Department of Molecular and Cellular Pharmacology, Pharmacogenomics and Pharmacoinformatics, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie, 514-8507, Japan.
Mol Biotechnol. 2013 Oct;55(2):131-42. 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.
斑马鱼作为一种模式生物，其显著优势之一是可以使用针对目标基因的 Morpholino 反义寡核苷酸（MO）进行基因敲低。然而，尽管我们控制了所有胚胎中 MO 溶液的注射体积，但用 MO 注射针对目标蛋白的斑马鱼突变体通常表现出异质表型。我们开发了一种基于荧光素标记的对照 MO 共注射的方法，用于估计每个活体突变体中注入的 MO 量。通过将这种方法应用于斑马鱼心脏肌钙蛋白 T（tnnt2a）的敲低，我们能够有效地选择心率降低和心脏收缩功能受损的部分 tnnt2a 耗尽的斑马鱼。为了研究 tnnt2a 突变体的心脏损伤，我们使用 Bodipy-ceramide 进行了荧光心脏成像。心脏图像分析显示 tnnt2a 受损的舒张延展性适度降低，收缩和松弛速度降低。据我们所知，这是首次在 tnnt2a 耗尽的活体动物中分析 tnnt2a 在心脏功能中的作用的报告。我们的组合方法可用于分析与人类心脏疾病相关的任何蛋白质的分子功能。
