{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,11,7]],"date-time":"2026-11-07T07:14:33Z","timestamp":1794035673332,"version":"4.8.0"},"reference-count":40,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2024,1,23]],"date-time":"2024-01-23T00:00:00Z","timestamp":1705968000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["RF1MH120016"],"award-info":[{"award-number":["RF1MH120016"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["G20RR024000"],"award-info":[{"award-number":["G20RR024000"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["P30EY028102"],"award-info":[{"award-number":["P30EY028102"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["P30EY007551"],"award-info":[{"award-number":["P30EY007551"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["F31EY034793"],"award-info":[{"award-number":["F31EY034793"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["TL1TR003169"],"award-info":[{"award-number":["TL1TR003169"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Cell. Neurosci."],"abstract":"<jats:p>High throughput sequencing has generated an enormous amount of information about the genes expressed in various cell types and tissues throughout the body, and about how gene expression changes over time and in diseased conditions. This knowledge has made targeted gene knockdowns an important tool in screening and identifying the roles of genes that are differentially expressed among specific cells of interest. While many approaches are available and optimized in mammalian models, there are still several limitations in the zebrafish model. In this article, we describe two approaches to target specific genes in the retina for knockdown: cell-penetrating, translation-blocking Vivo-Morpholino oligonucleotides and commercially available lipid nanoparticle reagents to deliver siRNA. We targeted expression of the PCNA gene in the retina of a P23H rhodopsin transgenic zebrafish model, in which rapidly proliferating progenitor cells replace degenerated rod photoreceptors. Retinas collected 48 h after intravitreal injections in adult zebrafish reveal that both Vivo-Morpholinos and lipid encapsulated siRNAs were able to successfully knock down expression of PCNA. However, only retinas injected with Vivo-Morpholinos showed a significant decrease in the formation of P23H rhodopsin-expressing rods, a downstream effect of PCNA inhibition. Surprisingly, Vivo-Morpholinos were able to exit the injected eye and enter the contralateral non-injected eye to inhibit PCNA expression. In this article we describe the techniques, concentrations, and considerations we found necessary to successfully target and inhibit genes through Vivo-Morpholinos and lipid encapsulated siRNAs.<\/jats:p>","DOI":"10.3389\/fncel.2023.1321337","type":"journal-article","created":{"date-parts":[[2024,1,23]],"date-time":"2024-01-23T11:51:26Z","timestamp":1706010686000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":6,"title":["Cost-effective strategies to knock down genes of interest in the retinas of adult zebrafish"],"prefix":"10.3389","volume":"17","author":[{"given":"Eyad","family":"Shihabeddin","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Abirami","family":"Santhanam","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexandra L.","family":"Aronowitz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John","family":"O\u2019Brien","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2024,1,23]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"67","DOI":"10.1007\/s40139-017-0127-9","article-title":"Retinal degeneration and regeneration-lessons from fishes and amphibians.","volume":"5","author":"Ail","year":"2017","journal-title":"Curr. Pathobiol. Rep."},{"key":"B2","doi-asserted-by":"publisher","DOI":"10.3390\/ijms222011217","article-title":"Progranulin a promotes compensatory hepatocyte proliferation via HGF\/c-met signaling after partial hepatectomy in zebrafish.","volume":"22","author":"Chiang","year":"2021","journal-title":"Int. J. Mol. Sci."},{"key":"B3","doi-asserted-by":"publisher","first-page":"3244","DOI":"10.1167\/iovs.09-4879","article-title":"The zebrafish galectin Drgal1-l2 is expressed by proliferating M\u00fcller glia and photoreceptor progenitors and regulates the regeneration of rod photoreceptors.","volume":"51","author":"Craig","year":"2010","journal-title":"Invest. Ophthalmol. Vis. Sci."},{"key":"B4","doi-asserted-by":"crossref","first-page":"1712","DOI":"10.1523\/JNEUROSCI.5317-06.2007","article-title":"Regeneration of inner retinal neurons after intravitreal injection of ouabain in zebrafish.","volume":"27","author":"Fimbel","year":"2007","journal-title":"J. Neurosci."},{"key":"B5","doi-asserted-by":"crossref","first-page":"431","DOI":"10.1038\/nrn3723","article-title":"Muller glial cell reprogramming and retina regeneration.","volume":"15","author":"Goldman","year":"2014","journal-title":"Nat. Rev. Neurosci."