DNA transfer method

Since the mechanism for the transfer of DNA to cells can vary widely from one method to another, one suspects that very different consequences may result within the cell - events over which the experimentation has no or little control. For example, the DNA transfer method may affect the plasmid copy number average in individual clones, depending on the amount of DNA transferred to the nucleus. Electroporation may result in a lower copy number of integrated DNA than calcium phosphate-based transfection. In view of the applied selection procedures for the generation of recombinant cell lines, very different structures of integrated plasmid DNA may result... 

Shen W, Li L, Pan Q et al (2006) Efficient and simple production of transgenic mice and rabbits using the new DMSO-sperm mediated exogenous DNA transfer method. Mol Reprod Dev 73 589-594... 

Developing a genetic system for lactic acid bacteria was initially dependent upon optimizing a DNA transfer method. 

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. 

Agrobacterium-mQdi iQd DNA transfer technique and direct gene transfer into protoplasts on the basis of the electroporation technique have frequently been used for the gene transfer methods in plants. However, these methods were hardly adapted to monocotyledonous plants or to varieties in which regeneration system was not established. These deficiencies found in gene transfer techniques will be largely overcome if pollen could be used as a DNA vector. 

Despite the almost 100% efficient transfection rates that can be achieved using vims-mediated DNA delivery, there are a number of concerns over the therapeutic use of this method for DNA transfer in humans. One concern is the toxicity induced by the viral infection and the potential for the generation of a strong immune... 

A wide variety of methodologies and reagents are currently employed to introduce different molecules into eukaryotic cells. The incorporation of DNA can be achieved by two different mechanisms infection or transfection. The first consists of a biological process mediated by a virus (the viral infection of cells is mediated by receptors), while the second makes use of physical or biochemical methods to incorporate the DNA into the cell. Although the virus-mediated methods are more efficient, they are more laborious and time-consuming compared with transfection. Additionally, the nature of the infection process requires the presence of virus-specific receptors in the host cell to allow viral penetration, which restricts the spectrum of possible host cells. Another limitation of viral infection as a method for DNA transfer is that, unlike plasmid transfection, it is not possible to simultaneously transfer multiple recombinant viruses into the cell (Wurm and Bernard, 1999). 

The construct requirements for the transfer of DNA to plant cells vary depending on the transfer method. Obviously, the foreign DNA must be genetically compatible with the plant cell host and there must be some means of differen-... 

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... 

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