexpression of As1_flv4 led to a decrease in flv4-2 mRNA. The promoter activity of as1_flv4 was transiently stimulated by Ci limitation and negatively regulated by the AbrB-like transcription regulator Sll0822, whereas the flv4-2 operon was positively regulated by the transcription factor NdhR. The results indicate that the tightly regulated antisense RNA As1_flv4 establishes a transient threshold for flv4-2 expression in the early phase after a change in Ci conditions. Thus, it prevents unfavorable synthesis of the proteins from the flv4-2 operon. PMID:22854963
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Expression and characterization of a brain-specific protein kinase BSK146 from zebrafish.
Chou, Chih-Ming; Chen, Yi-Chung; Lee, Ming-Ting; Chen, Gen-Der; Lu, I-Ching; Chen, Shui-Tsung; Huang, Chang-Jen
2006-02-17
We have previously identified a novel protein kinase, pk146, in the brain of Tetraodon. In the present study, we cloned the homologous protein kinase gene encoding a protein of 385 amino acid residues from zebrafish. The overall amino acid sequence and the kinase domain of zebrafish BSK146 shows 48% and 69% identity to that of rat sbk, a SH3-containing serine/threonine protein kinase. By whole-mount in situ hybridization and RT-PCR, the expression of bsk146 mRNA was mainly in the brain. To explore the in vivo function of BSK146 during zebrafish development, we used morpholino knockdown approach and found that BSK146 morphants displayed enlarged hindbrain ventricle and smaller eyes. Whole-mount in situ hybridization was further performed to analyze the brain defects in BSK146-MO-injected embryos. The expression of brain-specific markers, such as otx2, pax2.1, and krox20, was found normal in morphant embryos at 24hpf, while expression of pax2.1 exerted changes in midbrain-hindbrain boundary and hindbrain in morphant embryos at 48hpf. These data suggest that BSK146 may play an important role in later ventricle expansion in zebrafish brain development. Although the recombinant BSK146 protein produced in insect cells was active and could phosphorylate both histone H1 and histone 2B, the endogenous substrate of BSK146 in the embryonic brain of zebrafish is not clear at the present time and needs further investigation.
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Antisense Treatments for Biothreat Agents
2006-08-01
2001) 19(4):360-364. 82. Nekhotiaeva N, Awasthi SK, Nielsen PE, Good L: Inhibition of Staphylococcus aureus gene expression and growth using...to PNA enhanced the entry of the antisense molecules and reduced expression of the bacterial target genes both in E coli [81] and Staphylococcus ... aureus [82]. Peptide-tagged PMOs can also efficiently inhibit bacterial growth in pure and infected cultures [75]. In a recent study, we observed that
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Selective Androgen Receptor Down-Regulators (SARDs): A New Prostate Cancer Therapy
2007-10-01
PCa (9). Thus far, the techniques that have been used to down-regulate the AR include antisense oligonucleotides (10, 11), ribozyme treatments (12...Our findings suggest that ICI may present a useful treatment option for patients with AR-dependent PCa. Unlike the ribozyme , antisense, siRNA, or...Catalytic cleavage of the androgen receptor messenger RNA and functional inhibition of androgen receptor activity by a hammerhead ribozyme . Mol Endocrinol
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Steven L. Voelker; Barbara Lachenbruch; Frederick C. Meinzer; Peter Kitin; Steven H. Strauss
2011-01-01
Reduced lignin content in perennial crops has been sought as a means to improve biomass processability for paper and biofuels production, but it is unclear how this could affect wood properties and tree form. Here, we studied a nontransgenic control and 14 transgenic events containing an antisense 4-coumarate:coenzyme A ligase (4CL) to discern the...
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Antisense Oligonucleotide Therapy for Patients with Advanced Cancer | Center for Cancer Research
Colorectal cancer (CRC) is the second leading cause of cancer-related death in the U.S. Improvements in therapy have increased the survival of patients with CRC from 10 months to two years, but for patients who stop responding to treatments, such as irinotecan, options for additional therapy are limited. Antisense oligonucleotides (ASOs) may offer advantages over traditional therapies if an appropriate target can be identified.
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Annexin II-Dependent Mechanism of Breast Cancer Progression
2008-06-01
and migratory capacities of the annexin II-suppressed cells. Methods: We used antisense RNA technology to silence the annexin II gene in MDA...gene in mDA-MB231 cells using polymerase chain reaction-based short hairpin RNA (1–7 months) b) Characterize the proliferative, invasive, and...MB231 cells according to methods described by Li et al. (24). Briefly, three different diothionated antisense nucleotides (ODN) were synthesized
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Law, Lee Yong; Zhang, Wei V; Stott, N Susan; Becker, David L; Green, Colin R
2006-09-01
The completion of the human and mouse genomes has identified at least 20 connexin isomers in this family of intercellular channel proteins. However, there are no specific gap junction blockers or channel-blocking mimetic peptides available for the study of specific connexins. We designed antisense oligodeoxynucleotides that functionally reduce targeted connexin protein expression and can be used to reveal the biological function of individual connexins in vivo. Connexin mRNA was firstly exposed in vitro to deoxyribozymes complementing the sense coding sequence. Those that cleaved the target connexin mRNA in defined regions were used as the basis to design oligodeoxynucleotides to the accessible sites, thus taking into account tertiary mRNA configurations rather than relying on computed predictions. Antisense oligodeoxynucleotides designed to bind to accessible mRNA sites selectively reduced connexin26 and -43 mRNA expression in a corneal epithelium ex vivo model. Connexin43 protein levels were reduced correlating with the knockdown in mRNA and the protein's rapid turnover; protein levels of connexin26 did not alter, supporting lower turnover rates reported for that protein. We show, for the first time, an inexpensive and empirical approach to the preparation of specific and functional antisense oligodeoxynucleotides against known gene targets in the post-genomic era.
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ISS-N1 makes the First FDA-approved Drug for Spinal Muscular Atrophy
Ottesen, Eric W.
2017-01-01
Abstract Spinal muscular atrophy (SMA) is one of the leading genetic diseases of children and infants. SMA is caused by deletions or mutations of Survival Motor Neuron 1 (SMN1) gene. SMN2, a nearly identical copy of SMN1, cannot compensate for the loss of SMN1 due to predominant skipping of exon 7. While various regulatory elements that modulate SMN2 exon 7 splicing have been proposed, intronic splicing silencer N1 (ISS-N1) has emerged as the most promising target thus far for antisense oligonucleotide-mediated splicing correction in SMA. Upon procuring exclusive license from the University of Massachussets Medical School in 2010, Ionis Pharmaceuticals (formerly ISIS Pharamaceuticals) began clinical development of Spinraza™ (synonyms: Nusinersen, IONIS-SMNRX, ISIS-SMNRX), an antisense drug based on ISS-N1 target. Spinraza™ showed very promising results at all steps of the clinical development and was approved by US Food and Drug Administration (FDA) on December 23, 2016. Spinraza™ is the first FDA-approved treatment for SMA and the first antisense drug to restore expression of a fully functional protein via splicing correction. The success of Spinraza™ underscores the potential of intronic sequences as promising therapeutic targets and sets the stage for further improvement of antisense drugs based on advanced oligonucleotide chemistries and delivery protocols. PMID:28400976
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Carlson, Jonathan; Yan, Jiyu; Akinsiku, Olusimidele T.; Schaefer, Malinda; Sabbaj, Steffanie; Bet, Anne; Levy, David N.; Heath, Sonya; Tang, Jianming; Kaslow, Richard A.; Walker, Bruce D.; Ndung’u, Thumbi; Goulder, Philip J.; Heckerman, David; Hunter, Eric; Goepfert, Paul A.
2010-01-01
Retroviruses pack multiple genes into relatively small genomes by encoding several genes in the same genomic region with overlapping reading frames. Both sense and antisense HIV-1 transcripts contain open reading frames for known functional proteins as well as numerous alternative reading frames (ARFs). At least some ARFs have the potential to encode proteins of unknown function, and their antigenic properties can be considered as cryptic epitopes (CEs). To examine the extent of active immune response to virally encoded CEs, we analyzed human leukocyte antigen class I–associated polymorphisms in HIV-1 gag, pol, and nef genes from a large cohort of South Africans with chronic infection. In all, 391 CEs and 168 conventional epitopes were predicted, with the majority (307; 79%) of CEs derived from antisense transcripts. In further evaluation of CD8 T cell responses to a subset of the predicted CEs in patients with primary or chronic infection, both sense- and antisense-encoded CEs were immunogenic at both stages of infection. In addition, CEs often mutated during the first year of infection, which was consistent with immune selection for escape variants. These findings indicate that the HIV-1 genome might encode and deploy a large potential repertoire of unconventional epitopes to enhance vaccine-induced antiviral immunity. PMID:20065064
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Cocaine alters Homer1 natural antisense transcript in the nucleus accumbens.
Sartor, Gregory C; Powell, Samuel K; Velmeshev, Dmitry; Lin, David Y; Magistri, Marco; Wiedner, Hannah J; Malvezzi, Andrea M; Andrade, Nadja S; Faghihi, Mohammad A; Wahlestedt, Claes
2017-12-01
Natural antisense transcripts (NATs) are an abundant class of long noncoding RNAs that have recently been shown to be key regulators of chromatin dynamics and gene expression in nervous system development and neurological disorders. However, it is currently unclear if NAT-based mechanisms also play a role in drug-induced neuroadaptations. Aberrant regulation of gene expression is one critical factor underlying the long-lasting behavioral abnormalities that characterize substance use disorder, and it is possible that some drug-induced transcriptional responses are mediated, in part, by perturbations in NAT activity. To test this hypothesis, we used an automated algorithm that mines the NCBI AceView transcriptomics database to identify NAT overlapping genes linked to addiction. We found that 22% of the genes examined contain NATs and that expression of Homer1 natural antisense transcript (Homer1-AS) was altered in the nucleus accumbens (NAc) of mice 2h and 10days following repeated cocaine administration. In in vitro studies, depletion of Homer1-AS lead to an increase in the corresponding sense gene expression, indicating a potential regulatory mechanisms of Homer1 expression by its corresponding antisense transcript. Future in vivo studies are needed to definitely determine a role for Homer1-AS in cocaine-induced behavioral and molecular adaptations. Copyright © 2017 Elsevier Inc. All rights reserved.
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cis-antisense RNA, another level of gene regulation in bacteria.
Georg, Jens; Hess, Wolfgang R
2011-06-01
A substantial amount of antisense transcription is a hallmark of gene expression in eukaryotes. However, antisense transcription was first demonstrated in bacteria almost 50 years ago. The transcriptomes of bacteria as different as Helicobacter pylori, Bacillus subtilis, Escherichia coli, Synechocystis sp. strain PCC6803, Mycoplasma pneumoniae, Sinorhizobium meliloti, Geobacter sulfurreducens, Vibrio cholerae, Chlamydia trachomatis, Pseudomonas syringae, and Staphylococcus aureus have now been reported to contain antisense RNA (asRNA) transcripts for a high percentage of genes. Bacterial asRNAs share functional similarities with trans-acting regulatory RNAs, but in addition, they use their own distinct mechanisms. Among their confirmed functional roles are transcription termination, codegradation, control of translation, transcriptional interference, and enhanced stability of their respective target transcripts. Here, we review recent publications indicating that asRNAs occur as frequently in simple unicellular bacteria as they do in higher organisms, and we provide a comprehensive overview of the experimentally confirmed characteristics of asRNA actions and intimately linked quantitative aspects. Emerging functional data suggest that asRNAs in bacteria mediate a plethora of effects and are involved in far more processes than were previously anticipated. Thus, the functional impact of asRNAs should be considered when developing new strategies against pathogenic bacteria and when optimizing bacterial strains for biotechnology.
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cis-Antisense RNA, Another Level of Gene Regulation in Bacteria
Georg, Jens; Hess, Wolfgang R.
2011-01-01
Summary: A substantial amount of antisense transcription is a hallmark of gene expression in eukaryotes. However, antisense transcription was first demonstrated in bacteria almost 50 years ago. The transcriptomes of bacteria as different as Helicobacter pylori, Bacillus subtilis, Escherichia coli, Synechocystis sp. strain PCC6803, Mycoplasma pneumoniae, Sinorhizobium meliloti, Geobacter sulfurreducens, Vibrio cholerae, Chlamydia trachomatis, Pseudomonas syringae, and Staphylococcus aureus have now been reported to contain antisense RNA (asRNA) transcripts for a high percentage of genes. Bacterial asRNAs share functional similarities with trans-acting regulatory RNAs, but in addition, they use their own distinct mechanisms. Among their confirmed functional roles are transcription termination, codegradation, control of translation, transcriptional interference, and enhanced stability of their respective target transcripts. Here, we review recent publications indicating that asRNAs occur as frequently in simple unicellular bacteria as they do in higher organisms, and we provide a comprehensive overview of the experimentally confirmed characteristics of asRNA actions and intimately linked quantitative aspects. Emerging functional data suggest that asRNAs in bacteria mediate a plethora of effects and are involved in far more processes than were previously anticipated. Thus, the functional impact of asRNAs should be considered when developing new strategies against pathogenic bacteria and when optimizing bacterial strains for biotechnology. PMID:21646430
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Crooke, Stanley T; Baker, Brenda F; Kwoh, T Jesse; Cheng, Wei; Schulz, Dan J; Xia, Shuting; Salgado, Nelson; Bui, Huynh-Hoa; Hart, Christopher E; Burel, Sebastien A; Younis, Husam S; Geary, Richard S; Henry, Scott P; Bhanot, Sanjay
2016-01-01
The common chemical and biological properties of antisense oligonucleotides provide the opportunity to identify and characterize chemical class effects across species. The chemical class that has proven to be the most versatile and best characterized is the 2′-O-methoxyethyl chimeric antisense oligonucleotides. In this report we present an integrated safety assessment of data obtained from controlled dose-ranging studies in nonhuman primates (macaques) and healthy human volunteers for 12 unique 2′-O-methoxyethyl chimeric antisense oligonucleotides. Safety was assessed by the incidence of safety signals in standardized laboratory tests for kidney and liver function, hematology, and complement activation; as well as by the mean test results as a function of dose level over time. At high doses a number of toxicities were observed in nonhuman primates. However, no class safety effects were identified in healthy human volunteers from this integrated data analysis. Effects on complement in nonhuman primates were not observed in humans. Nonhuman primates predicted safe doses in humans, but over predicted risk of complement activation and effects on platelets. Although limited to a single chemical class, comparisons from this analysis are considered valid and accurate based on the carefully controlled setting for the specified study populations and within the total exposures studied. PMID:27357629
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Wang, Judy; Chen, Paul; Mrkobrada, Marko; Hu, Meiduo; Vallis, Katherine A; Reilly, Raymond M
2003-09-01
Molecular imaging of the expression of key genes which determine the response to DNA damage following cancer treatment may predict the effectiveness of a particular treatment strategy. A prominent early response gene for DNA damage is the gene encoding p21(WAF-1/CIP-1), a cyclin-dependent kinase inhibitor that regulates progression through the cell cycle. In this study, we explored the feasibility of imaging p21(WAF-1/CIP-1) gene expression at the mRNA level using an 18-mer phosphorothioated antisense oligodeoxynucleotide (ODN) labeled with (111)In. The known induction of the p21(WAF-1/CIP-1) gene in MDA-MB-468 human breast cancer cells following exposure to epidermal growth factor (EGF) was used as an experimental tool. Treatment of MDA-MB-468 cells in vitro with EGF (20 n M) increased the ratio of p21(WAF-1/CIP-1) mRNA/beta-actin mRNA threefold within 2 h as measured by the reverse transcription polymerase chain reaction (RT-PCR). A concentration-dependent inhibition of EGF-induced p21(WAF-1/CIP-1) protein expression was achieved in MDA-MB-468 cells by treatment with antisense ODNs with up to a tenfold decrease observed at 1 microM. There was a fourfold lower inhibition of p21(WAF-1/CIP-1) protein expression by control sense or random sequence ODNs. Intratumoral injections of EGF (15 microg/dayx3 days) were employed to induce p21(WAF-1/CIP-1) gene expression in MDA-MB-468 xenografts implanted subcutaneously into athymic mice. RT-PCR of explanted tumors showed a threefold increased level of p21(WAF-1/CIP-1) mRNA compared with normal saline-treated tumors. Successful imaging of EGF-induced p21(WAF-1/CIP-1) gene expression in MDA-MB-468 xenografts was achieved at 48 h post injection of (111)In-labeled antisense ODNs (3.7 MBq; 2 microg). Tumors displaying basal levels of p21(WAF-1/CIP-1) gene expression in the absence of EGF treatment could not be visualized. Biodistribution studies showed a significantly higher tumor accumulation of (111)In-labeled antisense ODNs in the presence of EGF induction of the p21(WAF-1/CIP-1) gene (0.32%+/-0.06% injected dose/g) compared with normal saline-treated control mice (0.11%+/-0.07% injected dose/g). The tumor/blood ratio for antisense ODNs in the presence of EGF induction of the p21(WAF-1/CIP-1) gene (4.87+/-0.87) was also significantly higher than for control random sequence ODNs (2.14+/-0.69) or for mice receiving antisense ODNs but not treated with EGF (2.07+/-0.37). We conclude that antisense imaging of upregulated p21(WAF-1/CIP-1) gene expression is feasible and could represent a promising new molecular imaging strategy for monitoring tumor response in cancer patients. To our knowledge, this study also describes the first report of molecular imaging of the upregulated expression of a downstream gene target of the EGFR, a transmembrane tyrosine kinase receptor.
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Anti-NGF Local Therapy for Autonomic Dysreflexia in Spinal Cord Injury
2013-10-01
growth factor in the urothelium of sham treated rats, which was decreased 5 by antisense treatment (Fig. 4A) and (5) increased nerve growth...sensitization. B Figure 4. A: Antisense OND mediated suppression of acetic acid (AA) induced NGF protein expression in urothelium . AA exposure...upregulation in the bladder urothelium of SCI rats Detection of hyperexcitability of bladder afferent neurons due to the reduction of A-type K+ channel
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Growth Suppression and Therapy Sensitization of Breast Cancer
2000-07-01
determined by performed on two independent occasions. PCR amplification of a given housekeeping gene have been shown to correspond to determinations of...h incubation in the presence or absence of 1 mM cisplatin expressed housekeeping gene, dihydrofolate reductase (DHFR). (Platinol, aqueous solution at... G3PDH :j G3PDH Figure 9. A549 cells were treated with 3 different antisense oligonucleotides complementary to JNKI mRNA (including the active antisense
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2009-10-01
differentiation and activation of osteoclast precursors. Targeting RANKL expression with antisense oligonucleotides (RANKL- ASO ) decreased RANKL expression and...1,175 Ci/mmol at 10 mCi/mL). Two microliters of the reaction mixture were separated on a 12% SDS-polyacrylamide gel and subsequently visualized...OPG), a decoy receptor for RANKL, at the TB-interface was also increased. Targeting RANKL expression with antisense oligonucleotides (RANKL- ASO
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Mukherjee, Archana; Wickstrom, Eric
2009-01-01
This review briefly outlines the importance of molecular imaging, particularly imaging of endogenous gene expression for noninvasive genetic analysis of radiographic masses. The concept of antisense imaging agents and the advantages and challenges in the development of hybridization probes for in vivo imaging are described. An overview of the investigations on oncogene expression imaging is given. Finally, the need for further improvement in antisense-based imaging agents and directions to improve oncogene mRNA targeting is stated. PMID:19264436
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Ebenryter-Olbińska, Katarzyna; Kaniowski, Damian; Sobczak, Milena; Wojtczak, Błażej A; Janczak, Sławomir; Wielgus, Ewelina; Nawrot, Barbara; Leśnikowski, Zbigniew J
2017-11-21
A general and convenient approach for the incorporation of different types of boron clusters into specific locations of the DNA-oligonucleotide chain based on the automated phosphoramidite method of oligonucleotide synthesis and post-synthetic "click chemistry" modification has been developed. Pronounced effects of boron-cluster modification on the physico- and biochemical properties of the antisense oligonucleotides were observed. The silencing activity of antisense oligonucleotides bearing a single boron cluster modification in the middle of the oligonucleotide chain was substantially higher than that of unmodified oligonucleotides. This finding may be of importance for the design of therapeutic nucleic acids with improved properties. The proposed synthetic methodology broadens the availability of nucleic acid-boron cluster conjugates and opens up new avenues for their potential practical use. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Wu, Q; Qiao, H; Wang, Z; Zhang, H; Liu, P; Xu, M; Ren, G; Zhao, S; She, M
2000-04-01
To study the mechanism of monocyte recruitment in atherogenesis and to clarify the effect of monocyte chemotactic protein-1 (MCP-1) in this process. Femoral arteries isolated from the rabbits which had been fed with a high cholesterol diet and locally perfused with MM-LDL within the artery beforehand, were used as the models. Antisense MCP-1cDNA was transferred into the arterial wall by injecting recombinant LNCX-anti-MCP-1/liposomal complex in the femoral sheath and the periarterial tissue. Expression of antisense MCP-1 mediated by recombinant LNCX plasmid/lipsomal complex gene transfer enabled to inhibit MCP-1 gene expression and adhesion of monocyte to the intima. MCP-1 plays an important role on the recruitment of monocytes in the arterial wall, which provides a potential clue in developing a gene therapy project for the prevention and treatment of atherogenesis.
