123 results on '"Tocchini-Valentini GP"'
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2. How to build a kangaroo the way a kangaroo builds itself
- Author
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Tocchini-Valentini GP, Tocchini-Valentini MA, and Schlessinger D
- Published
- 2009
3. The mammalian gene function resource: the international knockout mouse consortium
- Author
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Bradley, A, Anastassiadis, K, Ayadi, Azzeddine, Battey, JF, Bell, C, Birling, MC, Bottomley, J, Brown, SD, Burger, A, Bult, CJ, Bushell, W, Collins, FS, Desaintes, C, Doe, B, Economides, A, Eppig, JT, Finnell, RH, D Fletcher, C, Fray, M, Frendewey, D, Friedel, RH, Grosveld, Frank, Hansen, J, Herault, Y, Hicks, G, Horlein, A, Houghton, R, de Angelis, MH, Huylebroeck, Danny, Iyer, V, de Jong, PJ, Kadin, JA, Kaloff, C, Kennedy, K, Koutsourakis, M (Manousos), Lloyd, KCK, Marschall, S, Mason, J, McKerlie, C, McLeod, MP, von Melchner, H, Moore, M, Mujica, AO, Nagy, A, Nefedov, M, Nutter, LM, Pavlovic, G, Peterson, JL, Pollock, J, Ramirez-Solis, R, Rancourt, DE, Raspa, M, Remacle, JE, Ringwald, M, Rosen, B, Rosenthal, N, Rossant, J, Noppinger, PR, Ryder, E, Schick, JZ, Schnutgen, F, Schofield, P, Seisenberger, C, Selloum, M, Simpson, EM, Skarnes, WC, Smedley, D, Stanford, WL, Stewart, AF, Stone, K, Swan, K, Tadepally, H, Teboul, L, Tocchini-Valentini, GP, Valenzuela, D, West, AP, Yamamura, K, Yoshinaga, Y, Wurst, W, Bradley, A, Anastassiadis, K, Ayadi, Azzeddine, Battey, JF, Bell, C, Birling, MC, Bottomley, J, Brown, SD, Burger, A, Bult, CJ, Bushell, W, Collins, FS, Desaintes, C, Doe, B, Economides, A, Eppig, JT, Finnell, RH, D Fletcher, C, Fray, M, Frendewey, D, Friedel, RH, Grosveld, Frank, Hansen, J, Herault, Y, Hicks, G, Horlein, A, Houghton, R, de Angelis, MH, Huylebroeck, Danny, Iyer, V, de Jong, PJ, Kadin, JA, Kaloff, C, Kennedy, K, Koutsourakis, M (Manousos), Lloyd, KCK, Marschall, S, Mason, J, McKerlie, C, McLeod, MP, von Melchner, H, Moore, M, Mujica, AO, Nagy, A, Nefedov, M, Nutter, LM, Pavlovic, G, Peterson, JL, Pollock, J, Ramirez-Solis, R, Rancourt, DE, Raspa, M, Remacle, JE, Ringwald, M, Rosen, B, Rosenthal, N, Rossant, J, Noppinger, PR, Ryder, E, Schick, JZ, Schnutgen, F, Schofield, P, Seisenberger, C, Selloum, M, Simpson, EM, Skarnes, WC, Smedley, D, Stanford, WL, Stewart, AF, Stone, K, Swan, K, Tadepally, H, Teboul, L, Tocchini-Valentini, GP, Valenzuela, D, West, AP, Yamamura, K, Yoshinaga, Y, and Wurst, W
- Abstract
In 2007, the International Knockout Mouse Consortium (IKMC) made the ambitious promise to generate mutations in virtually every protein-coding gene of the mouse genome in a concerted worldwide action. Now, 5 years later, the IKMC members have developed high-throughput gene trapping and, in particular, gene-targeting pipelines and generated more than 17,400 mutant murine embryonic stem (ES) cell clones and more than 1,700 mutant mouse strains, most of them conditional. A common IKMC web portal (www.knockoutmouse.org) has been established, allowing easy access to this unparalleled biological resource. The IKMC materials considerably enhance functional gene annotation of the mammalian genome and will have a major impact on future biomedical research.
- Published
- 2012
4. Extensive identification of genes involved in congenital and structural heart disorders and cardiomyopathy.
- Author
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Spielmann N, Miller G, Oprea TI, Hsu CW, Fobo G, Frishman G, Montrone C, Haseli Mashhadi H, Mason J, Munoz Fuentes V, Leuchtenberger S, Ruepp A, Wagner M, Westphal DS, Wolf C, Görlach A, Sanz-Moreno A, Cho YL, Teperino R, Brandmaier S, Sharma S, Galter IR, Östereicher MA, Zapf L, Mayer-Kuckuk P, Rozman J, Teboul L, Bunton-Stasyshyn RKA, Cater H, Stewart M, Christou S, Westerberg H, Willett AM, Wotton JM, Roper WB, Christiansen AE, Ward CS, Heaney JD, Reynolds CL, Prochazka J, Bower L, Clary D, Selloum M, Bou About G, Wendling O, Jacobs H, Leblanc S, Meziane H, Sorg T, Audain E, Gilly A, Rayner NW, Hitz MP, Zeggini E, Wolf E, Sedlacek R, Murray SA, Svenson KL, Braun RE, White JK, Kelsey L, Gao X, Shiroishi T, Xu Y, Seong JK, Mammano F, Tocchini-Valentini GP, Beaudet AL, Meehan TF, Parkinson H, Smedley D, Mallon AM, Wells SE, Grallert H, Wurst W, Marschall S, Fuchs H, Brown SDM, Flenniken AM, Nutter LMJ, McKerlie C, Herault Y, Lloyd KCK, Dickinson ME, Gailus-Durner V, and Hrabe de Angelis M
- Abstract
Clinical presentation of congenital heart disease is heterogeneous, making identification of the disease-causing genes and their genetic pathways and mechanisms of action challenging. By using in vivo electrocardiography, transthoracic echocardiography and microcomputed tomography imaging to screen 3,894 single-gene-null mouse lines for structural and functional cardiac abnormalities, here we identify 705 lines with cardiac arrhythmia, myocardial hypertrophy and/or ventricular dilation. Among these 705 genes, 486 have not been previously associated with cardiac dysfunction in humans, and some of them represent variants of unknown relevance (VUR). Mice with mutations in Casz1, Dnajc18, Pde4dip, Rnf38 or Tmem161b genes show developmental cardiac structural abnormalities, with their human orthologs being categorized as VUR. Using UK Biobank data, we validate the importance of the DNAJC18 gene for cardiac homeostasis by showing that its loss of function is associated with altered left ventricular systolic function. Our results identify hundreds of previously unappreciated genes with potential function in congenital heart disease and suggest causal function of five VUR in congenital heart disease., (© 2022. The Author(s).)
- Published
- 2022
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5. Archaeal tRNA-Splicing Endonuclease as an Effector for RNA Recombination and Novel Trans-Splicing Pathways in Eukaryotes.
- Author
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Tocchini-Valentini GD and Tocchini-Valentini GP
- Abstract
We have characterized a homodimeric tRNA endonuclease from the euryarchaeota Ferroplasma acidarmanus (FERAC), a facultative anaerobe which can grow at temperatures ranging from 35 to 42 °C. This enzyme, contrary to the eukaryal tRNA endonucleases and the homotetrameric Methanocaldococcus jannaschii (METJA) homologs, is able to cleave minimal BHB (bulge-helix-bulge) substrates at 30 °C. The expression of this enzyme in Schizosaccharomyces pombe (SCHPO) enables the use of its properties as effectors by inserting BHB motif introns into hairpin loops normally seen in mRNA transcripts. In addition, the FERAC endonuclease can create proteins with new functionalities through the recombination of protein domains.
- Published
- 2021
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6. Three-Dimensional X-ray Imaging of β-Galactosidase Reporter Activity by Micro-CT: Implication for Quantitative Analysis of Gene Expression.
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Ermakova O, Orsini T, Fruscoloni P, Chiani F, Gambadoro A, Putti S, Cirilli M, Mezzi A, Kaciulis S, Pasquini M, Raspa M, Scavizzi F, and Tocchini-Valentini GP
- Abstract
Acquisition of detailed anatomical and molecular knowledge from intact biological samples while preserving their native three-dimensional structure is still a challenging issue for imaging studies aiming to unravel a system's functions. Three-dimensional micro-CT X-ray imaging with a high spatial resolution in minimally perturbed naive non-transparent samples has recently gained increased popularity and broad application in biomedical research. Here, we describe a novel X-ray-based methodology for analysis of β-galactosidase ( lacZ ) reporter-driven gene expression in an intact murine brain ex vivo by micro-CT. The method relies on detection of bromine molecules in the product of the enzymatic β-galactosidase reaction. Enhancement of the X-ray signal is observed specifically in the regions of the murine brain where expression of the lacZ reporter gene is also detected histologically. We performed quantitative analysis of the expression levels of lacZ reporter activity by relative radiodensity estimation of the β-galactosidase/X-gal precipitate in situ. To demonstrate the feasibility of the method, we performed expression analysis of the Tsen54-lacZ reporter gene in the murine brain in a semi-quantitative manner. Human mutations in the Tsen54 gene cause pontocerebellar hypoplasia (PCH), a group of severe neurodegenerative disorders with both mental and motor deficits. Comparing relative levels of Tsen54 gene expression, we demonstrate that the highest Tsen54 expression is observed in anatomical brain substructures important for the normal motor and memory functions in mice.
- Published
- 2021
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7. A resource of targeted mutant mouse lines for 5,061 genes.
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Birling MC, Yoshiki A, Adams DJ, Ayabe S, Beaudet AL, Bottomley J, Bradley A, Brown SDM, Bürger A, Bushell W, Chiani F, Chin HG, Christou S, Codner GF, DeMayo FJ, Dickinson ME, Doe B, Donahue LR, Fray MD, Gambadoro A, Gao X, Gertsenstein M, Gomez-Segura A, Goodwin LO, Heaney JD, Hérault Y, de Angelis MH, Jiang ST, Justice MJ, Kasparek P, King RE, Kühn R, Lee H, Lee YJ, Liu Z, Lloyd KCK, Lorenzo I, Mallon AM, McKerlie C, Meehan TF, Fuentes VM, Newman S, Nutter LMJ, Oh GT, Pavlovic G, Ramirez-Solis R, Rosen B, Ryder EJ, Santos LA, Schick J, Seavitt JR, Sedlacek R, Seisenberger C, Seong JK, Skarnes WC, Sorg T, Steel KP, Tamura M, Tocchini-Valentini GP, Wang CL, Wardle-Jones H, Wattenhofer-Donzé M, Wells S, Wiles MV, Willis BJ, Wood JA, Wurst W, Xu Y, Teboul L, and Murray SA
- Subjects
- Animals, Information Dissemination, International Cooperation, Internet, Mice, Mice, Knockout, Mouse Embryonic Stem Cells cytology, Mouse Embryonic Stem Cells metabolism, Mutagenesis, Phenotype, Gene Deletion, Genetic Association Studies, Genome, Genotype
- Published
- 2021
- Full Text
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8. Gpr37l1/prosaposin receptor regulates Ptch1 trafficking, Shh production, and cell proliferation in cerebellar primary astrocytes.
