细胞技术

This chapter presents a qualitative description of the freeze-drying process as it pertains to the development of stable, dry polycation-DNA complex formulations. It is not intended to be a comprehensive treatise on freeze-drying. Readers are referred to a series of excellent papers by Pi ...

A powerful, yet often underutilized tool available to probe the structure of macromolecules is absorption spectroscopy. The first of the various spectroscopic techniques to be widely developed, data from absorption spectroscopy is commonly viewed as low in overall information co ...

For many decades, infrared (IR) spectroscopy has been used to characterize the structure of molecules. In IR spectroscopy, absorption of light, corresponding to vibrational and rotational transitions of a molecule, is measured. For a transition to be IR-active, a change in the dipole moment ...

Within the past 10 years, major advances in the design and development of differential scanning calorimeters (DSC) (1) and isothermal titration calorimeters (ITC) (2) have resulted in an unparalleled level of sensitivity, stability, and reproducibility in calorimetric measurem ...

Until now there is no renal gene therapy available for clinical use, however, gene therapy for several experimental renal diseases has been tested with promising results. The kidney is a well-differentiated organ with a variety of specialized compartments, i.e., vascular, glomerular, tu ...

The colloidal properties of delivery systems currently being developed for nonviral gene therapy are extremely important. The physical stability of these systems on the shelf, as well as in the biological milieu, is mostly based on their size and interfacial properties. The size and surface ...

Development of nonviral gene transfer techniques has progressed, particularly the use of several kinds of cationic lipids and cationic polymers such as polylysine derivatives, polyethyleneimines, polyamidoamine dendrimers, and so on, which electrostatically form a compl ...

Research and development related to nonviral gene carriers comprising chemically synthesized molecules has increased enormously during the past decade. Polycationic polymers and cationic lipids have constituted the main themes of the studies. Various polymers from synthe ...

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.