遗传学实验技术

DNA methylation is an epigenetic modification of DNA that leads to heritable alterations in transcriptional regulation and conformational changes in chromatin structure of higher eukaryotes. Mammalian DNA methyltransferases, which are the enzymes responsible for DNA meth ...

Chromatin immunoprecipitation (ChIP) is a powerful tool to study protein-DNA interaction and is widely used in many fields to study proteins associated with chromatin, such as histone and its isoforms and transcription factors, across a defined DNA domain. Here, we show the step-by-step me ...

Active genes in eukaryotic genomes are typically found in open, nuclease-sensitive regions of chromatin. This chapter presents an overview of the techniques used to assay restriction endonuclease cleavage of chromosomal DNA as an approach to assess general accessibility at a genom ...

This chapter documents a simple protocol to identify the nucleosome positioning of any given genes. The procedure includes partitioning 200-bp DNA fragments constituting the nucleosomal core region by micrococcal nuclease digestion and semiquantitative polymerase chain r ...

Transcriptionally inactive DNA is packaged into condensed chromatin such that it is unavailable to the transcription initiation complex. Activation of the silenced genes during processes such as differentiation first requires that the chromatin structure be remodeled into a t ...

Covalent modifications on the nucleosomal histones are essential in chromatin regulation and gene expression. Patterns of histone modifications may be somatically maintained and can thereby maintain locus-specific repression/activity in defined lineages or througho ...

The ability to introduce new gene sequences into the tissues of experimental animals allows investigations into the regulation and effect of gene expression in that tissue as well as providing model systems for human gene therapy. In this regard, there has been considerable interest in the ex ...

Introduction of DNA into mammalian cells is a powerful tool for studying the function of various DNA sequences, and for gene therapy. The process of introducing DNA into cells for the purpose of gene expression is called transfection or gene delivery. Synthetic compounds used to facilitate DNA ...

Simplicity and cost are just two of the factors that have sustained the popularity of calcium phosphate and, to a lesser extent, DEAE-dextran transfection methods. However, notwithstanding these factors, the calcium phosphate method, especially use of N,N-bis(2-hydroxyethyl)-2-a ...

There are now several cationic peptide carriers that efficiently import plasmids and oligonucleotides into cells. As a result, we anticipate that cationic peptides will play an increasingly important role with in vitro and in vivo gene delivery systems. Cationic peptides usually bind ...

In recent years, Starburst� PAMAM dendrimers, a class of polyamidoamine polymers, have become an interesting alternative vector for nonviral delivery of DNA in vitro and in vivo (1-6). These nanoscopic polymers, characterized by regular dendritic branching, radial symmetry, and unif ...

Peptide nucleic acid (PNA) is a DNA mimic in which the deoxyribose phosphate backbone has been replaced by N-(2-aminoethyl) glycine linkages. PNAs, first described by Nielsen et al. in 1991 (1), possess a number of useful properties including rapid and high-affinity binding to DNA, RNA, and PNA, resi ...

The ability to mediate gene transfer to the lumen of the lung offers opportunities to treat diseases that affect the airways. Currently, several different gene-transfer vectors are being evaluated for delivery of a variety of therapeutic genes to the airways. These include recombinant vi ...

Following Fraley and colleagues’ initial discovery of liposomes (1), coupled with Behr’s discovery of the ability of cationic lipids to interact with and condense negatively charged DNA (2), Felgner et al. described the use of synthetic cationic lipids as a DNA delivery tool in 1987 (3). The autho ...

The lung endothelium has been studied as one of the most important sites for systemic DNA delivery using nonviral vectors. This is because not only does the lung play a critical role in maintaining the O2/CO2 concentration in blood, but it also has the largest capillary bed and an extensive cell surface ...

Cancer is a genetic disease. A cancer cell usually contains DNA abnormalities that may activate oncogenes or inactivate tumor suppressors or both. The identification of these cancer-causing genes and the signal-transduction pathways involved has allowed the development of cancer ...

The study of gene transfer has always been a major component of modern molecular biology. Initially, transfer of exogenous genes into cells was considered a critical technical step toward understanding the function and regulation of newly discovered genes. With the rapid advancement in ...

Microinjection—that is, the direct-pressure injection of a solution into a cell through a glass capillary—is an effective and reproducible method for introducing exogenous material into cells in culture. The method has been in existence almost as long as there have been microscopes to ob ...

Numerous chemical, physical, and viral methods have been developed for delivering genes to mammalian cells in culture. Physical methods of gene transfer have the advantage of transporting the genetic material directly into the cytoplasm or nucleus of the cell, bypassing the need for spec ...

Particle bombardment or particle-mediated DNA-delivery technologies were developed as physical gene-transfer methods for various in vivo, ex vivo, or in vitro applications. The basic concept is to directly deliver naked DNA plasmids into target cells by using accelerated particl ...