- Author
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La Sala G, Di Pietro C, Matteoni R, Bolasco G, Marazziti D, and Tocchini-Valentini GP
- Abstract
Mammalian cerebellar astrocytes critically regulate the differentiation and maturation of neuronal Purkinje cells and granule precursors. The G protein-coupled receptor 37-like 1 (Gpr37l1) is expressed by Bergmann astrocytes and interacts with patched 1 (Ptch1) at peri-ciliary membranes. Cerebellar primary astrocyte cultures from wild-type and Gpr37l1 null mutant mouse pups were established and studied. Primary cilia were produced by cultures of both genotypes, as well as Ptch1 and smoothened (Smo) components of the sonic hedgehog (Shh) mitogenic pathway. Compared to wild-type cells, Gpr37l1
-/- astrocytes displayed striking increases in proliferative activity, Ptch1 protein expression and internalization, intracellular cholesterol content, ciliary localization of Smo, as well as a marked production of active Shh. Similar effects were reproduced by treating wild-type astrocytes with a putative prosaptide ligand of Gpr37l1. These findings indicate that Gpr37l1-Ptch1 interactions specifically regulate Ptch1 internalization and trafficking, with consequent stimulation of Shh production and activation of proliferative signaling., (© 2020 Wiley Periodicals LLC.)- Published
- 2020
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9. Soft windowing application to improve analysis of high-throughput phenotyping data.
- Author
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Haselimashhadi H, Mason JC, Munoz-Fuentes V, López-Gómez F, Babalola K, Acar EF, Kumar V, White J, Flenniken AM, King R, Straiton E, Seavitt JR, Gaspero A, Garza A, Christianson AE, Hsu CW, Reynolds CL, Lanza DG, Lorenzo I, Green JR, Gallegos JJ, Bohat R, Samaco RC, Veeraragavan S, Kim JK, Miller G, Fuchs H, Garrett L, Becker L, Kang YK, Clary D, Cho SY, Tamura M, Tanaka N, Soo KD, Bezginov A, About GB, Champy MF, Vasseur L, Leblanc S, Meziane H, Selloum M, Reilly PT, Spielmann N, Maier H, Gailus-Durner V, Sorg T, Hiroshi M, Yuichi O, Heaney JD, Dickinson ME, Wolfgang W, Tocchini-Valentini GP, Lloyd KCK, McKerlie C, Seong JK, Yann H, de Angelis MH, Brown SDM, Smedley D, Flicek P, Mallon AM, Parkinson H, and Meehan TF
- Subjects
- Animals, Genetic Association Studies, Humans, Mice, Phenotype, Population Health, Software
- Abstract
Motivation: High-throughput phenomic projects generate complex data from small treatment and large control groups that increase the power of the analyses but introduce variation over time. A method is needed to utlize a set of temporally local controls that maximizes analytic power while minimizing noise from unspecified environmental factors., Results: Here we introduce 'soft windowing', a methodological approach that selects a window of time that includes the most appropriate controls for analysis. Using phenotype data from the International Mouse Phenotyping Consortium (IMPC), adaptive windows were applied such that control data collected proximally to mutants were assigned the maximal weight, while data collected earlier or later had less weight. We applied this method to IMPC data and compared the results with those obtained from a standard non-windowed approach. Validation was performed using a resampling approach in which we demonstrate a 10% reduction of false positives from 2.5 million analyses. We applied the method to our production analysis pipeline that establishes genotype-phenotype associations by comparing mutant versus control data. We report an increase of 30% in significant P-values, as well as linkage to 106 versus 99 disease models via phenotype overlap with the soft-windowed and non-windowed approaches, respectively, from a set of 2082 mutant mouse lines. Our method is generalizable and can benefit large-scale human phenomic projects such as the UK Biobank and the All of Us resources., Availability and Implementation: The method is freely available in the R package SmoothWin, available on CRAN http://CRAN.R-project.org/package=SmoothWin., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2019. Published by Oxford University Press.)
- Published
- 2020
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10. Functional loss of Ccdc1 51 leads to hydrocephalus in a mouse model of primary ciliary dyskinesia.
- Author
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Chiani F, Orsini T, Gambadoro A, Pasquini M, Putti S, Cirilli M, Ermakova O, and Tocchini-Valentini GP
- Subjects
- Animals, Animals, Newborn, Body Patterning, Ciliary Motility Disorders diagnostic imaging, Ciliary Motility Disorders genetics, Disease Models, Animal, Ependyma diagnostic imaging, Ependyma pathology, Gene Expression Regulation, Hydrocephalus diagnostic imaging, Hydrocephalus genetics, Imaging, Three-Dimensional, Male, Mice, Inbred C57BL, Mice, Knockout, Spermatogenesis, Testis metabolism, X-Ray Microtomography, Carrier Proteins metabolism, Ciliary Motility Disorders pathology, Hydrocephalus pathology
- Abstract
Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder affecting normal structure and function of motile cilia, phenotypically manifested as chronic respiratory infections, laterality defects and infertility. Autosomal recessive mutations in genes encoding for different components of the ciliary axoneme have been associated with PCD in humans and in model organisms. The CCDC151 gene encodes for a coiled-coil axonemal protein that ensures correct attachment of outer dynein arm (ODA) complexes to microtubules. A correct arrangement of dynein arm complexes is required to provide the proper mechanical force necessary for cilia beat. Loss-of-function mutations in CCDC151 in humans leads to PCD disease with respiratory distress and defective left-right body asymmetry. In mice with the Ccdc151
Snbl loss-of-function mutation ( Snowball mutant), left-right body asymmetry with heart defects have been observed. Here, we demonstrate that loss of Ccdc151 gene function via targeted gene deletion in mice leads to perinatal lethality and congenital hydrocephalus. Microcomputed tomography (microCT) X-ray imaging of Ccdc151-β-galactosidase reporter expression in whole-mount brain and histological analysis show that Ccdc151 is expressed in ependymal cells lining the ventricular brain system, further confirming the role of Ccdc151 dysfunction in hydrocephalus development. Analyzing the features of hydrocephalus in the Ccdc151 -knockout animals by microCT volumetric imaging, we observe continuity of the aqueduct of Sylvius, indicating the communicating nature of hydrocephalus in the Ccdc151 -knockout animals. Congenital defects in left-right asymmetry and male infertility have been also observed in Ccdc151- null animals. Ccdc151 gene deletion in adult animals results in abnormal sperm counts and defective sperm motility.This article has an associated First Person interview with the joint first authors of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)- Published
- 2019
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11. Genetic ablation of Gpr37l1 delays tumor occurrence in Ptch1 +/- mouse models of medulloblastoma.
- Author
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Di Pietro C, La Sala G, Matteoni R, Marazziti D, and Tocchini-Valentini GP
- Subjects
- Animals, Carcinogenesis genetics, Cell Proliferation physiology, Cerebellar Neoplasms genetics, Cerebellar Neoplasms pathology, Female, Male, Medulloblastoma genetics, Medulloblastoma pathology, Mice, Mice, Inbred C57BL, Mice, Transgenic, Patched-1 Receptor genetics, Receptors, G-Protein-Coupled genetics, Carcinogenesis metabolism, Cerebellar Neoplasms metabolism, Medulloblastoma metabolism, Patched-1 Receptor metabolism, Receptors, G-Protein-Coupled deficiency
- Abstract
The G-protein coupled receptor 37-like 1 (Gpr37l1) is specifically expressed in most astrocytic glial cells, including cerebellar Bergmann astrocytes and interacts with patched 1 (Ptch1), a co-receptor of the sonic hedgehog (Shh)-smoothened (Smo) signaling complex. Gpr37l1 null mutant mice exhibit precocious post-natal cerebellar development, with altered Shh-Smo mitogenic cascade and premature down-regulation of granule cell precursor (GCP) proliferation. Gpr37l1 expression is downregulated in medulloblastoma (MB) and upregulated in glioma and glioblastoma tumors. Shh-associated MBs originate postnatally, from dysregulated hyperproliferation of GCPs in developing cerebellum's external granular layer (EGL), as shown in heterozygous Ptch1
+/- knock-out mouse strains that model human MB occurrence and progression. This study investigates cerebellar MB phenotypes in newly produced Gpr37l1, Ptch1 double mutant mice. Natural history analysis shows that Gpr37l1 genetic ablation, in Ptch1+/- model animals, results in marked deferment of post-natal tumor occurrence and decreased incidence of more aggressive tumor types. It is also associated with the delayed and diminished presence of more severe types of hyperplastic lesions in Ptch1+/- mice. Consistently, during early post-natal development Gpr37l1-/- ;Ptch1+/- pups exhibit reduction in cerebellar GCP proliferation and EGL thickness and a precocious, sustained expression of wingless-type MMTV integration site member 3 (Wnt3), a specific inhibitor of Shh-induced neuronal mitogenesis, in comparison with Ptch1+/- heterozygous single mutants. These findings highlight the specific involvement of Gpr37l1 in modulating postnatal cerebellar Shh-Ptch1-Smo mitogenic signaling in both normal and pathological conditions. The novel Gpr37l1-/- ;Ptch1+/- mouse models may thus be instrumental in the detailed characterization of the initial phases of Shh-associated MB insurgence and development., (Copyright © 2018 Elsevier Inc. All rights reserved.)- Published
- 2019
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12. Three-dimensional microCT imaging of murine embryonic development from immediate post-implantation to organogenesis: application for phenotyping analysis of early embryonic lethality in mutant animals.
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Ermakova O, Orsini T, Gambadoro A, Chiani F, and Tocchini-Valentini GP
- Subjects
- Animals, Embryo Loss diagnostic imaging, Embryo, Mammalian diagnostic imaging, Female, Gastrulation, Mice, Inbred C57BL, Phenotype, Uterus diagnostic imaging, Embryo Implantation genetics, Embryo Loss genetics, Embryo Loss pathology, Imaging, Three-Dimensional, Mutation genetics, Organogenesis genetics, X-Ray Microtomography
- Abstract
In this work, we applied three-dimensional microCT imaging to study murine embryogenesis in the range from immediate post-implantation period (embryonic day 5.5) to mid-gestation (embryonic day 12.5) with the resolution up to 1.4 µm/voxel. Also, we introduce an imaging procedure for non-invasive volumetric estimation of an entire litter of embryos within the maternal uterine structures. This method allows for an accurate, detailed and systematic morphometric analysis of both embryonic and extra-embryonic components during embryogenesis. Three-dimensional imaging of unperturbed embryos was performed to visualize the egg cylinder, primitive streak, gastrulation and early organogenesis stages of murine development in the C57Bl6/N mouse reference strain. Further, we applied our microCT imaging protocol to determine the earliest point when embryonic development is arrested in a mouse line with knockout for tRNA splicing endonuclease subunit Tsen54 gene. Our analysis determined that the embryonic development in Tsen54 null embryos does not proceed beyond implantation. We demonstrated that application of microCT imaging to entire litter of non-perturbed embryos greatly facilitate studies to unravel gene function during early embryogenesis and to determine the precise point at which embryonic development is arrested in mutant animals. The described method is inexpensive, does not require lengthy embryos dissection and can be applicable for detailed analysis of mutant mice at laboratory scale as well as for high-throughput projects.
- Published
- 2018
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13. MicroRNA degradation by a conserved target RNA regulates animal behavior.
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Bitetti A, Mallory AC, Golini E, Carrieri C, Carreño Gutiérrez H, Perlas E, Pérez-Rico YA, Tocchini-Valentini GP, Enright AJ, Norton WHJ, Mandillo S, O'Carroll D, and Shkumatava A
- Subjects
- Animals, Anxiety, Binding Sites, Brain metabolism, Cerebellum metabolism, Mice, RNA, Long Noncoding chemistry, RNA, Long Noncoding metabolism, RNA, Messenger metabolism, Zebrafish genetics, Zebrafish metabolism, Behavior, Animal, MicroRNAs metabolism
- Abstract
microRNAs (miRNAs) repress target transcripts through partial complementarity. By contrast, highly complementary miRNA-binding sites within viral and artificially engineered transcripts induce miRNA degradation in vitro and in cell lines. Here, we show that a genome-encoded transcript harboring a near-perfect and deeply conserved miRNA-binding site for miR-29 controls zebrafish and mouse behavior. This transcript originated in basal vertebrates as a long noncoding RNA (lncRNA) and evolved to the protein-coding gene NREP in mammals, where the miR-29-binding site is located within the 3' UTR. We show that the near-perfect miRNA site selectively triggers miR-29b destabilization through 3' trimming and restricts its spatial expression in the cerebellum. Genetic disruption of the miR-29 site within mouse Nrep results in ectopic expression of cerebellar miR-29b and impaired coordination and motor learning. Thus, we demonstrate an endogenous target-RNA-directed miRNA degradation event and its requirement for animal behavior.