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Bell, Thomas A; Graham, Mark J; Lee, Richard G; Mullick, Adam E; Fu, Wuxia; Norris, Dan; Crooke, Rosanne M
2013-10-01
Due to their ability to promote positive effects across all of the lipoprotein classes, cholesteryl ester transfer protein (CETP) inhibitors are currently being developed as therapeutic agents for cardiovascular disease. In these studies, we compared an antisense oligonucleotide (ASO) inhibitor of CETP to the CETP small molecule inhibitor anacetrapib. In hyperlipidemic CETP transgenic (tg) mice, both drugs provided comparable reductions in total plasma cholesterol, decreases in CETP activity, and increases in HDL cholesterol. However, only mice treated with the antisense inhibitor showed an enhanced effect on macrophage reverse cholesterol transport, presumably due to differences in HDL apolipoprotein composition and decreases in plasma triglyceride. Additionally, the ASO-mediated reductions in CETP mRNA were associated with less accumulation of aortic cholesterol. These preliminary findings suggest that CETP ASOs may represent an alternative means to inhibit that target and to support their continued development as a treatment for cardiovascular disease in man.
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Antisense oligonucleotides as therapeutics for hyperlipidaemias.
Crooke, Rosanne M
2005-07-01
Hyperlipidaemia, due to elevations of low-density lipoprotein cholesterol (LDL-C) or triglycerides (TGs), is recognised as a significant risk factor contributing to the development of coronary heart disease (CHD), the leading cause of morbidity and mortality in the Western world. Even though a variety of established antihyperlipidaemic agents are available, the majority of high-risk patients do not reach their lipid goals, indicating the need for new and more effective therapeutics to be used alone or as combination agents with existing drugs. Antisense oligonucleotides (ASOs), designed to specifically and selectively inhibit novel targets involved in cholesterol/TG homeostasis, represent a new class of agents that may prove beneficial for the treatment of hyperlipidaemias resulting from various genetic, metabolic or behavioural factors. This article describes the antisense technology platform, highlights the advantages of these novel drugs for the treatment of hyperlipidaemia and reviews the current research in this area.
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Miroshnichenko, O I; Ponomareva, T I; Tikchonenko, T I
1989-12-07
To study the effect of antisense E1a RNA (asRNA) on adenovirus development, two types of adenovirus 5 E1a antisense constructs have been engineered. One was complementary to the viral DNA region [nucleotide (nt) positions 500-720] regulated by the metallothionein-I promoter, and the other was complementary to the DNA regions (nt positions 630-1570) under control of the long terminal repeat Moloney mouse leukosis virus promoter. Both asRNA constructs were cloned into a plasmid containing the simian virus 40 origin of replication, the gene controlling geneticin (G418) resistance (G418R), and other regulatory elements. The COS-1 cells, which contained up to 100 copies of the engineered plasmids, synthesized antiviral asRNAs, which provided 71 to over 95% inhibition of adenoviral replication, in comparison to the control cells not synthesizing asRNAs.
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Bacterial antisense RNAs are mainly the product of transcriptional noise.
Lloréns-Rico, Verónica; Cano, Jaime; Kamminga, Tjerko; Gil, Rosario; Latorre, Amparo; Chen, Wei-Hua; Bork, Peer; Glass, John I; Serrano, Luis; Lluch-Senar, Maria
2016-03-01
cis-Encoded antisense RNAs (asRNAs) are widespread along bacterial transcriptomes. However, the role of most of these RNAs remains unknown, and there is an ongoing discussion as to what extent these transcripts are the result of transcriptional noise. We show, by comparative transcriptomics of 20 bacterial species and one chloroplast, that the number of asRNAs is exponentially dependent on the genomic AT content and that expression of asRNA at low levels exerts little impact in terms of energy consumption. A transcription model simulating mRNA and asRNA production indicates that the asRNA regulatory effect is only observed above certain expression thresholds, substantially higher than physiological transcript levels. These predictions were verified experimentally by overexpressing nine different asRNAs in Mycoplasma pneumoniae. Our results suggest that most of the antisense transcripts found in bacteria are the consequence of transcriptional noise, arising at spurious promoters throughout the genome.
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Bacterial antisense RNAs are mainly the product of transcriptional noise
Lloréns-Rico, Verónica; Cano, Jaime; Kamminga, Tjerko; Gil, Rosario; Latorre, Amparo; Chen, Wei-Hua; Bork, Peer; Glass, John I.; Serrano, Luis; Lluch-Senar, Maria
2016-01-01
cis-Encoded antisense RNAs (asRNAs) are widespread along bacterial transcriptomes. However, the role of most of these RNAs remains unknown, and there is an ongoing discussion as to what extent these transcripts are the result of transcriptional noise. We show, by comparative transcriptomics of 20 bacterial species and one chloroplast, that the number of asRNAs is exponentially dependent on the genomic AT content and that expression of asRNA at low levels exerts little impact in terms of energy consumption. A transcription model simulating mRNA and asRNA production indicates that the asRNA regulatory effect is only observed above certain expression thresholds, substantially higher than physiological transcript levels. These predictions were verified experimentally by overexpressing nine different asRNAs in Mycoplasma pneumoniae. Our results suggest that most of the antisense transcripts found in bacteria are the consequence of transcriptional noise, arising at spurious promoters throughout the genome. PMID:26973873
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Method for phosphorothioate antisense DNA sequencing by capillary electrophoresis with UV detection.
Froim, D; Hopkins, C E; Belenky, A; Cohen, A S
1997-11-01
The progress of antisense DNA therapy demands development of reliable and convenient methods for sequencing short single-stranded oligonucleotides. A method of phosphorothioate antisense DNA sequencing analysis using UV detection coupled to capillary electrophoresis (CE) has been developed based on a modified chain termination sequencing method. The proposed method reduces the sequencing cost since it uses affordable CE-UV instrumentation and requires no labeling with minimal sample processing before analysis. Cycle sequencing with ThermoSequenase generates quantities of sequencing products that are readily detectable by UV. Discrimination of undesired components from sequencing products in the reaction mixture, previously accomplished by fluorescent or radioactive labeling, is now achieved by bringing concentrations of undesired components below the UV detection range which yields a 'clean', well defined sequence. UV detection coupled with CE offers additional conveniences for sequencing since it can be accomplished with commercially available CE-UV equipment and is readily amenable to automation.
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Method for phosphorothioate antisense DNA sequencing by capillary electrophoresis with UV detection.
Froim, D; Hopkins, C E; Belenky, A; Cohen, A S
1997-01-01
The progress of antisense DNA therapy demands development of reliable and convenient methods for sequencing short single-stranded oligonucleotides. A method of phosphorothioate antisense DNA sequencing analysis using UV detection coupled to capillary electrophoresis (CE) has been developed based on a modified chain termination sequencing method. The proposed method reduces the sequencing cost since it uses affordable CE-UV instrumentation and requires no labeling with minimal sample processing before analysis. Cycle sequencing with ThermoSequenase generates quantities of sequencing products that are readily detectable by UV. Discrimination of undesired components from sequencing products in the reaction mixture, previously accomplished by fluorescent or radioactive labeling, is now achieved by bringing concentrations of undesired components below the UV detection range which yields a 'clean', well defined sequence. UV detection coupled with CE offers additional conveniences for sequencing since it can be accomplished with commercially available CE-UV equipment and is readily amenable to automation. PMID:9336449
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Hu, Zhilian; Holzschuh, Jochen; Driever, Wolfgang
2015-01-01
DNA damage-binding protein 1 (DDB1) is a large subunit of the heterodimeric DDB complex that recognizes DNA lesions and initiates the nucleotide excision repair process. DDB1 is also a component of the CUL4 E3 ligase complex involved in a broad spectrum of cellular processes by targeted ubiquitination of key regulators. Functions of DDB1 in development have been addressed in several model organisms, however, are not fully understood so far. Here we report an ENU induced mutant ddb1 allele (ddb1m863) identified in zebrafish (Danio rerio), and analyze its effects on development. Zebrafish ddb1 is expressed broadly, both maternally and zygotically, with enhanced expression in proliferation zones. The (ddb1m863 mutant allele affects the splice acceptor site of exon 20, causing a splicing defect that results in truncation of the 1140 amino acid protein after residue 800, lacking part of the β-propeller domain BPC and the C-terminal helical domain CTD. ddb1m863 zygotic mutant embryos have a pleiotropic phenotype, including smaller and abnormally shaped brain, head skeleton, eyes, jaw, and branchial arches, as well as reduced dopaminergic neuron groups. However, early forming tissues develop normally in zygotic ddb1m863 mutant embryos, which may be due to maternal rescue. In ddb1m863 mutant embryos, pcna-expressing proliferating cell populations were reduced, concurrent with increased apoptosis. We also observed a concomitant strong up-regulation of transcripts of the tumor suppressor p53 (tp53) and the cell cycle inhibitor cdkn1a (p21a/bCIP1/WAF1) in proliferating tissues. In addition, transcription of cyclin genes ccna2 and ccnd1 was deregulated in ddb1m863 mutants. Reduction of p53 activity by anti-sense morpholinos alleviated the apoptotic phenotype in ddb1m863 mutants. These results imply that Ddb1 may be involved in maintaining proper cell cycle progression and viability of dividing cells during development through transcriptional mechanisms regulating genes involved in cell cycle control and cell survival.
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Vasil, Michael L.; Stonehouse, Martin J.; Vasil, Adriana I.; Wadsworth, Sandra J.; Goldfine, Howard; Bolcome, Robert E.; Chan, Joanne
2009-01-01
The hemolytic phospholipase C (PlcHR) expressed by Pseudomonas aeruginosa is the original member of a Phosphoesterase Superfamily, which includes phosphorylcholine-specific phospholipases C (PC-PLC) produced by frank and opportunistic pathogens. PlcHR, but not all its family members, is also a potent sphingomyelinase (SMase). Data presented herein indicate that picomolar (pM) concentrations of PlcHR are selectively lethal to endothelial cells (EC). An RGD motif of PlcHR contributes to this selectivity. Peptides containing an RGD motif (i.e., GRGDS), but not control peptides (i.e., GDGRS), block the effects of PlcHR on calcium signaling and cytotoxicity to EC. Moreover, RGD variants of PlcHR (e.g., RGE, KGD) are significantly reduced in their binding and toxicity, but retain the enzymatic activity of the wild type PlcHR. PlcHR also inhibits several EC-dependent in vitro assays (i.e., EC migration, EC invasion, and EC tubule formation), which represent key processes involved in angiogenesis (i.e., formation of new blood vessels from existing vasculature). Finally, the impact of PlcHR in an in vivo model of angiogenesis in transgenic zebrafish, and ones treated with an antisense morpholino to knock down a key blood cell regulator, were evaluated because in vitro assays cannot fully represent the complex processes of angiogenesis. As little as 2 ng/embryo of PlcHR was lethal to ∼50% of EGFP-labeled EC at 6 h after injection of embryos at 48 hpf (hours post-fertilization). An active site mutant of PlcHR (Thr178Ala) exhibited 120-fold reduced inhibitory activity in the EC invasion assay, and 20 ng/embryo elicited no detectable inhibitory activity in the zebrafish model. Taken together, these observations are pertinent to the distinctive vasculitis and poor wound healing associated with P. aeruginosa sepsis and suggest that the potent antiangiogenic properties of PlcHR are worthy of further investigation for the treatment of diseases where angiogenesis contributes pathological conditions (e.g., vascularization of tumors, diabetic retinopathy). PMID:19424430
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Mackereth, Melinda D; Kwak, Su-Jin; Fritz, Andreas; Riley, Bruce B
2005-01-01
Vertebrate Pax2 and Pax8 proteins are closely related transcription factors hypothesized to regulate early aspects of inner ear development. In zebrafish and mouse, Pax8 expression is the earliest known marker of otic induction, and Pax2 homologs are expressed at slightly later stages of placodal development. Analysis of compound mutants has not been reported. To facilitate analysis of zebrafish pax8, we completed sequencing of the entire gene, including the 5' and 3' UTRs. pax8 transcripts undergo complex alternative splicing to generate at least ten distinct isoforms. Two different subclasses of pax8 splice isoforms encode different translation initiation sites. Antisense morpholinos (MOs) were designed to block translation from both start sites, and four additional MOs were designed to target different exon-intron boundaries to block splicing. Injection of MOs, individually and in various combinations, generated similar phenotypes. Otic induction was impaired, and otic vesicles were small. Regional ear markers were expressed correctly, but hair cell production was significantly reduced. This phenotype was strongly enhanced by simultaneously disrupting either of the co-inducers fgf3 or fgf8, or another early regulator, dlx3b, which is thought to act in a parallel pathway. In contrast, the phenotype caused by disrupting foxi1, which is required for pax8 expression, was not enhanced by simultaneously disrupting pax8. Disrupting pax8, pax2a and pax2b did not further impair otic induction relative to loss of pax8 alone. However, the amount of otic tissue gradually decreased in pax8-pax2a-pax2b-deficient embryos such that no otic tissue was detectable by 24 hours post-fertilization. Loss of otic tissue did not correlate with increased cell death, suggesting that otic cells dedifferentiate or redifferentiate as other cell type(s). These data show that pax8 is initially required for normal otic induction, and subsequently pax8, pax2a and pax2b act redundantly to maintain otic fate.
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E2F8 as a Novel Therapeutic Target for Lung Cancer
Park, Sin-Aye; Platt, James; Lee, Jong Woo; López-Giráldez, Francesc; Herbst, Roy S.
2015-01-01
Background: The E2F members have been divided into transcription activators (E2F1-E2F3) and repressors (E2F4-E2F8). E2F8 with E2F7 has been known to play an important physiologic role in embryonic development and cell cycle regulation by repressing E2F1. However, the function of E2F8 in cancer cells is unknown. Methods: E2F8 expression was assessed by immunoblotting or immunofluorescence staining in human lung cancer (LC) cells and tissues from LC patients (n = 45). Cell proliferation, colony formation, and invasion analysis were performed to evaluate the role of E2F8 in LC. Microarray analysis was used to determine the target genes of E2F8. The regulation of E2F8 on the expression of ubiquitin-like PHD and RING domain-containing 1 (UHRF1), one of E2F8 target genes, was determined using chromatin immunoprecipitation and promoter activity assays. Human LC xenograft models were used to determine the effects of inhibiting E2F8 by siRNAs (n = 7 per group) or antisense morpholino (n = 8 per group) on tumor growth. Survival was analyzed using the Kaplan-Meier method and group differences by the Student’s t test. All statistical tests were two-sided. Results: LC tumors overexpressed E2F8 compared with normal lung tissues. Depletion of E2F8 inhibited cell proliferation and tumor growth. E2F8 knockdown statistically significantly reduced the expression of UHRF1 (~60%-70%, P < .001), and the direct binding of E2F8 on the promoter of UHRF1 was identified. Kaplan-Meier analysis with a public database showed prognostic significance of aberrant E2F8 expression in LC (HR = 1.91 95% CI = 1.21 to 3.01 in chemo-naïve patients, P = .0047). Conclusions: We demonstrated that E2F8 is overexpressed in LC and is required for the growth of LC cells. These findings implicate E2F8 as a novel therapeutic target for LC treatment. PMID:26089541
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Antisense RNA that Affects Rhodopseudomonas palustris Quorum-Sensing Signal Receptor Expression
2012-01-01
antisense molecules were produced, we performed a Northern blot analysis with RNA harvested from wild-type and rpaR-mutant R. palustris cells by using...aeruginosa, cells were grown to late-log phase, harvested by cen- trifugation, suspended in SDS/PAGE buffer, and lysed by boiling and sonication. Cell...a selectable DNA fragment. Gene 29:303–313. 17. Egland KA, Greenberg EP (1999) Quorum sensing in Vibrio fischeri: Elements of the luxl promoter. Mol
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Visual Servoing for Optimization of Anticancer Drug Uptake in Human Breast Cancer Cells
2000-09-01
successfully obtained new DOE Medical Applications Program funding for this research (included in Appendix G: Automated Imaging System for Guiding Antisense ...Guiding Antisense Compounds to Specific mRNVA targets in Living Cells ) that will support this integration and development work with Dr. Parvin and Deep...a DNA and RNA binding fluorescence probe with a very different emission wavelengths, depending on whether it is bound to DNA or RNA ). Cells were then
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ISS-N1 makes the First FDA-approved Drug for Spinal Muscular Atrophy.
Ottesen, Eric W
2017-01-01
Spinal muscular atrophy (SMA) is one of the leading genetic diseases of children and infants. SMA is caused by deletions or mutations of Survival Motor Neuron 1 ( SMN1 ) gene. SMN2 , a nearly identical copy of SMN1 , cannot compensate for the loss of SMN1 due to predominant skipping of exon 7. While various regulatory elements that modulate SMN2 exon 7 splicing have been proposed, intronic splicing silencer N1 (ISS-N1) has emerged as the most promising target thus far for antisense oligonucleotide-mediated splicing correction in SMA. Upon procuring exclusive license from the University of Massachussets Medical School in 2010, Ionis Pharmaceuticals (formerly ISIS Pharamaceuticals) began clinical development of Spinraza ™ (synonyms: Nusinersen, IONIS-SMN RX , ISIS-SMN RX ), an antisense drug based on ISS-N1 target. Spinraza ™ showed very promising results at all steps of the clinical development and was approved by US Food and Drug Administration (FDA) on December 23, 2016. Spinraza ™ is the first FDA-approved treatment for SMA and the first antisense drug to restore expression of a fully functional protein via splicing correction. The success of Spinraza ™ underscores the potential of intronic sequences as promising therapeutic targets and sets the stage for further improvement of antisense drugs based on advanced oligonucleotide chemistries and delivery protocols.
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Dong, Lei; Xia, Suhua; Chen, Huan; Chen, Jiangning; Zhang, Junfeng
2009-09-01
This study developed a transplantable platform based on cationic hydrogels to deliver antisense oligodeoxynucleotides (ASOs) targeting the mRNA of TNF-alpha. Cationic agarose (c-agarose) was obtained by conjugating ethylenediamine to agarose via an N,N'-carbonyldiimidazole (CDI)-activation method. ASO-c-agarose system was constructed by mixing ASO in cationic agarose gel of proper concentration and gelation temperature. In vivo assessment of ASO distribution suggested that the system specifically target to spleen, wherein the c-agarose-delivered ASO had a concentration remarkably 50-fold higher than that of the naked ASO. The distribution of c-agarose-delivered ASO was scarcely detectable in liver and kidney. Next, three types of animal models were setup to evaluate the therapeutic efficacies of ASO-Gel, including the adjuvant-induced arthritis (AA), carrageen/lipopolysaccharide (LPS)-induced arthritis (CLA) and collagen-induced arthritis (CIA) models. The effects of ASO-c-agarose in alleviating inflammation and tissue destruction were evidenced in more than 90% of the testing animals, with decrease of main inflammatory cytokines, lightening of joint swelling and tissue damage, as well as increase in their body weights. All these findings suggest that this highly operable devise for the conveyance of antisense nucleotides together with its spleen-targeting property, could become a useful means of antisense-based therapeutics against rheumatoid arthritis and other diseases.