},{"key":"B6","doi-asserted-by":"publisher","first-page":"259","DOI":"10.1002\/glia.24270","article-title":"Pten associates with important gene regulatory network to fine-tune M\u00fcller glia-mediated zebrafish retina regeneration.","volume":"71","author":"Gupta","year":"2023","journal-title":"Glia"},{"key":"B7","doi-asserted-by":"crossref","first-page":"727","DOI":"10.1002\/dvdy.24375","article-title":"\u00fcller glial cell-dependent regeneration of the neural retina: An overview across vertebrate model systems.","volume":"245","author":"Hamon","year":"2016","journal-title":"Dev. Dyn."},{"key":"B8","first-page":"293","article-title":"RNA interference to knock down gene expression.","volume":"2018","author":"Han","year":"2018","journal-title":"Dis. Gene Identif."},{"key":"B9","doi-asserted-by":"publisher","first-page":"646","DOI":"10.1038\/s41434-021-00224-2","article-title":"Highly efficient neuronal gene knockout in vivo by CRISPR-Cas9 via neonatal intracerebroventricular injection of AAV in mice.","volume":"28","author":"Hana","year":"2021","journal-title":"Gene Ther."},{"key":"B10","doi-asserted-by":"publisher","first-page":"189","DOI":"10.1186\/s13045-019-0839-x","article-title":"Assessment of in vivo siRNA delivery in cancer mouse models: Long Non-Coding RNAs.","volume":"2016","author":"Hatakeyama","year":"2016","journal-title":"Methods Protoc."},{"key":"B11","doi-asserted-by":"crossref","first-page":"521","DOI":"10.1007\/s00418-018-1764-8","article-title":"Differential expression of PKC\u03b1 and-\u03b2 in the zebrafish retina.","volume":"151","author":"Haug","year":"2019","journal-title":"Histochem. Cell Biol."},{"key":"B12","doi-asserted-by":"publisher","DOI":"10.3390\/pharmaceutics14102086","article-title":"Development of a combined lipid-based nanoparticle formulation for enhanced siRNA delivery to vascular endothelial cells.","volume":"14","author":"He","year":"2022","journal-title":"Pharmaceutics"},{"key":"B13","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0052622","article-title":"G6f-like is an ITAM-containing collagen receptor in thrombocytes.","volume":"7","author":"Hughes","year":"2012","journal-title":"PLoS One"},{"key":"B14","doi-asserted-by":"publisher","first-page":"1009","DOI":"10.1002\/dneu.20362","article-title":"Time course analysis of gene expression during light-induced photoreceptor cell death and regeneration in albino zebrafish.","volume":"67","author":"Kassen","year":"2007","journal-title":"Dev. Neurobiol."},{"key":"B15","doi-asserted-by":"crossref","first-page":"169","DOI":"10.1016\/j.bcmd.2009.12.004","article-title":"Vivo-Morpholino knockdown of \u03b1IIb: A novel approach to inhibit thrombocyte function in adult zebrafish.","volume":"44","author":"Kim","year":"2010","journal-title":"Blood Cells Mol. Dis."},{"key":"B16","doi-asserted-by":"publisher","DOI":"10.3791\/50415","article-title":"Micromanipulation of gene expression in the adult zebrafish brain using cerebroventricular microinjection of morpholino oligonucleotides.","volume":"75","author":"Kizil","year":"2013","journal-title":"JoVE"},{"key":"B17","doi-asserted-by":"publisher","DOI":"10.1016\/j.nano.2019.102077","article-title":"Single-cell kinetics of siRNA-mediated mRNA degradation.","volume":"21","author":"Krzyszto\u0144","year":"2019","journal-title":"Nanomedicine"},{"key":"B18","doi-asserted-by":"crossref","first-page":"4114","DOI":"10.4049\/jimmunol.138.12.4114","article-title":"Proliferating cell nuclear antigen (PCNA)\/cyclin in activated human T lymphocytes.","volume":"138","author":"Kurki","year":"1987","journal-title":"J. Immunol."},{"key":"B19","doi-asserted-by":"publisher","first-page":"1353","DOI":"10.1126\/science.1228773","article-title":"Acute inflammation initiates the regenerative response in the adult zebrafish brain.","volume":"338","author":"Kyritsis","year":"2012","journal-title":"Science"},{"key":"B20","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2022.1031962","article-title":"The effect and underlying mechanism of yeast \u03b2-glucan on antiviral resistance of zebrafish against spring viremia of carp virus infection.","volume":"13","author":"Liang","year":"2022","journal-title":"Front. Immunol."},{"key":"B21","doi-asserted-by":"publisher","DOI":"10.1186\/s12918-018-0544-3","article-title":"Uncovering the regeneration strategies of zebrafish organs: a comprehensive systems biology study on heart, cerebellum, fin, and retina regeneration.","volume":"12","author":"Liu","year":"2018","journal-title":"BMC Syst. Biol."},{"key":"B22","doi-asserted-by":"crossref","first-page":"343","DOI":"10.1038\/nature02873","article-title":"Mechanisms of gene silencing by double-stranded RNA.","volume":"431","author":"Meister","year":"2004","journal-title":"Nature"},{"key":"B23","doi-asserted-by":"publisher","first-page":"613","DOI":"10.2144\/000113005","article-title":"Vivo-Morpholinos: a non-peptide transporter delivers Morpholinos into a wide array of mouse tissues.","volume":"45","author":"Morcos","year":"2008","journal-title":"Biotechniques"},{"key":"B24","doi-asserted-by":"publisher","DOI":"10.1186\/1741-7007-12-30","article-title":"Specific NuRD components are required for fin regeneration in zebrafish.","volume":"12","author":"Pfefferli","year":"2014","journal-title":"BMC Biol."