基因表达差异显示实验

Transfection of postmitotic neurons remains one of the most challenging goals in the field of gene delivery. For many cell types, gene transfer using the various methods described in this book has become a routine tool for studying gene regulation and function. However, postmitotic neurons a ...

The easy accessibility and the large area of the skin make it a potential target for gene therapy (1) while its immune activity makes it an efficient site of DNA vaccination (2). Despite these potential advantages for the delivery of DNA into the skin, a significant physical barrier impedes the transf ...

Appropriate electric pulses can reversibly permeabilize living cells both in vitro and in vivo. Since the pioneering work of E. Neumann and colleagues (1), cell electroporation (also often termed cell electropermeabilization) has become the most frequent method for cell transfect ...

The study of gene expression and regulation relies on the introduction of DNA into cells, which can be accomplished by a procedure called transfection. In vitro studies with transfected cells expressing the introduced genes (trans-genes) have provided many valuable insights into gene f ...

Electroporation is a physical method that delivers plasmid DNA in a variety of tissues. This technology is currently gaining recognition and acceptance in the scientific community as evidenced by the number of studies using in vivo electroporation published each year. One potential ap ...

Gene therapy—the goal of which is to cure inheritable and acquired diseases by supplying genetic information to various tissues—is a promising therapy of the new millennium. To date, many strategies have been attempted to cure a disease by adding a foreign gene or correcting a mutation in the gene ...

Polypeptides with therapeutic benefit and those for which a cellular and humoral immune response is desirable can be produced by injection of plasmid DNA into muscle (for review, see ref. ). The safety and efficacy of the DNA-based approach has resulted in several clinical trials with plasmids e ...

Enhanced chemiluminescence (ECL) is a generic detection system that has been applied to all standard, membrane-based molecular biology techniques (1). The technology is based on the well-characterized horseradish peroxidase (HRP) catalyzed oxidation of luminol in the presence ...

The field inversion gel electrophoresis (FIGE) is a special pulsed field gel electrophoresis technique that is based on the periodic inversion of a uniform electric field in one dimension (1). A net migration is achieved since the product of the duration and amplitude of the “forward” pulse is lar ...

The λgt11 system has been designed to allow expression of cDNAs either in phage plaques or in bacteria lysogenized with recombinant λgtll phages (1, 2). cDNAs are cloned into a unique EcoRl restriction site near the 3′ end of a truncated E. coli lactose operon (lacP/O-lacZ), allowing inducible, high- ...

Denaturing-gradient gel electrophoresis (DGGE) detects DNA sequence differences. Thus, it can be used to screen for point mutations or other types of mutation prior to DNA sequence analysis. The technique, first described by Fischer and Lerman (1), entails electrophoresis of DNA fragme ...

Until recently, most defects identified in mutant genes have been based on large size differences, as detected by Southern or Northern blotting, or by the sequencing of cloned DNA fragments. However, it is probable that a large proportion of disease-causing mutations are point mutations. As a c ...

At present, the most polymorphic genetic markers are DNA regions composed of a variable number of tandem repeats (VNTRs) (1,2). The detection of VNTRs is made possible by restriction fragment-length polymorphism (RFLP) analysis via Southern blotting (3). The length of each DNA fragment is a fu ...

Histopathology archive material constitutes an enormous resource of diseased tissues. It is composed of specimens that have usually been fixed with formalin to stop further tissue changes after removal from the body and subsequently embedded in a supporting material, such as paraffi ...

The majority of human-rodent somatic cell hybrids lose human chromosomes in a more or less random manner. It is possible to obtain a panel of hybrids in which each contains a unique subset of human chromosomes. In principle, human genes can be mapped in such hybrids by looking for correlation between the ...

The Southern blot technique, published by Southern in 1975 (1), is probably the most widely used method in molecular biology and, to date, has received in excess of 100,000 literature citations. The purpose of the method is the detection of gel-fractionated DNA molecules following transfer to a me ...

Blotting transfer techniques are well-established procedures for the immobilization of DNA onto solid matrices, typically nitrocellulose or nylon membranes. The Southern blotting technique (ref. 1 and Chapter 15) has found many research and, more recently, medical applicatio ...

Cloning from DNA fragments fractionated by pulse-field gel electrophoresis (PFGE) offers an opportunity to isolate markers from a specific region of a genome and thus forms part of the armory of the reverse geneticist. In particular, it can be used as an adjunct to chromosome walking and jumping s ...

Yeast artificial-chromosome (YAC) cloning systems are used to clone large contiguous segments of DNA from any organism into suitable vectors in such a way that the recombinants can be transformed into yeast (Saccharomyces cerevisiae) cells, where they are stably propagated. The basic s ...

The polymerase chain reaction (PCR) uses two oligonucleotide primers to direct the synthesis of specific sequences of DNA. One primer anneals to the coding strand of DNA and the other to the anticoding strand; the primer binding sites are typically separated by a few hundred base pairs (100–1000 b ...