2007 — 2011 |
Ekker, Stephen Carl |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Intron-Based Mutagenic Transposons For Zebrafish
DESCRIPTION (provided by applicant): One of the major next genomic goals is the functional annotation of the over tens of thousands of protein- encoding, non-coding RNA and micro-RNA genes of unknown in vivo function. We have developed intronic- based mutagenesis for the zebrafish as an method for effective and regulated loss-of-function approach for this model vertebrate. We have developed gene-breaking and translational gene trap vectors as insertional mutagens as new approaches for the genome-wide assignment of function using forward (phenotype- based), reverse (sequence-based) and expression-based genetic approaches in this model vertebrate. We will achieve our goal to functionally annotate unknown genes in zebrafish by accomplishing the following specific aims: Develop and test GBTs specifically designed for forward genetic screens to isolate novel genes required for zebrafish development and substance addiction. We will develop and test our 3'gene trap vector as an insertional mutagen suitable for forward genetic screening in zebrafish. Biological assays for the identification of visibly apparent defects in embryogenesis, vascular development, and a novel behavioral assay for non-visible effects of mutations on the addiction process will be employed to measure the functional mutagenicity of this vector. Develop and test GBTs specifically designed for expression-based genetic screens for the isolation of genes with restricted expression patterns that are required for zebrafish development and substance addiction. We will develop and test our red fluorescent protein (RFP)-based translational gene trap vector for the isolation of genes with tissue-specific expression patterns. . Develop and test gene-break transposons for sequence-based genetics in zebrafish. We will generate a sequence-based database of insertional alleles in genes of high interest from GBTs. These lines will be catalogued in silico and stored as cryopreserved sperm for subsequent use by the zebrafish community for sequence-based searches, regulated mutagenesis studies, and as the starting point for null allele generation. The biological reagents developed over the course of this project will meet the goals of PAR-05-080 by providing the zebrafish community with tools that will allow the functional annotation of hundreds of protein- encoding and ncRNA genes necessary for development and growth of zebrafish as well as novel genetic loci involved with vascular biology, substance addiction and vertebrate development.
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2009 — 2013 |
Ekker, Stephen |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Genetics Core
The Gl Genetics Core will serve a critical role in facilitating access to genetic tools for Center-based research projects. Genetic manipulation is a critical approach from biological modeling to establishing and testing critical molecular signaling pathways in both the normal and clinically relevant disease state. Genetic tools are extremely dynamic, with new technologies coming on line every year. The Genetics Core will serve as a hub for current, emerging and future genetic technologies, advising and helping Center members properly assess and keep abreast of these often disruptive scientific advances. The Genetics Core will balance its efforts on current (such as siRNA, in vivo imaging, microarray, mouse ES cells), emerging (such as transcriptome analyses via next-generation sequencing and zebrafish knockouts) and near-future (such as new DNA delivery platforms including silica nanopartides) genetics tools. As a hub for access to the latest information on genetics technologies, this core will represent an important and central interface to this dynamic research area through its training opportunities, Web site and individual consultation. The Genetics Core Director (Stephen C. Ekker, Ph.D., 15% time requested) is a recent recruit to the Mayo Clinic with extensive molecular genetics expertise. To achieve the goal of this Core, part-time support for bioinformatics, biostatistics and genetics instruction is requested.
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2011 — 2016 |
Ekker, Stephen Carl |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
International Zebrafish Mutagenic Protein Trap
DESCRIPTION (provided by applicant): The zebrafish (Danio rerio) is the preeminent non-mammalian vertebrate system for the study of core vertebrate biology and behavior and for the modeling of human disease. Collections of molecularly characterized mutant lines have been powerful enabling tools for the nematode, fly and mouse fields. Genomic approaches have robustly characterized the nuclear genome and transcriptome. However, the genomic assessment of the full complexity of the proteome in a dynamic context and in vivo is still largely unknown. The zebrafish offers the first comprehensive analysis of the proteome using as template an entire vertebrate genome. We show that mutagenic protein trap gene-breaking transposons (GBTs) are effective and revertible loss- of-function tools for exploring traditional areas such as the biology of development (including organogenesis) but also open up new options including the genetics of behavior such as the biology of the nicotine response. A panel of Cre recombinase regulatable mutant zebrafish alleles offers the opportunity to explore critical questions in vertebrate biology at the tissue, organ and cellular level of resolution. This application is in response to PAR-08-139 Enhancing Zebrafish Research with Research Tools and Techniques (R01) to complete progress on the development of a large-scale collection of these revertible mutant alleles for the zebrafish community through a coordinated international effort. We will deliver the following at project's end: I. 1000 new mutagenic protein trap GBT zebrafish lines with basic description of the tagged expression patterns will be established and made available in real-time through the searchable online database zfishbook.org. These lines are immediately available through distribution by the Mayo Clinic Zebrafish Core Facility. II. We will conduct molecular analyses of these lines and subsequent annotation on the zebrafish genome project to identify the trapped proteins in this collection. We will continue to use our successful next generation sequence techniques followed by linkage analysis to isolate and map GBT insertions on the zebrafish genome. All sequence information will be initially distributed through zfishbook.org and then integrated in ZFIN, Fishmap and other zebrafish genomic databases. III. We will regularly synchronize this collection with the Zebrafish International Resource Center (ZIRC) for long-term archival and distribution to the zebrafish community. We will use a recently established 2D barcoding inventory system with a state-of-the-art sperm cryopreservation approach to facilitate regular shipping of new lines to ZIRC. This proposal is to complete this molecularly characterized collection, in silico catalog and distribution network of revertible mutations for the preeminent non-mammalian vertebrate, the zebrafish. These dominantly marked mutant lines also represent an ideal substrate for large-scale behavioral and other phenotypic assessment of the genome as a part of the broader initiative to functionally annotate the vertebrate genome using the zebrafish.
