细胞功能测定

The aim of gene therapy is to treat inherited or acquired genetic deficiencies (e.g., cystic fibrosis) or viral diseases (e.g., hepatitis B, HIV) by introduction of DNA encoding a therapeutic protein or a specific virus antigen, respectively, into the nucleus of the target cell. Because naked DNA wi ...

Polycation-DNA complexes represent promising synthetic vectors for gene delivery, showing good transfection activities in vitro and safety in vivo. However, simple polycation-DNA complexes suffer from several disadvantages that limit their potential usefulness in vivo. A ...

Gene therapy provides a paradigm of the treatment of human diseases. The ultimate goal of gene therapy is to cure both inherited and acquired disorders by removing the original causes, i.e., adding, blocking, correcting, or replacing genes. Although gene therapy trials have been initiated wo ...

The ability of antisense oligonucleotides to interdict, sequence-specifically, the expression of pathogenic genes affords an exciting new strategy for therapeutic intervention (1–3). Oligonucleotides with physiological phosphodiester internucleotide bonds are r ...

This chapter focuses on a methodology for covalently associating nuclear localization signal (NLS) peptides to DNA, in which cationic NLS peptides are covalently bound to plasmid DNA by photoactivation. Described here are the synthesis and characterization of these conjugates.

Vectors for gene transfer can be categorized as viral and nonviral. The advantages of nonviral carriers are their ease of preparation and scale-up, flexibility regarding the size of DNA to be transferred, and safety in vivo. Despite these advantages, nonviral vectors need to be further optimi ...

The isolation and characterization of RNA polymerases from the Salmonella phage SP6 and the E. coli phages T7 and T3 has revolutionized all aspects of the study of RNA metabolism (1–6). Indeed, it is now possible to generate unlimited quantities of virtually any RNA molecule in a chemically pure form. ...

Recently, nonradioactive techniques for detection of DNA and RNA have been developed (1,2). The most commonly used labels are “digoxigenin,” “fluorescein,” and “biotin” which are linked through a spacer to a nucleotide (in most cases UTP or dUTP) and are incorporated into specific gene-prob ...

RNA dot hybridizations were first described by Kafatos et al. (1). They enable rapid detection of transcription from a number of different mRNA populations and are particularly useful in the initial characterization of cDNA clones isolated by differential screening. In cases in which many ...

A mammalian cell contains approx 10−5 μg of RNA which consists mainly of rRNA and in smaller amounts of a variety of low-mol-wt RNA species. These RNAs are of defined size and sequence. The ability to isolate clean, intact, and DNA-free RNA is a prerequisite in analyzing gene expression and cloning genes. T ...

The ability to quantify nucleic acids accurately and rapidly is a prerequisite for many of the methods used in biochemistry and molecular biology. In the majority of situations this is carried out using spectrophotometry, which is nondestructive and allows the sample to be recovered for fur ...

mRNA comprises approx l–5% of total cellular RNA. Although the actual amount depends on the type of cell and its physiological state, at any given time approx 12,000 genes are being transcribed with approx 500,000 mRNA molecules present in each mammalian cell.

The purity and integrity of Isolated RNA IS a critical determinant of its effectiveness in such molecular biological procedures as Northern blot, poly A+ RNA separation, cDNA synthesis, and in vitro transcription and translation The successful isolation of total RNA from tissues or cell li ...

The susceptibihty of RNA to degradation by exogenous and endogenous RNase activity following cell lysis has been well documented (1,2). Moreover RNA usually occurs complexed with protein from which it must be released. Precautions to be taken against exogenous RNase include the use of plas ...

Whole blood contains nucleated white cells that constitute an easily accessible source from which RNA can be extracted, without the need for prior homogemzation as is necessary with solid tissues. However, blood is a particularly problematic tissue from which to isolate RNA because RNA is e ...

In plant cells, mitochondrial RNA (mtRNA) constitutes about only 1% of the total RNA. From this, most are ribosomal RNAs. Thus, isolation of high-purified mtRNA is necessary not only for construction of a mitochondrial cDNA library, but also for the analysis of plant mitochondrial transcript ...

The isolation of uncontaminated, intact RNA is essential for analyzing gene expression and for cloning genes. However, plant tissue is notorious for being a difficult source from which to isolate high-quality RNA with good yield. This difficulty is primarily due to the presence of naturally ...

Fixed and paraffin-embedded tissues from pathology department archives can be available for RNA expression analysis. We have already shown that RNA isolated from biopsy, surgical, or autopsy tissue, routinely processed by fixation and paraffin embedding, is not completely degrad ...

Cationic surfactants precipitate nucleic acids, presumably by forming reverse micelles in which the ahphatic tails of the surfactant face the aqueous phase, and the cationic head groups bind to the nucleic acid electrostatically. The precipitate can be dissolved in organic solvents, ...

With the introduction of polymerase chain reaction (PCR), analysis on even minute amounts of DNA and RNA from biological samples became practicable. Conventional methods of mRNA analysis are often not sensitive enough for detection of low-abundance transcripts or broad examination ...