Gene transfer techniques

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. 

All together, these findings have encouraged the development of neutral and cationic lipospheres (CLS) as nonviral DNA-mediated gene transfer techniques because CLS enable the extemporaneous production of pharmaceutical formulations. Like emulsions and liposomes, lipospheres (LS) consist of physiologically well-tolerated ingredients that have often already been approved for pharmaceutical use... 

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]. 

Genetic Basis of Lung Cancer Gene Transfer Techniques Adenoviral Delivery Systems p53 Gene Transfer in Lung Cancer Preliminary Clinical Trials with p53 Gene Transfer in Lung Cancer Conclusions References... 

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. 

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... 

The intensity of the search for new alkaloids of the ajmaline type will certinly not abate. The research on cell culture methods to prepare gmaline alkaloids is likely to continue. So far, however, the alkaloid content in cultured cells has almost always been low. For the future, we can expect the production of ajmaline derivatives by gene transfer techniques. 

Tomita, N., Higaki, J., Ogihara, T., Kondo, T and Kaneda, Y. (1994) A novel gene-transfer technique mediated by HVJ (Sendai virus), nuclear protein, and liposomes. Ca. Detec. Preven. 18(6), 485 491. 

Among the different gene transfer techniques, microinjection is by far the most efficient procedure. Only microinjection allows the transfer of a known number of test molecules either into the cytoplasm or into the nuclei of the recipient cell Up to 100% of the recipient cells support expression of the transferred material, and stable transformed cell lines can be isolated with a frequency of 20-30%. Biochemical studies can be performed with 50-100 injected cells, and the injected material (e.g., DNA, RNA) can be reisolated and further analyzed by standard techniques (e.g., Southern and Northern blots, electron microscopy) (for review, see Graessmann and Graessmann, 1983 Graessmann et al., 1983 Ceiis et al., 1986). 

Morishita, R., Gibbons, G., Kaneda, Y., Ogihara, T., and Dzau, V. (1993b) Novel and effective gene transfer technique for study of vascular renin angiotensin system. /. Clin. Invest. 91, 2580-2585. 

The two most often used methods for DNA delivery into protoplasts are electroporation and treatment with polyethylene glycol (Paszkowski et al., 1984). The observation that short electric pulses of high field strength transiently permeabilize cell membranes led to the development of electroporation-mediated gene transfer techniques for mammalian cells (Neumann et al., 1982) and plant cell protoplasts (Fromm et al., 1985). Electroporation is now the preferred technique for direct DNA transfer to plant protoplasts. 

Another method of increasing resistance to a PSII herbicide is to promote the breakdown of the molecule within the plant before it reaches the active site. This has been achieved in an effective way in the case of bromoxynil resistance in tobacco. Bromoxynil is broken down by a nitrilase enzyme to ammonia and a herbicidally inactive benzoic acid moiety (Figure 1.6). A strain of Klebsiella pneumoniae has been isolated which uses bromoxynil as a primary nitrogen source by virtue of its nitrilase activity. The gene has been characterized and transferred into tobacco using conventional gene transfer techniques. The transgenic tobacco thus produced was able to tolerate more than 20 times the normally lethal dose of bromoxynil. ... 

---