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.Electronic supplementary materialThe online version of this article (doi:10.1007/s12033-013-9664-6) contains supplementary material, which is available to authorized users.
Highlights
Gene targeting technology, such as gene knockout (KO) and gene knockdown, is used in the targeted analysis of specific gene function in human disease
The Knockdown Level of tnnt2a in Zebrafish Injected with tnnt2a-morpholino antisense oligonucleotide (MO) Can be Estimated by the fluorescent intensity (FI) of Co-injected Lissamine-MO
To estimate the amount of MO present in each embryo after microinjection, a mixture of an MO targeted to the gene of interest and lissamine attached to the end (Lis-MO) was injected into embryos at the 1–4-cell stage
Summary
Gene targeting technology, such as gene knockout (KO) and gene knockdown, is used in the targeted analysis of specific gene function in human disease. Reduced activity, of a gene product is inferred to be associated with the cause and progress of a disease, a KO animal for that gene may be developed to evaluate phenotypic similarity to the human disease. A particular difficulty arises when the homozygous KO results in embryonic lethality. To overcome the lethality of the homozygous KO, the phenotype of the heterozygous KO animal can be evaluated. A 50 % reduction in the expression of a targeted gene in heterozygous mutants rarely results in a. 0
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