生物化学技术

Trans-sialidase (TS; E.C. 3.2.1.18) catalyzes the transfer of preferably α2,3-linked sialic acid to another glycan or glycoconjugate, forming a new α2,3-linkage to galactose or N-acetylgalactosamine. In the absence of an appropriate acceptor, TS acts as a sialidase, hydrolytically re ...

In contrast to the linear sequences of protein and DNA, oligosaccharides are branched structures. In addition, almost all glycoproteins consist of a heterogeneous collection of differently glycosylated variants. Glycan analysis therefore requires high-resolution separ ...

ENU (N-ethyl-N-nitrosourea) mutagenesis is a widely accepted and proven method to introduce random point mutations in the genome. Because there are no targeted knockout strategies available for zebrafish so far, random mutagenesis is currently the preferred method in both forward and ...

This chapter describes a method for generation of the high-titer pseudotyped Moloney murine leukemia virus (MLV) that efficiently infects zebrafish embryos (i.e., more than 25 retroviral copies per cell). Injection techniques are also described for production of the retrovirus-in ...

Chemical mutagenesis using N-ethyl-N-nitrosourea is the current method of choice for dense mutagenesis in zebrafish. Methods are available for both pre-meiotic and post-meiotic sperm mutagenesis; in this chapter, pre-meiotic mutagenesis is described. Mutated males are crossed ...

In recent decades, laboratories throughout the world generated several thousand mutant, transgenic, and wild-type zebrafish lines and more lines continue to be produced. At the same time, relatively little effort has been expended to develop reliable, high-throughput, standardi ...

Transgenesis is an important methodology for studying the function of genes and genomes in model plants and animals. For the model vertebrate zebrafish, methods using the Tol2 transposable element have been developed for this purpose. With these methods, the function of the transgene can be ...

Transgenesis using bacterial artificial chromosomes (BAC) allows greater fidelity in directing desirable expression of transgenes. Application of this technology in the optically transparent zebrafish with fluorescent protein reporters enables unparalleled visu ...

The Tol2 transposon system can create insertions in the zebrafish genome efficiently. By using this system, the gene trap and enhancer trap methods have been developed. The gene trap and enhancer trap constructs contain the green fluorescent protein (GFP) reporter gene or the yeast Gal4 tran ...

In the past, microinjection of plasmid DNA into early embryos represented the state of the art to generate transgenic zebrafish. However, this approach suffers significant drawbacks (mosaic distribution of the injected transgene, late transgene integration at high copy numbers, l ...

A method is described that allows the introduction by electroporation of either small dyes or larger RNA, DNA, or morpholino constructs into single cells or small groups of cells in zebrafish embryos or larvae. The dye or construct is delivered to cells via a patch-like microelectrode that also de ...

Prodrug dependent cell ablation is a method that allows inducible and spatially restricted cell destruction. We describe transgenic methods to express the Escherichia coli nfsB in a tissue restricted manner in the zebrafish. This bacterial gene encodes a nitroreductase (NTR) enzyme ...

Although a common approach in large vertebrate embryos such as chick or frog, manipulation at the tissue level is only rarely applied to zebrafish embryos. Despite its relatively small size, the zebrafish embryo can be readily used for micromanipulations such as tissue and organ primordium ...

The availability of microarray technology for zebrafish research has enabled the expression of tens of thousands of genes to be studied simultaneously in one experiment. The experiment usually involves measuring and comparing the relative abundance of tens of thousands of mRNA spec ...

In expression microarray experiments, post-hybridization procedures involving data acquisition and analysis are mainly computational and hence are described as “dry-lab procedures.” The aim of these procedures is to acquire the fluorescent signals representing the relat ...

Hundreds of tiny noncoding RNAs known as microRNAs (miRNAs) have been identified in the genomes of plants and animals. Studies are increasingly demonstrating that individual miRNAs are important in normal development and physiology. miRNAs are regulators of gene expression that bi ...

Understanding the dynamics of developmental and cellular processes requires documentation of their changes with appropriate temporal and spatial resolution. Furthermore, simultaneous recording from a population of embryos under identical conditions allows statist ...

Zebrafish are ideally suited for the live imaging of early immune cell compartments. Macrophages that initially appear on the yolk surface prior to the onset of circulation are the first functional immune cells within the embryo, predating the emergence of the first granulocytic cells—t ...

In correlative microscopy, light microscopy provides the overview and orientation in the complex cells and tissue, whereas electron microscopy offers the detailed localization and correlation to subcellular structures. In this chapter, we offer the detailed high-quality ele ...

Immunocytochemistry for transmission electron microscopy provides important information on the location and relative abundance of proteins inside cells. Gaining access to this information without extracting or disrupting the location of target proteins requires spec ...