细胞功能测定

This chapter describes the protocol for preparation of recombinant adenoviruses and infection of target cells to express transiently G-protein-coupled receptors or other proteins of interest. Adenoviruses are nonenveloped viruses containing a linear double-stranded DNA ...

The molecular cloning of the cDNA sequences encoding most G-protein-coupled receptors, including those from humans, allows their study in a variety of recombinant systems. In this respect, transfected mammalian cell lines constitute the most frequently used model for investigati ...

The localization and density of G-protein-coupled receptors (GPCRs) on the cell surface is a critical factor for specifying signaling within and between neurons. GPCRs and their associated signaling components are localized to specific neuronal compartments that give rise to a vari ...

G-protein-coupled receptor mRNAs are expressed at low levels and therefore present a challenge for the study of their sites and levels of expression. In situ hybridization (ISH) and Northern blotting are powerful methods for the localization of mRNAs and the study of regulation of mRNA expre ...

Epitope tagging of a receptor involves introducing a defined amino acid sequence, to which an antibody has already been produced, into the primary amino acid sequence of the receptor. The new sequence can be as short as 10–15 amino acids and the method allows the receptor to be monitored without having ...

Immunocytochemistry exploits the incomparable specificity of the antibody-antigen interaction to form the basis of a flexible approach to the study of expression and localization of proteins both in model systems and their physiological context. This chapter details the theory a ...

Antibodies have proved invaluable in the study of G-protein-coupled receptors (GPCRs). The utility of these immunoglobulin probes for investigation of protein structures and functions arises from their selectivity as well as their versatility. Antibodies can be used to analyze GP ...

Methods are presented for identifying and quantifying allosteric interactions of G-protein-coupled receptors with labeled and unlabeled ligands using radioligand-binding assays. The experimental designs and analyses are based on the simplest ternary complex alloster ...

The radioligand-binding assay is a relatively simple but powerful tool for studying G-protein-coupled receptors. There are three basic types of radioligand-binding experiments: (1) saturation experiments from which the affinity of the radioligand for the receptor and the bindi ...

The misexpression and overexpression of proteins by injection of synthetic mRNA into Xenopus oocytes and, more particularly, embryos has provided the experimental basis for a considerable portion of our current knowledge of early vertebrate development (1, and references there ...

Immunohistochemistry is a very powerful technique for determining both the tissue-specific and subcellular location of endogenous and exogenous proteins within an embryo. The technique is relatively simple and when used on its own or in conjunction with other immunological techn ...

Immunohistochemistry is a powerful technique for determining both the presence of and the subcellular location of proteins within tissues. Zebrafish are particularly amenable to this technique and it is possible to localize proteins both in whole embryos and larvae, as well as section ...

Analysis of the spatial and temporal regulation of genes during embryogenesis is necessary if we are to understand their roles in developmental processes. In situ hybridization is the standard procedure used for describing the patterns of gene expression in embryos. In this technique, an ...

Wholemount in situ hybridization (WISH) is a technique widely used to study the expression patterns of developmentally regulated genes. The last few years have seen massive improvements in the protocol. Not only can we now detect weak signals much more clearly but we can also visualize two, or even ...

Over the last few years, RT-PCR (1,2) has become a widely accepted method for quantitation of steady-state mRNA levels, particularly in Xenopus. Its unmatched sensitivity and swiftness allows for a high sample throughput with minimal amounts of starting material—considerable advant ...

When characterizing the developmental expression of a novel gene, or when examining the response of a known gene to experimental manipulations, it is important to be able to assay mRNA transcript levels accurately. Although a number of techniques for transcript analysis are available, one ...

In vivo footprinting is a technique that enables one to detect protein-DNA interactions as they are occurring in a cell. The principle behind this technique is similar to the principle behind the in vitro footprinting technique: Both rely on the fact that a bound protein often causes its binding si ...

The interactions of trans-acting factors with cis-acting DNA elements have been a mainstay of molecular biology. With the emergence of new techniques, it is becoming easier to investigate how specific genes are regulated in differentiating cells. In 1978, Galas and Schmitz developed a mod ...

The DNA-binding proteins play a pivotal role in the cell, regulating gene transcription, DNA replication and repair, it is therefore of fundamental importance that the mechanisms controlling these factors and their interactions be investigated. The study of DNA-binding proteins in t ...

A common approach to the study of gene function in Xenopus embryos is to ectopically express the gene of interest, or a mutant of that gene, by injecting either RNA or DNA, encoding the gene into early-stage embryos. Both of these approaches have disadvantages (1). RNA injection results in little tempo ...