Fluorescent-Based Methods for Gene Knockdown and Functional Cardiac Imaging in Zebrafish
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RESEARCH
Fluorescent-Based Methods for Gene Knockdown and Functional Cardiac Imaging in Zebrafish Noriko Umemoto • Yuhei Nishimura • Yasuhito Shimada • Yukiko Yamanaka • Seiya Kishi • Saki Ito • Kana Okamori • Yuuki Nakamura • Junya Kuroyanagi • Zi Zhang • Liqing Zang • Zhipeng Wang • Norihiro Nishimura • Toshio Tanaka
Ó The Author(s) 2013. This article is published with open access at Springerlink.com
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
Electronic supplementary material The online version of this article (doi:10.1007/s12033-013-9664-6) contains supplementary material, which is available to authorized users. N. Umemoto Y. Nishimura Y. Shimada Y. Yamanaka S. Kishi S. Ito K. Okamori Y. Nakamura J. Kuroyanagi Z. Zhang L. Zang Z. Wang T. Tanaka (&) Department of Molecular and Cellular Pharmacology, Pharmacogenomics and Pharmacoinformatics, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan e-mail: [email protected] Y. Nishimura Y. Shimada T. Tanaka Mie University Medical Zebrafish Research Center, 2-174 Edobashi, Tsu, Mie 514-8507, Japan Y. Nishimura Y. Shimada T. Tanaka Department of Bioinformatics, Mie University Life Science Research Center, 2-174 Edobashi, Tsu, Mie 514-8507, Japan Y. Nishimura Y. Shimada T. Tanaka Department of Omics Medicine, Mie University Industrial Technology Innovation Institute, 2-174 Edobashi, Tsu, Mie 514-8507, Japan L. Zang N. Nishimura Department of Translational Medical Science, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan
this method for knockdown of cardiac troponin T (tnnt2a) in zebrafish, we could efficiently select the partial tnnt2adepleted 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. Keywords Zebrafish Gene knockdown Morpholino Lissamine Cardiac function Bodipy-ceramide Cardiac troponin T Cardiomyopathy Gene dosage effect Human disease model
Introduction Gene targeting technology, such as gene knockout (KO) and
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