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Foerst-Potts, L; Sadler, T W
1997-05-01
In mouse embryos, the muscle segment homeobox genes, Msx-1 and Msx-2 are expressed during critical stages of neural tube, neural crest, and craniofacial development, suggesting that these genes play important roles in organogenesis and cell differentiation. Although the patterns of expression are intriguing, little is known about the function of these genes in vertebrate embryonic development. Therefore, the expression of both genes, separately and together, was disrupted using antisense oligodeoxynucleotides and whole embryo culture techniques. Antisense attenuation of Msx-1 during early stages of neurulation produced hypoplasia of the maxillary, mandibular, and frontonasal prominences, eye anomalies, and somite and neural tube abnormalities. Eye defects consisted of enlarged optic vesicles, which may ultimately result in micropthalmia similar to that observed in Small eye mice homozygous for mutations in the Pax-6 gene. Histological sections and SEM analysis revealed a thinning of the neuroepithelium in the diencephalon and optic vesicle and mesenchymal deficiencies in the craniofacial region. Injections of Msx-2 antisense oligodeoxynucleotides produced similar malformations as those targeting Msx-1, with the exception that there was an increase in number and severity of neural tube and somite defects. Embryos injected with the combination of Msx-1 + Msx-2 antisense oligodeoxynucleotides showed no novel abnormalities, suggesting that the genes do not operate in a redundant manner.
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Li, Xiao-guang; Wang, Xu-xia; Li, Teng-yu; Wang, Yan-xiu; Gao, Jing; Ni, Chun-xiao
2012-12-01
To investigate the inhibitory effect of VEGF antisense phosphorothioate oligodeoxynucleoiides on the growth of human salivary adenoid cystic carcinoma (SACC) xenografts in nude mice. The VEGF-ASODN was synthesised artificially. After the model of human SACC xenografts in nude mice was established, they were random1y divided into three groups: antisense group, scrambled group and normal saline group. A control group without cancer was also established. Antisense(66 μg), scrambled sequence(66 μg) and normal saline(once every 3 days and 7 times in all) were injected in three experimental groups, respectively. Two days after therapy, the mice were sacrificed. Serums were used for detection of VEGF protein. All tumors were measured and weighted. The quantity of VEGF mRNA and protein and PLI, MVD was detected by hybridization in situ and immunohistochemistry. SPSS13.0 software package was used for statistical analysis. The VEGF-ASODN could suppress the expression of VEGF in human SACC xenografts in nude mice and reduce VEGF protein in serum of nude mice significantly. It cou1d also reduce the volume and weight of xenografts and could reduce the expression of VEGF mRNA and its protein, PCNA and CD34. By inhibiting the expression of VEGF, VEGF-ASODN can inhabit proliferation of human SACC xenografts in nude mice.
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Konishi, Hirosato; Yamane, Hisakazu; Maeshima, Masayoshi; Komatsu, Setsuko
2004-12-01
Fructose-bisphosphate aldolase is a glycolytic enzyme whose activity increases in rice roots treated with gibberellin (GA). To investigate the relationship between aldolase and root growth, GA-induced root aldolase was characterized. GA3 promoted an increase in aldolase accumulation when 0.1 microM GA3 was added exogenously to rice roots. Aldolase accumulated abundantly in roots, especially in the apical region. To examine the effect of aldolase function on root growth, transgenic rice plants expressing antisense aldolase were constructed. Root growth of aldolase-antisense transgenic rice was repressed compared with that of the vector control transgenic rice. Although aldolase activity increased by 25% in vector control rice roots treated with 0.1 microM GA3, FBPA activity increased very little by 0.1 microM GA3 treatment in the root of aldolase-antisense transgenic rice. Furthermore, aldolase co-immunoprecipitated with antibodies against vacuolar H+ -ATPase in rice roots. In the root of OsCDPK13-antisense transgenic rice, aldolase did not accumulate even after treatment with GA3. These results suggest that the activation of glycolytic pathway function accelerates root growth and that GA3-induced root aldolase may be modulated through OsCDPK13. Aldolase physically associates with vacuolar H-ATPase in roots and may regulate the vacuolar H-ATPase mediated control of cell elongation that determines root length.
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Ralph, J; Hatfield, R D; Piquemal, J; Yahiaoui, N; Pean, M; Lapierre, C; Boudet, A M
1998-10-27
Homologous antisense constructs were used to down-regulate tobacco cinnamyl-alcohol dehydrogenase (CAD; EC 1.1.1.195) and cinnamoyl-CoA reductase (CCR; EC 1.2.1.44) activities in the lignin monomer biosynthetic pathway. CCR converts activated cinnamic acids (hydroxycinnamoyl-SCoAs) to cinnamaldehydes; cinnamaldehydes are then reduced to cinnamyl alcohols by CAD. The transformations caused the incorporation of nontraditional components into the extractable tobacco lignins, as evidenced by NMR. Isolated lignin of antisense-CAD tobacco contained fewer coniferyl and sinapyl alcohol-derived units that were compensated for by elevated levels of benzaldehydes and cinnamaldehydes. Products from radical coupling of cinnamaldehydes, particularly sinapaldehyde, which were barely discernible in normal tobacco, were major components of the antisense-CAD tobacco lignin. Lignin content was reduced in antisense-CCR tobacco, which displayed a markedly reduced vigor. That lignin contained fewer coniferyl alcohol-derived units and significant levels of tyramine ferulate. Tyramine ferulate is a sink for the anticipated build-up of feruloyl-SCoA, and may be up-regulated in response to a deficit of coniferyl alcohol. Although it is not yet clear whether the modified lignins are true structural components of the cell wall, the findings provide further indications of the metabolic plasticity of plant lignification. An ability to produce lignin from alternative monomers would open new avenues for manipulation of lignin by genetic biotechnologies.
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Song, W; Koh, S; Czako, M; Marton, L; Drenkard, E; Becker, J M; Stacey, G
1997-01-01
Previously, we identified a peptide transport gene, AtPTR2-B, from Arabidopsis thaliana that was constitutively expressed in all plant organs, suggesting an important physiological role in plant growth and development. To evaluate the function of this transporter, transgenic Arabidopsis plants were constructed expressing antisense or sense AtPTR2-B. Genomic Southern analysis indicated that four independent antisense and three independent sense AtPTR2-B transgenic lines were obtained, which was confirmed by analysis of the segregation of the kanamycin resistance gene carried on the T-DNA. RNA blot data showed that the endogenous AtPTR2-B mRNA levels were significantly reduced in transgenic leaves and flowers, but not in transgenic roots. Consistent with this reduction in endogenous AtPTR2-B mRNA levels, all four antisense lines and one sense line exhibited significant phenotypic changes, including late flowering and arrested seed development. These phenotypic changes could be explained by a defect in nitrogen nutrition due to the reduced peptide transport activity conferred by AtPTR2-B. These results suggest that AtPTR2-B may play a general role in plant nutrition. The AtPTR2-B gene was mapped to chromosome 2, which is closely linked to the restriction fragment length polymorphism marker m246. PMID:9232875
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Bey, E A; Cathcart, M K
2000-03-01
We previously reported that superoxide dismutase (SOD) blocked human monocyte oxidation of LDL and therefore concluded that superoxide anion (O(2)(.-)) was required for oxidation. Others, however, have suggested that SOD may inhibit by mechanisms alternative to the dismutation of O(2)(.-). This study definitively addresses the involvement of O(2)(.-) in monocyte oxidation of LDL. Using an antisense ODN designed to target p47phox mRNA, we found that treatment of monocytes with antisense ODN caused a substantial and selective decrease in expression of p47phox protein, whereas sense ODN was without effect. Corresponding functional assays demonstrated that antisense ODN inhibited production of O(2)(.-). As sense ODN caused no inhibition of O(2)(.-) production, these results suggested that inhibition of p47phox expression caused reduction in O(2)(.-) production. Evaluation of the contribution of O(2)(.-) production to monocyte-mediated oxidation of LDL lipids confirmed that O(2)(.-) production is required for LDL lipid oxidation as antisense ODN treatment significantly inhibited LDL oxidation whereas sense ODN treatment caused no inhibition. This is the first report of the reduction of NADPH oxidase activity in intact human monocytes by directly targeting the mRNA of a significant member of this enzyme complex. Our results provide convincing data that O(2)(.-) is indeed required for monocyte-mediated LDL oxidation.
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Lindholm, Marie W; Elmén, Joacim; Fisker, Niels; Hansen, Henrik F; Persson, Robert; Møller, Marianne R; Rosenbohm, Christoph; Ørum, Henrik; Straarup, Ellen M; Koch, Troels
2012-02-01
Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a therapeutic target for the reduction of low-density lipoprotein cholesterol (LDL-C). PCSK9 increases the degradation of the LDL receptor, resulting in high LDL-C in individuals with high PCSK9 activity. Here, we show that two locked nucleic acid (LNA) antisense oligonucleotides targeting PCSK9 produce sustained reduction of LDL-C in nonhuman primates after a loading dose (20 mg/kg) and four weekly maintenance doses (5 mg/kg). PCSK9 messenger RNA (mRNA) and serum PCSK9 protein were reduced by 85% which resulted in a 50% reduction in circulating LDL-C. Serum total cholesterol (TC) levels were reduced to the same extent as LDL-C with no reduction in high-density lipoprotein levels, demonstrating a specific pharmacological effect on LDL-C. The reduction in hepatic PCSK9 mRNA correlated with liver LNA oligonucleotide content. This verified that anti-PCSK9 LNA oligonucleotides regulated LDL-C through an antisense mechanism. The compounds were well tolerated with no observed effects on toxicological parameters (liver and kidney histology, alanine aminotransferase, aspartate aminotransferase, urea, and creatinine). The pharmacologic evidence and initial safety profile of the compounds used in this study indicate that LNA antisense oligonucleotides targeting PCSK9 provide a viable therapeutic strategy and are potential complements to statins in managing high LDL-C.
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Stefan, Alessandra; Schwarz, Flavio; Bressanin, Daniela; Hochkoeppler, Alejandro
2010-11-01
Silencing of the lacZ gene in Escherichia coli was attempted by means of the expression of antisense RNAs (asRNAs) in vivo. A short fragment of lacZ was cloned into the pBAD expression vector, in reverse orientation, using the EcoRI and PstI restriction sites. This construct (pBAD-Zcal1) was used to transform E. coli cells, and the antisense transcription was induced simply by adding arabinose to the culture medium. We demonstrated that the Zcal1 asRNA effectively silenced lacZ using β-galactosidase activity determinations, SDS-PAGE, and Western blotting. Because the concentration of the lac mRNA was always high in cells that expressed Zcal1, we hypothesize that this antisense acts by inhibiting messenger translation. Similar analyses, performed with a series of site-specific Zcal1 mutants, showed that the Shine-Dalgarno sequence, which is conferred by the pBAD vector, is an essential requisite for silencing competence. Indeed, the presence of the intact Shine-Dalgarno sequence positively affects asRNA stability and, hence, silencing effectiveness. Our observations will contribute to the understanding of the main determinants of silencing as exerted by asRNAs as well as provide useful support for the design of robust and efficient prokaryotic gene silencers. Copyright © 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.
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Islet-1 is required for ventral neuron survival in Xenopus
DOE Office of Scientific and Technical Information (OSTI.GOV)
Shi, Yu; Zhao, Shuhua; Li, Jiejing
Islet-1 is a LIM domain transcription factor involved in several processes of embryonic development. Xenopus Islet-1 (Xisl-1) has been shown to be crucial for proper heart development. Here we show that Xisl-1 and Xisl-2 are differentially expressed in the nervous system in Xenopus embryos. Knock-down of Xisl-1 by specific morpholino leads to severe developmental defects, including eye and heart failure. Staining with the neuronal markers N-tubulin and Xisl-1 itself reveals that the motor neurons and a group of ventral interneurons are lost in the Xisl-1 morphants. Terminal dUTP nick-end labeling (TUNEL) analysis shows that Xisl-1 morpholino injection induces extensive apoptosismore » in the ventral neural plate, which can be largely inhibited by the apoptosis inhibitor M50054. We also find that over-expression of Xisl-1 is able to promote cell proliferation and induce Xstat3 expression in the injected side, suggesting a potential role for Xisl-1 in the regulation of cell proliferation in co-operation with the Jak-Stat pathway.« less
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ISIS 301012 gene therapy for hypercholesterolemia: sense, antisense, or nonsense?
Ito, Matthew K
2007-10-01
To present an overview of antisense technology and to review and assess available literature on the chemistry, pharmacology, pharmacokinetics, drug interactions, preclinical and clinical studies, dosing, and adverse events of ISIS 301012 in the treatment of hyperlipidemia. PubMed database searches were conducted from 1966 to May 2007 using the search terms ISIS 301012, antisense, oligonucleotide, hypercholesterolemia, hyperlipidemia, and apolipoprotein B. Bibliographies of relevant review articles and information from the manufacturer were reviewed for additional references. Available English-language literature, including abstracts, preclinical, and clinical trials, review articles, and scientific presentations were examined. Apolipoprotein B is an important structural protein on the surface of atherogenic lipoproteins such as remnant very-low-density lipoprotein and low-density lipoprotein and facilitates the clearance of these particles from the circulation by binding to the low-density lipoprotein receptor. Overproduction of apolipoprotein B or reduced receptor-mediated clearance of lipoproteins leads to elevated serum cholesterol levels and premature atherosclerosis. ISIS 301012 is an antisense oligonucleotide that inhibits apolipoprotein B production by binding directly to and reducing the expression of apolipoprotein B messenger RNA. In a clinical trial, ISIS 301012 50-400 mg administered weekly via subcutaneous injection for 4 weeks reduced apolipoprotein B by 14.3-47.4% and low-density lipoprotein cholesterol by 5.9-40% at 55 days. The most frequent adverse event was injection-site erythema that resolved spontaneously. Studies are ongoing to further define the safety, efficacy, and pharmacokinetics of ISIS 301012 as add-on therapy in patients with heterozygous and homozygous familial hypercholesterolemia. No pharmacokinetic interactions have been demonstrated with ezetimibe and simvastatin. ISIS 301012 is the first agent to enter clinical trials utilizing an antisense mechanism for reducing the production of apolipoprotein B. Further studies are needed to verify its safety, efficacy, and position of therapy in the dyslipidemic patient.
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López-Aguilar, Celeste; Romero-López, Cristina; Espinosa, Manuel; Berzal-Herranz, Alfredo; del Solar, Gloria
2015-01-01
Rolling-circle replication of streptococcal plasmid pMV158 is controlled by the concerted action of two trans-acting elements, namely transcriptional repressor CopG and antisense RNAII, which inhibit expression of the repB gene encoding the replication initiator protein. The pMV158-encoded antisense RNAII exerts its activity of replication control by inhibiting translation of the essential repB gene. RNAII is the smallest and simplest among the characterized antisense RNAs involved in control of plasmid replication. Structure analysis of RNAII revealed that it folds into an 8-bp-long stem containing a 1-nt bulge and closed by a 6-nt apical loop. This hairpin is flanked by a 17-nt-long single-stranded 5′-tail and an 8-nt-long 3′-terminal U-rich stretch. Here, the 3′ and 5′ regions of the 5′-tail of RNAII are shown to play a critical role in the binding to the target mRNA and in the inhibition of repB translation, respectively. In contrast, the apical loop of the single hairpin of RNAII plays a rather secondary role and the upper stem region hardly contributes to the binding or inhibition processes. The entire 5′-tail is required for efficient inhibition of repB translation, though only the 8-nt-long region adjacent to the hairpin seems to be essential for rapid binding to the mRNA. These results show that a “kissing” interaction involving base-pairing between complementary hairpin loops in RNAII and mRNA is not critical for efficient RNA/RNA binding or repB translation inhibition. A singular binding mechanism is envisaged whereby initial pairing between complementary single-stranded regions in the antisense and sense RNAs progresses upwards into the corresponding hairpin stems to form the intermolecular duplex. PMID:26175752
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Li, D; Mehta, J L
2000-04-01
A specific lectin-like endothelial receptor for oxidized low density lipoprotein (LOX-1), distinct from the scavenger receptor in monocytes/macrophages, has been identified and cloned. In this study, we examined the regulation of LOX-1 by oxidized low density lipoprotein (ox-LDL) and determined the role of LOX-1 in ox-LDL-induced apoptosis of cultured human coronary artery endothelial cells (HCAECs). Incubation of HCAECs with ox-LDL (40 microg/mL), but not native LDL, for 24 hours markedly increased LOX-1 expression (mRNA and protein). After 48 hours of preincubation of HCAECs with a specific antisense to LOX-1 mRNA (antisense LOX-1), ox-LDL-mediated upregulation of LOX-1 was suppressed (P<0.01). In contrast, treatment of HCAECs with sense LOX-1 had no effect. Ox-LDL also induced apoptosis (determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling and DNA laddering) of HCAECs in a concentration- and time-dependent fashion. LOX-1 played an important role in ox-LDL-mediated apoptosis of HCAECs because antisense LOX-1 inhibited this effect of ox-LDL. Polyinosinic acid and carrageenan, 2 different chemical inhibitors of LOX-1, also decreased ox-LDL-mediated apoptosis of HCAECs. Nuclear factor (NF)-kappaB was markedly activated in ox-LDL-treated HCAECs. The critical role of NF-kappaB activation became evident in experiments with antisense LOX-1, which abolished ox-LDL-mediated NF-kappaB activation. In this process, an NF-kappaB inhibitor, caffeic acid phenethyl ester, also inhibited ox-LDL-mediated apoptosis of HCAECs. These findings indicate that ox-LDL upregulates its own endothelial receptor. Ox-LDL-induced apoptosis is mediated by the action of LOX-1. In this process, NF-kappaB activation may play an important role as a signal transduction mechanism.
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Sensible use of antisense: how to use oligonucleotides as research tools.
Myers, K J; Dean, N M
2000-01-01
In the past decade, there has been a vast increase in the amount of gene sequence information that has the potential to revolutionize the way diseases are both categorized and treated. Old diagnoses, largely anatomical or descriptive in nature, are likely to be superceded by the molecular characterization of the disease. The recognition that certain genes drive key disease processes will also enable the rational design of gene-specific therapeutics. Antisense oligonucleotides represent a technology that should play multiple roles in this process.
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2013-03-01
oligonucleotide-based drug approaches (better than ribozymes, antisense oligonucleotides ( ASO ), or microRNAs). (4) To accomplish these objectives, we...negative control scrambled ASO (designated NC). The combination of siRNAs T1 and R1 produced a knockdown of ~80% of TGFb1 protein in the conditioned...sequences (antisense oligonucleotides, ASOs ) into rabbit corneal cells and found that technique was very effective in delivering ASOs into the stroma and
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NASA Technical Reports Server (NTRS)
Reinsch, Sigrid; Conway, Gregory; Dalton, Bonnie P. (Technical Monitor)
2002-01-01
In a differential RNA display screen we have isolated a zebrafish gene, G12, for which homologs can only be found in DNA databases for vertebrates, but not invertebrates. This suggests that this is a gene required specifically in vertebrates. G12 expression is upregulated at mid-blastula transition (MBT). Morpholino inactivation of this gene by injection into 1-cell embryos results in mitotic defects and apoptosis shortly after MBT. Nuclei in morpholino treated embryos also display segregation defects. We have characterized the localization of this gene as a GFP fusion in live and fixed embryos. Overexpression of G12-GFP is non-toxic. Animals retain GFP expression for at least 7 days with no developmental defects, Interestingly in these animals G12-GFP is never detectable in blood cells though blood is present. In the deep cells of early embryos, G 12GFP is localized to nuclei and cytoskeletal elements in interphase and to the centrosome and spindle apparatus during mitosis. In the EVL, G12-GFP shows additional localization to the cell periphery, especially in mitosis. In the yolk syncytium, G12-GFP again localizes to nuclei and strongly to cytoplasmic microtubules of migrating nuclei at the YSL margin. Morpholinc, injection specifically into the YSL after cellularization blocks epiboly and nuclei of the YSL show mitotic defects while deep cells show no mitotic defects and continue to divide. Rescue experiments in which morpholino and G12-GFP RNA are co-injected indicate partial rescue by the G12-GFP. The rescue is cell autonomous; that is, regions of the embryo with higher G12-GFP expression show fewer mitotic defects. Spot 14, the human bomolog of G12, has been shown to be amplified in aggressive breast tumors. This finding, along with our functional and morphological data suggest that G12 and spot 14 are vertebrate-specific and may function either as mitotic checkpoints or as structural components of the spindle apparatus.
