Fluorescent-Based Methods for Gene Knockdown and Functional Cardiac Imaging in Zebrafish

• Published in: Molecular biotechnology Vol. 55; no. 2; pp. 131 - 142
• Main Authors: Umemoto, Noriko, Nishimura, Yuhei, Shimada, Yasuhito, Yamanaka, Yukiko, Kishi, Seiya, Ito, Saki, Okamori, Kana, Nakamura, Yuuki, Kuroyanagi, Junya, Zhang, Zi, Zang, Liqing, Wang, Zhipeng, Nishimura, Norihiro, Tanaka, Toshio
• Format: Journal Article
• Language: English
• Published: Boston Springer-Verlag 01.10.2013; Springer US; Springer Nature B.V
• Subjects: Animals; Biochemistry; Biological Techniques; Biotechnology; Cardiac Imaging Techniques; cardiac output; Cardiomyopathy; Cell Biology; Chemistry; Chemistry and Materials Science; Danio rerio; Embryo, Nonmammalian; Fluorescence; Fluorescent Dyes; Gene Knockdown Techniques; genes; Genetics; Heart - physiology; Heart Rate; Human Genetics; Humans; image analysis; Injection; Models, Animal; Morpholinos - administration & dosage; Morpholinos - metabolism; Mutation; Myocardial Contraction; Oligonucleotides, Antisense - administration & dosage; Oligonucleotides, Antisense - metabolism; Phenotype; Protein Science; troponin T; Troponin T - genetics; Troponin T - metabolism; Zebrafish; Zebrafish - embryology; Zebrafish - genetics; Zebrafish - metabolism; Zebrafish Proteins - genetics; Zebrafish Proteins - metabolism; Cardiomyopathy; Cardiac function; Lissamine; Zebrafish; Gene dosage effect; Morpholino; Cardiac troponin T; Human disease model; Gene knockdown; Bodipy-ceramide
• ISSN: 1073-6085; 1559-0305
• DOI: 10.1007/s12033-013-9664-6

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.