title: Three-Dimensional Imaging of the Intracellular Fate of Plasmid DNA and Transgene Expression: ZsGreen1 and Tissue Clearing Method CUBIC Are an Optimal Combination for Multicolor Deep Imaging in Murine Tissues.
abstract: Evaluation methods for determining the distribution of transgene expression in the body and the in vivo fate of viral and non-viral vectors are necessary for successful development of in vivo gene delivery systems. Here, we evaluated the spatial distribution of transgene expression using tissue clearing methods. After hydrodynamic injection of plasmid DNA into mice, whole tissues were subjected to tissue clearing. Tissue clearing followed by confocal laser scanning microscopy enabled evaluation of the three-dimensional distribution of transgene expression without preparation of tissue sections. Among the tested clearing methods (ClearT2, SeeDB, and CUBIC), CUBIC was the most suitable method for determining the spatial distribution of transgene expression in not only the liver but also other tissues such as the kidney and lung. In terms of the type of fluorescent protein, the observable depth for green fluorescent protein ZsGreen1 was slightly greater than that for red fluorescent protein tdTomato. We observed a depth of ~1.5 mm for the liver and 500 μm for other tissues without preparation of tissue sections. Furthermore, we succeeded in multicolor deep imaging of the intracellular fate of plasmid DNA in the murine liver. Thus, tissue clearing would be a powerful approach for determining the spatial distribution of plasmid DNA and transgene expression in various murine tissues.
authors.affiliation: Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.
authors.forename: Shintaro
authors.initials: S
authors.lastname: Fumoto
authors.affiliation: Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.
authors.forename: Koyo
authors.initials: K
authors.lastname: Nishimura
authors.affiliation: Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.
authors.forename: Koyo
authors.initials: K
authors.lastname: Nishida
authors.affiliation: Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Kawakami
mesh_terms: D000818:Animals; D005557:Formaldehyde / Q000737:chemistry; D005559:Formamides / Q000737:chemistry; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D021621:Imaging, Three-Dimensional / Q000379:methods*; D007668:Kidney / Q000378:metabolism / Q000648:ultrastructure; D008099:Liver / Q000378:metabolism* / Q000648:ultrastructure; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008168:Lung / Q000378:metabolism / Q000648:ultrastructure; D008297:Male; D051379:Mice; D057054:Molecular Imaging / Q000379:methods*; D017830:Octoxynol / Q000737:chemistry; D009928:Organ Specificity; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics*; D011092:Polyethylene Glycols / Q000737:chemistry; D016707:Tissue Fixation; D014162:Transfection; D019076:Transgenes; D014508:Urea / Q000737:chemistry; D000097573:Red Fluorescent Protein
chemical_list: D005557:Formaldehyde; D005559:Formamides; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D017830:Octoxynol; D011092:Polyethylene Glycols; D014508:Urea; D000097573:Red Fluorescent Protein; C031066:formamide
references.citation: Science. 1990 Apr 6;248(4951):73-6
references.pmid: 2321027
references.citation: Cell. 2014 Aug 14;158(4):945-58
references.pmid: 25088144
references.citation: Int J Nanomedicine. 2014;9:4267-76
references.pmid: 25228805
references.citation: Gene Ther. 2004 Apr;11(8):675-82
references.pmid: 14724673
references.citation: Mol Ther. 2006 Apr;13(4):786-94
references.pmid: 16364692
references.citation: Cell. 2014 Apr 24;157(3):726-39
references.pmid: 24746791
references.citation: PLoS Pathog. 2012;8(10):e1002971
references.pmid: 23071441
references.citation: Biochem Biophys Res Commun. 2002 May 31;294(1):46-50
references.pmid: 12054738
references.citation: Nat Commun. 2014;5:3545
references.pmid: 24694458
references.citation: Nat Methods. 2005 Dec;2(12):905-9
references.pmid: 16299475
references.citation: Comput Struct Biotechnol J. 2012 Oct 09;2:e201209004
references.pmid: 24688645
references.citation: Nat Neurosci. 2015 Oct;18(10):1518-29
references.pmid: 26368944
references.citation: Mol Pharm. 2012 Jul 2;9(7):1962-70
references.pmid: 22670625
references.citation: J Control Release. 2014 Sep 28;190:542-55
references.pmid: 24915504
references.citation: Nat Neurosci. 2013 Aug;16(8):1154-61
references.pmid: 23792946
references.citation: J Biol Chem. 1999 Jul 30;274(31):22025-32
references.pmid: 10419528
references.citation: Biophys J. 2012 Feb 22;102(4):934-44
references.pmid: 22385865
references.citation: Cell. 2014 Nov 6;159(4):911-24
references.pmid: 25417165
references.citation: Development. 2013 Mar;140(6):1364-8
references.pmid: 23444362
references.citation: Biotech Histochem. 2010 Dec;85(6):379-87
references.pmid: 20109099
references.citation: ACS Nano. 2013 Jun 25;7(6):5376-84
references.pmid: 23647441
references.citation: Exp Cell Res. 1999 Dec 15;253(2):713-22
references.pmid: 10585295
references.citation: Cytometry. 2002 Jun 1;48(2):106-12
references.pmid: 12116372
references.citation: Gene Ther. 1999 Jun;6(6):1006-14
references.pmid: 10455402
references.citation: Laser Photon Rev. 2013 Sep;7(5):732-757
references.pmid: 24348874
references.citation: Nature. 2013 May 16;497(7449):332-7
references.pmid: 23575631
references.citation: Pharm Res. 2000 Mar;17(3):306-13
references.pmid: 10801219
references.citation: J Control Release. 2014 Feb 28;176:24-34
references.pmid: 24384299
references.citation: PLoS One. 2012;7(7):e41319
references.pmid: 22844458
references.citation: Gene Ther. 2003 Dec;10(25):2074-80
references.pmid: 14595380
references.citation: Biomaterials. 2010 Oct;31(30):7813-26
references.pmid: 20656348
references.citation: Pharm Res. 2003 Sep;20(9):1452-9
references.pmid: 14567641
references.citation: Mol Ther Methods Clin Dev. 2014 May 07;1:14014
references.pmid: 26015958