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2014 — 2018 |
Ekker, Stephen Carl |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Genetics and Model Systems Core
The C-SiG Genetics and Model Systems Core facilitates access to genetic tools and model systems for digestive disease-related research projects. Genetic manipulation is a critical approach from biological modeling to establishing and testing critical molecular signaling pathways in both normal and clinically relevant disease states. Genetic tools are extremely dynamic, with new technologies emerging every year. The Core provides current, emerging, and future genetic technologies while facilitating access to a full range of model systems including mouse, zebrafish, and rat. The Core Director, Dr. Stephen Ekker, is a well-established geneticist with extensive expertise using a variety of model systems. The Core has focused our original Specific Aims on tangible deliverables to better serve the C-SiG membership. Thus, the current SPECIFIC AIMS of the C-SiG Genetics and Model Systems Core are three-fold. First, to accelerate research by connecting and educating members to genetics and model systems tools. Second, to deliver new genetics and model systems tools/technologies that are needed by C-SiG members. Third, to establish cutting-edge genetic tools for genome editing including zinc finger nucleases (ZFNs), TALENs, Cas9 Custom Restriction Enzyme System (CRlSPRs) and future locus-specific genome editing tools that can be applied to model organism development including zebrafish, rats, mice and Drosophila. These aims will be accomplished by: i) Directly generating custom reagents including transposon clones, BAC clones, and TALENs for top-priority projects; ii) Providing education through core-sponsored seminars, Web site, and presentations to C-SiG Member laboratories; iii) Providing consultative services by connecting C-SiG members to genetics tools and institutional infrastructures directly and through a novel online reagent hub; and, iv) Developing model experimental systems, including zebrafish and genetically manipulated mice, flies, and rats, directly and by facilitating internal and external collaborative partnerships that benefit C-SiG members. Tiie C-SiG Genetics and Model Systems Core services have been used by 55% of Center members and supported 18 publications.
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2015 — 2019 |
Ekker, Stephen Carl |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Model Systems Core
MODEL SYSTEMS CORE PROJECT SUMMARY/ABSTRACT The overall goal of the Model Systems Core (MSC) is to develop, provide, and study PKD-relevant models as well as test therapeutic interventions in order to facilitate the translation of basic research findings of Mayo and non-Mayo investigators into clinical practice. This is achieved using three models, nematode, zebrafish and rodent, which work separately and in an integrated way to advance basic and translational PKD research. By spanning from simple to complex organisms, the MSC incorporates expertise in gene and protein function discovery through forward genetic or non-targeted chemical screens (nematodes), PKD pathway discovery and therapeutic testing in small, easily maintained vertebrates (zebrafish), and anatomical, physiological and pharmacological evaluation of mammalian pathomechanisms (rodents). The core is directed by Dr. Stephen C. Ekker who is a leading expert in genome editing, a key component of new model development. Further, MSC Associate Directors provide expertise in each individual model system. Dr. Jinghua Hu (nematode), Dr. Caroline R. Sussman (zebrafish), and Dr. Jan van Deursen (rodent), assure organism-specific advances in accordance with developing technologies in each field. During its four years, the MSC has fine-tuned its services based on PKD researchers' needs in order to provide maximum functionality. The current service aims are: (I) Generate, maintain and distribute PKD models, (II) Characterize PKD models, and (III) Chemical and therapeutic testing in PKD models. Within each model system, service lines are available spanning these three aims, and PKD researchers can make use of >150 nematode, >10 zebrafish, or >20 rodent knock-out/knock-in/transgenic ADPKD/ARPKD/ciliopathy lines, the majority of which have been developed and characterized in the MSC. In the last funding period these lines were used by 52 investigators to support 106 projects at 22 sites. Projects under development in nematode and zebrafish will allow higher throughput screening of pathways and therapeutics, which can then be tested in rodent preclinical trials, with which we have tremendous experience (>25 trials within the last funding period). The expertise and resources of the MSC are enhanced by expertise and facilities for rodent imaging (Dr. Slobodan I. Macura [Co-Investigator] and Dr. Maria V. Irazabal [Consultant]) and development/characterization of new rodent models as well as execution of preclinical trials (Dr. Katharina Hopp [Consultant]).