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Neural Crest Migration and Survival Are Susceptible to Morpholino-Induced Artifacts
Jette, Cicely A.
2016-01-01
The neural crest (NC) is a stem cell-like embryonic population that is essential for generating and patterning the vertebrate body, including the craniofacial skeleton and peripheral nervous system. Defects in NC development underlie many birth defects and contribute to formation of some of the most malignant cancers in humans, such as melanoma and neuroblastoma. For these reasons, significant research efforts have been expended to identify genes that control NC development, as it is expected to lead to a deeper understanding of the genetic mechanisms controlling vertebrate development and identify new treatments for NC-derived diseases and cancers. However, a number of inconsistencies regarding gene function during NC development have emerged from comparative analyses of gene function between mammalian and non-mammalian systems (chick, frog, zebrafish). This poses a significant barrier to identification of single genes and/or redundant pathways to target in NC diseases. Here, we determine whether technical differences, namely morpholino-based approaches used in non-mammalian systems, could contribute to these discrepancies, by examining the extent to which NC phenotypes in fascin1a (fscn1a) morphant embryos are similar to or different from fscn1a null mutants in zebrafish. Analysis of fscn1a morphants showed that they mimicked early NC phenotypes observed in fscn1a null mutants; however, these embryos also displayed NC migration and derivative phenotypes not observed in null mutants, including accumulation of p53-independent cell death. These data demonstrate that morpholinos can cause seemingly specific NC migration and derivative phenotypes, and thus have likely contributed to the inconsistencies surrounding NC gene function between species. We suggest that comparison of genetic mutants between different species is the most rigorous method for identifying conserved genetic mechanisms controlling NC development and is critical to identify new treatments for NC diseases. PMID:28005909
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Hennessy, Elizabeth J; Moore, Kathryn J
2013-09-01
It is now appreciated that over 90% of the human genome is comprised of noncoding RNAs that have the ability to affect other components of the genome and regulate gene expression. This has galvanized the development of RNA-based therapeutics for a myriad of diseases, including cancer, inflammatory conditions, and cardiovascular disease. Several classes of RNA therapeutics are currently under clinical development, including antisense oligonucleotides, small interfering RNA, and microRNA mimetics and inhibitors. The field of antisense technology saw a huge leap forward with the recent Food and Drug Administration approval of the first antisense therapy, directed against apolipoprotein B, for the treatment of familial hypercholesterolemia. In addition, recent progress in the development of approaches to inhibit microRNAs has helped to illuminate their roles in repressing gene networks and also revealed their potential as therapeutic targets. In this review, these exciting opportunities in the field of drug discovery, with a focus on emerging therapeutics in the field of cardiovascular disease, are summarized.
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Bhargava-Shah, Aarohi; Foygel, Kira; Devulapally, Rammohan; Paulmurugan, Ramasamy
2016-01-01
Background: This study explores the use of hydrophilic poly(ethylene glycol)-conjugated poly(lactic-co-glycolic acid) nanoparticles (PLGA-PEG-NPs) as delivery system to improve the antitumor effect of antiobesity drug orlistat for triple-negative breast cancer (TNBC) therapy by improving its bioavailability. Materials & methods: PLGA-PEG-NPs were synthesized by emulsion-diffusion-evaporation method, and the experiments were conducted in vitro in MDA-MB-231 and SKBr3 TNBC and normal breast fibroblast cells. Results: Delivery of orlistat via PLGA-PEG-NPs reduced its IC50 compared with free orlistat. Combined treatment of orlistat-loaded NPs and doxorubicin or antisense-miR-21-loaded NPs significantly enhanced apoptotic effect compared with independent doxorubicin, anti-miR-21-loaded NPs, orlistat-loaded NPs or free orlistat treatments. Conclusion: We demonstrate that orlistat in combination with antisense-miR-21 or current chemotherapy holds great promise as a novel and versatile treatment agent for TNBC. PMID:26787319
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Vickers, Timothy A.; Freier, Susan M.; Bui, Huynh-Hoa; Watt, Andrew; Crooke, Stanley T.
2014-01-01
A new strategy for identifying potent RNase H-dependent antisense oligonucleotides (ASOs) is presented. Our analysis of the human transcriptome revealed that a significant proportion of genes contain unique repeated sequences of 16 or more nucleotides in length. Activities of ASOs targeting these repeated sites in several representative genes were compared to those of ASOs targeting unique single sites in the same transcript. Antisense activity at repeated sites was also evaluated in a highly controlled minigene system. Targeting both native and minigene repeat sites resulted in significant increases in potency as compared to targeting of non-repeated sites. The increased potency at these sites is a result of increased frequency of ASO/RNA interactions which, in turn, increases the probability of a productive interaction between the ASO/RNA heteroduplex and human RNase H1 in the cell. These results suggest a new, highly efficient strategy for rapid identification of highly potent ASOs. PMID:25334092
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Gifford, Lida K.; Opalinska, Joanna B.; Jordan, David; Pattanayak, Vikram; Greenham, Paul; Kalota, Anna; Robbins, Michelle; Vernovsky, Kathy; Rodriguez, Lesbeth C.; Do, Bao T.; Lu, Ponzy; Gewirtz, Alan M.
2005-01-01
We describe a physical mRNA mapping strategy employing fluorescent self-quenching reporter molecules (SQRMs) that facilitates the identification of mRNA sequence accessible for hybridization with antisense nucleic acids in vitro and in vivo, real time. SQRMs are 20–30 base oligodeoxynucleotides with 5–6 bp complementary ends to which a 5′ fluorophore and 3′ quenching group are attached. Alone, the SQRM complementary ends form a stem that holds the fluorophore and quencher in contact. When the SQRM forms base pairs with its target, the structure separates the fluorophore from the quencher. This event can be reported by fluorescence emission when the fluorophore is excited. The stem–loop of the SQRM suggests that SQRM be made to target natural stem–loop structures formed during mRNA synthesis. The general utility of this method is demonstrated by SQRM identification of targetable sequence within c-myb and bcl-6 mRNA. Corresponding antisense oligonucleotides reduce these gene products in cells. PMID:15718294
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COOLAIR Antisense RNAs Form Evolutionarily Conserved Elaborate Secondary Structures
Hawkes, Emily J.; Hennelly, Scott P.; Novikova, Irina V.; ...
2016-09-20
There is considerable debate about the functionality of long non-coding RNAs (lncRNAs). Lack of sequence conservation has been used to argue against functional relevance. Here, we investigated antisense lncRNAs, called COOLAIR, at the A. thaliana FLC locus and experimentally determined their secondary structure. The major COOLAIR variants are highly structured, organized by exon. The distally polyadenylated transcript has a complex multi-domain structure, altered by a single non-coding SNP defining a functionally distinct A. thaliana FLC haplotype. The A. thaliana COOLAIR secondary structure was used to predict COOLAIR exons in evolutionarily divergent Brassicaceae species. These predictions were validated through chemical probingmore » and cloning. Despite the relatively low nucleotide sequence identity, the structures, including multi-helix junctions, show remarkable evolutionary conservation. In a number of places, the structure is conserved through covariation of a non-contiguous DNA sequence. This structural conservation supports a functional role for COOLAIR transcripts rather than, or in addition to, antisense transcription.« less
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Kawano, Mitsuoki
2012-12-01
Toxin-antitoxin (TA) systems are categorized into three classes based on the type of antitoxin. In type I TA systems, the antitoxin is a small antisense RNA that inhibits translation of small toxic proteins by binding to the corresponding mRNAs. Those type I TA systems were originally identified as plasmid stabilization modules rendering a post-segregational killing (PSK) effect on the host cells. The type I TA loci also exist on the Escherichia coli chromosome but their biological functions are less clear. Genetic organization and regulatory elements of hok/sok and ldr/rdl families are very similar and the toxins are predicted to contain a transmembrane domain, but otherwise share no detectable sequence similarity. This review will give an overview of the type I TA modules of E. coli K-12, especially hok/sok, ldr/rdl and SOS-inducible symE/symR systems, which are regulated by divergently overlapping cis-encoded antisense RNAs.
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COOLAIR Antisense RNAs Form Evolutionarily Conserved Elaborate Secondary Structures
DOE Office of Scientific and Technical Information (OSTI.GOV)
Hawkes, Emily J.; Hennelly, Scott P.; Novikova, Irina V.
There is considerable debate about the functionality of long non-coding RNAs (lncRNAs). Lack of sequence conservation has been used to argue against functional relevance. Here, we investigated antisense lncRNAs, called COOLAIR, at the A. thaliana FLC locus and experimentally determined their secondary structure. The major COOLAIR variants are highly structured, organized by exon. The distally polyadenylated transcript has a complex multi-domain structure, altered by a single non-coding SNP defining a functionally distinct A. thaliana FLC haplotype. The A. thaliana COOLAIR secondary structure was used to predict COOLAIR exons in evolutionarily divergent Brassicaceae species. These predictions were validated through chemical probingmore » and cloning. Despite the relatively low nucleotide sequence identity, the structures, including multi-helix junctions, show remarkable evolutionary conservation. In a number of places, the structure is conserved through covariation of a non-contiguous DNA sequence. This structural conservation supports a functional role for COOLAIR transcripts rather than, or in addition to, antisense transcription.« less
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Skalickova, Sylvie; Nejdl, Lukas; Kudr, Jiri; Ruttkay-Nedecky, Branislav; Jimenez, Ana Maria Jimenez; Kopel, Pavel; Kremplova, Monika; Masarik, Michal; Stiborova, Marie; Eckschlager, Tomas; Adam, Vojtech; Kizek, Rene
2016-02-25
Liposome-based drug delivery systems hold great potential for cancer therapy. The aim of this study was to design a nanodevice for targeted anchoring of liposomes (with and without cholesterol) with encapsulated anticancer drugs and antisense N-myc gene oligonucleotide attached to its surface. To meet this main aim, liposomes with encapsulated doxorubicin, ellipticine and etoposide were prepared. They were further characterized by measuring their fluorescence intensity, whereas the encapsulation efficiency was estimated to be 16%. The hybridization process of individual oligonucleotides forming the nanoconstruct was investigated spectrophotometrically and electrochemically. The concentrations of ellipticine, doxorubicin and etoposide attached to the nanoconstruct in gold nanoparticle-modified liposomes were found to be 14, 5 and 2 µg·mL(-1), respectively. The study succeeded in demonstrating that liposomes are suitable for the transport of anticancer drugs and the antisense oligonucleotide, which can block the expression of the N-myc gene.
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The Role of Mesopontine NGF in Sleep and Wakefulness
Ramos, Oscar V.; Torterolo, Pablo; Lim, Vincent; Chase, Michael H.; Sampogna, Sharon; Yamuy, Jack
2011-01-01
The microinjection of nerve growth factor (NGF) into the cat pontine tegmentum rapidly induces rapid eye movement (REM) sleep. To determine if NGF is involved in naturally-occurring REM sleep, we examined whether it is present in mesopontine cholinergic structures that promote the initiation of REM sleep, and whether the blockade of NGF production in these structures suppresses REM sleep. We found that cholinergic neurons in the cat dorsolateral mesopontine tegmentum exhibited NGF-like immunoreactivity. In addition, the microinjection of an oligodeoxyribonucleotide (OD) directed against cat NGF mRNA into this region resulted in a reduction in the time spent in REM sleep in conjunction with an increase in the time spent in wakefulness. Sleep and wakefulness returned to baseline conditions 2 to 5 days after antisense OD administration. The preceding antisense OD-induced effects occurred in conjunction with the suppression of NGF-like immunoreactivity within the site of antisense OD injection. These data support the hypothesis that NGF is involved in the modulation of naturally-occurring sleep and wakefulness. PMID:21840513
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NASA Astrophysics Data System (ADS)
Hashimoto, Muneaki; Nara, Takeshi; Hirawake, Hiroko; Morales, Jorge; Enomoto, Masahiro; Mikoshiba, Katsuhiko
2014-02-01
Chagas disease is caused by an intracellular parasitic protist, Trypanosoma cruzi. As there are no highly effective drugs against this agent that also demonstrate low toxicity, there is an urgent need for development of new drugs to treat Chagas disease. We have previously demonstrated that the parasite inositol 1,4,5-trisphosphate receptor (TcIP3R) is crucial for invasion of the mammalian host cell by T. cruzi. Here, we report that TcIP3R is a short-lived protein and that its expression is significantly suppressed in trypomastigotes. Treatment of trypomastigotes, an infective stage of T. cruzi, with antisense oligonucleotides specific to TcIP3R deceased TcIP3R protein levels and impaired trypomastigote invasion of host cells. Due to the resulting instability and very low expression level of TcIP3R in trypomastigotes indicates that TcIP3R is a promising target for antisense therapy in Chagas disease.
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Graham, Mark J; Lemonidis, Kristina M; Whipple, Charles P; Subramaniam, Amuthakannan; Monia, Brett P; Crooke, Stanley T; Crooke, Rosanne M
2007-04-01
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of a family of proteases that is thought to promote the degradation of the low density lipoprotein receptor (LDLR) through an as yet undefined mechanism. We developed second generation antisense oligonucleotide (ASO) inhibitors targeting murine PCSK9 to determine their potential as lipid-lowering agents. Administration of a PCSK9 ASO to high fat-fed mice for 6 weeks reduced total cholesterol and LDL by 53% and 38%, respectively. Moreover, inhibition of PCSK9 expression resulted in a 2-fold increase in hepatic LDLR protein levels. This phenotype closely resembles that reported previously in Pcsk9-deficient mice. The absence of cholesterol lowering in Ldlr-deficient mice effectively demonstrated a critical role for this receptor in mediating the lipid-lowering effects of PCSK9 inhibition. Antisense inhibition of PCSK9 is an attractive and novel therapeutic approach for treating hypercholesterolemia in human.
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Crooke, Stanley T; Geary, Richard S
2013-01-01
Mipomersen is a second generation antisense oligonucleotide that targets apolipoprotein B. It has been studied thoroughly in clinical trials (more than 800 subjects), including four randomized double-blind placebo controlled phase 3 studies involving 391 patients, and is in registration for the treatment of severe hypercholesterolaemia. The pharmacokinetic and pharmacodynamic properties of mipomersen are well characterized. Mipomersen is rapidly and extensively absorbed after subcutaneous administration and has an elimination half-life of approximately 30 days across species. It is cleared by nuclease metabolism and renal excretion of the metabolites. Mipomersen reduces all apolipoprotein B containing atherogenic particles and displays dose dependent reductions between 50–400 mg week−1, both as a single agent and in the presence of maximal lipid lowering therapy. No drug–drug interactions have been identified. Mipomersen is a representative of second generation antisense drugs, all of which have similar properties, and is thus representative of the behaviour of the class of drugs. PMID:23013161
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Staphylococcus aureus Transcriptome Architecture: From Laboratory to Infection-Mimicking Conditions
Depke, Maren; Pané-Farré, Jan; Debarbouille, Michel; van der Kooi-Pol, Magdalena M.; Guérin, Cyprien; Dérozier, Sandra; Hiron, Aurelia; Jarmer, Hanne; Leduc, Aurélie; Michalik, Stephan; Reilman, Ewoud; Schaffer, Marc; Schmidt, Frank; Bessières, Philippe; Noirot, Philippe; Hecker, Michael; Msadek, Tarek; Völker, Uwe; van Dijl, Jan Maarten
2016-01-01
Staphylococcus aureus is a major pathogen that colonizes about 20% of the human population. Intriguingly, this Gram-positive bacterium can survive and thrive under a wide range of different conditions, both inside and outside the human body. Here, we investigated the transcriptional adaptation of S. aureus HG001, a derivative of strain NCTC 8325, across experimental conditions ranging from optimal growth in vitro to intracellular growth in host cells. These data establish an extensive repertoire of transcription units and non-coding RNAs, a classification of 1412 promoters according to their dependence on the RNA polymerase sigma factors SigA or SigB, and allow identification of new potential targets for several known transcription factors. In particular, this study revealed a relatively low abundance of antisense RNAs in S. aureus, where they overlap only 6% of the coding genes, and only 19 antisense RNAs not co-transcribed with other genes were found. Promoter analysis and comparison with Bacillus subtilis links the small number of antisense RNAs to a less profound impact of alternative sigma factors in S. aureus. Furthermore, we revealed that Rho-dependent transcription termination suppresses pervasive antisense transcription, presumably originating from abundant spurious transcription initiation in this A+T-rich genome, which would otherwise affect expression of the overlapped genes. In summary, our study provides genome-wide information on transcriptional regulation and non-coding RNAs in S. aureus as well as new insights into the biological function of Rho and the implications of spurious transcription in bacteria. PMID:27035918
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Dorion, Sonia; Clendenning, Audrey; Rivoal, Jean
2017-03-01
Nucleoside diphosphate kinase (NDPK) is a ubiquitous enzyme that catalyzes the transfer of the γ-phosphate from a donor nucleoside triphosphate to an acceptor nucleoside diphosphate. In this study we used a targeted metabolomic approach and measurement of physiological parameters to report the effects of the genetic manipulation of cytosolic NDPK (NDPK1) expression on physiology and carbon metabolism in potato (Solanum tuberosum) roots. Sense and antisense NDPK1 constructs were introduced in potato using Agrobacterium rhizogenes to generate a population of root clones displaying a 40-fold difference in NDPK activity. Root growth, O 2 uptake, flux of carbon between sucrose and CO 2 , levels of reactive oxygen species and some tricarboxylic acid cycle intermediates were positively correlated with levels of NDPK1 expression. In addition, NDPK1 levels positively affected UDP-glucose and cellulose contents. The activation state of ADP-glucose pyrophosphorylase, a key enzyme in starch synthesis, was higher in antisense roots than in roots overexpressing NDPK1. Further analyses demonstrated that ADP-glucose pyrophosphorylase was more oxidized, and therefore less active, in sense clones than antisense clones. Consequently, antisense NDPK1 roots accumulated more starch and the starch to cellulose ratio was negatively affected by the level of NDPK1. These data support the idea that modulation of NDPK1 affects the distribution of carbon between starch and cellulose biosynthetic pathways. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.
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Song, Jiang-Hua; Cao, Jia-Shu; Wang, Cheng-Gang
2013-01-01
KEY MESSAGE : BcMF11 as a non-coding RNA gene has an essential role in pollen development, and might be useful for regulating the pollen fertility of crops by antisense RNA technology. We previously identified a 828-bp full-length cDNA of BcMF11, a novel pollen-specific non-coding mRNA-like gene from Chinese cabbage (Brassica campestris L. ssp. chinensis Makino). However, little information is known about the function of BcMF11 in pollen development. To investigate its exact biological roles in pollen development, the BcMF11 cDNA was antisense inhibited in transgenic Chinese cabbage under the control of a tapetum-specific promoter BcA9 and a constitutive promoter CaMV 35S. Antisense RNA transgenic plants displayed decreasing expression of BcMF11 and showed distinct morphological defects. Pollen germination test in vitro and in vivo of the transgenic plants suggested that inhibition of BcMF11 decreased pollen germination efficiency and delayed the pollen tubes' extension in the style. Under scanning electron microscopy, many shrunken and collapsed pollen grains were detected in the antisense BcMF11 transgenic Chinese cabbage. Further cytological observation revealed abnormal pollen development process in transgenic plants, including delayed degradation of tapetum, asynchronous separation of microspore, and aborted development of pollen grain. These results suggest that BcMF11, as a non-coding RNA, plays an essential role in pollen development and male fertility.
