The main challenge of the post-genomic era is to functionally characterize genes identified by the genome sequencing projects. Model organisms, including zebrafish, are indispensable for this demanding task. Zebrafish has been successfully incorporated into large-scale genetic screens due to the optical clarity of the embryos and their accessibility to various experimental techniques throughout development. The attractiveness of the zebrafish as a model organism is enhanced by the availability of continuously improving genomic tools and methodologies for functional characterization of the gene. This article will highlight the current techniques used in the field, with the focus on transgenesis.

https://www.future-science.com/doi/pdf/10.2144/05386RV01

New ways to admire zebrafish: progress in functional genomics research methodology

Publisher Summary This chapter focuses on the nonconventional antisense in zebrafish for functional genomics applications. The ability of the various genome projects to acquire gene sequence data has far outpaced the ability to ascribe biological functions to these new genes. For the many biological processes that are well conserved in evolution, model systems with rapid genetic tools such as D. melanogaster have opened the door to functional genomics. In this paradigm, genes with specific biological roles are identified first in the model organism, and genome databases are subsequently used to identify human homologs. Many biological and biochemical pathways are not conserved between flies and vertebrates, and, consequently, studies directly using biologically more complex model systems such as the mouse and the fish are warranted. Examples include neural crest formation, most organogenesis pathways, and signaling cascades such as those induced by vascular endothelial growth factor. Genetic approaches have been developed to approach this problem, but each is a significant cost in terms of animal husbandry needs and time for large-scale analyses in vertebrates. The chapter discusses the use of morpholinos (MOs) as antisense tools in zebrafish.

Nonconventional Antisense in Zebrafish for Functional Genomics Applications

The invention provides sequence specific polynucleotide analogues, especially morpholino-modified polynucleotide analogues, and methods for determining the function of a nucleic acid of known sequence in a teleost embryo, especially a zebrafish embryo.

Inhibition of teleost gene expression using morpholino modified polynucleotide analogues

Genetic model organisms are increasingly valuable in the post-genomics era to provide a basis for comparative analysis of the human genome. For higher order processes of vertebrate pigment cell biology and development, the mouse has historically been the model of choice. A complementary organism, the zebrafish (Danio rerio), shares many of the signaling and biological processes of vertebrates, e.g. neural crest development. The zebrafish has a number of characteristics that make it an especially valuable model for the study of pigment cell biology and disease. Large-scale genetic screens have identified a collection of pigmentation mutants that have already made valuable contributions to pigment research. An increasing repertoire of genomic resources such as an expressed sequence tag-based Gene Index (The Institute for Genomic Research) and improving methods of mutagenesis, transgenesis, and gene targeting make zebrafish a particularly attractive model. Morpholino phosphorodiamidate oligonucleotide (MO) 'knockdown' of pigment gene expression provides a non-conventional antisense tool for the analysis of genes involved in pigment cell biology and disease. In addition, an ongoing, reverse-genetic, MO-based screen for the rapid identification of gene function promises to be a valuable complement to other high-throughput microarray and proteomic approaches for understanding pigment cell biology. Novel reagents for zebrafish transgenesis, such as the Sleeping Beauty transposon system, continue to improve the capacity for genetic analysis in this system and ensure that the zebrafish will be a valuable genetic model for understanding a variety of biological processes and human diseases for years to come.

Functional Genomics Tools for the Analysis of Zebrafish Pigment

Gene 'knockdown' approaches using unconventional antisense oligonucleotides

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Gene 'knockdown' approaches using unconventional antisense oligonucleotides