细胞功能测定

This chapter is an overview of basic principles of fluorescence microscopy, including a brief history on the invention of this type of microscopy. The chapter highlights important points related to properties of fluorochromes, resolution in fluorescence microscopy, phase contra ...

Activation of the canonical Wnt pathway leads to the transcriptional activation of a particular subset of downstream Wnt target genes. To track this localized cellular output in a living organism, reporter constructs can be designed containing multimerized consensus lymphoid enh ...

Immunodetection of β-catenin accumulation in the nucleus is the most direct and reliable method to determine the intensity and the spatial/temporal patterns of Wnt-dependent signaling activity. Due to the large size of the Xenopus embryo, staining must be done on sections. We present here a s ...

Determining the expression pattern of a gene of interest is critical to understanding when, where, and how it may function during development. This chapter describes methods for determining the localization and expression levels for both mRNA and protein. Some of these methods can be descr ...

Xenopus is an established and powerful model system for the study of Wnt signaling in vertebrates. Above all, the relatively large size of the embryos enables microinjection experiments, which have led to key discoveries not only about the functional role of Wnt signaling in vertebrate embr ...

Wnt signaling plays several important roles in the development of the zebrafish central nervous system (CNS). This chapter outlines both the known and postulated roles of Wnts from the earliest step of neural plate induction to relatively late events such as axon pathfinding and synaptoge ...

The rapid embryonic development and high fecundity of zebrafish contribute to the great advantages of this model for the study of developmental genetics. Transient disruption of the normal function of a gene during development can be achieved by microinjecting mRNA, DNA or short chemica ...

Optically transparent zebrafish embryos provide an excellent vertebrate model system in which to reveal specific mRNA and protein expression patterns during development. Whole-mount preparations can be used to generate three-dimensional color or fluorescent readouts of t ...

Biological cells accomplish their physiological functions using interconnected networks of genes, proteins, and other biomolecules. Most interactions in biological signaling networks, such as bimolecular association or covalent modification, can be modeled in a physic ...

Several strategies based on synthetic oligonucleotides (ON) have been proposed to control gene expression. As for most biomolecules, however, delivery has remained a major roadblock for in vivo applications. Conjugation of steric-block neutral DNA mimics, such as peptide nucleic a ...

The methods for evaluating internalization pathways of cellular CPP-mediated ON delivery utilizing a pre-mRNA splice correction assay and fluorescence-based quantification are described. Examples for characterization of CPP uptake routes, employing various endocyt ...

It is now clear that the major mechanism by which cellular function is switched from one state to another in eukaryotic cells, including the response to cellular stress (1), is via changes in phosphorylation of key proteins. This is usually achieved by modulation of the activity of pre-existing pr ...

Research over the past decade has focused increasingly on the photoreceptor mechanisms that regulate the circadian system in all forms of life. Some of the results to emerge are surprising. For example, the rods and cones within the mammalian eye are not required for the alignment (entrainment) ...

Triplex DNA has attracted considerable interest recently because of its possible biological functions in vivo and its wide variety of potential applications, such as regulation of gene expression, site-specific cleavage of duplex DNA, mapping of genomic DNA, and gene-targeted muta ...

Gene therapy holds great promise for the treatment of disease by delivering genes encoding for therapeutic proteins. Although it was originally devised for the treatment of inherited genetic disorders, such as cystic fibrosis, recent work has expanded the applications of gene therapy ...

This chapter describes the methods we use to maintain and expand undifferentiated human embryonic stem (hES) cells on human and mouse feeder cells. All of the available hES cells have been derived and propagated on primary mouse embryonic fibroblasts as feeder cells that have been mitotical ...

Human embryonic stem cells provide both an in vitro model of human development and a potential source of cells for treatment of degenerative, metabolic, or traumatic disorders. This chapter describes techniques for routine maintenance and differentiation of human embryonic stem ce ...

Massively parallel signature sequencing is an ultra-high throughput sequencing technology. It can simultaneously sequence millions of sequence tags, and, therefore, is ideal for whole genome analysis. When applied to expression profiling, it reveals almost every transcript in ...

Microarray technology is a powerful tool that allows for simultaneous assessment of the expression of thousands of genes and identification of gene expression patterns associated with specific cell types. Here we describe a protocol using this method to examine stem cells.

Human embryonic stem (hES) cell lines are usually derived and propagated on inactivated murine embryonic fibroblast (MEF) feeders. The use of MEFs and culture ingredients of animal origin for hES cell support increases the risk of cross-contamination of the hES cells with infectious anim ...