细胞技术

In situ hybridization is a basic method in modern plant cell and molecular biology. It is used to locate the chromosomal position of genomic DNA sequences. It is able to determine the patterns of gene transcription in mature tissues and during development. In situ hybridization, in combination w ...

The potential of cell and gene therapy has generated extensive interest over the past several years. More recently, identification of stem cells of various types, especially embryonic stem cells, reinforced this interest. Systematic studies are now being launched to define the biology ...

In this chapter, we describe a simple and relatively rapid technique for detecting lowabundance slug mRNA in cultured cells. The procedure uses nonradioactive digoxigeninlabeled RNA probes that are more sensitive than deoxyribonucleic probes and simpler to detect than radioact ...

A method is described for in situ hybridization of riboprobes to free-floating brain sections. Brain sections are hybridized and processed free-floating in buffer, i.e., without attachment to a support such as a slide. To withstand the extra wear compared with sections processed on-slide, t ...

This chapter describes a pre-embedding in situ hybridization method utilizing an immunogold-silver intensification step to identify P2Y2 receptor mRNA transcripts in the adult rat cerebellum. The method was applied for ultrastructural (electron microscopic) examinatio ...

The term in situ hybridization (ISH) refers to all methods allowing the detection of specific DNA (gene loci) or RNA (gene expression products) sequences, using molecular hybridization (base pairing) of labeled nucleic acid probes to target molecules within “intact” cell populations in ...

In situ hybridization (ISH) has significantly advanced the study of gene structure and expression in cells and tissues, but its application is often limited by detection sensitivity. The introduction of signal amplification after ISH using tyramides has greatly advanced in situ dete ...

In this chapter we describe the use of cRNA (riboprobes) in the detection of gene expression in tissue sections. Riboprobes offer good sensitivity and allow the detection of low-level mRNA expression. In some cases, the use of radiolabeling is justified because this method is still sensitive. H ...

A variety of probes can be used for in situ hybridization, depending on the application and the labeling strategy. In general, RNA probes (riboprobes) are now more commonly used. However, some laboratories may still prefer to use DNA probes for in situ hybridization, and a number of techniques for th ...

The treatment of tissue sections to enhance probe access to target mRNA is a critical step in the methodology of in situ hybridization. We have overcome some of the problems encountered in enzyme-based treatment of tissue sections by the application of microwave oven heating. Microwave trea ...

Nonradioactive in situ hybridization offers a unique opportunity to study gene expression on samples with preserved histological information. This method makes it possible to locate not only where in a tissue a particular gene is expressed, but in many cases also in which specific cell type it ...

Epstein-Barr virus (EBV) establishes a lifelong infection of B cells. Consequently, EBV-carrying B cells are present in the peripheral blood as well as in lymphoid and nonlymphoid tissues of most individuals. As a result, the detection by polymerase chain reaction of EBV genomes in DNA extrac ...

Liposomes can serve as carriers of radionuclides for diagnostic imaging and therapeutic applications. Herein, procedures are outlined for radiolabeling liposomes with the gamma-emitting radionuclide, technetium-99m (99mTc), for non-invasive detection of disease and for ...

The intracellular distribution of nanoparticular drug delivery systems is very complex, but its investigation yields high potential for further development and optimization of these systems. In the following chapter, we introduce the application of fluorescent imaging tech ...

By monitoring the efficiency of fluorescence resonance energy transfer of dyes attached to the different strands of siRNA, the structural integrity of the latter can be traced inside cells. Here, the experimental details of dye-labeled siRNA construction, tissue culture, and transfe ...

Although lipids are essential for the optimal activity of the cytochromes P450 monooxygenase system, relatively little is known about the membrane environment in which these enzymes function. One approach used to mimic the structural arrangement of lipids and enzymes within the endo ...

The biological activity of cationic liposome/DNA complexes (“lipoplexes”) is strongly dependent on their ability to protect DNA and to interact with cells, including binding to the cell surface, internalization via endocytosis and cytoplasmic delivery of the DNA. In this chapter, we de ...

Efficient delivery of genetic material to cells is needed for tasks of utmost importance in the laboratory and clinic, such as gene transfection and gene silencing. Synthetic cationic lipids can be used as delivery vehicles for nucleic acids and are now considered the most promising nonviral ...

We describe three applications of the FRET technique to analysis of structural and thermodynamic properties of cationic lipids and their complexes with DNA. (1) Lipid mixing assay to determine the degree to which individual vesicles undergo fusion upon complex formation. (2) DNA binding ...

Lipoplex preparations are heterogeneous mixtures of lipoplex particles of different structures. As these structures determine the efficiency of the delivery of genetic material, it is important to characterize the distribution of particles of different types in lipoplex prep ...