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Kojima, Yasutaka; Otsuki, Naoki; Kubo, Mie; Kitamoto, Junko; Takata, Eri; Saito, Hiroki; Kosaka, Kyoko; Morishita, Naoya; Uehara, Natsumi; Shirakawa, Toshiro; Nibu, Ken-Ich
2018-05-24
Human papillomavirus (HPV) infection has been identified as an etiologic factor of head and neck cancers (HNCs). We explored the potential use of antisense HPV RNA transcripts for gene therapy and its effect in combination with cisplatin (CDDP) for HPV-positive HNCs. We introduced the antisense RNA transcripts of the E6 and E7 genes of HPV type 16 into UM-SCC-47 cells harboring HPV 16 and YCU-T892 cells that were HPV-negative using a recombinant adenoviral vector, Ad-E6/E7-AS. We then analyzed the effects of the introduction of Ad-E7-AS on cell and tumor growth and the synergistic effect with CDDP in vitro and in vivo. After infection of Ad-E6/E7-AS, the cellular growth of UM-SCC-47 cells were suppressed, but not that of YCU-T892 cells. E7 protein expression was suppressed, and p53 and pRb protein expression increased after infection of Ad-E7-AS. Cell growth and tumorigenicity were greatly suppressed in combination with CDDP compared with Ad-E7-AS or CDDP treatment alone in vitro. Ad-E7-AS combined with CDDP treatment significantly reduced the volumes of established subcutaneous tumors. Transfection with HPV 16 E7 antisense RNA combined with CDDP treatment might be a potentially useful approach to the therapy of HPV 16-positive HNC.
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How the discovery of ISS-N1 led to the first medical therapy for spinal muscular atrophy
Singh, Natalia N.; Howell, Matthew D.; Androphy, Elliot J.; Singh, Ravindra N.
2017-01-01
Spinal muscular atrophy (SMA), a prominent genetic disease of infant mortality, is caused by low levels of survival motor neuron (SMN) protein owing to deletions or mutations of the SMN1 gene. SMN2, a nearly identical copy of SMN1 present in humans, cannot compensate for the loss of SMN1 due to predominant skipping of exon 7 during pre-mRNA splicing. With the recent FDA approval of nusinersen (Spinraza™), the potential for correction of SMN2 exon 7 splicing as a SMA therapy has been affirmed. Nusinersen is an antisense oligonucleotide that targets intronic splicing silencer N1 (ISS-N1) discovered in 2004 at the University of Massachusetts Medical School. ISS-N1 has emerged as the model target for testing the therapeutic efficacy of antisense oligonucleotides using different chemistries as well as different mouse models of SMA. Here we provide a historical account of events that led to the discovery of ISS-N1 and describe the impact of independent validations that raised the profile of ISS-N1 as one of the most potent antisense targets for the treatment of a genetic disease. Recent approval of nusinersen provides a much-needed boost for antisense technology that is just beginning to realize its potential. Beyond treating SMA, the ISS-N1 target offers myriad potentials for perfecting various aspects of the nucleic-acid-based technology for the amelioration of the countless number of pathological conditions. PMID:28485722
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How the discovery of ISS-N1 led to the first medical therapy for spinal muscular atrophy.
Singh, N N; Howell, M D; Androphy, E J; Singh, R N
2017-09-01
Spinal muscular atrophy (SMA), a prominent genetic disease of infant mortality, is caused by low levels of survival motor neuron (SMN) protein owing to deletions or mutations of the SMN1 gene. SMN2, a nearly identical copy of SMN1 present in humans, cannot compensate for the loss of SMN1 because of predominant skipping of exon 7 during pre-mRNA splicing. With the recent US Food and Drug Administration approval of nusinersen (Spinraza), the potential for correction of SMN2 exon 7 splicing as an SMA therapy has been affirmed. Nusinersen is an antisense oligonucleotide that targets intronic splicing silencer N1 (ISS-N1) discovered in 2004 at the University of Massachusetts Medical School. ISS-N1 has emerged as the model target for testing the therapeutic efficacy of antisense oligonucleotides using different chemistries as well as different mouse models of SMA. Here, we provide a historical account of events that led to the discovery of ISS-N1 and describe the impact of independent validations that raised the profile of ISS-N1 as one of the most potent antisense targets for the treatment of a genetic disease. Recent approval of nusinersen provides a much-needed boost for antisense technology that is just beginning to realize its potential. Beyond treating SMA, the ISS-N1 target offers myriad potentials for perfecting various aspects of the nucleic-acid-based technology for the amelioration of the countless number of pathological conditions.
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Dissecting the hybridization of oligonucleotides to structured complementary sequences.
Peracchi, Alessio
2016-06-01
When oligonucleotides hybridize to long target molecules, the process is slowed by the secondary structure in the targets. The phenomenon has been analyzed in several previous studies, but many details remain poorly understood. I used a spectrofluorometric strategy, focusing on the formation/breaking of individual base pairs, to study the kinetics of association between a DNA hairpin and >20 complementary oligonucleotides ('antisenses'). Hybridization rates differed by over three orders of magnitude. Association was toehold-mediated, both for antisenses binding to the target's ends and for those designed to interact with the loop. Binding of these latter, besides being consistently slower, was affected to variable, non-uniform extents by the asymmetric loop structure. Divalent metal ions accelerated hybridization, more pronouncedly when nucleation occurred at the loop. Incorporation of locked nucleic acid (LNA) residues in the antisenses substantially improved the kinetics only when LNAs participated to the earliest hybridization steps. The effects of individual LNAs placed along the antisense indicated that the reaction transition state occurred after invading at least the first base pair of the stem. The experimental approach helps dissect hybridization reactions involving structured nucleic acids. Toehold-dependent, nucleation-invasion models appear fully appropriate for describing such reactions. Estimating the stability of nucleation complexes formed at internal toeholds is the major hurdle for the quantitative prediction of hybridization rates. While analyzing the mechanisms of a fundamental biochemical process (hybridization), this work also provides suggestions for the improvement of technologies that rely on such process. Copyright © 2016 Elsevier B.V. All rights reserved.
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Nanjo, T; Kobayashi, M; Yoshiba, Y; Sanada, Y; Wada, K; Tsukaya, H; Kakubari, Y; Yamaguchi-Shinozaki, K; Shinozaki, K
1999-04-01
Many organisms, including higher plants, accumulate free proline (Pro) in response to osmotic stress. Although various studies have focused on the ability of Pro as a compatible osmolyte involved in osmotolerance, its specific role throughout plant growth is still unclear. It has been reported that Pro is synthesized from Glu catalyzed by a key enzyme, delta 1-pyrroline-5-carboxylate synthetase (P5CS), in plants. To elucidate essential roles of Pro, we generated antisense transgenic Arabidopsis plants with a P5CS cDNA. Several transgenics accumulated Pro at a significantly lower level than wild-type plants, providing direct evidence for a key role of P5CS in Pro production in Arabidopsis. These antisense transgenics showed morphological alterations in leaves and a defect in elongation of inflorescences. Furthermore, transgenic leaves were hypersensitive to osmotic stress. Microscopic analysis of transgenic leaves, in which the mutated phenotype clearly occurred, showed morphological abnormalities of epidermal and parenchymatous cells and retardation of differentiation of vascular systems. These phenotypes were suppressed by exogenous L-Pro but not by D-Pro or other Pro analogues. In addition, Pro deficiency did not broadly affect all proteins but specifically affected structural proteins of cell walls in the antisense transgenic plants. These results indicate that Pro is not just an osmoregulator in stressed plants but has a unique function involved in osmotolerance as well as in morphogenesis as a major constituent of cell wall structural proteins in plants.
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Celton, Jean-Marc; Gaillard, Sylvain; Bruneau, Maryline; Pelletier, Sandra; Aubourg, Sébastien; Martin-Magniette, Marie-Laure; Navarro, Lionel; Laurens, François; Renou, Jean-Pierre
2014-07-01
Characterizing the transcriptome of eukaryotic organisms is essential for studying gene regulation and its impact on phenotype. The realization that anti-sense (AS) and noncoding RNA transcription is pervasive in many genomes has emphasized our limited understanding of gene transcription and post-transcriptional regulation. Numerous mechanisms including convergent transcription, anti-correlated expression of sense and AS transcripts, and RNAi remain ill-defined. Here, we have combined microarray analysis and high-throughput sequencing of small RNAs (sRNAs) to unravel the complexity of transcriptional and potential post-transcriptional regulation in eight organs of apple (Malus × domestica). The percentage of AS transcript expression is higher than that identified in annual plants such as rice and Arabidopsis thaliana. Furthermore, we show that a majority of AS transcripts are transcribed beyond 3'UTR regions, and may cover a significant portion of the predicted sense transcripts. Finally we demonstrate at a genome-wide scale that anti-sense transcript expression is correlated with the presence of both short (21-23 nt) and long (> 30 nt) siRNAs, and that the sRNA coverage depth varies with the level of AS transcript expression. Our study provides a new insight on the functional role of anti-sense transcripts at the genome-wide level, and a new basis for the understanding of sRNA biogenesis in plants. © 2014 INRA. New Phytologist © 2014 New Phytologist Trust.
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Ralph, John; Hatfield, Ronald D.; Piquemal, Joël; Yahiaoui, Nabila; Pean, Michel; Lapierre, Catherine; Boudet, Alain M.
1998-01-01
Homologous antisense constructs were used to down-regulate tobacco cinnamyl-alcohol dehydrogenase (CAD; EC 1.1.1.195) and cinnamoyl-CoA reductase (CCR; EC 1.2.1.44) activities in the lignin monomer biosynthetic pathway. CCR converts activated cinnamic acids (hydroxycinnamoyl–SCoAs) to cinnamaldehydes; cinnamaldehydes are then reduced to cinnamyl alcohols by CAD. The transformations caused the incorporation of nontraditional components into the extractable tobacco lignins, as evidenced by NMR. Isolated lignin of antisense-CAD tobacco contained fewer coniferyl and sinapyl alcohol-derived units that were compensated for by elevated levels of benzaldehydes and cinnamaldehydes. Products from radical coupling of cinnamaldehydes, particularly sinapaldehyde, which were barely discernible in normal tobacco, were major components of the antisense-CAD tobacco lignin. Lignin content was reduced in antisense-CCR tobacco, which displayed a markedly reduced vigor. That lignin contained fewer coniferyl alcohol-derived units and significant levels of tyramine ferulate. Tyramine ferulate is a sink for the anticipated build-up of feruloyl–SCoA, and may be up-regulated in response to a deficit of coniferyl alcohol. Although it is not yet clear whether the modified lignins are true structural components of the cell wall, the findings provide further indications of the metabolic plasticity of plant lignification. An ability to produce lignin from alternative monomers would open new avenues for manipulation of lignin by genetic biotechnologies. PMID:9788995
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Osteopontin regulates adhesion of calcium oxalate crystals to renal epithelial cells.
Yasui, Takahiro; Fujita, Keiji; Asai, Kiyofumi; Kohri, Kenjiro
2002-02-01
The association of calcium crystals with renal tubular cells is an important factor during the formation of urinary stones. We previously reported the strong expression of osteopontin (OPN) on renal tubular cells in the stone-forming kidney, suggesting that OPN plays a role in the crystal-cell interaction. In the present study, we examined the biological consequences of inhibiting OPN expression at the translational level on the formation and adhesion of crystals. We synthesized antisense OPN expression vector (pTet-OPNas) using the tetracycline-regulated expression system. The pTet-OPNas was constructed using a mouse OPN cDNA sequence in an inverted (antisense) orientation. Two clones (NRK-52E/ASs) were identified by transfection of pTet-OPNas into NRK-52E cells and they showed a marked reduction of OPN synthesis in the absence of tetracycline. Calcium oxalate (CaOx) crystal suspension was spread homogeneously on top of the NRK-52E cells. After incubation, the association of CaOx crystals and cells was visualized by scanning electron microscopy. Intact NRK-52E cells, NRK-52E cells transfected with empty vector and tetracycline-treated antisense clones (NRK-52E/ASs), under identical conditions, were associated with CaOx crystals. In contrast, the expression of antisense OPN prevented the association of CaOx crystals with NRK-52E cells. Osteopontin plays a crucial role in the adhesion process of CaOx crystals to renal tubular cells in stone formation.
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Yu, Bo; Mao, Yicheng; Bai, Li-Yuan; Herman, Sarah E. M.; Wang, Xinmei; Ramanunni, Asha; Jin, Yan; Mo, Xiaokui; Cheney, Carolyn; Chan, Kenneth K.; Jarjoura, David; Marcucci, Guido; Lee, Robert J.; Byrd, John C.
2013-01-01
Several RNA-targeted therapeutics, including antisense oligonucleotides (ONs), small interfering RNAs, and miRNAs, constitute immunostimulatory CpG motifs as an integral part of their design. The limited success with free antisense ONs in hematologic malignancies in recent clinical trials has been attributed to the CpG motif–mediated, TLR-induced prosurvival effects and inefficient target modulation in desired cells. In an attempt to diminish their off-target prosurvival and proinflammatory effects and specific delivery, as a proof of principle, in the present study, we developed an Ab-targeted liposomal delivery strategy using a clinically relevant CD20 Ab (rituximab)–conjugated lipopolyplex nanoparticle (RIT-INP)– and Bcl-2–targeted antisense G3139 as archetypical antisense therapeutics. The adverse immunostimulatory responses were abrogated by selective B cell–targeted delivery and early endosomal compartmentalization of G3139-encapsulated RIT-INPs, resulting in reduced NF-κB activation, robust Bcl-2 down-regulation, and enhanced sensitivity to fludarabine-induced cytotoxicity. Furthermore, significant in vivo therapeutic efficacy was noted after RIT-INP–G3139 administration in a disseminated xenograft leukemia model. The results of the present study demonstrate that CD20-targeted delivery overcomes the immunostimulatory properties of CpG-containing ON therapeutics and improves efficient gene silencing and in vivo therapeutic efficacy for B-cell malignancies. The broader implications of similar approaches in overcoming immunostimulatory properties of RNA-directed therapeutics in hematologic malignancies are also discussed. PMID:23165478
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Yamamoto, Tsuyoshi; Harada-Shiba, Mariko; Nakatani, Moeka; Wada, Shunsuke; Yasuhara, Hidenori; Narukawa, Keisuke; Sasaki, Kiyomi; Shibata, Masa-Aki; Torigoe, Hidetaka; Yamaoka, Tetsuji; Imanishi, Takeshi; Obika, Satoshi
2012-05-15
Recent findings in molecular biology implicate the involvement of proprotein convertase subtilisin/kexin type 9 (PCSK9) in low-density lipoprotein receptor (LDLR) protein regulation. The cholesterol-lowering potential of anti-PCSK9 antisense oligonucleotides (AONs) modified with bridged nucleic acids (BNA-AONs) including 2',4'-BNA (also called as locked nucleic acid (LNA)) and 2',4'-BNA(NC) chemistries were demonstrated both in vitro and in vivo. An in vitro transfection study revealed that all of the BNA-AONs induce dose-dependent reductions in PCSK9 messenger RNA (mRNA) levels concomitantly with increases in LDLR protein levels. BNA-AONs were administered to atherogenic diet-fed C57BL/6J mice twice weekly for 6 weeks; 2',4'-BNA-AON that targeted murine PCSK9 induced a dose-dependent reduction in hepatic PCSK9 mRNA and LDL cholesterol (LDL-C); the 43% reduction of serum LDL-C was achieved at a dose of 20 mg/kg/injection with only moderate increases in toxicological indicators. In addition, the serum high-density lipoprotein cholesterol (HDL-C) levels increased. These results support antisense inhibition of PCSK9 as a potential therapeutic approach. When compared with 2',4'-BNA-AON, 2',4'-BNA(NC)-AON showed an earlier LDL-C-lowering effect and was more tolerable in mice. Our results validate the optimization of 2',4'-BNA(NC)-based anti-PCSK9 antisense molecules to produce a promising therapeutic agent for the treatment of hypercholesterolemia.
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Parent, Jean-Sébastien; Jauvion, Vincent; Bouché, Nicolas; Béclin, Christophe; Hachet, Mélanie; Zytnicki, Matthias; Vaucheret, Hervé
2015-01-01
Although post-transcriptional gene silencing (PTGS) has been studied for more than a decade, there is still a gap in our understanding of how de novo silencing is initiated against genetic elements that are not supposed to produce double-stranded (ds)RNA. Given the pervasive transcription occurring throughout eukaryote genomes, we tested the hypothesis that unintended transcription could produce antisense (as)RNA molecules that participate to the initiation of PTGS triggered by sense transgenes (S-PTGS). Our results reveal a higher level of asRNA in Arabidopsis thaliana lines that spontaneously trigger S-PTGS than in lines that do not. However, PTGS triggered by antisense transgenes (AS-PTGS) differs from S-PTGS. In particular, a hypomorphic ago1 mutation that suppresses S-PTGS prevents the degradation of asRNA but not sense RNA during AS-PTGS, suggesting a different treatment of coding and non-coding RNA by AGO1, likely because of AGO1 association to polysomes. Moreover, the intended asRNA produced during AS-PTGS is capped whereas the asRNA produced during S-PTGS derives from 3′ maturation of a read-through transcript and is uncapped. Thus, we propose that uncapped asRNA corresponds to the aberrant RNA molecule that is converted to dsRNA by RNA-DEPENDENT RNA POLYMERASE 6 in siRNA-bodies to initiate S-PTGS, whereas capped asRNA must anneal with sense RNA to produce dsRNA that initiate AS-PTGS. PMID:26209135
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Burton, Derek F; Zhang, Chengjin; Boa-Amponsem, Oswald; Mackinnon, Shanta; Cole, Gregory J
2017-05-01
Developmental exposure to ethanol is recognized to produce long-term neurobehavioral impairment in multiple animal models. However, the molecular mechanisms underlying these deficits remain poorly understood. The present study was undertaken to ascertain whether two well-characterized targets of prenatal alcohol exposure, sonic hedgehog (Shh) and retinoic acid (RA), that induce the hallmark morphological phenotypes of fetal alcohol spectrum disorders (FASD), are involved in the generation of behavioral alterations as a result of alcohol exposure. Zebrafish embryos were exposed to ethanol (0%, 1%, 3%) at either 8-10 or 24-27h post-fertilization (hpf) and then evaluated during adolescence in the novel tank dive test to assess anxiety and risk-taking behavior. Overt signs of dysmorphogenesis were also scored and behavioral and morphological changes were compared for embryos treated with alcohol alone or in combination with subthreshold doses of shh or alhh1a3 morpholinos (MOs). Ethanol treated fish displayed altered tank diving behavior that was not exacerbated by combined MO treatment. While treatment of embryos with either shha mRNA or RA prior to ethanol exposure only ameliorated the altered tank diving response in the case of shha mRNA overexpression, dysmorphogenesis was rescued by both treatments. These results suggest that the effects of ethanol exposure on changes in anxiety and risk-taking behavior in adolescent zebrafish is manifested by a blunting of Shh, but not RA, signaling during early development. Copyright © 2017 Elsevier Inc. All rights reserved.
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ERIC Educational Resources Information Center
Withey, Jonathan M.; Bajic, Andrea
2015-01-01
A novel procedure is described where students use COMU [(1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate], as a nonhazardous partner, in the one-pot coupling of a carboxylic acid and amine producing N,N-diethyl-3-methylbenzamide (DEET). Fundamental principles of carbonyl reactivity are understood,…
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A Morpholino Strategy to Assess TSC Gene Function in Zebrafish
2006-11-01
ABSTRACT Tuberous sclerosis complex (TSC) is a genetic disorder associated with severe neurological symptoms including mental retardation, autism ...ABSTRACT Tuberous sclerosis complex (TSC) is a genetic disorder associated with severe neurological symptoms including mental retardation, autism ...devastating autosomal dominant disease that results in severe neurological symptoms including mental retardation, autism , and seizures (DiMario, 2004
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NASA Astrophysics Data System (ADS)
Azeke, John Imuetinyan-Jesu, Jr.