- Published
- 2018
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14. Identification of genetic elements in metabolism by high-throughput mouse phenotyping.
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Rozman J, Rathkolb B, Oestereicher MA, Schütt C, Ravindranath AC, Leuchtenberger S, Sharma S, Kistler M, Willershäuser M, Brommage R, Meehan TF, Mason J, Haselimashhadi H, Hough T, Mallon AM, Wells S, Santos L, Lelliott CJ, White JK, Sorg T, Champy MF, Bower LR, Reynolds CL, Flenniken AM, Murray SA, Nutter LMJ, Svenson KL, West D, Tocchini-Valentini GP, Beaudet AL, Bosch F, Braun RB, Dobbie MS, Gao X, Herault Y, Moshiri A, Moore BA, Kent Lloyd KC, McKerlie C, Masuya H, Tanaka N, Flicek P, Parkinson HE, Sedlacek R, Seong JK, Wang CL, Moore M, Brown SD, Tschöp MH, Wurst W, Klingenspor M, Wolf E, Beckers J, Machicao F, Peter A, Staiger H, Häring HU, Grallert H, Campillos M, Maier H, Fuchs H, Gailus-Durner V, Werner T, and Hrabe de Angelis M
- Subjects
- Animals, Area Under Curve, Gene Regulatory Networks, Genome-Wide Association Study, High-Throughput Screening Assays, Humans, Metabolic Diseases genetics, Mice, Mice, Knockout, Phenotype, Basal Metabolism genetics, Blood Glucose metabolism, Body Weight genetics, Diabetes Mellitus, Type 2 genetics, Obesity genetics, Oxygen Consumption genetics, Triglycerides metabolism
- Abstract
Metabolic diseases are a worldwide problem but the underlying genetic factors and their relevance to metabolic disease remain incompletely understood. Genome-wide research is needed to characterize so-far unannotated mammalian metabolic genes. Here, we generate and analyze metabolic phenotypic data of 2016 knockout mouse strains under the aegis of the International Mouse Phenotyping Consortium (IMPC) and find 974 gene knockouts with strong metabolic phenotypes. 429 of those had no previous link to metabolism and 51 genes remain functionally completely unannotated. We compared human orthologues of these uncharacterized genes in five GWAS consortia and indeed 23 candidate genes are associated with metabolic disease. We further identify common regulatory elements in promoters of candidate genes. As each regulatory element is composed of several transcription factor binding sites, our data reveal an extensive metabolic phenotype-associated network of co-regulated genes. Our systematic mouse phenotype analysis thus paves the way for full functional annotation of the genome.
- Published
- 2018
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15. Corrigendum: High-throughput discovery of novel developmental phenotypes.
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Dickinson ME, Flenniken AM, Ji X, Teboul L, Wong MD, White JK, Meehan TF, Weninger WJ, Westerberg H, Adissu H, Baker CN, Bower L, Brown JM, Caddle LB, Chiani F, Clary D, Cleak J, Daly MJ, Denegre JM, Doe B, Dolan ME, Edie Helmut Fuchs SM, Gailus-Durner V, Galli A, Gambadoro A, Gallegos J, Guo S, Horner NR, Hsu CW, Johnson SJ, Kalaga S, Keith LC, Lanoue L, Lawson TN, Lek M, Mark M, Marschall S, Mason J, McElwee ML, Nutter SNLMJ, Peterson KA, Ramirez-Solis R, Rowland DJ, Ryder E, Samocha KE, Seavitt JR, Selloum M, Szoke-Kovacs Z, Tamura M, Trainor AG, Tudose I, Wakana S, Warren J, Wendling O, West DB, Wong L, Yoshiki A, Wurst W, MacArthur DG, Tocchini-Valentini GP, Gao X, Flicek P, Bradley A, Skarnes WC, Justice MJ, Parkinson HE, Moore M, Wells S, Braun RE, Svenson KL, de Angelis MH, Herault Y, Mohun T, Mallon AM, Henkelman RM, Brown SDM, Adams DJ, Lloyd KCK, McKerlie C, Beaudet AL, and Murray MBSA
- Abstract
This corrects the article DOI: 10.1038/nature19356.
- Published
- 2017
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16. A large scale hearing loss screen reveals an extensive unexplored genetic landscape for auditory dysfunction.
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Bowl MR, Simon MM, Ingham NJ, Greenaway S, Santos L, Cater H, Taylor S, Mason J, Kurbatova N, Pearson S, Bower LR, Clary DA, Meziane H, Reilly P, Minowa O, Kelsey L, Tocchini-Valentini GP, Gao X, Bradley A, Skarnes WC, Moore M, Beaudet AL, Justice MJ, Seavitt J, Dickinson ME, Wurst W, de Angelis MH, Herault Y, Wakana S, Nutter LMJ, Flenniken AM, McKerlie C, Murray SA, Svenson KL, Braun RE, West DB, Lloyd KCK, Adams DJ, White J, Karp N, Flicek P, Smedley D, Meehan TF, Parkinson HE, Teboul LM, Wells S, Steel KP, Mallon AM, and Brown SDM
- Subjects
- Animals, Datasets as Topic, Genetic Testing, Hearing Loss epidemiology, Hearing Tests, Mice, Mice, Knockout, Phenotype, Hearing Loss genetics, Protein Interaction Maps genetics
- Abstract
The developmental and physiological complexity of the auditory system is likely reflected in the underlying set of genes involved in auditory function. In humans, over 150 non-syndromic loci have been identified, and there are more than 400 human genetic syndromes with a hearing loss component. Over 100 non-syndromic hearing loss genes have been identified in mouse and human, but we remain ignorant of the full extent of the genetic landscape involved in auditory dysfunction. As part of the International Mouse Phenotyping Consortium, we undertook a hearing loss screen in a cohort of 3006 mouse knockout strains. In total, we identify 67 candidate hearing loss genes. We detect known hearing loss genes, but the vast majority, 52, of the candidate genes were novel. Our analysis reveals a large and unexplored genetic landscape involved with auditory function.The full extent of the genetic basis for hearing impairment is unknown. Here, as part of the International Mouse Phenotyping Consortium, the authors perform a hearing loss screen in 3006 mouse knockout strains and identify 52 new candidate genes for genetic hearing loss.
- Published
- 2017
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17. Disease model discovery from 3,328 gene knockouts by The International Mouse Phenotyping Consortium.
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Meehan TF, Conte N, West DB, Jacobsen JO, Mason J, Warren J, Chen CK, Tudose I, Relac M, Matthews P, Karp N, Santos L, Fiegel T, Ring N, Westerberg H, Greenaway S, Sneddon D, Morgan H, Codner GF, Stewart ME, Brown J, Horner N, Haendel M, Washington N, Mungall CJ, Reynolds CL, Gallegos J, Gailus-Durner V, Sorg T, Pavlovic G, Bower LR, Moore M, Morse I, Gao X, Tocchini-Valentini GP, Obata Y, Cho SY, Seong JK, Seavitt J, Beaudet AL, Dickinson ME, Herault Y, Wurst W, de Angelis MH, Lloyd KCK, Flenniken AM, Nutter LMJ, Newbigging S, McKerlie C, Justice MJ, Murray SA, Svenson KL, Braun RE, White JK, Bradley A, Flicek P, Wells S, Skarnes WC, Adams DJ, Parkinson H, Mallon AM, Brown SDM, and Smedley D
- Subjects
- Animals, Female, Genetic Diseases, Inborn, Genetic Predisposition to Disease, Humans, Male, Mice, Mice, Knockout, Phenotype, Disease Models, Animal, Gene Knockout Techniques
- Abstract
Although next-generation sequencing has revolutionized the ability to associate variants with human diseases, diagnostic rates and development of new therapies are still limited by a lack of knowledge of the functions and pathobiological mechanisms of most genes. To address this challenge, the International Mouse Phenotyping Consortium is creating a genome- and phenome-wide catalog of gene function by characterizing new knockout-mouse strains across diverse biological systems through a broad set of standardized phenotyping tests. All mice will be readily available to the biomedical community. Analyzing the first 3,328 genes identified models for 360 diseases, including the first models, to our knowledge, for type C Bernard-Soulier, Bardet-Biedl-5 and Gordon Holmes syndromes. 90% of our phenotype annotations were novel, providing functional evidence for 1,092 genes and candidates in genetically uncharacterized diseases including arrhythmogenic right ventricular dysplasia 3. Finally, we describe our role in variant functional validation with The 100,000 Genomes Project and others.
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- 2017
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18. Primary Cilia in the Murine Cerebellum and in Mutant Models of Medulloblastoma.
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Di Pietro C, Marazziti D, La Sala G, Abbaszadeh Z, Golini E, Matteoni R, and Tocchini-Valentini GP
- Subjects
- Animals, Animals, Newborn, Cerebellum pathology, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Mutation genetics, Cerebellar Neoplasms genetics, Cerebellar Neoplasms pathology, Cilia genetics, Cilia pathology, Medulloblastoma genetics, Medulloblastoma pathology
- Abstract
Cellular primary cilia crucially sense and transduce extracellular physicochemical stimuli. Cilium-mediated developmental signaling is tissue and cell type specific. Primary cilia are required for cerebellar differentiation and sonic hedgehog (Shh)-dependent proliferation of neuronal granule precursors. The mammalian G-protein-coupled receptor 37-like 1 is specifically expressed in cerebellar Bergmann glia astrocytes and participates in regulating postnatal cerebellar granule neuron proliferation/differentiation and Bergmann glia and Purkinje neuron maturation. The mouse receptor protein interacts with the patched 1 component of the cilium-associated Shh receptor complex. Mice heterozygous for patched homolog 1 mutations, like heterozygous patched 1 humans, have a higher incidence of Shh subgroup medulloblastoma (MB) and other tumors. Cerebellar cells bearing primary cilia were identified during postnatal development and in adulthood in two mouse strains with altered Shh signaling: a G-protein-coupled receptor 37-like 1 null mutant and an MB-susceptible, heterozygous patched homolog 1 mutant. In addition to granule and Purkinje neurons, primary cilia were also expressed by Bergmann glia astrocytes in both wild-type and mutant animals, from birth to adulthood. Variations in ciliary number and length were related to the different levels of neuronal and glial cell proliferation and maturation, during postnatal cerebellar development. Primary cilia were also detected in pre-neoplastic MB lesions in heterozygous patched homolog 1 mutant mice and they could represent specific markers for the development and analysis of novel cerebellar oncogenic models.
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- 2017
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19. High-throughput discovery of novel developmental phenotypes.
