title: Fluorescent-based methods for gene knockdown and functional cardiac imaging in zebrafish.
abstract: A notable advantage of zebrafish as a model organism is the ease of gene knockdown using morpholino antisense oligonucleotide (MO). However, zebrafish morphants injected with MO for a target protein often show heterogeneous phenotypes, despite controlling the injection volume of the MO solution in all embryos. We developed a method for estimating the quantity of MO injected into each living morphant, based on the co-injection of a control MO labeled with the fluorophore lissamine. By applying this method for knockdown of cardiac troponin T (tnnt2a) in zebrafish, we could efficiently select the partial tnnt2a-depleted zebrafish with a decreased heart rate and impairment of cardiac contraction. To investigate cardiac impairment of the tnnt2a morphant, we performed fluorescent cardiac imaging using Bodipy-ceramide. Cardiac image analysis showed moderate reduction of tnnt2a impaired diastolic distensibility and decreased contraction and relaxation velocities. To the best of our knowledge, this is the first report to analyze the role of tnnt2a in cardiac function in tnnt2a-depleted living animals. Our combinatorial approach can be applied for analyzing the molecular function of any protein associated with human cardiac diseases.
authors.affiliation: Department of Molecular and Cellular Pharmacology, Pharmacogenomics and Pharmacoinformatics, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie, 514-8507, Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Umemoto
authors.forename: Yuhei
authors.initials: Y
authors.lastname: Nishimura
authors.forename: Yasuhito
authors.initials: Y
authors.lastname: Shimada
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Yamanaka
authors.forename: Seiya
authors.initials: S
authors.lastname: Kishi
authors.forename: Saki
authors.initials: S
authors.lastname: Ito
authors.forename: Kana
authors.initials: K
authors.lastname: Okamori
authors.forename: Yuuki
authors.initials: Y
authors.lastname: Nakamura
authors.forename: Junya
authors.initials: J
authors.lastname: Kuroyanagi
authors.forename: Zi
authors.initials: Z
authors.lastname: Zhang
authors.forename: Liqing
authors.initials: L
authors.lastname: Zang
authors.forename: Zhipeng
authors.initials: Z
authors.lastname: Wang
authors.forename: Norihiro
authors.initials: N
authors.lastname: Nishimura
authors.forename: Toshio
authors.initials: T
authors.lastname: Tanaka
mesh_terms: D000818:Animals; D057791:Cardiac Imaging Techniques; D004625:Embryo, Nonmammalian; D005456:Fluorescent Dyes; D055785:Gene Knockdown Techniques; D006321:Heart / Q000502:physiology*; D006339:Heart Rate; D006801:Humans; D023421:Models, Animal; D060172:Morpholinos / Q000008:administration & dosage* / Q000378:metabolism; D009154:Mutation; D009200:Myocardial Contraction; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage* / Q000378:metabolism; D010641:Phenotype; D020107:Troponin T / Q000235:genetics* / Q000378:metabolism*; D015027:Zebrafish / Q000196:embryology / Q000235:genetics* / Q000378:metabolism; D029961:Zebrafish Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D060172:Morpholinos; D016376:Oligonucleotides, Antisense; D020107:Troponin T; D029961:Zebrafish Proteins