Cell transfer technique

Classical gene transfer methods still in use today are diethylamino ethyl (DEAE)-dextran and calcium phosphate precipitation, electroporation, and microinjection. Introduced in 1965, DEAE-dextran transfection is one of the oldest gene transfer techniques [2]. It is based on the interaction of positive charges on the DEAE-dextran molecule with the negatively charged backbone of nucleic acids. The DNA-DEAE-dextran complexes appear to adsorb onto cell surfaces and be taken up by endocytosis. 

Several enveloped viruses, and some physical gene transfer techniques such as electroporation, deliver the nucleic acid into the cell by direct crossing of the cell membrane. Lipid-based, enveloped systems can do this by a physiological, selfsealing membrane fusion process, avoiding physical damage of the cell membrane. For cationic lipid-mediated delivery of siRNA, most material is taken up by endo-cytotic processes. Recently, direct transfer into the cytosol has been demonstrated to be the bioactive delivery principle for certain siRNA lipid formulations [151]. 

The degree of exposure of the fetus to a particular substance can be best assessed in human subjects, but concerns of fetal safety have restricted the use of this approach. Moreover, clinical studies cannot elucidate the various mechanisms that contribute to transplacental transport of a particular compound. There are many structural differences between the human placenta and the placenta of other mammalian species, which complicates extrapolation of data obtained from in vivo animal models to humans [7], Thus, several ex vivo and in vitro techniques have been developed to study the placental role in drug transfer and metabolism during pregnancy and there are some excellent articles that discuss these systems in detail [7], Both isolated tissues and various cell culture techniques are currently in use and these have been summarized below. 

The first successful attempts to study RNA evolution in vitro were carried out in the late sixties by Sol Spiegelman9 and his group at Columbia University (Spiegel-man, 1971). They made use of an RNA replicase isolated from Escherichia coli cells infected by the RNA bacteriophage QP and prepared a medium for replication by adding the four ribonucleoside triphosphates (GTP, ATP, CTP, and UTP) in a suitable buffer solution. QP RNA, when transferred into this medium, instantaneously started to replicate. Evolutionary experiments were carried out by means of the serial transfer technique (Figure 4). Materials consumed in RNA replication... 

W. C. Heiser, Ed., Gene Delivery to Mammalian Cells. Vol. 1. Nonviral Gene Transfer Techniques, Totowa, NJ Humana, 2004. 

Heiser WC (2004), Gene delivery to mammalian cells, vol. 2 Viral gene transfer techniques, In Heiser W (Ed.), Methods in Molecular Biology, vol. 246, Humana Press, Totowa. 

Between the methods of Agrobacterium and microprojectile transfer, nearly every plant species can be transformed effectively [35]. However, these methods are covered by patent claims and may result in limited transformation efficiency for some cell types. For this reason, the use of alternative gene-transfer methods is an active area of research for all cell types. Alternative gene-transfer techniques include electroporation, microinjection, liposome fusion, direct transfer into protoplasts, and laser treatment [38]. In electroporation, DNA is transferred into the cell using a high-voltage electrical pulse [39]. Standard... 

In addition to the vascular perfusion system studies, we have employed brush border membrane vesicles. Isolated from rat mucosa, to determine more closely the parameters of mucosal zinc transport (43). These vesicle preparations represent the best means currently available to delineate the characteristics of zinc transfer into mucosal cells. The technique permits isolation of the microvillous membrane free of other cellular contaminants, as determined by established procedures (44). 

The three kinds of reactors already described in this section are all traditional cross-flow reactors with permeable plates or membranes. The electrochemical filter-press cell reactors used, e.g., for electrosynthesis, are equipped with cation-selective membranes to prevent mixing of the anolyte and the catholyte. These cell reactors are therefore good examples of the extended type of cross-flow reactors according to the definition transferred from the filtration field. The application of the electrochemical filter-press cell reactor technique... 

Several studies have demonstrated that enzyme preparations with reproducible properties and purity and having assured stability can be made. These may he from animal sources in which the enzymes closely resemble their human counterparts, although new possibilities have been created by gene-transfer and mass-cell culture techniques ( recombinant proteins). 

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