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Dickinson ME, Flenniken AM, Ji X, Teboul L, Wong MD, White JK, Meehan TF, Weninger WJ, Westerberg H, Adissu H, Baker CN, Bower L, Brown JM, Caddle LB, Chiani F, Clary D, Cleak J, Daly MJ, Denegre JM, Doe B, Dolan ME, Edie SM, Fuchs H, Gailus-Durner V, Galli A, Gambadoro A, Gallegos J, Guo S, Horner NR, Hsu CW, Johnson SJ, Kalaga S, Keith LC, Lanoue L, Lawson TN, Lek M, Mark M, Marschall S, Mason J, McElwee ML, Newbigging S, Nutter LM, Peterson KA, Ramirez-Solis R, Rowland DJ, Ryder E, Samocha KE, Seavitt JR, Selloum M, Szoke-Kovacs Z, Tamura M, Trainor AG, Tudose I, Wakana S, Warren J, Wendling O, West DB, Wong L, Yoshiki A, MacArthur DG, Tocchini-Valentini GP, Gao X, Flicek P, Bradley A, Skarnes WC, Justice MJ, Parkinson HE, Moore M, Wells S, Braun RE, Svenson KL, de Angelis MH, Herault Y, Mohun T, Mallon AM, Henkelman RM, Brown SD, Adams DJ, Lloyd KC, McKerlie C, Beaudet AL, Bućan M, and Murray SA
- Subjects
- Animals, Conserved Sequence genetics, Disease, Genome-Wide Association Study, High-Throughput Screening Assays, Humans, Imaging, Three-Dimensional, Mice, Mice, Inbred C57BL, Mice, Knockout, Penetrance, Polymorphism, Single Nucleotide genetics, Sequence Homology, Embryo, Mammalian embryology, Embryo, Mammalian metabolism, Genes, Essential genetics, Genes, Lethal genetics, Mutation genetics, Phenotype
- Abstract
Approximately one-third of all mammalian genes are essential for life. Phenotypes resulting from knockouts of these genes in mice have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5,000 knockout mouse lines, here we identify 410 lethal genes during the production of the first 1,751 unique gene knockouts. Using a standardized phenotyping platform that incorporates high-resolution 3D imaging, we identify phenotypes at multiple time points for previously uncharacterized genes and additional phenotypes for genes with previously reported mutant phenotypes. Unexpectedly, our analysis reveals that incomplete penetrance and variable expressivity are common even on a defined genetic background. In addition, we show that human disease genes are enriched for essential genes, thus providing a dataset that facilitates the prioritization and validation of mutations identified in clinical sequencing efforts.
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- 2016
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20. Analysis of random PCR-originated mutants of the yeast Ste2 and Ste3 receptors.
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Gastaldi S, Zamboni M, Bolasco G, Di Segni G, and Tocchini-Valentini GP
- Subjects
- Amino Acid Sequence, Base Sequence, DNA, Fungal genetics, Genes, Fungal, Homeodomain Proteins genetics, Mating Factor metabolism, Polymerase Chain Reaction, Repressor Proteins genetics, Sequence Analysis, DNA, Signal Transduction genetics, Receptors, Mating Factor genetics, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins genetics
- Abstract
The G protein-coupled receptors Ste2 and Ste3 bind α- and a-factor, respectively, in Saccharomyces cerevisiae. These receptors share a similar conformation, with seven transmembrane segments, three intracellular loops, a C-terminus tail, and three extracellular loops. However, the amino acid sequences of these two receptors bear no resemblance to each other. Coincidently the two ligands, α- and a-factor, have different sequences. Both receptors activate the same G protein. To identify amino acid residues that are important for signal transduction, the STE2 and STE3 genes were mutagenized by a random PCR-based method. Mutant receptors were analyzed in MATα cells mutated in the ITC1 gene, whose product represses transcription of a-specific genes in MATα. Expression of STE2 or STE3 in these cells results in autocrine activation of the mating pathway, since this strain produces the Ste2 receptor in addition to its specific ligand, α-factor. It also produces a-factor in addition to its specific receptor, Ste3. Therefore, this strain provides a convenient model to analyze mutants of both receptors in the same background. Many hyperactive mutations were found in STE3, whereas none was detected in STE2. This result is consistent with the different strategies that the two genes have adopted to be expressed., (© 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)
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- 2016
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21. Genome Wide Conditional Mouse Knockout Resources.
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Kaloff C, Anastassiadis K, Ayadi A, Baldock R, Beig J, Birling MC, Bradley A, Brown S, Bürger A, Bushell W, Chiani F, Collins FS, Doe B, Eppig JT, Finnel RH, Fletcher C, Flicek P, Fray M, Friedel RH, Gambadoro A, Gates H, Hansen J, Herault Y, Hicks GG, Hörlein A, Hrabé de Angelis M, Iyer V, de Jong PJ, Koscielny G, Kühn R, Liu P, Lloyd KC, Lopez RG, Marschall S, Martínez S, McKerlie C, Meehan T, von Melchner H, Moore M, Murray SA, Nagy A, Nutter L, Pavlovic G, Pombero A, Prosser H, Ramirez-Solis R, Ringwald M, Rosen B, Rosenthal N, Rossant J, Ruiz Noppinger P, Ryder E, Skarnes WC, Schick J, Schnütgen F, Schofield P, Seisenberger C, Selloum M, Smedley D, Simpson EM, Stewart AF, Teboul L, Tocchini Valentini GP, Valenzuela D, West A, and Wurst W
- Abstract
The International Knockout Mouse Consortium (IKMC) developed high throughput gene trapping and gene targeting pipelines that produced mostly conditional mutations of more than 18,500 genes in C57BL/6N mouse embryonic stem (ES) cells which have been archived and are freely available to the research community as a frozen resource. From this unprecedented resource more than 6,000 mutant mouse strains have been produced by the IKMC and mostly the International Mouse Phenotyping Consortium (IMPC). In addition, a cre-driver resource was established including 250 inducible cre-driver mouse strains in a C57BL/6 background. Complementing the cre-driver resource, a collection of comprising 27 cre-driver rAAVs has also been produced. The resources can be easily accessed at the IKMC/IMPC web portal (www.mousephenotype.org). The IKMC/IMPC resource is a standardized reference library of mouse models with defined genetic backgrounds that enables the analysis of gene-disease associations in mice of different genetic makeup and should therefore have a major impact on biomedical research., Competing Interests: Conflict of interest: EMS, and the University of British Columbia have filed for US patent protection on a subset of the MiniPs used in cre-driver strains developed by Pleiades/CanEuCre.
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- 2016
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22. Analysis of mammalian gene function through broad-based phenotypic screens across a consortium of mouse clinics.
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de Angelis MH, Nicholson G, Selloum M, White J, Morgan H, Ramirez-Solis R, Sorg T, Wells S, Fuchs H, Fray M, Adams DJ, Adams NC, Adler T, Aguilar-Pimentel A, Ali-Hadji D, Amann G, André P, Atkins S, Auburtin A, Ayadi A, Becker J, Becker L, Bedu E, Bekeredjian R, Birling MC, Blake A, Bottomley J, Bowl M, Brault V, Busch DH, Bussell JN, Calzada-Wack J, Cater H, Champy MF, Charles P, Chevalier C, Chiani F, Codner GF, Combe R, Cox R, Dalloneau E, Dierich A, Di Fenza A, Doe B, Duchon A, Eickelberg O, Esapa CT, El Fertak L, Feigel T, Emelyanova I, Estabel J, Favor J, Flenniken A, Gambadoro A, Garrett L, Gates H, Gerdin AK, Gkoutos G, Greenaway S, Glasl L, Goetz P, Da Cruz IG, Götz A, Graw J, Guimond A, Hans W, Hicks G, Hölter SM, Höfler H, Hancock JM, Hoehndorf R, Hough T, Houghton R, Hurt A, Ivandic B, Jacobs H, Jacquot S, Jones N, Karp NA, Katus HA, Kitchen S, Klein-Rodewald T, Klingenspor M, Klopstock T, Lalanne V, Leblanc S, Lengger C, le Marchand E, Ludwig T, Lux A, McKerlie C, Maier H, Mandel JL, Marschall S, Mark M, Melvin DG, Meziane H, Micklich K, Mittelhauser C, Monassier L, Moulaert D, Muller S, Naton B, Neff F, Nolan PM, Nutter LM, Ollert M, Pavlovic G, Pellegata NS, Peter E, Petit-Demoulière B, Pickard A, Podrini C, Potter P, Pouilly L, Puk O, Richardson D, Rousseau S, Quintanilla-Fend L, Quwailid MM, Racz I, Rathkolb B, Riet F, Rossant J, Roux M, Rozman J, Ryder E, Salisbury J, Santos L, Schäble KH, Schiller E, Schrewe A, Schulz H, Steinkamp R, Simon M, Stewart M, Stöger C, Stöger T, Sun M, Sunter D, Teboul L, Tilly I, Tocchini-Valentini GP, Tost M, Treise I, Vasseur L, Velot E, Vogt-Weisenhorn D, Wagner C, Walling A, Weber B, Wendling O, Westerberg H, Willershäuser M, Wolf E, Wolter A, Wood J, Wurst W, Yildirim AÖ, Zeh R, Zimmer A, Zimprich A, Holmes C, Steel KP, Herault Y, Gailus-Durner V, Mallon AM, and Brown SD
- Subjects
- Animals, Female, Heterozygote, Homozygote, Humans, Male, Mice, Inbred C57BL, Mice, Knockout, Molecular Sequence Annotation, Mutation, Phenotype, Genetic Association Studies
- Abstract
The function of the majority of genes in the mouse and human genomes remains unknown. The mouse embryonic stem cell knockout resource provides a basis for the characterization of relationships between genes and phenotypes. The EUMODIC consortium developed and validated robust methodologies for the broad-based phenotyping of knockouts through a pipeline comprising 20 disease-oriented platforms. We developed new statistical methods for pipeline design and data analysis aimed at detecting reproducible phenotypes with high power. We acquired phenotype data from 449 mutant alleles, representing 320 unique genes, of which half had no previous functional annotation. We captured data from over 27,000 mice, finding that 83% of the mutant lines are phenodeviant, with 65% demonstrating pleiotropy. Surprisingly, we found significant differences in phenotype annotation according to zygosity. New phenotypes were uncovered for many genes with previously unknown function, providing a powerful basis for hypothesis generation and further investigation in diverse systems.
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- 2015
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23. Applying the ARRIVE Guidelines to an In Vivo Database.
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Karp NA, Meehan TF, Morgan H, Mason JC, Blake A, Kurbatova N, Smedley D, Jacobsen J, Mott RF, Iyer V, Matthews P, Melvin DG, Wells S, Flenniken AM, Masuya H, Wakana S, White JK, Lloyd KC, Reynolds CL, Paylor R, West DB, Svenson KL, Chesler EJ, de Angelis MH, Tocchini-Valentini GP, Sorg T, Herault Y, Parkinson H, Mallon AM, and Brown SD
- Subjects
- Animals, Mice, Animal Experimentation standards, Databases as Topic, Guidelines as Topic, Phenotype
- Abstract
The Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines were developed to address the lack of reproducibility in biomedical animal studies and improve the communication of research findings. While intended to guide the preparation of peer-reviewed manuscripts, the principles of transparent reporting are also fundamental for in vivo databases. Here, we describe the benefits and challenges of applying the guidelines for the International Mouse Phenotyping Consortium (IMPC), whose goal is to produce and phenotype 20,000 knockout mouse strains in a reproducible manner across ten research centres. In addition to ensuring the transparency and reproducibility of the IMPC, the solutions to the challenges of applying the ARRIVE guidelines in the context of IMPC will provide a resource to help guide similar initiatives in the future.
- Published
- 2015
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24. Modulation of Dhh signaling and altered Sertoli cell function in mice lacking the GPR37-prosaposin receptor.
