细胞技术

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 ...

Analysis of promoter regulation is a powerful strategy for the study of regulatory interactions between genes that play a role in embryonic development. It allows investigation of the molecular mechanisms that underlay temporal and spatial expression of a specific gene. In addition, o ...

Zebrafish (Danio rerio) embryos have gained considerable popularity in recent years because they offer several advantages for developmental studies. The embryos are easy to manipulate, develop quite rapidly, and many genetic mutations are now becoming available. Classical cell ...

Expression of genes by introduction of DNA into developing embryos can be used to analyze promoter elements that are developmentally regulated or to ectopically express proteins to test their function during development. Because of its large size and resilience to manipulation, the Xe ...

Microinjection remains the most popular and effective of the methods to introduce DNA, RNA, and proteins into fertilized zebrafish eggs. The method is simple and reliable. A microinjection pipet is filled with the DNA or RNA solution and attached to an apparatus that forces the solution out of the p ...

The ease of obtaining large numbers of Xenopus laevis eggs and oocytes together with their size and robust nature have made them a popular choice when microinjection of macromolecules into a cell is required for protein expression and modification studies, protein function analysis, RNA p ...

Over the last 10 years, the animal cap of the Xenopus laevis embryo has proved to be a versatile test tissue for a variety of molecules involved not only in animal development but also vertebrate cell regulation in general. These molecules include growth factors (1–3), cell surface receptors (4–6), si ...

The authors have reported several comb-type copolymers, consisting of a polycation backbone such as poly-L-lysine (PLL) and hydrophilic side chains of polysaccharides, for controling the assembling structure of DNA-copolymer complexes (1–3). The DNA-copolymer system consis ...

There is an urgent requirement in the field of gene therapy for gene transfer vectors that are both safe to use and able to efficiently deliver therapeutic genes to target cells in vivo. Viral vectors, such as retrovirus, adenovirus, and herpes simplex virus, are efficient in transducing a broad ran ...

In recent years, interest in in vitro DNA condensation has been revived by efforts to develop vectors for nonviral gene therapy (1,2). One of the critical elements for successful and versatile delivery of specific genes into targeted cells is that DNA vectors of several kilobase pairs must be comp ...

Studies of the human genome have prompted the development of several cloning systems that can manipulate large fragments of human DNA as functional units. The discoveries in yeast of the sequences for replication (autonomously replicating sequence ), centromeres, and telomeres have ...

In vivo delivery of a cytokine gene to treat a tumor has usually involved either injection of ex vivo transfected cells around the tumor site or direct intratumoral injection of a virus or plasmid DNA (pDNA) vector encoding the cytokine gene (1,2). In this manner, transfected cells in or around the tumor ...

There are several strategies by which one may deliver a plasmid DNA (pDNA) encoding a therapeutic gene to a tumor. One may transfect cells ex vivo, single cell clone, expand the clone in vitro, and reinject the cells at the tumor site. This is a labor-intensive process and is especially impractical for hum ...