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2018 — 2021 |
Clark, Karl J (co-PI) [⬀] Ekker, Stephen Carl |
U01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Building the Mitochondrial Genome Editing Repertoire
Abstract Mitochondria have critical normal roles in metabolism, organ homeostasis, apoptosis and aging. Mitochondria play important but still largely mysterious roles in human physiology. Mutations in both nuclear DNA associated with proteins imported into mitochondria as well as mitochondrial DNA (mtDNA) are pathogenic. Despite this clear association of genotype with disease, there are no current treatments for patients with mitochondrial disease. Mitochondria represent a unique cellular compartment with different DNA and RNA repair and editing rules. For example, DNA nucleases that introduce double strand breaks and subsequent repair in nuclear DNA induce the degradation of mtDNA. Indeed, none of the common repair pathways found in the nucleus are active in mitochondria. This proposal uses the well-established TALE-based programmable DNA binding system for targeting mtDNA and the single-tube FUSX TALE assembly system to rapidly generate any protein-based genome engineering reagents. Similarly, we use a new, protein-based and programmable RNA binding system based on PPR proteins, a class of naturally occurring, mitochondrially localized RNA editors from plants. This application harnesses the unique environment of mitochondria to generate a new toolbox to expand the repertoire of tools to edit the human genome (RFA-RM-18-017). To develop these new molecular reagents for mtDNA and mtRNA editing of somatic cells, we will conduct the following aims: I. Develop new classes of mtDNA editing tools. Enhanced approaches to the use of mitoTALENs for preferential degradation of pathogenic mtDNA variants for MELAS and KSS will be developed, including novel nuclearly encoded reporters to detect non-mitochondrial off-targeting gene editing events. A new class of TALE mitochondrial base editors will be developed to directly edit mtDNA for pathogenic variants. II. mtRNA editing tools will be generated through harnessing the PPR family of naturally occurring programmable RNA editors. We will use our new FUSR assembly system to rapidly develop optimal RNA binding reagents, including the fusion to a set of test RNA nuclease or editing protein domains. Errant fusion transcripts in mtDNA deletion or single base variants in heteroplasmic cells will be used as the test paradigm for potential RNA editing platform development with the potential use as a therapeutic. Milestones for initial stages include the establishment of novel mtDNA heteroplasmy converting mitoTALENs against MELAS and KSS followed by testing of the new mtDNA base editor. For mtRNA editing, establishing PPR scaffolding rules for mtRNA binding followed by new programmable RNA nucleases and editors will be established. Deliverables include these novel mtDNA and mtRNA editing systems as well as humans cells with matched nuclearly encoded off-target reporter cassettes for use by any mitochondrial gene editing therapeutic system.
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2019 — 2021 |
Ekker, Stephen Carl |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Gene Editing and Epigenomics Core
PROJECT SUMMARY: GENE EDITING AND EPIGENOMICS CORE The C-SiG Gene Editing and Epigenomics Core provides current, emerging, and future gene editing and epigenomic technologies and expertise to center members. The organization and infrastructure of the core provides essential gene editing and epigenomics expertise and dedicated personnel to advise, interact with, provide reagents, and directly support C-SiG members in pursuit of the Specific Aims described below. The core will continue to be led by the Core Director, Dr. Stephen Ekker, who is a well-established molecular geneticist and gene editor and the Associate Core Director, Dr. Tamas Ordog, who is the founding Director of the Epigenomics Program of the Mayo Clinic Center for Individualized Medicine (CIM). The CIM Epigenomics Program develops, implements, and validates epigenomic technology and offering these methods to investigators. This resource will be leveraged to provide access to a broader segment of the C-SiG membership to state-of-the-art epigenomic methods at a discounted price, through a C-SiG supported technologist (50% FTE). The rapidly evolving disciplines of gene editing and epigenomics provide powerful tools to investigate the complex coordination of gene expression, which is modulated via a web of mechanistic relationships involving signaling pathways, transcription factors, and chromatin packaging. Epigenomics analysis when paired with genetic studies facilitated by gene editing technologies, greatly enhances identification of contributing molecular signaling pathways and environmental factors. Thus, provision of integrated access to gene editing and epigenomics expertise, tools, and assays, provides C-SiG members with a powerful and robust set of technologies to support digestive disease-related cell signaling research. Accordingly, we have defined and will pursue the following Specific Aims: i) Deliver state-of-the-art gene editing and epigenomics tools and assays that are needed by C-SiG members, including custom plasmids, custom nucleases and access to epigenomics assays including chromatin immunoprecipitation coupled with next generation sequencing (ChIP-seq assays), DNA immunoprecipitation-sequencing-based (DIP-seq); ii) Accelerate research by connecting members to gene editing and epigenomics-related consultation and educational opportunities such as seminars, journal clubs, and conferences; iii) Establish cutting-edge molecular tools and methods for custom genome and RNA editing including new DNA base editors, new RNA editing tools, and for epitranscriptomics (RNA modification assays). The C-SiG Gene Editing and Epigenomics Core services have been used by 53% of Center members and supported 29 publications during the past funding cycle.
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