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La Sala G, Marazziti D, Di Pietro C, Golini E, Matteoni R, and Tocchini-Valentini GP
- Subjects
- Animals, Apoptosis, Blotting, Western, Cell Differentiation, Cell Proliferation, Cells, Cultured, Hedgehog Proteins genetics, Immunoenzyme Techniques, Immunoprecipitation, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Patched Receptors, Patched-1 Receptor, RNA, Messenger genetics, Real-Time Polymerase Chain Reaction, Receptors, Cell Surface genetics, Receptors, Cell Surface metabolism, Reverse Transcriptase Polymerase Chain Reaction, Sertoli Cells cytology, Signal Transduction, Testis cytology, Gene Expression Regulation, Hedgehog Proteins metabolism, Receptors, G-Protein-Coupled physiology, Saposins metabolism, Sertoli Cells metabolism, Spermatogenesis physiology, Testis metabolism
- Abstract
The mammalian G-protein-coupled receptor 37 (GPR37) is expressed in brain, in adult testis, and during the early phase of gonad differentiation. Somatic Sertoli cells (SCs) are located within the seminiferous tubules where they support the germinal epithelium. An adequate number of SCs is required for the complete prepubertal differentiation of germ cells and adult fertility. This study shows that Gpr37 and its ligand prosaposin are both postnatally expressed by SCs, whose proliferation and maturation are affected in Gpr37-null mutant mice during postnatal testicular development. Mutant pups show a delayed timing in sperm cell development, with a partial arrest of spermatocytes at the meiotic pachytene (e.g., 1.5-fold increase in Gpr37(-/-) P21 pups) and their increased apoptosis (e.g., 1.8-fold and 3.5-fold increase in Gpr37(-/-) P21 and adult mice, respectively). Mutant adults have reduced testis weight (wild type, 299 ± 5 mg; knockout, 258 ± 16 mg; P < 0.05) and epididymal sperm count and motility (e.g., 1.5-fold and 1.45-fold decrease in Gpr37(-/-) mice, respectively). Lack of Gpr37 results in the reduction in androgen receptor levels during prepubertal testis development, alongside the altered expression of SC maturation markers. It also affects the prepubertal testis expression of desert hedgehog (Dhh) mitogenic cascade components (Dhh, 1.3-fold increase in Gpr37(-/-) P10 and P21 pups; Gli2, 1.4-fold and 1.6-fold increase in Gpr37(-/-) P10 and P21 pups, respectively) including patched homolog 1 (1.3-fold increase in Gpr37(-/-) P10 and P21 pups), which is found localized in prepubertal SCs and is associated with Gpr37 in cultured primary SC samples. These results indicate that Gpr37 is a specific modulator of murine testis Dhh mitogenic signaling and SC proliferation and maturation., (© FASEB.)
- Published
- 2015
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25. Early motor deficits in mouse disease models are reliably uncovered using an automated home-cage wheel-running system: a cross-laboratory validation.
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Mandillo S, Heise I, Garbugino L, Tocchini-Valentini GP, Giuliani A, Wells S, and Nolan PM
- Subjects
- Animals, Automation, Behavior, Animal, Humans, Male, Mice, Inbred C57BL, Mice, Neurologic Mutants, Mice, Transgenic, Principal Component Analysis, Reference Standards, Reproducibility of Results, Rotarod Performance Test, Superoxide Dismutase metabolism, Disease Models, Animal, Huntington Disease physiopathology, Laboratories, Motor Activity, Physical Conditioning, Animal, Running
- Abstract
Deficits in motor function are debilitating features in disorders affecting neurological, neuromuscular and musculoskeletal systems. Although these disorders can vary greatly with respect to age of onset, symptomatic presentation, rate of progression and severity, the study of these disease models in mice is confined to the use of a small number of tests, most commonly the rotarod test. To expand the repertoire of meaningful motor function tests in mice, we tested, optimised and validated an automated home-cage-based running-wheel system, incorporating a conventional wheel with evenly spaced rungs and a complex wheel with particular rungs absent. The system enables automated assessment of motor function without handler interference, which is desirable in longitudinal studies involving continuous monitoring of motor performance. In baseline studies at two test centres, consistently significant differences in performance on both wheels were detectable among four commonly used inbred strains. As further validation, we studied performance in mutant models of progressive neurodegenerative diseases--Huntington's disease [TgN(HD82Gln)81Dbo; referred to as HD mice] and amyotrophic lateral sclerosis [Tg(SOD1G93A)(dl)1/GurJ; referred to as SOD1 mice]--and in a mutant strain with subtle gait abnormalities, C-Snap25(Bdr)/H (Blind-drunk, Bdr). In both models of progressive disease, as with the third mutant, we could reliably and consistently detect specific motor function deficits at ages far earlier than any previously recorded symptoms in vivo: 7-8 weeks for the HD mice and 12 weeks for the SOD1 mice. We also conducted longitudinal analysis of rotarod and grip strength performance, for which deficits were still not detectable at 12 weeks and 23 weeks, respectively. Several new parameters of motor behaviour were uncovered using principal component analysis, indicating that the wheel-running assay could record features of motor function that are independent of rotarod performance. This represents a powerful new method to detect motor deficits at pre-symptomatic stages in mouse disease models and should be considered as a valid tool to investigate the efficacy of therapeutic agents.
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- 2014
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26. Precocious cerebellum development and improved motor functions in mice lacking the astrocyte cilium-, patched 1-associated Gpr37l1 receptor.
- Author
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Marazziti D, Di Pietro C, Golini E, Mandillo S, La Sala G, Matteoni R, and Tocchini-Valentini GP
- Subjects
- Animals, Blotting, Western, Cell Proliferation, Cerebellum cytology, DNA Primers genetics, Fluorescent Antibody Technique, Gene Deletion, Genetic Vectors genetics, Hedgehog Proteins metabolism, Immunoprecipitation, In Situ Hybridization, Mice, Mice, Knockout, Mitogens metabolism, Patched Receptors, Patched-1 Receptor, Receptors, Cell Surface metabolism, Cerebellum growth & development, Neuroglia physiology, Psychomotor Performance physiology, Purkinje Cells physiology, Receptors, G-Protein-Coupled genetics
- Abstract
In the developing cerebellum, the proliferation and differentiation of glial and neuronal cell types depend on the modulation of the sonic hedgehog (Shh) signaling pathway. The vertebrate G-protein-coupled receptor 37-like 1 (GPR37L1) gene encodes a putative G-protein-coupled receptor that is expressed in newborn and adult cerebellar Bergmann glia astrocytes. This study shows that the ablation of the murine Gpr37l1 gene results in premature down-regulation of proliferation of granule neuron precursors and precocious maturation of Bergmann glia and Purkinje neurons. These alterations are accompanied by improved adult motor learning and coordination. Gpr37l1(-/-) mice also exhibit specific modifications of the Shh signaling cascade. Specific assays show that in Bergmann glia cells Gpr37l1 is associated with primary cilium membranes and it specifically interacts and colocalizes with the Shh primary receptor, patched 1. These findings indicate that the patched 1-associated Gpr37l1 receptor participates in the regulation of postnatal cerebellum development by modulating the Shh pathway.
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- 2013
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27. Highly efficient, in vivo optimized, archaeal endonuclease for controlled RNA splicing in mammalian cells.
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Putti S, Calandra P, Rossi N, Scarabino D, Deidda G, and Tocchini-Valentini GP
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- Archaeal Proteins genetics, Blotting, Western, Cell Line, Endonucleases genetics, Fluorescent Antibody Technique, Humans, Polymerase Chain Reaction, Archaea enzymology, Archaea genetics, Archaeal Proteins metabolism, Endonucleases metabolism, RNA Splicing genetics
- Abstract
ARCHAEA-ExPRESs is an mRNA modification technology that makes use of components derived from the Archaeon Methanocaldococcus jannaschii, namely the tRNA splicing endonuclease (MJ-EndA) and its natural substrate, the bulge-helix-bulge (BHB) structure (1). These components can perform both cis- and trans-splicing in cellular and animal models and may provide a convenient way to modulate gene expression using components independent of cellular regulatory networks. To use MJ-EndA in stable expression mammalian systems, we developed variants characterized by high efficiency and sustainable in vivo activity. The MJ-EndA variants were created by the introduction of proper localization signals followed by mutagenesis and direct selection in mammalian cells. Of note, enzyme selection used an in vivo selection method based on puromycin resistance conferred to cells by BHB-mediated intron splicing from an out-of-frame puromycin N-acetyl transferase (PAC) gene. This approach yielded several endonuclease variants, the best of which showed 40-fold higher activity compared to the parental enzyme and stable processing of 30% of the target mRNA. Notably, these variants showed complete compatibility with long-term expression in mammalian cells, suggesting that they may be usefully applied in functional genomics and genetically modified animal models.
- Published
- 2013
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28. A comparative phenotypic and genomic analysis of C57BL/6J and C57BL/6N mouse strains.
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Simon MM, Greenaway S, White JK, Fuchs H, Gailus-Durner V, Wells S, Sorg T, Wong K, Bedu E, Cartwright EJ, Dacquin R, Djebali S, Estabel J, Graw J, Ingham NJ, Jackson IJ, Lengeling A, Mandillo S, Marvel J, Meziane H, Preitner F, Puk O, Roux M, Adams DJ, Atkins S, Ayadi A, Becker L, Blake A, Brooker D, Cater H, Champy MF, Combe R, Danecek P, di Fenza A, Gates H, Gerdin AK, Golini E, Hancock JM, Hans W, Hölter SM, Hough T, Jurdic P, Keane TM, Morgan H, Müller W, Neff F, Nicholson G, Pasche B, Roberson LA, Rozman J, Sanderson M, Santos L, Selloum M, Shannon C, Southwell A, Tocchini-Valentini GP, Vancollie VE, Westerberg H, Wurst W, Zi M, Yalcin B, Ramirez-Solis R, Steel KP, Mallon AM, de Angelis MH, Herault Y, and Brown SD
- Subjects
- Animals, Behavior, Animal, Disease Resistance immunology, Eye pathology, Female, Femur diagnostic imaging, Hypersensitivity immunology, INDEL Mutation genetics, Killer Cells, Natural immunology, Listeriosis immunology, Listeriosis microbiology, Male, Maze Learning, Mice, Inbred C57BL, Phenotype, Polymorphism, Single Nucleotide genetics, Spleen immunology, X-Ray Microtomography, Genome genetics
- Abstract
Background: The mouse inbred line C57BL/6J is widely used in mouse genetics and its genome has been incorporated into many genetic reference populations. More recently large initiatives such as the International Knockout Mouse Consortium (IKMC) are using the C57BL/6N mouse strain to generate null alleles for all mouse genes. Hence both strains are now widely used in mouse genetics studies. Here we perform a comprehensive genomic and phenotypic analysis of the two strains to identify differences that may influence their underlying genetic mechanisms., Results: We undertake genome sequence comparisons of C57BL/6J and C57BL/6N to identify SNPs, indels and structural variants, with a focus on identifying all coding variants. We annotate 34 SNPs and 2 indels that distinguish C57BL/6J and C57BL/6N coding sequences, as well as 15 structural variants that overlap a gene. In parallel we assess the comparative phenotypes of the two inbred lines utilizing the EMPReSSslim phenotyping pipeline, a broad based assessment encompassing diverse biological systems. We perform additional secondary phenotyping assessments to explore other phenotype domains and to elaborate phenotype differences identified in the primary assessment. We uncover significant phenotypic differences between the two lines, replicated across multiple centers, in a number of physiological, biochemical and behavioral systems., Conclusions: Comparison of C57BL/6J and C57BL/6N demonstrates a range of phenotypic differences that have the potential to impact upon penetrance and expressivity of mutational effects in these strains. Moreover, the sequence variants we identify provide a set of candidate genes for the phenotypic differences observed between the two strains.
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- 2013
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29. Mice lacking the Parkinson's related GPR37/PAEL receptor show non-motor behavioral phenotypes: age and gender effect.
