Nucleic acids introduction into cells

Epoxides are often encountered in nature, both as intermediates in key biosynthetic pathways and as secondary metabolites. The selective epoxidation of squa-lene, resulting in 2,3-squalene oxide, for example, is the prelude to the remarkable olefin oligomerization cascade that creates the steroid nucleus [7]. Tetrahydrodiols, the ultimate products of metabolism of polycyclic aromatic hydrocarbons, bind to the nucleic acids of mammalian cells and are implicated in carcinogenesis [8], In organic synthesis, epoxides are invaluable building blocks for introduction of diverse functionality into the hydrocarbon backbone in a 1,2-fashion. It is therefore not surprising that chemistry of epoxides has received much attention [9]. 

Transfection—the introduction of a nucleic acid construct into a cell(s) so that it remains intact and maintains its function. 

Early development of nucleic acid therapy was aimed at genetic deficiency diseases which were amenable to replacement therapy, or correction by administration of the protein products of the defective genes, as described in Chapter 3. The introduction of the normal counterpart of a defective gene into cells of the patient, resulting in the long-term production of the missing protein, appeared as a desirable alternative to the repeated parenteral administration of purified protein (Verma, 1990 Caskey, 1992). However, the current span of nucleic acid-based therapies far exceeds this limited application conceived initially (Riordan and Martin, 1991 Crooke, 1992a). 

TECHNOLOGIES AND TECHNIQUES Introduction of nucleic acids into cells... 

The delivery of preformed siRNA duplexes offers an attractive alternative to the introduction of large DNA or RNA which requires processing to become active siRNA. The delivery of siRNA into the cell faces similar problems to the delivery of most macromolecules intracellulary. Delivery vectors used to address larger nucleic acids have been adapted for the siRNA delivery and will be discussed below. 

Gene therapy is another technique to correct imperfect organ operation, albeit at the basic level of cell function (Le Doux et al., 1995). Gene therapy is the introduction of nucleic acids into cells for a therapeutic effect. Portions of the DNA strand are transferred into the host cell to produce proteins that the unaided cell cannot produce. There are over 4000 known human genetic diseases, and it is likely several hundred of these will be able to be successfully treated with gene therapy (Le Doux et al., 1995). Gene therapy is still in its infancy, and its success is still largely dependent upon chance. Steps in this process involve... 

Gene therapy refers to the introduction of nucleic acids, e.g., DNAs or RNAs, into the cells of target tissues with a therapeutic purpose. Gene therapy can be applied as a gene replacement... 

Rnally, for this introduction, metabolic processes are, in short, all of the reactions that must occur for life to be maintained. The processes themselves are generally divided into catabolic (or exergonic [but not necessarily in the form of work] or energy releasing) paths as in the breakdown of food in cellular respiration and anabolic (or endergonic [but not necessarily in the form of work] or energy utilizing) paths that are used to construct proteins and nucleic acids, cell walls, and so on. 

It is expected that binding to a membrane is also an essential step for penetration of nucleic acids into the cell [53, 54]. Such binding of nucleic acids may be achieved by chemical modification with hydrophobic residues as well. Thus, the general strategy described in this Section arises from the results obtained in [50-52]. However, in the case of nucleic acids, which represent polyanions with relatively high charge density, another route for introduction of a hydrophobic moiety can be proposed. 

Transfection techniques in vitro and ex vivo (organotypic cultures) offer an array of possibilities to investigate the consequence) s) of the introduction of foreign nucleic acids (DNA but also RNA) and or other biologically active molecules into neurons, and to combine observations with immunocytochemistry. In particular, a wide number of fluorescent reporter proteins (FRPs) can be employed for multicolor fluorescence imaging. Here we present a series of protocols for in vitro and ex vivo transfection of DNA or RNA sequences into cerebellar neuron cultures and organotypic slices based on the use of plasmid vectors and multicolor laser scanning confocal microscopy. These protocols allow analysis of live transfected cells, and, after fixation, correlative neurochemical studies. 

Transfection is tfie introduction of foreign nucleic acids into the cells. Originally the procedure was developed for DNA, but, more recently, successful delivery of RNA to live cells has also been made possible, so that, today, one (or more) gene(s) (DNA transfer) or foreign RNA(s) can be introduced into cells by a number of well-established protocols. In a broader sense, the term transfection can be even used to indicate the direct introduction of proteins (e.g., antibodies) into cells (also referred to as proteofection). 

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