Abdominal adhesions are the aberrant result of peritoneal wound healing commonly associated with surgery and inflammation. A subject of a large number of studies since the first half of the last century, peritoneal adhesion prevention has, for the most part, evaded the scientific community and continues to cost Americans an estimated $2-4 billion annually. It is known that transforming growth factor-beta (TGF-beta) plays a key role in the wound healing cascade; however, suppression of this multifunctional growth factor's activity may have more harmful consequences than can be tolerated. As a result, much attention has fallen on connective tissue growth factor (CTGF), a downstream mediator of TGF-beta's fibrotic action. It has been demonstrated in several in vitro models, that the suppression of CTGF hinders fibroblast proliferation, a necessary condition for fibrosis. Furthermore, antisense oligonucleotides (antisense oligos, AO) to CTGF have been shown to knock down CTGF mRNA levels by specifically hindering the translation of CTGF protein. Antisense technologies have met with a great deal of excitement as a viable means of preventing diseases such as adhesions by hindering protein translation at the mRNA level. However, the great challenge associated with the use of these drugs lies in the short circulation time when administered "naked". Viral delivery systems, although excellent platforms in metabolic studies, are not ideal for diagnostic use because of the inherent danger associated with viral vectors. Microparticles made of biodegradable polymers have therefore presented themselves as a viable means of delivering these drugs to target cells over extended periods. Herein, we present two in vivo studies confirming the up-regulation of TGF-beta protein and CTGF mRNA following injury to the uterine tissues of female rats. We were able to selectively knockdown post-operative CTGF protein levels following surgery, however, our observations led us to conclude that, while both cytokines are over-expressed within the first day following injury, CTGF protein levels could not be correlated with observed adhesion development. In addition, we synthesized linear triblock copolymers of polyethylene glycol (PEG) and poly(D,L-lactide-co-glycolide) (PLGA), two of the most widely studied biodegradable polymers in use today. Bulk gels and microparticles of the copolymers were then evaluated for gelling behavior, temperature stability, and drug loading and release kinetics in order assess their suitability as potential carriers of antisense therapeutics. A novel approach to affecting the antisense oligonucleotide release kinetics by varying the relative concentrations of co-encapsulated cationic lipid transfection agents was also presented.
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Oligonucleotides as antivirals: dream or realistic perspective?
Van Aerschot, Arthur
2006-09-01
Many reports have been published on antiviral activity of synthetic oligonucleotides, targeted to act either by a true antisense effect or via non-sequence specific interactions. This short review will try to evaluate the current status of the field by focusing on the effects as reported for inhibition of either HSV-1, HCMV or HIV-1. Following an introduction with a historical background and a brief discussion on the different types of constructs and mechanisms of action, the therapeutic potential of antisense oligonucleotides as antivirals, as well as possible pitfalls upon their evaluation will be discussed.
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Yao, Yong-Xing; Jiang, Zhen; Zhao, Zhi-Qi
2011-12-01
Previous studies have demonstrated that Homer 1b/c, a postsynaptic molecular scaffolding protein that binds and clusters metabotropic glutamate receptors at neuronal synapses, has an important role in the metabotropic glutamate receptor signaling process. In the current study, we investigated the possible involvement of Homer 1b/c in secondary hyperalgesia induced by complete Freund's adjuvant (CFA). Chronic inflammation was induced by injecting CFA into the left hind ankle of Wistar rats. Homer 1b/c antisense or missense oligonucleotides were intrathecally administrated (antisense, 10 μg/10 μL, 5 μg/10 μL, or 2.5 μg/10 μL, once a day; missense, 10 μg/10 μL) from 5 to 8 days after the onset of inflammation. The withdrawal threshold and withdrawal latency to mechanical or thermal stimuli were determined before and after the intrathecal administration. The expression and distribution of Homer 1b/c were examined in the spinal cord using immunological techniques. Mechanical allodynia and thermal hyperalgesia were induced within 24 hours and maintained for >2 weeks after the CFA injection. The expression of Homer 1b/c reached the highest level 7 days after inflammation and returned to baseline at day 28. Intrathecal administration of Homer 1b/c antisense oligonucleotides markedly reduced the expression of Homer 1b/c protein in the spinal cord. Additionally, administration of Homer 1b/c antisense oligonucleotides attenuated secondary mechanical hypersensitization on days 2 to 5 and reduced thermal hypersensitization on days 3 to 4. There were no effects of missense oligonucleotides on hypersensitization and the expression of Homer 1b/c. In the naïve rats, Homer 1b/c antisense oligonucleotides did not affect the mechanical and thermal responses or locomotor activity. These novel results demonstrate that Homer 1b/c in the spinal cord contributes to the maintenance of secondary hyperalgesia induced by CFA and suggest that Homer 1b/c may be a novel target for pain therapy.
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Sawada, S; Sugano, M; Makino, N; Okamoto, H; Tsuchida, K
1999-10-01
Prebeta HDL are small, protein rich lipoproteins that are predominantly composed of apo A-I, without apo A-II. Prebeta HDL are secreted from the liver as nascent HDL and/or are produced in the incubated plasma by cholesteryl ester transfer protein (CETP). However, the role of CETP in the secretion of HDL from the liver has yet to be determined. In the present study, we examined the effect of the suppression of hepatic CETP by antisense oligodeoxynucleotides (ODNs) against CETP targeted to the liver on the secretion of apo A-I using a Hep G2 cell culture. The ODNs against CETP were coupled to asialoglycoprotein (ASOR) carrier molecules, which serve as an important method for the regulation of liver gene expression. Hep G2 cells were cultured in DMEM supplemented with 10 FBS. After 2 days, the medium was changed to DMEM with EGF and the cells were divided into three groups. The control group received saline, while the sense group was mixed with the sense ODNs complex and the antisense group was mixed with the antisense ODNs complex, respectively, for 2 days. Both the hepatic CETP mRNA and the CETP mass in the medium in the antisense group decreased significantly more than in the sense and the control groups (CETP mass: 1.697 + /- 0.410 ng/mg cell protein vs. 2.367 + /- 0.22 and 2.360 + /- 0.139, n = 3 in each determination). In contrast, both the hepatic apo A-I mRNA and the apo A-I mass in the medium in the antisense group were significantly higher than those in the sense and the control groups (apo A-I mass; 1.877 + /- 0.215 micro/mg cell protein vs. 1.213 + /- 0.282 and 1.097 + /- 0.144, n = 3 in each determination). The increase in apo A-I was mainly due to the increase in prebeta apo A-I. These findings may partly explain why HDL and apo A-I increase in patients with CETP deficiency, while also indicating the possibility that the original level of prebeta HDL is sufficient in such patients.
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A facile synthesis, antibacterial activity of pulvinone and its derivatives.
Xu, Hai-Wei; Xu, Chao; Fan, Zi-qi; Zhao, Ling-Jie; Liu, Hong-Min
2013-02-01
Pulvinone and several 3-fluoro-4-morpholino substituted pulvinone derivatives were synthesized in five steps from a common precursor, phenyl acetic acid. Most of synthetic morpholine substituted pulvinones showed inhibitory activity against Esherichia coli. For the first time, the inhibition of pulvinone and its derivatives against Gram-negative bacteria was reported. Copyright © 2012 Elsevier Ltd. All rights reserved.
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Comparison of small molecules and oligonucleotides that target a toxic, non-coding RNA.
Costales, Matthew G; Rzuczek, Suzanne G; Disney, Matthew D
2016-06-01
Potential RNA targets for chemical probes and therapeutic modalities are pervasive in the transcriptome. Oligonucleotide-based therapeutics are commonly used to target RNA sequence. Small molecules are emerging as a modality to target RNA structures selectively, but their development is still in its infancy. In this work, we compare the activity of oligonucleotides and several classes of small molecules that target the non-coding r(CCUG) repeat expansion (r(CCUG)(exp)) that causes myotonic dystrophy type 2 (DM2), an incurable disease that is the second-most common cause of adult onset muscular dystrophy. Small molecule types investigated include monomers, dimers, and multivalent compounds synthesized on-site by using RNA-templated click chemistry. Oligonucleotides investigated include phosphorothioates that cleave their target and vivo-morpholinos that modulate target RNA activity via binding. We show that compounds assembled on-site that recognize structure have the highest potencies amongst small molecules and are similar in potency to a vivo-morpholino modified oligonucleotide that targets sequence. These studies are likely to impact the design of therapeutic modalities targeting other repeats expansions that cause fragile X syndrome and amyotrophic lateral sclerosis, for example. Copyright © 2016. Published by Elsevier Ltd.
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Coleman, James R; Thompson, Karen C; Wilson, Marlene A; Wilson, Steven P
2017-06-01
Herpes virus technology involving manipulation of GAD65 was used to study effects on audiogenic seizures (AGS). Audiogenic seizure behaviors were examined following injections of replication-defective herpes simplex virus (HSV-1) vectors incorporating sense or antisense toward GAD65 along with 10% lac-Z into the central nucleus of inferior colliculus (CNIC) of Long-Evans rats. In seizure-sensitive animals developmentally primed by intense sound exposure, injection of GAD65 in the sense orientation increased wild running latencies and reduced incidence of clonus compared with lac-Z only, unoperated, and vehicle seizure groups. In contrast, infection of CNIC with GAD65 antisense virus resulted in 100% incidence of wild running and clonus behaviors in AGS animals. Unprimed animals not operated continued to show uniform absence of seizure activity. Administration of GAD65 antisense virus into CNIC produced novel wild running and clonus behaviors in some unprimed animals. Staining for β-galactosidase in all vector animals revealed no differences in pattern or numbers of immunoreactive cells at injection sites. Qualitatively, typical small and medium multipolar/stellate and medium fusiform neurons appeared in the CNIC of vector animals. These results demonstrate that HSV-1 vector constructs implanted into the CNIC can predictably influence incidence and severity of AGS and suggest that viral vectors can be useful in studying GABA mechanisms with potential for therapeutic application in epilepsy. This article is part of a Special Issue entitled "Genetic and Reflex Epilepsies, Audiogenic Seizures and Strains: From Experimental Models to the Clinic". Copyright © 2016 Elsevier Inc. All rights reserved.
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Marsollier, Anne-Charlotte; Ciszewski, Lukasz; Mariot, Virginie; Popplewell, Linda; Voit, Thomas; Dickson, George; Dumonceaux, Julie
2016-04-15
Defects in mRNA 3'end formation have been described to alter transcription termination, transport of the mRNA from the nucleus to the cytoplasm, stability of the mRNA and translation efficiency. Therefore, inhibition of polyadenylation may lead to gene silencing. Here, we choose facioscapulohumeral dystrophy (FSHD) as a model to determine whether or not targeting key 3' end elements involved in mRNA processing using antisense oligonucleotide drugs can be used as a strategy for gene silencing within a potentially therapeutic context. FSHD is a gain-of-function disease characterized by the aberrant expression of the Double homeobox 4 (DUX4) transcription factor leading to altered pathogenic deregulation of multiple genes in muscles. Here, we demonstrate that targeting either the mRNA polyadenylation signal and/or cleavage site is an efficient strategy to down-regulate DUX4 expression and to decrease the abnormally high-pathological expression of genes downstream of DUX4. We conclude that targeting key functional 3' end elements involved in pre-mRNA to mRNA maturation with antisense drugs can lead to efficient gene silencing and is thus a potentially effective therapeutic strategy for at least FSHD. Moreover, polyadenylation is a crucial step in the maturation of almost all eukaryotic mRNAs, and thus all mRNAs are virtually eligible for this antisense-mediated knockdown strategy. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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D'Aoust, M A; Yelle, S; Nguyen-Quoc, B
1999-01-01
The role of sucrose synthase (SuSy) in tomato fruit was studied in transgenic tomato (Lycopersicon esculentum) plants expressing an antisense fragment of fruit-specific SuSy RNA (TOMSSF) under the control of the cauliflower mosaic virus 35S promoter. Constitutive expression of the antisense RNA markedly inhibited SuSy activity in flowers and fruit pericarp tissues. However, inhibition was only slight in the endosperm and was undetectable in the embryo, shoot, petiole, and leaf tissues. The activity of sucrose phosphate synthase decreased in parallel with that of SuSy, but acid invertase activity did not increase in response to the reduced SuSy activity. The only effect on the carbohydrate content of young fruit was a slight reduction in starch accumulation. The in vitro sucrose import capacity of fruits was not reduced by SuSy inhibition at 23 days after anthesis, and the rate of starch synthesized from the imported sucrose was not lessened even when SuSy activity was decreased by 98%. However, the sucrose unloading capacity of 7-day-old fruit was substantially decreased in lines with low SuSy activity. In addition, the SuSy antisense fruit from the first week of flowering had a slower growth rate. A reduced fruit set, leading to markedly less fruit per plant at maturity, was observed for the plants with the least SuSy activity. These results suggest that SuSy participates in the control of sucrose import capacity of young tomato fruit, which is a determinant for fruit set and development. PMID:10590167
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Hara, Takamitsu; Omura-Minamisawa, Motoko; Chao Cheng
Purpose: Bcl-2, an inhibitor of apoptosis frequently shows elevated expression in human tumors, thus resulting in resistance to radiation therapy. Therefore, inhibiting Bcl-2 function may enhance the radiosensitivity of tumor cells. Tetrocarcin A (TC-A) and bcl-2 antisense oligonucleotides exhibit antitumor activity by inhibiting Bcl-2 function and transcription, respectively. We investigated whether these antitumor agents would enhance the cytotoxic effects of radiation in tumor cells overexpressing Bcl-2. Methods and materials: We used HeLa/bcl-2 cells, a stable Bcl-2-expressing cell line derived from wild-type HeLa (HeLa/wt) cells. Cells were incubated with TC-A and bcl-2 antisense oligonucleotides for 24 h after irradiation, and cellmore » viability was then determined. Apoptotic cells were quantified by flow cytometric assay. Results: The HeLa/bcl-2 cells were more resistant to radiation than HeLa/wt cells. At concentrations that are not inherently cytotoxic, both TC-A and bcl-2 antisense oligonucleotides increased the cytotoxic effects of radiation in HeLa/bcl-2 cells, but not in HeLa/wt cells. However, in HeLa/bcl-2 cells, additional treatment with TC-A in combination with radiation did not significantly increase apoptosis. Conclusions: The present results suggest that TC-A and bcl-2 antisense oligonucleotides reduce radioresistance of tumor cells overexpressing Bcl-2. Therefore, a combination of radiotherapy and Bcl-2 inhibitors may prove to be a useful therapeutic approach for treating tumors that overexpress Bcl-2.« less
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Hfq restructures RNA-IN and RNA-OUT and facilitates antisense pairing in the Tn10/IS10 system
Ross, Joseph A.; Ellis, Michael J.; Hossain, Shahan; Haniford, David B.
2013-01-01
Hfq functions in post-transcriptional gene regulation in a wide range of bacteria, usually by promoting base-pairing of mRNAs and trans-encoded sRNAs that share partial sequence complementarity. It is less clear if Hfq is required for pairing of cis-encoded RNAs (i.e., antisense RNAs) with their target mRNAs. In the current work, we have characterized the interactions between Escherichia coli Hfq and the components of the Tn10/IS10 antisense system, RNA-IN and RNA-OUT. We show that Hfq interacts with RNA-OUT through its proximal RNA-binding surface, as is typical for Hfq and trans-encoded sRNAs. In contrast, RNA-IN binds both proximal and distal RNA-binding surfaces in Hfq with a higher affinity for the latter, as is typical for mRNA interactions in canonical sRNA-mRNA pairs. Importantly, an amino acid substitution in Hfq that interferes with RNA binding to the proximal site negatively impacts RNA-IN:OUT pairing in vitro and suppresses the ability of Hfq to negatively regulate IS10 transposition in vivo. We also show that Hfq binding to RNA-IN and RNA-OUT alters secondary structure elements in both of these RNAs and speculate that this could be important in how Hfq facilitates RNA-IN:OUT pairing. Based on the results presented here, we suggest that Hfq could be involved in regulating RNA pairing in other antisense systems, including systems encoded by other transposable elements. PMID:23510801
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Parent, Jean-Sébastien; Jauvion, Vincent; Bouché, Nicolas; Béclin, Christophe; Hachet, Mélanie; Zytnicki, Matthias; Vaucheret, Hervé
2015-09-30
Although post-transcriptional gene silencing (PTGS) has been studied for more than a decade, there is still a gap in our understanding of how de novo silencing is initiated against genetic elements that are not supposed to produce double-stranded (ds)RNA. Given the pervasive transcription occurring throughout eukaryote genomes, we tested the hypothesis that unintended transcription could produce antisense (as)RNA molecules that participate to the initiation of PTGS triggered by sense transgenes (S-PTGS). Our results reveal a higher level of asRNA in Arabidopsis thaliana lines that spontaneously trigger S-PTGS than in lines that do not. However, PTGS triggered by antisense transgenes (AS-PTGS) differs from S-PTGS. In particular, a hypomorphic ago1 mutation that suppresses S-PTGS prevents the degradation of asRNA but not sense RNA during AS-PTGS, suggesting a different treatment of coding and non-coding RNA by AGO1, likely because of AGO1 association to polysomes. Moreover, the intended asRNA produced during AS-PTGS is capped whereas the asRNA produced during S-PTGS derives from 3' maturation of a read-through transcript and is uncapped. Thus, we propose that uncapped asRNA corresponds to the aberrant RNA molecule that is converted to dsRNA by RNA-DEPENDENT RNA POLYMERASE 6 in siRNA-bodies to initiate S-PTGS, whereas capped asRNA must anneal with sense RNA to produce dsRNA that initiate AS-PTGS. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
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Improved silencing properties using small internally segmented interfering RNAs
Bramsen, Jesper B.; Laursen, Maria B.; Damgaard, Christian K.; Lena, Suzy W.; Ravindra Babu, B.; Wengel, Jesper; Kjems, Jørgen
2007-01-01
RNA interference is mediated by small interfering RNAs (siRNAs) that upon incorporation into the RNA-induced silencing complex (RISC) can target complementary mRNA for degradation. Standard siRNA design usually feature a 19–27 base pair contiguous double-stranded region that is believed to be important for RISC incorporation. Here, we describe a novel siRNA design composed of an intact antisense strand complemented with two shorter 10–12 nt sense strands. This three-stranded construct, termed small internally segmented interfering RNA (sisiRNA), is highly functional demonstrating that an intact sense strand is not a prerequisite for RNA interference. Moreover, when using the sisiRNA design only the antisense strand is functional in activated RISC thereby completely eliminating unintended mRNA targeting by the sense strand. Interestingly, the sisiRNA design supports the function of chemically modified antisense strands, which are non-functional within the context of standard siRNA designs. This suggests that the sisiRNA design has a clear potential of improving the pharmacokinetic properties of siRNA in vivo. PMID:17726057
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Kozhuharova, Ana; Sharma, Harshita; Ohyama, Takako; Fasolo, Francesca; Yamazaki, Toshio; Cotella, Diego; Santoro, Claudio; Zucchelli, Silvia; Gustincich, Stefano; Carninci, Piero
2018-01-01
SINEUPs are antisense long noncoding RNAs, in which an embedded SINE B2 element UP-regulates translation of partially overlapping target sense mRNAs. SINEUPs contain two functional domains. First, the binding domain (BD) is located in the region antisense to the target, providing specific targeting to the overlapping mRNA. Second, the inverted SINE B2 represents the effector domain (ED) and enhances translation. To adapt SINEUP technology to a broader number of targets, we took advantage of a high-throughput, semi-automated imaging system to optimize synthetic SINEUP BD and ED design in HEK293T cell lines. Using SINEUP-GFP as a model SINEUP, we extensively screened variants of the BD to map features needed for optimal design. We found that most active SINEUPs overlap an AUG-Kozak sequence. Moreover, we report our screening of the inverted SINE B2 sequence to identify active sub-domains and map the length of the minimal active ED. Our synthetic SINEUP-GFP screening of both BDs and EDs constitutes a broad test with flexible applications to any target gene of interest. PMID:29414979
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Sequence-Specific Targeting of Bacterial Resistance Genes Increases Antibiotic Efficacy
Wong, Michael; Daly, Seth M.; Greenberg, David E.; Toprak, Erdal
2016-01-01
The lack of effective and well-tolerated therapies against antibiotic-resistant bacteria is a global public health problem leading to prolonged treatment and increased mortality. To improve the efficacy of existing antibiotic compounds, we introduce a new method for strategically inducing antibiotic hypersensitivity in pathogenic bacteria. Following the systematic verification that the AcrAB-TolC efflux system is one of the major determinants of the intrinsic antibiotic resistance levels in Escherichia coli, we have developed a short antisense oligomer designed to inhibit the expression of acrA and increase antibiotic susceptibility in E. coli. By employing this strategy, we can inhibit E. coli growth using 2- to 40-fold lower antibiotic doses, depending on the antibiotic compound utilized. The sensitizing effect of the antisense oligomer is highly specific to the targeted gene’s sequence, which is conserved in several bacterial genera, and the oligomer does not have any detectable toxicity against human cells. Finally, we demonstrate that antisense oligomers improve the efficacy of antibiotic combinations, allowing the combined use of even antagonistic antibiotic pairs that are typically not favored due to their reduced activities. PMID:27631336
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Akagi, H; Patton, D E; Miledi, R
1989-01-01
Three synthetic oligodeoxynucleotides complementary to different parts of an RNA encoding a glycine receptor subunit were used to discriminate heterogenous mRNAs coding for glycine receptors in adult and neonatal rat spinal cord. Injection of the three antisense oligonucleotides into Xenopus oocytes specifically inhibited the expression of glycine receptors by adult spinal cord mRNA. In contrast, the antisense oligonucleotides were much less potent in inhibiting the expression of glycine receptors encoded by neonatal spinal cord mRNA. Northern blot analysis revealed that the oligonucleotides hybridized mostly to an adult cord transcript of approximately 10 kilobases in size. This band was also present in neonatal spinal cord mRNA but its density was about one-fourth of the adult cord message. There was no intense band in the low molecular weight position (approximately 2 kilobases), the existence of which was expected from electrophysiological studies with size-fractionated mRNA of neonatal spinal cord. Our results suggest that in the rat spinal cord there are at least three different types of mRNAs encoding functional strychnine-sensitive glycine receptors. Images PMID:2479016
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Zinker, Bradley A; Rondinone, Cristina M; Trevillyan, James M; Gum, Rebecca J; Clampit, Jill E; Waring, Jeffrey F; Xie, Nancy; Wilcox, Denise; Jacobson, Peer; Frost, Leigh; Kroeger, Paul E; Reilly, Regina M; Koterski, Sandra; Opgenorth, Terry J; Ulrich, Roger G; Crosby, Seth; Butler, Madeline; Murray, Susan F; McKay, Robert A; Bhanot, Sanjay; Monia, Brett P; Jirousek, Michael R
2002-08-20
The role of protein-tyrosine phosphatase 1B (PTP1B) in diabetes was investigated using an antisense oligonucleotide in ob/ob and db/db mice. PTP1B antisense oligonucleotide treatment normalized plasma glucose levels, postprandial glucose excursion, and HbA(1C). Hyperinsulinemia was also reduced with improved insulin sensitivity. PTP1B protein and mRNA were reduced in liver and fat with no effect in skeletal muscle. Insulin signaling proteins, insulin receptor substrate 2 and phosphatidylinositol 3 (PI3)-kinase regulatory subunit p50alpha, were increased and PI3-kinase p85alpha expression was decreased in liver and fat. These changes in protein expression correlated with increased insulin-stimulated protein kinase B phosphorylation. The expression of liver gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase was also down-regulated. These findings suggest that PTP1B modulates insulin signaling in liver and fat, and that therapeutic modalities targeting PTP1B inhibition may have clinical benefit in type 2 diabetes.
