基因表达差异显示实验

RNase protection provides a sensitive method for detecting and quantitating specific RNAs. The method relies on the ability of ribonuclease A and ribonuclease T1 to digest single-stranded RNA, but not perfectly base-paired double-stranded RNA. In this respect RNA:RNA hybrids are more ...

The techniques described in this chapter have proven useful in the investigation of intracellular protein transport in plants and, in particular, transport of plastid proteins. The method of posttranslational transport, originally elaborated by Chua and Schmidt (1) and Highfield ...

Pioneering work leading to the elucidation of the genetic code and the characterization of the protein synthesis machinery relied heavily on in vitro transcription and translation systems. Such systems allowed the use of defined templates to direct the synthesis of proteins, which cou ...

The rabbit reticulocyte in vitro translation system, originally described by Pelham and Jackson (1), is widely used to identify mRNA species and characterize their products. Extensive descriptive protocols for the preparation of rabbit reticulocyte lysates have been previously ...

The general features of the organization of in vivo transcription of the mammalian mitochondrial DNA (mtDNA) were delineated in the 1970s and early 1980s by a detailed analysis of the mapping and metabolic properties of the transcripts of the two strands of this DNA (1, 2), the elucidation of the RNA pr ...

The oocyte is a useful model for the investigation of the function of various genes, especially those involved in meiosis. Since meiosis and mitosis are very similar processes, oocytes could prove useful in elucidating the role of genes in cellular proliferation. Indeed, the identificati ...

In vivo gene delivery has proven to be much more difficult than in vitro delivery. Many techniques have worked in vitro only to fail in vivo. Techniques such as electroporation require invasive and painful procedures to deliver genes. Ultrasound-mediated delivery has the potential to help ov ...

The Du Pont Genesis™ 2000 DNA Analysis System consists of an instrument, reagents capable of labeling DNA with novel fluorescent dideoxynucleotides (1), and a Macintosh® II computer to operate the instrument and perform all subsequent data analysis The instrument houses a vertical elec ...

The following view of transgenic studies applied to the understanding of brain function is written as a guide for neuroscientists who may be considering transgenesis techniques in the pursuit of their research. Although most important studies are covered, the chapter is not a comprehens ...

The commercial potential for transgenesis techniques is substantial, particularly in the fields of animal and plant agriculture. This results from productivity being a function of genetic potential and interaction with the environment, but environmental factors being only pa ...

Prior to the introduction of transgene constructs into animals, it is useful first to determine the presence of a functional promoter and transcription unit. This can be achieved by transient transfection of the construct into heterologous cell lines and subsequent measurement of repo ...

This chapter describes a standard method for the hybridization of labeled DNA probes to nucleic acids bound to a nylon matrix (1). Filters bearing bound nucleic acids produced by Northern blotting of RNA (Chapter 39), Southern blotting of DNA (Chapter 37), and slot blotting of DNA (Chapters 35) or RNA ...

Reporter genes code for proteins that have a unique enzymatic activity and are used to assess the transcriptional properties of DNA elements. The use of reporter genes in transgenic animals provides a rapid method for the detection of transgene expression, which is easily distinguishable ...

Immunohistochemistry can be used to localize the peptide or protein product of the transgene, or to identify particular cell types within tissue sections by labeling a specific protein. There are many ways to do this. Today many involve a primary antibody to the protein of interest, and a secondary ...

In situ hybridization (ISH), also called in situ hybridohistochemistry (ISHH), has been used since the 1970s to localize mRNA within a tissue. This technique is now routine in many laboratories, and some standard procedures are emerging.

Transgenic technology, the introduction of genetic information into the germ line of an animal to alter its genetic constitution stably, provides a powerful tool for studying the development, functions, and malfunctions of complex biological systems (1–3). The main advantage has been ...

Although subsequent generations can be rapidly screened using slot blotting (Chapter 35) or the polymerase chain reaction (PCR; Chapter 36), founder transgenic animals should initially be analyzed by Southern blotting for two reasons. First, low copy number transgenics or mosaic tra ...

Once transgenic animals have been made and founder animals identified, it is usually necessary to establish lines of animals carrying the transgene. Since the founder animal will be hemizygous with respect to the transgene, at most 50% of its offspring will be transgenic. If the original animal ...

Slot or dot blotting is a technique whereby nucleic acids can be applied to a solid matrix, unfractionated, using a vacuum manifold. Slot blotting is the quickest, easiest, and, apart from polymerase chain reaction, probably the most sensitive assay of transgenic animal genotype (1). However, it ...

Transgenic animals are identified by the presence of transgene sequences in genomic DNA isolated from biopsy material. A suitable method of obtaining this genomic DNA must fulfill three criteria: 1.The method must be rapid and easily performed. Up to 100 genomic DNA preparations may have to be p ...