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      Zinc-Finger Nuclease Knockout of Dual-Specificity Protein Phosphatase-5 Enhances the Myogenic Response and Autoregulation of Cerebral Blood Flow in FHH.1 BN Rats

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          Abstract

          We recently reported that the myogenic responses of the renal afferent arteriole (Af-Art) and middle cerebral artery (MCA) and autoregulation of renal and cerebral blood flow (RBF and CBF) were impaired in Fawn Hooded hypertensive (FHH) rats and were restored in a FHH.1 BN congenic strain in which a small segment of chromosome 1 from the Brown Norway (BN) containing 15 genes including dual-specificity protein phosphatase-5 (Dusp5) were transferred into the FHH genetic background. We identified 4 single nucleotide polymorphisms in the Dusp5 gene in FHH as compared with BN rats, two of which altered CpG sites and another that caused a G155R mutation. To determine whether Dusp5 contributes to the impaired myogenic response in FHH rats, we created a Dusp5 knockout (KO) rat in the FHH.1 BN genetic background using a zinc-finger nuclease that introduced an 11 bp frame-shift deletion and a premature stop codon at AA121. The expression of Dusp5 was decreased and the levels of its substrates, phosphorylated ERK1/2 (p-ERK1/2), were enhanced in the KO rats. The diameter of the MCA decreased to a greater extent in Dusp5 KO rats than in FHH.1 BN and FHH rats when the perfusion pressure was increased from 40 to 140 mmHg. CBF increased markedly in FHH rats when MAP was increased from 100 to 160 mmHg, and CBF was better autoregulated in the Dusp5 KO and FHH.1 BN rats. The expression of Dusp5 was higher at the mRNA level but not at the protein level and the levels of p-ERK1/2 and p-PKC were lower in cerebral microvessels and brain tissue isolated from FHH than in FHH.1 BN rats. These results indicate that Dusp5 modulates myogenic reactivity in the cerebral circulation and support the view that a mutation in Dusp5 may enhance Dusp5 activity and contribute to the impaired myogenic response in FHH rats.

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          Most cited references47

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          An improved zinc-finger nuclease architecture for highly specific genome editing.

          Genome editing driven by zinc-finger nucleases (ZFNs) yields high gene-modification efficiencies (>10%) by introducing a recombinogenic double-strand break into the targeted gene. The cleavage event is induced using two custom-designed ZFNs that heterodimerize upon binding DNA to form a catalytically active nuclease complex. Using the current ZFN architecture, however, cleavage-competent homodimers may also form that can limit safety or efficacy via off-target cleavage. Here we develop an improved ZFN architecture that eliminates this problem. Using structure-based design, we engineer two variant ZFNs that efficiently cleave DNA only when paired as a heterodimer. These ZFNs modify a native endogenous locus as efficiently as the parental architecture, but with a >40-fold reduction in homodimer function and much lower levels of genome-wide cleavage. This architecture provides a general means for improving the specificity of ZFNs as gene modification reagents.
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            Knockout rats via embryo microinjection of zinc-finger nucleases.

            The toolbox of rat genetics currently lacks the ability to introduce site-directed, heritable mutations into the genome to create knockout animals. By using engineered zinc-finger nucleases (ZFNs) designed to target an integrated reporter and two endogenous rat genes, Immunoglobulin M (IgM) and Rab38, we demonstrate that a single injection of DNA or messenger RNA encoding ZFNs into the one-cell rat embryo leads to a high frequency of animals carrying 25 to 100% disruption at the target locus. These mutations are faithfully and efficiently transmitted through the germline. Our data demonstrate the feasibility of targeted gene disruption in multiple rat strains within 4 months time, paving the way to a humanized monoclonal antibody platform and additional human disease models.
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              Dual-specificity phosphatases: critical regulators with diverse cellular targets.

              DUSPs (dual-specificity phosphatases) are a heterogeneous group of protein phosphatases that can dephosphorylate both phosphotyrosine and phosphoserine/phosphothreonine residues within the one substrate. DUSPs have been implicated as major modulators of critical signalling pathways that are dysregulated in various diseases. DUSPs can be divided into six subgroups on the basis of sequence similarity that include slingshots, PRLs (phosphatases of regenerating liver), Cdc14 phosphatases (Cdc is cell division cycle), PTENs (phosphatase and tensin homologues deleted on chromosome 10), myotubularins, MKPs (mitogen-activated protein kinase phosphatases) and atypical DUSPs. Of these subgroups, a great deal of research has focused on the characterization of the MKPs. As their name suggests, MKPs dephosphorylate MAPK (mitogen-activated protein kinase) proteins ERK (extracellular-signal-regulated kinase), JNK (c-Jun N-terminal kinase) and p38 with specificity distinct from that of individual MKP proteins. Atypical DUSPs are mostly of low-molecular-mass and lack the N-terminal CH2 (Cdc25 homology 2) domain common to MKPs. The discovery of most atypical DUSPs has occurred in the last 6 years, which has initiated a large amount of interest in their role and regulation. In the past, atypical DUSPs have generally been grouped together with the MKPs and characterized for their role in MAPK signalling cascades. Indeed, some have been shown to dephosphorylate MAPKs. The current literature hints at the potential of the atypical DUSPs as important signalling regulators, but is crowded with conflicting reports. The present review provides an overview of the DUSP family before focusing on atypical DUSPs, emerging as a group of proteins with vastly diverse substrate specificity and function.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2014
                14 November 2014
                : 9
                : 11
                : e112878
                Affiliations
                [1 ]Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi, United States of America
                [2 ]Human and Molecular Genetics Center, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America
                [3 ]Department of Physiology and Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America
                University Medical Center Utrecht, Netherlands
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: FF DRH HJ RJR. Performed the experiments: FF AMG MRP. Analyzed the data: FF AMG MRP SVS RJR. Contributed reagents/materials/analysis tools: AMG MRP HJ RJR. Wrote the paper: FF SVS RJR.

                Article
                PONE-D-14-32128
                10.1371/journal.pone.0112878
                4232417
                25397684
                48bea1e4-8b96-4cca-a36e-c3cc440bbd20
                Copyright @ 2014

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 18 July 2014
                : 18 October 2014
                Page count
                Pages: 10
                Funding
                This work was funded in part by National Institutes of Health R01 HL36279 and DK104184 (RJR), H105997 (Harder), GO grant HL-101681 (Jacob) and New innovator award OD-8396 (Geurts), VA Research Career Scientist Award (Harder) and Scientist Development Grant 13SDG14000006 (Pabbidi) from American Heart Association. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Biochemistry
                Genetics
                Molecular Biology
                Neuroscience
                Physiology
                Medicine and Health Sciences
                Congenital Disorders
                Research and Analysis Methods
                Animal Studies
                Custom metadata
                The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper.

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