基因表达差异显示实验

Gene targeting in mice has become a commonly used technique to elucidate gene function. The principles for the generation of such gene knockout mice are relatively straightforward and many of the experimental procedures are standardised (1–3). A further step toward increasing the effic ...

In recent years, gene targeting technology has been rapidly evolving and has been used by a number of laboratories to systematically alter the mouse genome and to generate mice with specific targeted gene mutations. The improvement in the quality of reagents (embryonic stem (ES) cells and med ...

Conventional gene knockout technology by homologous recombination can provide important information toward elucidating the function of some genes; however, the role of many genes cannot be investigated due to early embryonic lethality. Alternatively, the role of a particular ge ...

Embryonic stem (ES) cell-mediated transgenic approaches have revolutionized mammalian genetics in the last decade. Close to 4000 genes have been targeted to date. Analysis of these genetic alterations has provided an unprecedented understanding of critical gene functions that u ...

In designing and planning the generation of a gene-targeted mouse, there are several stages at which the choice of mouse strain is critical. Consideration must be given to the genetic strain from which (1) the genomic DNA library, (2) the embryonic stem cells, and (3) the recipient embryos are derived, ...

Over the last half century, the efforts of several pioneering developmental biologists have established the conditions to culture, manipulate, and reintroduce mouse embryos that will successfully develop to term. The isolation of teratocarcinoma cell lines, first, and later of bla ...

The recombinant DNA revolution has, since its very birth, been accompanied by many thorny ethical problems; some are new, but most are variations on dilemmas that have confronted scientists and medical practitioners for a very long time.

Normal and disease-associated gene sequences may be rapidly and accurately characterized at the molecular level using the procedures described here. First, a modification of the polymerase chain reaction (PCR) technique (1,2) provides a simple method of template preparation star ...

The identification and use of restriction fragment length polymorphisms (RFLPs) (1) detected by Southern blotting (2), using known or anonymous DNA probes, has provided the means for development of genetic markers that are distributed throughout the genome and that form the basis for lin ...

This chapter deals with the use of synthetic oligonucleotides as probes for the detection of allelic sequence variation and point mutations by use of a dot-blot procedure in DNA enzymatically amplified in vitro (polymerase chain reaction). The approach is based on the principle that a fully m ...

The amplification refractory mutation system (ARMS) is a simple and rapid method of detecting point mutations, restriction fragment length polymorphisms (RFLPs), and small nucleotide insertions or deletions. The method was first described by Newton et al. (1) for analyzing single DNA ...

Routine DNA analysis is assuming an increasing importance in clinical medicine as well as in basic research. The expanding field of DNA diagnostics will take advantage of the growing understanding of the molecular basis of human disease. Similarly, extensive studies of the structure of la ...

The introduction of double-stranded RNA (dsRNA) into Caenorhabditis elegans hermaphrodites results in the rapid and sequence-specific degradation of endogenous mRNAs (1,2). This RNA-mediated interference, or RNAi, effectively shuts down expression of the target gene and can p ...

RNA interference (RNAi) in Trypanosoma brucei was first reported in 1998 (1). As in other eukaryotes, interference involves digestion of the interfering double-stranded RNA into short fragments (2), a polysome-associated complex (3), and Argonaute protein (4,5). T. brucei is an ideal orga ...

RNA-mediated gene silencing is triggered by double-stranded RNAs (dsRNAs) that are processed to short RNAs approx 21–25 nucleotides in length. The short RNAs act as guides for enzyme complexes that degrade, modify, or inhibit the function of homologous target nucleic acids. There are three g ...

Various methods have been devised to elucidate gene function in a highthroughput format. With the potential to silence any gene once the sequence is known, small, interfering RNAs (siRNAs) have been considered ideal for functional analysis and gene target validation (1–3). Furthermore, t ...

With the explosion of genomic information, there is an increasing need to analyze gene function in a high-throughput fashion. This makes reverse genetic approaches extremely attractive; however, in most mammalian and vertebrate systems it has been difficult and time-consuming to dev ...

Angiogenesis, also known as new blood vessel formation, is a critical process during physiological and pathological conditions (1,2). During normal development, the physiological wound healing process, and the female menstrual cycle, blood vessel growth is coordinated concomi ...

DNA fragments larger than 25 kb are not resolved effectively by standard gel electrophoresis. Pulsed field gel electrophoresis (PFGE), first described by Schwartz and Cantor in 1984 (1), is a technique that allows for the separation of large DNA fragments of up to several mega base pairs (mbp) in len ...

In the previous chapter we described linker scanning mutagenesis by oligonucleotide ligation. In this chapter we describe a more recent and versatile procedure that makes use of amplification by the polymerase chain reaction (PCR). For a description of the traditional method of genera ...