},{"key":"B25","doi-asserted-by":"crossref","first-page":"8","DOI":"10.1016\/j.nano.2008.06.001","article-title":"Drug delivery of siRNA therapeutics: potentials and limits of nanosystems.","volume":"5","author":"Reischl","year":"2009","journal-title":"Nanomedicine"},{"key":"B26","doi-asserted-by":"crossref","DOI":"10.3390\/cells9102242","article-title":"Zebrafish model of retinitis pigmentosa shows continuous degeneration and regeneration of rod photoreceptors.","volume":"9","author":"Santhanam","year":"2020","journal-title":"Cells"},{"key":"B27","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1007\/s00018-023-05021-1","article-title":"Molecular basis of retinal remodeling in a zebrafish model of retinitis pigmentosa.","volume":"80","author":"Santhanam","year":"2023","journal-title":"Cell. Mol. Life Sci."},{"key":"B28","author":"Schnell","year":"2019","journal-title":"Targeted siRNA delivery in vivo."},{"key":"B29","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0008835","article-title":"Let it flow: Morpholino knockdown in zebrafish embryos reveals a pro-angiogenic effect of the metalloprotease meprin \u03b12.","volume":"5","author":"Sch\u00fctte","year":"2010","journal-title":"PLoS One"},{"key":"B30","doi-asserted-by":"crossref","first-page":"1127","DOI":"10.1093\/aob\/mcq243","article-title":"Proliferating cell nuclear antigen (PCNA): a key factor in DNA replication and cell cycle regulation.","volume":"107","author":"Strzalka","year":"2011","journal-title":"Ann. Bot."},{"key":"B31","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1186\/s12951-020-00745-9","article-title":"Distribution of polymeric nanoparticles in the eye: implications in ocular disease therapy.","volume":"19","author":"Swetledge","year":"2021","journal-title":"J. Nanobiotechnol."},{"key":"B32","doi-asserted-by":"publisher","first-page":"336","DOI":"10.1002\/dvdy.20630","article-title":"Inhibition of zebrafish fin regeneration using in vivo electroporation of morpholinos against fgfr1 and msxb.","volume":"235","author":"Thummel","year":"2006","journal-title":"Dev. Dyn."},{"key":"B33","article-title":"In vivo electroporation of morpholinos into the adult zebrafish retina.","volume":"58","author":"Thummel","year":"2011","journal-title":"J. Vis. Exp."},{"key":"B34","doi-asserted-by":"publisher","first-page":"572","DOI":"10.1016\/j.exer.2010.02.001","article-title":"Pax6a and Pax6b are required at different points in neuronal progenitor cell proliferation during zebrafish photoreceptor regeneration.","volume":"90","author":"Thummel","year":"2010","journal-title":"Exp. Eye Res."},{"key":"B35","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2415-10-25","article-title":"Non-viral siRNA delivery into the mouse retina in vivo.","volume":"10","author":"Turchinovich","year":"2010","journal-title":"BMC Ophthalmol."},{"key":"B36","doi-asserted-by":"publisher","first-page":"33529","DOI":"10.1021\/acsami.6b13295","article-title":"A neutralized noncharged polyethylenimine-based system for efficient delivery of siRNA into heart without toxicity.","volume":"8","author":"Wang","year":"2016","journal-title":"ACS Appl. Mater. Interf."},{"key":"B37","doi-asserted-by":"publisher","DOI":"10.7150\/thno.15230","article-title":"Cell-specific promoters enable lipid-based nanoparticles to deliver genes to specific cells of the retina in vivo.","volume":"6","author":"Wang","year":"2016","journal-title":"Theranostics"},{"key":"B38","author":"Westerfield","year":"2000","journal-title":"The zebrafish book: a guide for the laboratory use of zebrafish."},{"key":"B39","doi-asserted-by":"publisher","DOI":"10.3791\/58054","article-title":"Nanoparticle-mediated sirna gene-silencing in adult zebrafish heart.","volume":"137","author":"Xiao","year":"2018","journal-title":"JoVE"},{"key":"B40","doi-asserted-by":"crossref","first-page":"4381","DOI":"10.1016\/j.csbj.2022.08.003","article-title":"Exploring long non-coding RNA networks from single cell omics data.","volume":"20","author":"Zhao","year":"2022","journal-title":"Comput. Struct. Biotechnol. J."}],"container-title":["Frontiers in Cellular Neuroscience"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fncel.2023.1321337\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,1,23]],"date-time":"2024-01-23T11:51:37Z","timestamp":1706010697000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fncel.2023.1321337\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,1,23]]},"references-count":40,"alternative-id":["10.3389\/fncel.2023.1321337"],"URL":"https:\/\/doi.org\/10.3389\/fncel.2023.1321337","relation":{},"ISSN":["1662-5102"],"issn-type":[{"value":"1662-5102","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,1,23]]},"article-number":"1321337"}}