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Zinker, Bradley A.; Rondinone, Cristina M.; Trevillyan, James M.; Gum, Rebecca J.; Clampit, Jill E.; Waring, Jeffrey F.; Xie, Nancy; Wilcox, Denise; Jacobson, Peer; Frost, Leigh; Kroeger, Paul E.; Reilly, Regina M.; Koterski, Sandra; Opgenorth, Terry J.; Ulrich, Roger G.; Crosby, Seth; Butler, Madeline; Murray, Susan F.; McKay, Robert A.; Bhanot, Sanjay; Monia, Brett P.; Jirousek, Michael R.
2002-01-01
The role of protein-tyrosine phosphatase 1B (PTP1B) in diabetes was investigated using an antisense oligonucleotide in ob/ob and db/db mice. PTP1B antisense oligonucleotide treatment normalized plasma glucose levels, postprandial glucose excursion, and HbA1C. Hyperinsulinemia was also reduced with improved insulin sensitivity. PTP1B protein and mRNA were reduced in liver and fat with no effect in skeletal muscle. Insulin signaling proteins, insulin receptor substrate 2 and phosphatidylinositol 3 (PI3)-kinase regulatory subunit p50α, were increased and PI3-kinase p85α expression was decreased in liver and fat. These changes in protein expression correlated with increased insulin-stimulated protein kinase B phosphorylation. The expression of liver gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase was also down-regulated. These findings suggest that PTP1B modulates insulin signaling in liver and fat, and that therapeutic modalities targeting PTP1B inhibition may have clinical benefit in type 2 diabetes. PMID:12169659
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Kadakkuzha, Beena M; Liu, Xin-An; Narvaez, Maria; Kaye, Alexandra; Akhmedov, Komolitdin; Puthanveettil, Sathyanarayanan V
2014-01-01
Despite the advances in our understanding of transcriptome, regulation and function of its non-coding components continue to be poorly understood. Here we searched for natural antisense transcript for sensorin (NAT-SRN), a neuropeptide expressed in the presynaptic sensory neurons of gill-withdrawal reflex of the marine snail Aplysia californica. Sensorin (SRN) has a key role in learning and long-term memory storage in Aplysia. We have now identified NAT-SRN in the central nervous system (CNS) and have confirmed its expression by northern blotting and fluorescent RNA in situ hybridization. Quantitative analysis of NAT-SRN in micro-dissected cell bodies and processes of sensory neurons suggest that NAT-SRN is present in the distal neuronal processes along with sense transcripts. Importantly, aging is associated with reduction in levels of NAT-SRN in sensory neuron processes. Furthermore, we find that forskolin, an activator of CREB signaling, differentially alters the distribution of SRN and NAT-SRN. These studies reveal novel insights into physiological regulation of natural antisense RNAs.
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Inhaled ENaC antisense oligonucleotide ameliorates cystic fibrosis-like lung disease in mice.
Crosby, Jeff R; Zhao, Chenguang; Jiang, Chong; Bai, Dong; Katz, Melanie; Greenlee, Sarah; Kawabe, Hiroshi; McCaleb, Michael; Rotin, Daniela; Guo, Shuling; Monia, Brett P
2017-11-01
Epithelial sodium channel (ENaC, Scnn1) hyperactivity in the lung leads to airway surface dehydration and mucus accumulation in cystic fibrosis (CF) patients and in mice with CF-like lung disease. We identified several potent ENaC specific antisense oligonucleotides (ASOs) and tested them by inhalation in mouse models of CF-like lung disease. The inhaled ASOs distributed into lung airway epithelial cells and decreased ENaC expression by inducing RNase H1-dependent degradation of the targeted Scnn1a mRNA. Aerosol delivered ENaC ASO down-regulated mucus marker expression and ameliorated goblet cell metaplasia, inflammation, and airway hyper-responsiveness. Lack of systemic activity of ASOs delivered via the aerosol route ensures the safety of this approach. Our results demonstrate that antisense inhibition of ENaC in airway epithelial cells could be an effective and safe approach for the prevention and reversal of lung symptoms in CF and potentially other inflammatory diseases of the lung. Copyright © 2017 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.
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Bramson, J L; Bodner, C A; Johnson, J; Semple, S; Hope, M J
2000-06-01
Stabilized antisense lipid particles (SALP) have been developed for the systemic delivery of oligonucleotides. The impact of intravenous SALP administration was measured with respect to activation of natural killer (NK) and NK1.1+ T (NKT) cells in the livers of immunocompetent mice. Treatment with a SALP containing a highly mitogenic oligonucleotide (INX-6295) generated an increase in NK cytolytic activity and cell number within the liver but did not appear to affect the number of hepatic NKT cells or their cytolytic activity. The same results were observed after intravenous administration of the mitogenic oligonucleotide alone. Interestingly, treatment with a SALP containing a weakly mitogenic oligonucleotide (INX-6300) also activated the liver NK cells, whereas the oligonucleotide alone was unable to elicit these effects. The NK stimulatory activity of a SALP containing INX-6300 required both lipid and oligonucleotide components. These results demonstrate that in addition to modifying the pharmacokinetics and biodistribution of intravenously administered oligonucleotides, SALP possess immunostimulatory activity independent of oligonucleotide mitogenicity, which can serve as an adjuvant to antisense therapies for cancer.
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Coetzer, C; Corsini, D; Love, S; Pavek, J; Tumer, N
2001-02-01
Polyphenol oxidase (PPO) activity of Russet Burbank potato was inhibited by sense and antisense PPO RNAs expressed from a tomato PPO cDNA under the control of the 35S promoter from the cauliflower mosaic virus. Transgenic Russet Burbank potato plants from 37 different lines were grown in the field. PPO activity and the level of enzymatic browning were measured in the tubers harvested from the field. Of the tubers from 28 transgenic lines that were sampled, tubers from 5 lines exhibited reduced browning. The level of PPO activity correlated with the reduction in enzymatic browning in these lines. These results indicate that expression of tomato PPO RNA in sense or antisense orientation inhibits PPO activity and enzymatic browning in the major commercial potato cultivar. Expression of tomato PPO RNA in sense orientation led to the greatest decrease in PPO activity and enzymatic browning, possibly due to cosuppression. These results suggest that expression of closely related heterologous genes can be used to prevent enzymatic browning in a wide variety of food crops without the application of various food additives.
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Crooke, Stanley T; Geary, Richard S
2013-08-01
Mipomersen is a second generation antisense oligonucleotide that targets apolipoprotein B. It has been studied thoroughly in clinical trials (more than 800 subjects), including four randomized double-blind placebo controlled phase 3 studies involving 391 patients, and is in registration for the treatment of severe hypercholesterolaemia. The pharmacokinetic and pharmacodynamic properties of mipomersen are well characterized. Mipomersen is rapidly and extensively absorbed after subcutaneous administration and has an elimination half-life of approximately 30 days across species. It is cleared by nuclease metabolism and renal excretion of the metabolites. Mipomersen reduces all apolipoprotein B containing atherogenic particles and displays dose dependent reductions between 50-400 mg week⁻¹ , both as a single agent and in the presence of maximal lipid lowering therapy. No drug-drug interactions have been identified. Mipomersen is a representative of second generation antisense drugs, all of which have similar properties, and is thus representative of the behaviour of the class of drugs. © 2012 The Authors. British Journal of Clinical Pharmacology © 2012 The British Pharmacological Society.
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Mutations in RAD21 Disrupt Regulation of APOB in Patients with Chronic Intestinal Pseudo-obstruction
Bonora, Elena; Bianco, Francesca; Cordeddu, Lina; Bamshad, Michael; Francescatto, Ludmila; Dowless, Dustin; Stanghellini, Vincenzo; Cogliandro, Rosanna F.; Lindberg, Greger; Mungan, Zeynel; Cefle, Kivanc; Ozcelik, Tayfun; Palanduz, Sukru; Ozturk, Sukru; Gedikbasi, Asuman; Gori, Alessandra; Pippucci, Tommaso; Graziano, Claudio; Volta, Umberto; Caio, Giacomo; Barbara, Giovanni; D'Amato, Mauro; Seri, Marco; Katsanis, Nicholas; Romeo, Giovanni; De Giorgio, Roberto
2015-01-01
Background & Aims Chronic intestinal pseudo-obstruction (CIPO) is characterized by severe intestinal dysmotility that mimicks a mechanical sub-occlusion with no evidence of gut obstruction. We searched for genetic variants associated with CIPO to increase our understanding of its pathogenesis and indentify potential biomarkers. Methods We performed whole-exome sequencing of genomic DNA from patients with familial CIPO syndrome. Blood and lymphoblastoid cells were collected from patients and controls (individuals without CIPO); levels of mRNA and proteins were analyzed by quantitative reverse transcription PCR, immunoblot, and mobility shift assays. cDNAs were transfected into HEK293 cells. Expression of rad21 was suppressed in zebrafish embryos using a splice-blocking morpholino (rad21a MO). Gut tissues were collected and analyzed. Results We identified a homozygous mutation (p.622, encodes Ala>Thr) in RAD21 in patients from a consanguineous family with CIPO. Expression of RUNX1, a target of RAD21, was reduced in cells from patients with CIPO compared with controls. In zebrafish, suppression of rad21a reduced expression of runx1; this phenotype was corrected by injection of human RAD21 mRNA, but not with the mRNA from the mutated p.622 allele. rad21a MO zebrafish had delayed intestinal transit and greatly reduced numbers of enteric neurons, similar to patients with CIPO. This defect was greater in zebrafish with suppressed expression of ret and rad21, indicating their interaction in regulation of gut neurogenesis. The promoter region of APOB bound RAD21 but not RAD21 p.622 Ala>Thr; expression of wild-type RAD21 in HEK293 cells repressed expression of APOB, compared with control vector. The gut-specific isoform of APOB (APOB48) is overexpressed in sera from patients with CIPO who carry the RAD21 mutation. APOB48 is also overexpressed in sporadic CIPO in sera and gut biopsies. Conclusions Some patients with CIPO carry mutations in RAD21 that disrupt the ability of its product to regulate genes such as RUNX1 and APOB. Reduced expression of rad21 in zebrafish, and dysregulation of these target genes, disrupts intestinal transit and development of enteric neurons. PMID:25575569
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Boneva, Neli; Hamra-Amitay, Yasmine; Wirguin, Itzhak; Brenner, Talma
2006-05-01
The neuromuscular weakness associated with myasthenia gravis (MG) can be transiently relieved by pharmacological inhibitors of acetylcholinesterase (AChE). Here, we expand the anticholinesterase repertoire to include 2'-O-methyl-protected antisense oligonucleotides targeted to AChE mRNA (EN101). Using stimulated-single fiber electromyography, we show that EN101 treatment of rats with experimental autoimmune myasthenia gravis (EAMG), improved the mean consecutive difference (MCD) and blocking for 24h. This treatment was more efficient than pyridostigmine and was accompanied by marked improvement in stamina and clinical profile.
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Zizioli, Daniela; Tiso, Natascia; Guglielmi, Adele; Saraceno, Claudia; Busolin, Giorgia; Giuliani, Roberta; Khatri, Deepak; Monti, Eugenio; Borsani, Giuseppe; Argenton, Francesco; Finazzi, Dario
2016-01-01
Pantothenate Kinase Associated Neurodegeneration (PKAN) is an autosomal recessive disorder with mutations in the pantothenate kinase 2 gene (PANK2), encoding an essential enzyme for Coenzyme A (CoA) biosynthesis. The molecular connection between defects in this enzyme and the neurodegenerative phenotype observed in PKAN patients is still poorly understood. We exploited the zebrafish model to study the role played by the pank2 gene during embryonic development and get new insight into PKAN pathogenesis. The zebrafish orthologue of hPANK2 lies on chromosome 13, is a maternal gene expressed in all development stages and, in adult animals, is highly abundant in CNS, dorsal aorta and caudal vein. The injection of a splice-inhibiting morpholino induced a clear phenotype with perturbed brain morphology and hydrocephalus; edema was present in the heart region and caudal plexus, where hemorrhages with reduction of blood circulation velocity were detected. We characterized the CNS phenotype by studying the expression pattern of wnt1 and neurog1 neural markers and by use of the Tg(neurod:EGFP/sox10:dsRed) transgenic line. The results evidenced that downregulation of pank2 severely impairs neuronal development, particularly in the anterior part of CNS (telencephalon). Whole-mount in situ hybridization analysis of the endothelial markers cadherin-5 and fli1a, and use of Tg(fli1a:EGFP/gata1a:dsRed) transgenic line, confirmed the essential role of pank2 in the formation of the vascular system. The specificity of the morpholino-induced phenotype was proved by the restoration of a normal development in a high percentage of embryos co-injected with pank2 mRNA. Also, addition of pantethine or CoA, but not of vitamin B5, to pank2 morpholino-injected embryos rescued the phenotype with high efficiency. The zebrafish model indicates the relevance of pank2 activity and CoA homeostasis for normal neuronal development and functioning and provides evidence of an unsuspected role for this enzyme and its product in vascular development. PMID:26476142
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Apoptosis is rapidly triggered by antisense depletion of MCL-1 in differentiating U937 cells.
Moulding, D A; Giles, R V; Spiller, D G; White, M R; Tidd, D M; Edwards, S W
2000-09-01
Mcl-1 is a member of the Bcl-2 protein family, which has been shown to delay apoptosis in transfection and/or overexpression experiments. As yet no gene knockout mice have been engineered, and so there is little evidence to show that loss of Mcl-1 expression is sufficient to trigger apoptosis. U937 cells constitutively express the antiapoptotic protein Bcl-2; but during differentiation, in response to the phorbol ester PMA (phorbol 12 beta-myristate 13 alpha-acetate), Mcl-1 is transiently induced. The purpose of this investigation was to determine the functional role played by Mcl-1 in this differentiation program. Mcl-1 expression was specifically disrupted by chimeric methylphosphonate/phosphodiester antisense oligodeoxynucleotides to just 5% of control levels. The depletion of Mcl-1 messenger RNA (mRNA) and protein was both rapid and specific, as indicated by the use of control oligodeoxynucleotides and analysis of the expression of other BCL2 family members and PMA-induced tumor necrosis factor-alpha (TNF-alpha). Specific depletion of Mcl-1 mRNA and protein, in the absence of changes in cellular levels of Bcl-2, results in a rapid entry into apoptosis. Levels of the proapoptotic protein Bax remained unchanged during differentiation, while Bak expression doubled within 24 hours. Apoptosis was detected within 4 hours of Mcl-1 antisense treatment by a variety of parameters including a novel live cell imaging technique allowing correlation of antisense treatment and apoptosis in individual cells. The induction of Mcl-1 is required to prevent apoptosis during differentiation of U937 cells, and the constitutive expression of Bcl-2 is unable to compensate for the loss of Mcl-1. (Blood. 2000;96:1756-1763)
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Size-uniform 200 nm particles: fabrication and application to magnetofection.
Mair, Lamar; Ford, Kris; Alam, M d Rowshon; Kole, Ryszard; Fisher, Michael; Superfine, Richard
2009-04-01
We report on the fabrication of arrays of mono- and multimetallic particles via metal evaporation onto lithographically patterned posts, as well as the magnetic force calibration and successful magnetofection of iron particles grown via this method. This work represents the first instance in which metal evaporation onto post structures was used for the formation of released, shape-defined metal particles. Also, our work represents the first use of lithographically defined particles as agents of magnetofection. Using these techniques it is possible to create particles with complex shapes and lateral dimensions as small as 40 nm. Our demonstrated compositionally flexible particles are highly size-uniform due to their photolithographically defined growth substrates, with particle dimensions along two axes fixed at 200 nm; the third axis dimension can be varied from 20 nm to 300 nm during the deposition procedure. Atomic percent of metals incorporated into the particle volume is highly tunable and particles have been synthesized with as many as four different metals. We performed magnetic force calibrations on a single particle size for iron particles using an axially magnetized NeFeB permanent magnet and comparisons are made with commercially available magnetic beads. In order to evalutate their usefulness as magnetofection agents, an antisense oligonucleotide (ODN) designed to correct the aberrant splicing of enhanced green fluorescent protein mRNA, was successfully transfected into a modified HeLa cell line. Magnetically enhanced gene delivery was accomplished in vitro using antisense ODN-laden iron particles followed by application of a field gradient. Magnetically enhanced transfection resulted in a 76% and 139% increase in fluorescence intensity when compared to Lipofectamine and antisense ODN-loaded particles delivered without magnetic treatment, respectively. To our knowledge, these experiments constitute the first use of lithographically defined particles as successful agents for magnetically enhanced transfection of an antisense oligonucleotide.