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Mandillo S, Golini E, Marazziti D, Di Pietro C, Matteoni R, and Tocchini-Valentini GP
- Subjects
- Age Factors, Animals, Brain metabolism, Brain physiology, Female, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Neurotransmitter Agents genetics, Neurotransmitter Agents metabolism, Receptors, G-Protein-Coupled metabolism, Reflex, Startle genetics, Sex Factors, Smell genetics, Anxiety genetics, Depression genetics, Phenotype, Receptors, G-Protein-Coupled genetics
- Abstract
Non-motor symptoms in Parkinson's disease (PD) have been often described at different stages of the disease but they are poorly understood. We observed specific phenotypes related to these symptoms in mice lacking the PD-associated GPR37/PAEL receptor. GPR37 is an orphan G-protein-coupled receptor highly expressed in the mammalian central nervous system. It is a substrate of parkin and it is involved in the pathogenesis of PD. GPR37 interacts with the dopamine transporter (DAT), modulating nigro-striatal dopaminergic signaling and behavioral responses to amphetamine and cocaine. GPR37 knockout (KO) mice are resistant to MPTP and exhibit several motor behavioral abnormalities related to altered dopaminergic system function. To evaluate non-motor behavioral domains, adult and aged, male and female GPR37 KO mice and their wild-type (WT) littermates were analyzed in a series of cross-sectional studies. Aged GPR37 KO female mice showed mild improvements in olfactory function, while anxiety and depression-like behaviors appeared to be significantly increased. A reduction of the startle response to acoustic stimuli was observed only in adult GPR37 KO mice of both genders. Furthermore, HPLC analysis of major neurotransmitter levels revealed gender differences in the striatum, hippocampus and olfactory bulb of mutant mice. The absence of GPR37 receptor could have a neuroprotective effect in an age and gender-dependent manner, and the study of this receptor could be valuable in the search for novel therapeutic targets., (© 2013 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.)
- Published
- 2013
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30. Avatar pre-tRNAs help elucidate the properties of tRNA-splicing endonucleases that produce tRNA from permuted genes.
- Author
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Tocchini-Valentini GD and Tocchini-Valentini GP
- Subjects
- Base Sequence, Methanococcaceae enzymology, Molecular Sequence Data, Nucleic Acid Conformation, RNA Precursors chemistry, RNA Precursors genetics, RNA Processing, Post-Transcriptional genetics, RNA, Transfer chemistry, Schizosaccharomyces enzymology, Endoribonucleases metabolism, Genes genetics, RNA Precursors metabolism, RNA Splicing genetics, RNA, Transfer genetics, RNA, Transfer metabolism
- Abstract
Unusual tRNA genes, found in some algae, have their mature terminal 3' portion in front of their 5' portion in the genome. The transcripts from such genes must be cleaved by a pre-tRNA endonuclease to form a functional tRNA. We present a mechanism for the generation of "corrected" tRNAs from such a "permuted" pre-tRNA configuration. We used two avatar (av) or model pre-tRNAs and two splicing endonucleases with distinct mechanisms of recognition of the pre-tRNA. The splicing results are compatible with an evolutionary route in which permuted genes result from a duplication event followed by DNA rearrangement. The model pre-tRNAs permit description of the features that a transcript, derived from a rearranged duplicated gene, must have to give rise to functional tRNA. The two tRNA endonucleases are a eukaryal enzyme that normally acts in a mature domain-dependent mode and an archaeal enzyme that acts in a mature domain-independent mode. Both av pre-tRNAs are able to fold into two conformations: 1 and 2. We find that only conformation 2 can yield a corrected functional tRNA. This result is consistent with contemporary algae representing snapshots of different evolutionary stages, with duplicated genes preceding recombinatorial events generating a permutated gene. In a scenario elucidated by the use of the av pre-tRNAs, algal permuted tRNA genes could have further lost one of two mature domains, eliminating steric problems for the algal tRNA endonuclease, which remains a typical eukaryal enzyme capable of correcting the permuted transcript to a functional tRNA.
- Published
- 2012
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31. The mammalian gene function resource: the International Knockout Mouse Consortium.
- Author
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Bradley A, Anastassiadis K, Ayadi A, Battey JF, Bell C, Birling MC, Bottomley J, Brown SD, Bürger A, Bult CJ, Bushell W, Collins FS, Desaintes C, Doe B, Economides A, Eppig JT, Finnell RH, Fletcher C, Fray M, Frendewey D, Friedel RH, Grosveld FG, Hansen J, Hérault Y, Hicks G, Hörlein A, Houghton R, Hrabé de Angelis M, Huylebroeck D, Iyer V, de Jong PJ, Kadin JA, Kaloff C, Kennedy K, Koutsourakis M, Lloyd KC, Marschall S, Mason J, McKerlie C, McLeod MP, von Melchner H, Moore M, Mujica AO, Nagy A, Nefedov M, Nutter LM, Pavlovic G, Peterson JL, Pollock J, Ramirez-Solis R, Rancourt DE, Raspa M, Remacle JE, Ringwald M, Rosen B, Rosenthal N, Rossant J, Ruiz Noppinger P, Ryder E, Schick JZ, Schnütgen F, Schofield P, Seisenberger C, Selloum M, Simpson EM, Skarnes WC, Smedley D, Stanford WL, Stewart AF, Stone K, Swan K, Tadepally H, Teboul L, Tocchini-Valentini GP, Valenzuela D, West AP, Yamamura K, Yoshinaga Y, and Wurst W
- Subjects
- Animals, Internationality, Internet, Mice, Mice, Knockout genetics
- Abstract
In 2007, the International Knockout Mouse Consortium (IKMC) made the ambitious promise to generate mutations in virtually every protein-coding gene of the mouse genome in a concerted worldwide action. Now, 5 years later, the IKMC members have developed high-throughput gene trapping and, in particular, gene-targeting pipelines and generated more than 17,400 mutant murine embryonic stem (ES) cell clones and more than 1,700 mutant mouse strains, most of them conditional. A common IKMC web portal (www.knockoutmouse.org) has been established, allowing easy access to this unparalleled biological resource. The IKMC materials considerably enhance functional gene annotation of the mammalian genome and will have a major impact on future biomedical research.
- Published
- 2012
- Full Text
- View/download PDF
32. Comparative anatomy: Giorgione's Venus, Connoisseur Morelli, and the Reverend Bayes.
- Author
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Tocchini-Valentini GP and Tocchini-Valentini MA
- Subjects
- Anatomy, Artistic methods, History, 18th Century, History, 19th Century, History, 20th Century, Italy, Anatomy, Artistic history, Art history, Bayes Theorem, Fraud history, Mathematics history
- Published
- 2012
- Full Text
- View/download PDF
33. Yeast pheromone receptor genes STE2 and STE3 are differently regulated at the transcription and polyadenylation level.
- Author
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Di Segni G, Gastaldi S, Zamboni M, and Tocchini-Valentini GP
- Subjects
- Base Sequence, Binding Sites genetics, Gene Expression Regulation, Fungal, Models, Genetic, Molecular Sequence Data, Mutagenesis, Site-Directed, Polyadenylation, RNA, Fungal genetics, RNA, Fungal metabolism, RNA, Messenger genetics, RNA, Messenger metabolism, Saccharomyces cerevisiae metabolism, Transcription, Genetic, Genes, Fungal, Receptors, Mating Factor genetics, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics
- Abstract
The orderly expression of specific genes is the basis for cell differentiation. Saccharomyces cerevisiae has two haploid mating types, a and α cells, in which the mating-specific genes are differentially expressed. When a and α cells are committed to mate, their growth is arrested. Here we show that a cryptic polyadenylation site is present inside the coding region of the a-specific STE2 gene, encoding the receptor for the α-factor. The two cell types produce an incomplete STE2 transcript, but only a cells generate full-length STE2 mRNA. We eliminated the cryptic poly(A) signal, thereby allowing the production of a complete STE2 mRNA in α cells. We mutagenized α cells and isolated a mutant producing full-length STE2 mRNA. The mutation occurred in the ITC1 gene, whose product, together with the product of ISW2, is known to repress STE2 transcriptional initiation. We propose that the regulation of the yeast mating genes is achieved through a concerted mechanism involving transcriptional and posttranscriptional events. In particular, the early poly(A) site in STE2 could contribute to a complete shutoff of its expression in α cells, avoiding autocrine activation and growth arrest. Remarkably, no cryptic poly(A) sites are present in the a-factor receptor STE3 gene, indicating that S. cerevisiae has devised different strategies to regulate the two receptor genes. It is predictable that a correlation between the repression of a gene and the presence of a cryptic poly(A) site could also be found in other organisms, especially when expression of that gene may be harmful.
- Published
- 2011
- Full Text
- View/download PDF
34. Absence of the GPR37/PAEL receptor impairs striatal Akt and ERK2 phosphorylation, DeltaFosB expression, and conditioned place preference to amphetamine and cocaine.
- Author
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Marazziti D, Di Pietro C, Mandillo S, Golini E, Matteoni R, and Tocchini-Valentini GP
- Subjects
- Amphetamine metabolism, Animals, Central Nervous System Stimulants metabolism, Cocaine metabolism, Corpus Striatum drug effects, Corpus Striatum metabolism, Gene Expression Regulation physiology, Male, Mice, Mice, Knockout, Mitogen-Activated Protein Kinase 1 genetics, Phosphorylation, Proto-Oncogene Proteins c-akt genetics, Proto-Oncogene Proteins c-fos genetics, Receptors, G-Protein-Coupled genetics, Dopamine Uptake Inhibitors metabolism, Mitogen-Activated Protein Kinase 1 metabolism, Proto-Oncogene Proteins c-akt metabolism, Proto-Oncogene Proteins c-fos metabolism, Receptors, G-Protein-Coupled metabolism
- Abstract
The orphan G-protein-coupled receptor 37 (GPR37) colocalizes with the dopamine (DA) transporter (DAT) in mouse nigrostriatal presynaptic membranes, and its genetic ablation in homozygous null-mutant (GPR37-KO) mice provokes the marked increase of plasma membrane expression of DAT, alteration of psychostimulant-induced locomotor activity, and reduction of catalepsy induced by DA-receptor antagonists. We report that extracts from GPR37-KO mice displayed biochemical alterations of the nigrostriatal signaling pathways mediated by D1 and D2 dopaminergic receptors. Null-mutant mice showed an increase of the basal phosphorylation level of the D2-regulated Akt kinase. The basal phosphorylation of the D1-activated ERK2 kinase was not altered, but acute treatments with amphetamine or cocaine failed to produce its specific increase, as detected in samples from wild-type littermates. Furthermore, the chronic administration of cocaine to GPR37-KO mice did not increase the expression of the ΔFosB transcription factor isoforms. Consistently, behavioral analysis showed that null-mutant animals did not respond to the incentive properties of amphetamine or cocaine, in conditioned place preference tests. Thus, the lack of GPR37 affects both ERK2- and Akt-mediated striatal signaling pathways, impairing the biochemical and behavioral responses typically induced by acute and chronic administration of psychostimulant drugs.
- Published
- 2011
- Full Text
- View/download PDF
35. Evolution of introns in the archaeal world.
- Author
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Tocchini-Valentini GD, Fruscoloni P, and Tocchini-Valentini GP
- Subjects
- Archaeal Proteins genetics, Archaeal Proteins metabolism, Endonucleases genetics, Endonucleases metabolism, Archaea physiology, Evolution, Molecular, Introns physiology, RNA Precursors genetics, RNA Precursors metabolism, RNA Splicing physiology, RNA, Archaeal genetics, RNA, Archaeal metabolism, RNA, Ribosomal, 23S genetics, RNA, Ribosomal, 23S metabolism
- Abstract
The self-splicing group I introns are removed by an autocatalytic mechanism that involves a series of transesterification reactions. They require RNA binding proteins to act as chaperones to correctly fold the RNA into an active intermediate structure in vivo. Pre-tRNA introns in Bacteria and in higher eukaryote plastids are typical examples of self-splicing group I introns. By contrast, two striking features characterize RNA splicing in the archaeal world. First, self-splicing group I introns cannot be found, to this date, in that kingdom. Second, the RNA splicing scenario in Archaea is uniform: All introns, whether in pre-tRNA or elsewhere, are removed by tRNA splicing endonucleases. We suggest that in Archaea, the protein recruited for splicing is the preexisting tRNA splicing endonuclease and that this enzyme, together with the ligase, takes over the task of intron removal in a more efficient fashion than the ribozyme. The extinction of group I introns in Archaea would then be a consequence of recruitment of the tRNA splicing endonuclease. We deal here with comparative genome analysis, focusing specifically on the integration of introns into genes coding for 23S rRNA molecules, and how this newly acquired intron has to be removed to regenerate a functional RNA molecule. We show that all known oligomeric structures of the endonuclease can recognize and cleave a ribosomal intron, even when the endonuclease derives from a strain lacking rRNA introns. The persistence of group I introns in mitochondria and chloroplasts would be explained by the inaccessibility of these introns to the endonuclease.
