Other Non-viral Delivery Techniques

Calcium phosphate transfection

Calcium phosphate transfection, initially described in the early 1960s, was refined and systematically improved to result in a standard protocol which has changed little since the early 1970s. In many cases it remains the system of choice for transferring plasmid DNA into a variety of cell cultures and packaging cell lines. It is particularly important in the production of recombinant viral vectors.

Basics of the technique. The technique relies on precipitates of plasmid DNA formed by its interaction with calcium ions. It is a very inexpensive and simple technique to perform. Plasmid DNA is mixed in a solution of calcium chloride, and then is added to a phosphate- buffered solution. Over a period of 20 minutes a fine precipitate forms in the solution, and this solution is then added directly to the cells in culture. Transfer efficiency, the number of cells which express the desired gene, is usually quite limited and only reaches levels greater than 10% in a few specific cell lines. In many cases, the level is less than 1%. Transfection efficiencies can be improved in some cell lines by 'shocking' the cells with DMSO or glycerol. Cells can be either transiently or stably transfected using this technique. Levels of stable transfectants can be improved using bis-hydroxyethylaminoethansulfonate (BES). The DNA precipitates are thought to enter the cell by endocytosis. Although this technique has minimal cellular toxicity, and is both simple and inexpensive, the low level of transgene expression prompted development of other techniques. Little or no transgene expression has been observed in vivo following calcium phosphate transfection.

DEAE-dextran is another compound that interacts and delivers DNA into cells. It is more reproducible than calcium phosphate transfection but only works in a select few cell lines. Also, DEAE-dextran-mediated gene transfer results only in transient transfections. The exact mechanism of DNA uptake mediated by this technique is not understood.

Microinjection

Microinjection delivers plasmid DNA directly into the cell's nucleus. Using the light microscope, a glass pipette is guided into the nucleus and a small amount of DNA or RNA injected. In doing so, both cytoplasmic and lysosomal degradation of the injected material is avoided and efficient gene expression can be expected from the surviving cells. Unfortunately, this technique is extremely labor intensive and requires well isolated cells.

Since safety and efficiency can be closely monitored using microinjection, it may one day prove useful in germline gene therapy applications. Microinjection of a particular gene into a cell at a specific time during development has the potential to alter the genotype and phenotype of every cell formed thereafter. Germline modification has already been accomplished in many mammals, which have been termed transgenic animals, ranging in size from mice to cattle. Germline modifications in humans for the correction of specific disorders is far from a present reality and is fraught with ethical considerations.

Electroporation

Electroporation is the application of high voltage to a mixture of DNA and cells in suspension. The cell-DNA suspension is placed between two electrodes and subjected to an electrical pulse. The DNA enters the cells through holes formed in the cellular membrane during the electrical pulse. The DNA is trapped within the cytoplasm upon termination of the electrical pulse. For efficient gene transfer, electroporation depends upon the nature of the electrical pulse, the distance between the electrodes, the ionic strength of the suspension buffer, and the nature of the cells. Best results have often been obtained from rapidly proliferating cells. Electroporation of mammalian cells is an inefficient technique since many cells do not survive the high voltage nature of this procedure. Because of its unique characteristics, electroporation has been difficult to properly design for in vivo applications.

Latest Review: 

Nonviral Gene Therapy: promises and challenges 

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