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Rodermel, S; Haley, J; Jiang, C Z; Tsai, C H; Bogorad, L
1996-01-01
Multimeric protein complexes in chloroplasts and mitochondria are generally composed of products of both nuclear and organelle genes of the cell. A central problem of eukaryotic cell biology is to identify and understand the molecular mechanisms for integrating the production and accumulation of the products of the two separate genomes. Ribulose bisphosphate carboxylase (Rubisco) is localized in the chloroplasts of photosynthetic eukaryotic cells and is composed of small subunits (SS) and large subunits (LS) coded for by nuclear rbcS and chloroplast rbcL genes, respectively. Transgenic tobacco plants containing antisense rbcS DNA have reduced levels of rbcS mRNA, normal levels of rbcL mRNA, and coordinately reduced LS and SS proteins. Our previous experiments indicated that the rate of translation of rbcL mRNA might be reduced in some antisense plants; direct evidence is presented here. After a short-term pulse there is less labeled LS protein in the transgenic plants than in wild-type plants, indicating that LS accumulation is controlled in the mutants at the translational and/or posttranslational levels. Consistent with a primary restriction at translation, fewer rbcL mRNAs are associated with polysomes of normal size and more are free or are associated with only a few ribosomes in the antisense plants. Effects of the rbcS antisense mutation on mRNA and protein accumulation, as well as on the distribution of mRNAs on polysomes, appear to be minimal for other chloroplast and nuclear photosynthetic genes. Our results suggest that SS protein abundance specifically contributes to the regulation of LS protein accumulation at the level of rbcL translation initiation. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 6 Fig. 7 Fig. 8 PMID:8632983
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Jiang, Weihua; Qin, Anqi X.; Bodell, Paul W.; Baldwin, Kenneth M.; Haddad, Fadia
2012-01-01
Postnatal development of fast skeletal muscle is characterized by a transition in expression of myosin heavy chain (MHC) isoforms, from primarily neonatal MHC at birth to primarily IIb MHC in adults, in a tightly coordinated manner. These isoforms are encoded by distinct genes, which are separated by ∼17 kb on rat chromosome 10. The neonatal-to-IIb MHC transition is inhibited by a hypothyroid state. We examined RNA products [mRNA, pre-mRNA, and natural antisense transcript (NAT)] of developmental and adult-expressed MHC genes (embryonic, neonatal, I, IIa, IIx, and IIb) at 2, 10, 20, and 40 days after birth in normal and thyroid-deficient rat neonates treated with propylthiouracil. We found that a long noncoding antisense-oriented RNA transcript, termed bII NAT, is transcribed from a site within the IIb-Neo intergenic region and across most of the IIb MHC gene. NATs have previously been shown to mediate transcriptional repression of sense-oriented counterparts. The bII NAT is transcriptionally regulated during postnatal development and in response to hypothyroidism. Evidence for a regulatory mechanism is suggested by an inverse relationship between IIb MHC and bII NAT in normal and hypothyroid-treated muscle. Neonatal MHC transcription is coordinately expressed with bII NAT. A comparative phylogenetic analysis also suggests that bII NAT-mediated regulation has been a conserved trait of placental mammals for most of the eutherian evolutionary history. The evidence in support of the regulatory model implicates long noncoding antisense RNA as a mechanism to coordinate the transition between neonatal and IIb MHC during postnatal development. PMID:22262309
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Pandorf, Clay E; Jiang, Weihua; Qin, Anqi X; Bodell, Paul W; Baldwin, Kenneth M; Haddad, Fadia
2012-04-01
Postnatal development of fast skeletal muscle is characterized by a transition in expression of myosin heavy chain (MHC) isoforms, from primarily neonatal MHC at birth to primarily IIb MHC in adults, in a tightly coordinated manner. These isoforms are encoded by distinct genes, which are separated by ∼17 kb on rat chromosome 10. The neonatal-to-IIb MHC transition is inhibited by a hypothyroid state. We examined RNA products [mRNA, pre-mRNA, and natural antisense transcript (NAT)] of developmental and adult-expressed MHC genes (embryonic, neonatal, I, IIa, IIx, and IIb) at 2, 10, 20, and 40 days after birth in normal and thyroid-deficient rat neonates treated with propylthiouracil. We found that a long noncoding antisense-oriented RNA transcript, termed bII NAT, is transcribed from a site within the IIb-Neo intergenic region and across most of the IIb MHC gene. NATs have previously been shown to mediate transcriptional repression of sense-oriented counterparts. The bII NAT is transcriptionally regulated during postnatal development and in response to hypothyroidism. Evidence for a regulatory mechanism is suggested by an inverse relationship between IIb MHC and bII NAT in normal and hypothyroid-treated muscle. Neonatal MHC transcription is coordinately expressed with bII NAT. A comparative phylogenetic analysis also suggests that bII NAT-mediated regulation has been a conserved trait of placental mammals for most of the eutherian evolutionary history. The evidence in support of the regulatory model implicates long noncoding antisense RNA as a mechanism to coordinate the transition between neonatal and IIb MHC during postnatal development.
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Natural antisense transcripts are significantly involved in regulation of drought stress in maize.
Xu, Jie; Wang, Qi; Freeling, Micheal; Zhang, Xuecai; Xu, Yunbi; Mao, Yan; Tang, Xin; Wu, Fengkai; Lan, Hai; Cao, Moju; Rong, Tingzhao; Lisch, Damon; Lu, Yanli
2017-05-19
Natural antisense transcripts (NATs) are a prominent and complex class of regulatory RNAs. Using strand-specific RNA sequencing, we identified 1769 sense and antisense transcript pairs (NAT pairs) in two maize inbreds with different sensitivity to drought, as well as in two derivative recombination inbred lines (RILs). A significantly higher proportion of NATs relative to non-NATs are specifically expressed under water stress (WS). Surprisingly, expression of sense and antisense transcripts produced by NAT pairs is significantly correlated, particularly under WS. We found an unexpected large proportion of NATs with protein coding potential, as estimated by ribosome release scores. Small RNAs significantly accumulate within NAT pairs, with 21 nt smRNA particularly enriched in overlapping regions of these pairs of genes. The abundance of these smRNAs is significantly altered in the leafbladeless1 mutant, suggesting that these genes may be regulated by the tasiRNA pathway. Further, NATs are significantly hypomethylated and include fewer transposable element sequences relative to non-NAT genes. NAT gene regions also exhibit higher levels of H3K36me3, H3K9ac, and H3K4me3, but lower levels of H3K27me3, indicating that NAT gene pairs generally exhibit an open chromatin configuration. Finally, NAT pairs in 368 diverse maize inbreds and 19 segregating populations were specifically enriched for polymorphisms associated with drought tolerance. Taken together, the data highlight the potential impact of that small RNAs and histone modifications have in regulation of NAT expression, and the significance of NATs in response to WS. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
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Howard, Thomas P.; Fryer, Michael J.; Singh, Prashant; Metodiev, Metodi; Lytovchenko, Anna; Obata, Toshihiro; Fernie, Alisdair R.; Kruger, Nicholas J.; Quick, W. Paul; Lloyd, Julie C.; Raines, Christine A.
2011-01-01
The thioredoxin-regulated chloroplast protein CP12 forms a multienzyme complex with the Calvin-Benson cycle enzymes phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). PRK and GAPDH are inactivated when present in this complex, a process shown in vitro to be dependent upon oxidized CP12. The importance of CP12 in vivo in higher plants, however, has not been investigated. Here, antisense suppression of CP12 in tobacco (Nicotiana tabacum) was observed to impact on NAD-induced PRK and GAPDH complex formation but had little effect on enzyme activity. Additionally, only minor changes in photosynthetic carbon fixation were observed. Despite this, antisense plants displayed changes in growth rates and morphology, including dwarfism and reduced apical dominance. The hypothesis that CP12 is essential to separate oxidative pentose phosphate pathway activity from Calvin-Benson cycle activity, as proposed in cyanobacteria, was tested. No evidence was found to support this role in tobacco. Evidence was seen, however, for a restriction to malate valve capacity, with decreases in NADP-malate dehydrogenase activity (but not protein levels) and pyridine nucleotide content. Antisense repression of CP12 also led to significant changes in carbon partitioning, with increased carbon allocation to the cell wall and the organic acids malate and fumarate and decreased allocation to starch and soluble carbohydrates. Severe decreases were also seen in 2-oxoglutarate content, a key indicator of cellular carbon sufficiency. The data presented here indicate that in tobacco, CP12 has a role in redox-mediated regulation of carbon partitioning from the chloroplast and provides strong in vivo evidence that CP12 is required for normal growth and development in plants. PMID:21865489
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The, Frans O; de Jonge, Wouter J; Bennink, Roel J; van den Wijngaard, Rene M; Boeckxstaens, Guy E
2005-09-01
Intestinal manipulation (IM) during abdominal surgery triggers the influx of inflammatory cells, leading to postoperative ileus. Prevention of this local muscle inflammation, using intercellular adhesion molecule-1 (ICAM-1) and leukocyte function-associated antigen-1-specific antibodies, has been shown to shorten postoperative ileus. However, the therapeutic use of antibodies has considerable disadvantages. The aim of the current study was to evaluate the effect of ISIS-3082, a mouse-specific ICAM-1 antisense oligonucleotide, on postoperative ileus in mice. Mice underwent a laparotomy or a laparotomy combined with IM after treatment with ICAM-1 antibodies, 0.1-10 mg kg(-1) ISIS-3082, saline or ISIS-8997 (scrambled control antisense oligonucleotides, 1 and 3 mg kg(-1)). At 24 h after surgery, gastric emptying of a 99mTC labelled semi-liquid meal was determined using scintigraphy. Intestinal inflammation was assessed by myeloperoxidase (MPO) activity in ileal muscle whole mounts. IM significantly reduced gastric emptying compared to laparotomy. Pretreatment with ISIS-3082 (0.1-1 mg kg(-1)) as well as ICAM-1 antibodies (10 mg kg(-1)), but not ISIS-8997 or saline, improved gastric emptying in a dose-dependent manner. This effect diminished with higher doses of ISIS-3082 (3-10 mg kg(-1)). Similarly, ISIS-3082 (0.1-1 mg kg(-1)) and ICAM-1 antibodies, but not ISIS-8997 or higher doses of ISIS-3082 (3-10 mg kg(-1)), reduced manipulation-induced inflammation. Immunohistochemistry showed reduction of ICAM-1 expression with ISIS-3082 only. ISIS-3082 pretreatment prevents postoperative ileus in mice by reduction of manipulation-induced local intestinal muscle inflammation. Our data suggest that targeting ICAM-1 using antisense oligonucleotides may represent a new therapeutic approach to the prevention of postoperative ileus.
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Semple, S C; Klimuk, S K; Harasym, T O; Dos Santos, N; Ansell, S M; Wong, K F; Maurer, N; Stark, H; Cullis, P R; Hope, M J; Scherrer, P
2001-02-09
Typical methods used for encapsulating antisense oligodeoxynucleotides (ODN) and plasmid DNA in lipid vesicles result in very low encapsulation efficiencies or employ cationic lipids that exhibit unfavorable pharmacokinetic and toxicity characteristics when administered intravenously. In this study, we describe and characterize a novel formulation process that utilizes an ionizable aminolipid (1,2-dioleoyl-3-dimethylammonium propane, DODAP) and an ethanol-containing buffer system for encapsulating large quantities (0.15--0.25 g ODN/g lipid) of polyanionic ODN in lipid vesicles. This process requires the presence of up to 40% ethanol (v/v) and initial formulation at acidic pH values where the DODAP is positively charged. In addition, the presence of a poly(ethylene glycol)-lipid was required during the formulation process to prevent aggregation. The 'stabilized antisense-lipid particles' (SALP) formed are stable on adjustment of the external pH to neutral pH values and the formulation process allows encapsulation efficiencies of up to 70%. ODN encapsulation was confirmed by nuclease protection assays and (31)P NMR measurements. Cryo-electron microscopy indicated that the final particles consisted of a mixed population of unilamellar and small multilamellar vesicles (80--140 nm diameter), the relative proportion of which was dependent on the initial ODN to lipid ratio. Finally, SALP exhibited significantly enhanced circulation lifetimes in mice relative to free antisense ODN, cationic lipid/ODN complexes and SALP prepared with quaternary aminolipids. Given the small particle sizes and improved encapsulation efficiency, ODN to lipid ratios, and circulation times of this formulation compared to others, we believe SALP represent a viable candidate for systemic applications involving nucleic acid therapeutics.
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Yu, Rosie Z; Lemonidis, Kristina M; Graham, Mark J; Matson, John E; Crooke, Rosanne M; Tribble, Diane L; Wedel, Mark K; Levin, Arthur A; Geary, Richard S
2009-03-01
The in vivo pharmacokinetics/pharmacodynamics of 2'-O-(2-methoxyethyl) (2'-MOE) modified antisense oligonucleotides (ASOs), targeting apolipoprotein B-100 (apoB-100), were characterized in multiple species. The species-specific apoB antisense inhibitors demonstrated target apoB mRNA reduction in a drug concentration and time-dependent fashion in mice, monkeys, and humans. Consistent with the concentration-dependent decreases in liver apoB mRNA, reductions in serum apoB, and LDL-C, and total cholesterol were concurrently observed in animal models and humans. Additionally, the long duration of effect after cessation of dosing correlated well with the elimination half-life of 2'-MOE modified apoB ASOs studied in mice (t(1/2) congruent with 20 days) and humans (t(1/2) congruent with 30 days) following parental administrations. The plasma concentrations of ISIS 301012, observed in the terminal elimination phase of both mice and monkeys were in equilibrium with liver. The partition ratios between liver and plasma were similar, approximately 6000:1, across species, and thus provide a surrogate for tissue exposure in humans. Using an inhibitory E(max) model, the ASO liver EC(50s) were 101+/-32, 119+/-15, and 300+/-191 microg/g of ASO in high-fat-fed (HF) mice, transgenic mice containing the human apoB transgene, and monkeys, respectively. The estimated liver EC(50) in man, extrapolated from trough plasma exposure, was 81+/-122 microg/g. Therefore, extraordinary consistency of the exposure-response relationship for the apoB antisense inhibitor was observed across species, including human. The cross-species PK/PD relationships provide confidence in the use of pharmacology animal models to predict human dosing for second-generation ASOs targeting the liver.
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Darsonval, Maud; Msadek, Tarek; Alexandre, Hervé
2015-01-01
Oenococcus oeni is a wine-associated lactic acid bacterium mostly responsible for malolactic fermentation in wine. In wine, O. oeni grows in an environment hostile to bacterial growth (low pH, low temperature, and ethanol) that induces stress response mechanisms. To survive, O. oeni is known to set up transitional stress response mechanisms through the synthesis of heat stress proteins (HSPs) encoded by the hsp genes, notably a unique small HSP named Lo18. Despite the availability of the genome sequence, characterization of O. oeni genes is limited, and little is known about the in vivo role of Lo18. Due to the lack of genetic tools for O. oeni, an efficient expression vector in O. oeni is still lacking, and deletion or inactivation of the hsp18 gene is not presently practicable. As an alternative approach, with the goal of understanding the biological function of the O. oeni hsp18 gene in vivo, we have developed an expression vector to produce antisense RNA targeting of hsp18 mRNA. Recombinant strains were exposed to multiple stresses inducing hsp18 gene expression: heat shock and acid shock. We showed that antisense attenuation of hsp18 affects O. oeni survival under stress conditions. These results confirm the involvement of Lo18 in heat and acid tolerance of O. oeni. Results of anisotropy experiments also confirm a membrane-protective role for Lo18, as previous observations had already suggested. This study describes a new, efficient tool to demonstrate the use of antisense technology for modulating gene expression in O. oeni. PMID:26452552
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A riboswitch-regulated antisense RNA in Listeria monocytogenes.
Mellin, J R; Tiensuu, Teresa; Bécavin, Christophe; Gouin, Edith; Johansson, Jörgen; Cossart, Pascale
2013-08-06
Riboswitches are ligand-binding elements located in 5' untranslated regions of messenger RNAs, which regulate expression of downstream genes. In Listeria monocytogenes, a vitamin B12-binding (B12) riboswitch was identified, not upstream of a gene but downstream, and antisense to the adjacent gene, pocR, suggesting it might regulate pocR in a nonclassical manner. In Salmonella enterica, PocR is a transcription factor that is activated by 1,2-propanediol, and subsequently activates expression of the pdu genes. The pdu genes mediate propanediol catabolism and are implicated in pathogenesis. As enzymes involved in propanediol catabolism require B12 as a cofactor, we hypothesized that the Listeria B12 riboswitch might be involved in pocR regulation. Here we demonstrate that the B12 riboswitch is transcribed as part of a noncoding antisense RNA, herein named AspocR. In the presence of B12, the riboswitch induces transcriptional termination, causing aspocR to be transcribed as a short transcript. In contrast, in the absence of B12, aspocR is transcribed as a long antisense RNA, which inhibits pocR expression. Regulation by AspocR ensures that pocR, and consequently the pdu genes, are maximally expressed only when both propanediol and B12 are present. Strikingly, AspocR can inhibit pocR expression in trans, suggesting it acts through a direct interaction with pocR mRNA. Together, this study demonstrates how pocR and the pdu genes can be regulated by B12 in bacteria and extends the classical definition of riboswitches from elements governing solely the expression of mRNAs to a wider role in controlling transcription of noncoding RNAs.
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Huschka, Ryan; Barhoumi, Aoune; Liu, Qing; Roth, Jack A.; Ji, Lin; Halas, Naomi J.
2013-01-01
The approach of RNA interference (RNAi)- using antisense DNA or RNA oligonucleotides to silence activity of a specific pathogenic gene transcript and reduce expression of the encoded protein- is very useful in dissecting genetic function and holds significant promise as a molecular therapeutic. A major obstacle in achieving gene silencing with RNAi technology is the systemic delivery of therapeutic oligonucleotides. Here we demonstrate an engineered gold nanoshell (NS)-based therapeutic oligonucleotide delivery vehicle, designed to release its cargo on demand upon illumination with a near-infrared (NIR) laser. A poly(L)lysine peptide (PLL) epilayer covalently attached to the NS surface (NS-PLL) is used to capture intact, single-stranded antisense DNA oligonucleotides, or alternatively, double-stranded short-interfering RNA (siRNA) molecules. Controlled release of the captured therapeutic oligonucleotides in each case is accomplished by continuous wave NIR laser irradiation at 800 nm, near the resonance wavelength of the nanoshell. Fluorescently tagged oligonucleotides were used to monitor the time-dependent release process and light-triggered endosomal release. A green fluorescent protein (GFP)-expressing human lung cancer H1299 cell line was used to determine cellular uptake and gene silencing mediated by the NS-PLL carrying GFP gene-specific single-stranded DNA antisense oligonucleotide (AON-GFP), or a double-stranded siRNA (siRNA-GFP), in vitro. Light-triggered delivery resulted in ∼ 47% and ∼49% downregulation of the targeted GFP expression by AON-GFP and siRNA-GFP, respectively. Cytotoxicity induced by both the NS-PLL delivery vector and by laser irradiation is minimal, as demonstrated by a XTT cell proliferation assay. PMID:22862291
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Wilson, K S; Matrone, G; Livingstone, D E W; Al-Dujaili, E A S; Mullins, J J; Tucker, C S; Hadoke, P W F; Kenyon, C J; Denvir, M A
2013-01-01
While glucocorticoids (GCs) are known to be present in the zebrafish embryo, little is known about their physiological roles at this stage. We hypothesised that GCs play key roles in stress response, hatching and swim activity during early development. To test this, whole embryo cortisol (WEC) and corticosteroid-related genes were measured in embryos from 6 to 120 h post fertilisation (hpf) by enzyme linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR). Stress response was assessed by change in WEC following stirring, hypoxia or brief electrical impulses applied to the bathing water. The impact of pharmacological and molecular GC manipulation on the stress response, spontaneous hatching and swim activity at different stages of development was also assessed. WEC levels demonstrated a biphasic pattern during development with a decrease from 0 to 36 hpf followed by a progressive increase towards 120 hpf. This was accompanied by a significant and sustained increase in the expression of genes encoding cyp11b1 (GC biosynthesis), hsd11b2 (GC metabolism) and gr (GC receptor) from 48 to 120 hpf. Metyrapone (Met), an inhibitor of 11β-hydroxylase (encoded by cyp11b1), and cyp11b1 morpholino (Mo) knockdown significantly reduced basal and stress-induced WEC levels at 72 and 120 hpf but not at 24 hpf. Spontaneous hatching and swim activity were significantly affected by manipulation of GC action from approximately 48 hpf onwards. We have identified a number of key roles of GCs in zebrafish embryos contributing to adaptive physiological responses under adverse conditions. The ability to alter GC action in the zebrafish embryo also highlights its potential value for GC research. PMID:24167225