- Published
- 2011
- Full Text
- View/download PDF
36. ARCHAEA-ExPRESs targeting of alpha-tubulin 4 mRNA: a model for high-specificity trans-splicing.
- Author
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Deidda G, Rossi N, Putti S, and Tocchini-Valentini GP
- Subjects
- Animals, Archaea enzymology, Endonucleases, Liver metabolism, Methods, Mice, Genetic Techniques, RNA, Messenger genetics, Trans-Splicing, Tubulin genetics
- Abstract
Effectiveness of trans-splicing-mediated mRNA reprogramming depends on specificity and efficiency. We have previously developed a new strategy (ARCHAEA-ExPRESs) that uses a tRNA endonuclease derived from Archaea and its natural substrate, the bulge-helix-bulge (BHB) structure. ARCHAEA-ExPRESs provides increased specificity in functional targeting. In fact, this system is based on a double check, the base pairing and the formation of a BHB structure between the target mRNA and the targeting RNA. In this study, we demonstrate the high specificity of ARCHAEA-ExPRESs by tagging the endogenous alpha-tubulin 4 via trans-splicing. Alpha-tubulin 4 belongs to a gene family sharing high degree of nucleotide sequence homology. The formation of a perfect BHB structure between targeting RNAs and the isotype alpha-tubulin 4 enables selective trans-splicing. Most important, ARCHAEA-ExPRESs functionality is conserved in vivo following transient expression of archaeal tRNA endonuclease in mouse liver. Production of the recombinant protein is strictly dependent on the expression of the archaeal endonuclease, and the efficiency of the system depends on the relative amount of the target and targeting mRNAs. These data prove the effectiveness of ARCHAEA-ExPRESs in an endogenous highly demanding context and disclose the possibility to utilize this system in a variety of technological or therapeutic applications.
- Published
- 2010
- Full Text
- View/download PDF
37. EMMA--mouse mutant resources for the international scientific community.
- Author
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Wilkinson P, Sengerova J, Matteoni R, Chen CK, Soulat G, Ureta-Vidal A, Fessele S, Hagn M, Massimi M, Pickford K, Butler RH, Marschall S, Mallon AM, Pickard A, Raspa M, Scavizzi F, Fray M, Larrigaldie V, Leyritz J, Birney E, Tocchini-Valentini GP, Brown S, Herault Y, Montoliu L, de Angelis MH, and Smedley D
- Subjects
- Animals, Chromosomes, Computational Biology trends, Databases, Protein, Information Storage and Retrieval methods, Internet, Mice, Mice, Inbred C57BL, Mice, Knockout, Models, Genetic, Protein Structure, Tertiary, Software, User-Computer Interface, Computational Biology methods, Databases, Genetic, Databases, Nucleic Acid
- Abstract
The laboratory mouse is the premier animal model for studying human disease and thousands of mutants have been identified or produced, most recently through gene-specific mutagenesis approaches. High throughput strategies by the International Knockout Mouse Consortium (IKMC) are producing mutants for all protein coding genes. Generating a knock-out line involves huge monetary and time costs so capture of both the data describing each mutant alongside archiving of the line for distribution to future researchers is critical. The European Mouse Mutant Archive (EMMA) is a leading international network infrastructure for archiving and worldwide provision of mouse mutant strains. It operates in collaboration with the other members of the Federation of International Mouse Resources (FIMRe), EMMA being the European component. Additionally EMMA is one of four repositories involved in the IKMC, and therefore the current figure of 1700 archived lines will rise markedly. The EMMA database gathers and curates extensive data on each line and presents it through a user-friendly website. A BioMart interface allows advanced searching including integrated querying with other resources e.g. Ensembl. Other resources are able to display EMMA data by accessing our Distributed Annotation System server. EMMA database access is publicly available at http://www.emmanet.org.
- Published
- 2010
- Full Text
- View/download PDF
38. Splicing of mRNA mediated by tRNA sequences in mouse cells.
- Author
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Zamboni M, Scarabino D, and Tocchini-Valentini GP
- Subjects
- Animals, Base Sequence, Intracellular Signaling Peptides and Proteins genetics, Intracellular Signaling Peptides and Proteins metabolism, Methanococcus genetics, Methanococcus metabolism, Mice, Molecular Sequence Data, NF-kappa B metabolism, Nucleic Acid Conformation, RNA Precursors chemistry, RNA Precursors genetics, Signal Transduction, RNA Splicing, RNA, Messenger chemistry, RNA, Messenger genetics, RNA, Transfer chemistry, RNA, Transfer genetics
- Abstract
tRNA splicing is essential for the formation of tRNAs and therefore for gene expression. A circularly permuted sequence of an amber-suppressor pre-tRNA gene was inserted into the sequence encoding the mouse NEMO protein. We demonstrated that, in mouse cells, the hybrid pre-tRNA/pre-mRNAs can be spliced precisely at the sites of the pre-tRNA intron. This splicing reaction produces functional tRNAs that suppress amber codons as well as translatable mRNAs that sustain the NF-kappaB activation pathway. The RNA molecules extracted from mouse cells were amplified by RT-PCR, and their sequences were determined, confirming the identity of the splice junctions. We then applied the Archaea-express technology, in which an archaeal RNA endonuclease is expressed in mouse cells. We show that both the endogenous eukaryal endonuclease and the archaeal one cleave the hybrid pre-tRNA/pre-mRNAs in the same manner with an additive effect.
- Published
- 2009
- Full Text
- View/download PDF
39. Processing of multiple-intron-containing pretRNA.
- Author
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Tocchini-Valentini GD, Fruscoloni P, and Tocchini-Valentini GP
- Subjects
- Endoribonucleases genetics, Methanococcales, Models, Genetic, Sulfolobus solfataricus, Endoribonucleases metabolism, Introns physiology, Nucleic Acid Conformation, RNA Precursors metabolism, RNA Splicing physiology, RNA, Transfer metabolism
- Abstract
Computational studies predict the simultaneous presence of two and even three introns in certain crenarchaeal tRNA genes. In these multiple-intron-containing pretRNAs, the introns are nested one inside the other and the pretRNA folds into a conformation that is anticipated to allow splicing of the last intron only after splicing the others. A set of operations, each consisting of two cleavages and one ligation, therefore needs to be carried out sequentially. PretRNAs containing multiple introns are predicted to fold, forming bulge-helix-bulge (BHB) and BHB-like motifs. The tRNA splicing endonuclease should recognize these motifs. To test this hypothetical scenario, we used the homotetrameric enzyme from Methanocaldococcus jannaschii (METJA) and the heterotetrameric enzyme from Sulfolobus solfataricus (SULSO). On the basis of our previous studies, the METJA enzyme should cleave only the BHB structure motif, while the SULSO enzyme can in addition cleave variant substrate structures, like the bulge-helix-loop (BHL). We show here that the processing of multiple-intron-containing pretRNA can be observed in vitro.
- Published
- 2009
- Full Text
- View/download PDF
40. Macroautophagy of the GPR37 orphan receptor and Parkinson disease-associated neurodegeneration.
- Author
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Marazziti D, Di Pietro C, Golini E, Mandillo S, Matteoni R, and Tocchini-Valentini GP
- Subjects
- 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine, Animals, Gene Expression Regulation, Mice, Protein Structure, Quaternary, Autophagy, Nerve Degeneration complications, Nerve Degeneration pathology, Parkinson Disease complications, Parkinson Disease pathology, Receptors, G-Protein-Coupled metabolism
- Published
- 2009
- Full Text
- View/download PDF
41. Induction of macroautophagy by overexpression of the Parkinson's disease-associated GPR37 receptor.
- Author
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Marazziti D, Di Pietro C, Golini E, Mandillo S, Matteoni R, and Tocchini-Valentini GP
- Subjects
- Animals, Biomarkers metabolism, Cell Death physiology, Cell Line, Corpus Striatum cytology, Corpus Striatum metabolism, Endoplasmic Reticulum metabolism, Endoplasmic Reticulum Chaperone BiP, Heat-Shock Proteins genetics, Heat-Shock Proteins metabolism, Humans, Lactosylceramides chemistry, Lactosylceramides metabolism, Lysosomes metabolism, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Molecular Chaperones genetics, Molecular Chaperones metabolism, Oligopeptides metabolism, Proteasome Endopeptidase Complex metabolism, Proteasome Inhibitors, Receptors, G-Protein-Coupled genetics, Ubiquitin-Protein Ligases genetics, Ubiquitin-Protein Ligases metabolism, Vacuoles metabolism, Autophagy physiology, Parkinson Disease metabolism, Receptors, G-Protein-Coupled metabolism
- Abstract
The orphan G-protein-coupled receptor 37 (GPR37) is a substrate of parkin, and its insoluble aggregates accumulate in brain tissue samples of Parkinson's disease patients, including Lewy bodies and neurites. Parkin activates the clearance of the unfolded receptor, while the overexpression of GPR37, in the absence of parkin, can lead to unfolded protein-induced cell death. We found that overexpression of the human GPR37 receptor in HEK293 cells and consequent activation of an endoplasmic reticulum (ER) stress response had effects comparable to starvation, in inducing the cellular autophagic pathway. Treatment with specific modulators provided further evidence for the autophagic clearance of the overexpressed GPR37 protein, in detergent-soluble and -insoluble fractions, as confirmed by the conversion of the microtubule-associated protein 1, light chain 3 (LC3)-I marker to its LC3-II isoform. Furthermore, Gpr37-null mutant mice displayed consistent alterations of ER stress and autophagic pathway markers in brain tissue samples. These findings show that GPR37 overexpression per se can induce cellular autophagy, which may prevent the selective degeneration of GPR37-expressing neurons, as reported for Parkinson's and related neurodegenerative diseases.
- Published
- 2009
- Full Text
- View/download PDF
42. Cis- and trans-splicing of mRNAs mediated by tRNA sequences in eukaryotic cells.
- Author
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Di Segni G, Gastaldi S, and Tocchini-Valentini GP
- Subjects
- Base Sequence, Gene Expression Regulation, Fungal, Genes, Mating Type, Fungal, Molecular Sequence Data, Nucleic Acid Conformation, Nucleic Acid Heteroduplexes, Protein Biosynthesis, RNA Precursors chemistry, RNA Precursors genetics, RNA Splice Sites genetics, RNA, Messenger chemistry, RNA, Messenger genetics, RNA, Transfer chemistry, Reverse Transcriptase Polymerase Chain Reaction, Sequence Deletion, Signal Transduction, Suppression, Genetic, Eukaryotic Cells metabolism, RNA, Transfer genetics, Saccharomyces cerevisiae cytology, Saccharomyces cerevisiae genetics, Trans-Splicing genetics
- Abstract
The formation of chimeric mRNAs is a strategy used by human cells to increase the complexity of their proteome, as revealed by the ENCODE project. Here, we use Saccharomyces cerevisiae to show a way by which trans-spliced mRNAs can be generated. We demonstrate that a pretRNA inserted into a premRNA context directs the splicing reaction precisely to the sites of the tRNA intron. A suppressor pretRNA gene was inserted, in cis, into the sequence encoding the third cytoplasmic loop of the Ste2 or Ste3 G protein-coupled receptor. The hybrid RNAs are spliced at the specific pretRNA splicing sites, releasing both functional tRNAs that suppress nonsense mutations and translatable mRNAs that activate the signal transduction pathway. The RNA molecules extracted from yeast cells were amplified by RT-PCR, and their sequences were determined, confirming the identity of the splice junctions. We then constructed two fusions between the premRNA sequence (STE2 or STE3) and the 5'- or 3'-pretRNA half, so that the two hybrid RNAs can associate with each other, in trans, through their tRNA halves. Splicing occurs at the predicted pretRNA sites, producing a chimeric STE3-STE2 receptor mRNA. RNA trans-splicing mediated by tRNA sequences, therefore, is a mechanism capable of producing new kinds of RNAs, which could code for novel proteins.
- Published
- 2008
- Full Text
- View/download PDF
43. The dawn of dominance by the mature domain in tRNA splicing.
- Author
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Tocchini-Valentini GD, Fruscoloni P, and Tocchini-Valentini GP
- Subjects
- Amino Acid Sequence, Archaeal Proteins chemistry, Archaeal Proteins genetics, Archaeal Proteins metabolism, Archaeoglobus fulgidus enzymology, Base Sequence, Binding Sites, Humans, Models, Molecular, Molecular Sequence Data, Nucleic Acid Conformation, Protein Binding, Protein Structure, Tertiary, Static Electricity, Structural Homology, Protein, Substrate Specificity, RNA Splicing genetics, RNA, Transfer chemistry, RNA, Transfer genetics
- Abstract
The relationship between enzyme architecture and substrate specificity among archaeal pre-tRNA splicing endonucleases has been investigated more deeply, by using biochemical assays and model building. The enzyme from Archeoglobus fulgidus (AF) is particularly interesting: it cleaves the bulge-helix-bulge target without requiring the mature tRNA domain, but, when the target is a bulge-helix-loop, the mature domain is required. A model of AF based on its electrostatic potential shows three polar patches interacting with the pre-tRNA substrate. A simple deletion mutant of the AF endonuclease lacking two of the three polar patches no longer cleaves the bulge-helix-loop substrate with or without the mature domain. This single deletion shows a possible path for the evolution of eukaryal splicing endonucleases from the archaeal enzyme.
- Published
- 2007
- Full Text
- View/download PDF
44. GPR37 associates with the dopamine transporter to modulate dopamine uptake and behavioral responses to dopaminergic drugs.
- Author
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Marazziti D, Mandillo S, Di Pietro C, Golini E, Matteoni R, and Tocchini-Valentini GP
- Subjects
- Animals, Blotting, Western, Cell Membrane metabolism, Cocaine pharmacology, Corpus Striatum metabolism, Dopamine Antagonists pharmacology, Female, Haloperidol pharmacology, Immunoprecipitation, Male, Mice, Mice, Knockout, Presynaptic Terminals metabolism, Receptors, G-Protein-Coupled genetics, Dopamine metabolism, Dopamine Plasma Membrane Transport Proteins metabolism, Locomotion drug effects, Receptors, G-Protein-Coupled metabolism
- Abstract
The orphan G protein-coupled receptor 37 (GPR37) is a substrate of parkin; its insoluble aggregates accumulate in brain samples of Parkinson's disease patients. We report here that GPR37 interacts with the dopamine transporter (DAT) and modulates DAT activity. GPR37 and DAT were found colocalized in mouse striatal presynaptic membranes and in transfected cells and their interaction was confirmed by coimmunoprecipitation assays. Gpr37-null mutant mice showed enhanced DAT-mediated dopamine uptake in striatal membrane samples, with a significant increase in the number of plasma membrane DAT molecules. The null mutant mice also exhibited a decrease in cocaine-induced locomotor activity and in catalepsy induced by dopamine receptor antagonists. These results reveal the specific role of GPR37, a putative peptidergic G protein-coupled receptor, in modulating the functional expression of DAT and the behavioral responses to dopaminergic drugs.
- Published
- 2007
- Full Text
- View/download PDF
45. Laying solid foundations for Europe.
- Author
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Mattaj IW and Tocchini-Valentini GP
- Subjects
- Animals, Biological Science Disciplines instrumentation, Biological Science Disciplines trends, Europe, Humans, Mice, Research organization & administration, Research trends, Biological Science Disciplines economics, Biological Science Disciplines organization & administration, Research economics, Research Support as Topic trends
- Published
- 2007
- Full Text
- View/download PDF
46. Coevolution of tRNA intron motifs and tRNA endonuclease architecture in Archaea.
- Author
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Tocchini-Valentini GD, Fruscoloni P, and Tocchini-Valentini GP
- Subjects
- Dimerization, Protein Folding, Protein Subunits, RNA Splicing, Archaeal Proteins chemistry, Endoribonucleases chemistry, Introns, RNA Precursors chemistry, RNA, Archaeal chemistry
- Abstract
Members of the three kingdoms of life contain tRNA genes with introns. The introns in pre-tRNAs of Bacteria are self-splicing, whereas introns in archaeal and eukaryal pre-tRNAs are removed by splicing endonucleases. We have studied the structures of the endonucleases of Archaea and the architecture of the sites recognized in their pre-tRNA substrates. Three endonuclease structures are known in the Archaea: a homotetramer in some Euryarchaea, a homodimer in other Euryarchaea, and a heterotetramer in the Crenarchaeota. The homotetramer cleaves only the canonical bulge-helix-bulge structure in its substrates. Variants of the substrate structure, termed bulge-helix-loops, appear in the pre-tRNAs of the Crenarcheota and Nanoarcheota. These variant structures can be cleaved only by the homodimer or heterotetramer forms of the endonucleases. Thus, the structures of the endonucleases and their substrates appear to have evolved together.
- Published
- 2005
- Full Text
- View/download PDF
47. Structure, function, and evolution of the tRNA endonucleases of Archaea: an example of subfunctionalization.
- Author
-
Tocchini-Valentini GD, Fruscoloni P, and Tocchini-Valentini GP
- Subjects
- Archaea genetics, Archaeal Proteins genetics, Archaeal Proteins metabolism, Endoribonucleases chemistry, Models, Molecular, Protein Conformation, RNA, Archaeal genetics, RNA, Archaeal metabolism, Archaea enzymology, Endoribonucleases genetics, Endoribonucleases metabolism, Evolution, Molecular, RNA, Transfer genetics, RNA, Transfer metabolism
- Abstract
We have detected two paralogs of the tRNA endonuclease gene of Methanocaldococcus jannaschii in the genome of the crenarchaeote Sulfolobus solfataricus. This finding has led to the discovery of a previously unrecognized oligomeric form of the enzyme. The two genes code for two different subunits, both of which are required for cleavage of the pre-tRNA substrate. Thus, there are now three forms of tRNA endonuclease in the Archaea: a homotetramer in some Euryarchaea, a homodimer in other Euryarchaea, and a heterotetramer in the Crenarchaea and the Nanoarchaea. The last-named enzyme, arising most likely by gene duplication and subsequent "subfunctionalization," requires the products of both genes to be active.
- Published
- 2005
- Full Text
- View/download PDF
48. A pre-tRNA carrying intron features typical of Archaea is spliced in yeast.
- Author
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Di Segni G, Borghese L, Sebastiani S, and Tocchini-Valentini GP
- Subjects
- Base Sequence, Chimera genetics, Genes, Fungal, Introns, Molecular Sequence Data, Nucleic Acid Conformation, RNA Precursors chemistry, RNA Splicing, RNA, Archaeal chemistry, RNA, Archaeal genetics, RNA, Archaeal metabolism, RNA, Transfer, Tyr chemistry, RNA, Transfer, Tyr genetics, RNA, Transfer, Tyr metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Species Specificity, Suppression, Genetic, RNA Precursors genetics, RNA Precursors metabolism
- Abstract
Archaeal pre-tRNAs are characterized by the presence of the bulge-helix-bulge (BHB) structure in the intron stem-and-loop region. A chimeric pre-tRNA was constructed bearing an intron of the archaeal type and the mature domain of the Saccharomyces cerevisiae suppressor SUP4 tRNA(Tyr). This pre-tRNA(ArchEuka) is correctly cleaved in several cell-free extracts and by purified splicing endonucleases. It is also cleaved and ligated in S. cerevisiae cells, providing efficient suppression of nonsense mutations in various genes.
- Published
- 2005
- Full Text
- View/download PDF
49. Altered dopamine signaling and MPTP resistance in mice lacking the Parkinson's disease-associated GPR37/parkin-associated endothelin-like receptor.
- Author
-
Marazziti D, Golini E, Mandillo S, Magrelli A, Witke W, Matteoni R, and Tocchini-Valentini GP
- Subjects
- Amphetamine pharmacology, Animals, Corpus Striatum chemistry, Dopamine analysis, Female, Male, Mice, Mice, Inbred C57BL, Motor Activity drug effects, Parkinsonian Disorders metabolism, Dopamine metabolism, Parkinsonian Disorders prevention & control, Receptors, G-Protein-Coupled physiology
- Abstract
GPR37 is an orphan G protein-coupled receptor expressed in mammalian brain, and its insoluble aggregates are found in the brain samples of juvenile Parkinson's disease patients. We have produced a Gpr37 knock-out mouse strain and identified several phenotypic features that are similar to those reported for mutants of genes encoding components of synaptic dopamine vesicles. Our results reveal an unanticipated role of GPR37 in regulating substantia nigra-striatum dopaminergic signaling. Gpr37(-/-) mice are viable, with normal brain development and anatomy, but they exhibit reduced striatal dopamine content, enhanced amphetamine sensitivity, and specific deficits in motor behavior paradigms sensitive to nigrostriatal dysfunction. These functional alterations are not associated with any substantial loss of substantia nigra neurons or degeneration of striatal dopaminergic afferences, the main histological marks of Parkinson's disease. The inactivation of GPR37, in fact, has protective effects on substantia nigra neurons, causing resistance to treatment with the Parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.
- Published
- 2004
- Full Text
- View/download PDF
50. An archaeal endoribonuclease catalyzes cis- and trans- nonspliceosomal splicing in mouse cells.
- Author
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Deidda G, Rossi N, and Tocchini-Valentini GP
- Subjects
- 3T3 Cells, Animals, Humans, Mice, Mutation genetics, Endoribonucleases genetics, Gene Targeting methods, Gene Transfer Techniques, Genetic Therapy methods, Methanococcus enzymology, RNA, Messenger genetics, Recombination, Genetic genetics, Trans-Splicing genetics
- Abstract
The tRNA endonuclease from the archaebacterium Methanococcus jannaschii (MJ endonuclease) can cleave RNAs forming specific bulge-helix-bulge (BHB) structures recognized by the enzyme. The resulting cleavage products are subsequently joined together by an endogenous ligase. We demonstrate the potential of using this strategy for repairing RNA in higher organisms by expressing the enzyme in mouse cells. Reporter target mRNAs modified with 17-nucleotide introns, flanked by sequences capable of forming BHB structures in cis, were expressed in mouse cells. RNA molecules that can form BHB substrates in trans with targeted mRNAs were also designed. Co-transfection of mouse cells with plasmids expressing these RNAs and the MJ endonuclease led to formation of RNA chimeras in which the target and exogenous RNA were recombined across the BHB. This technology is not limited to mRNA, but could in principle be used to destroy, modify or restore the function of a vast repertoire of RNA species or to join selectable tags to target RNAs.
- Published
- 2003
- Full Text
- View/download PDF
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