Mechanism of transfection

The internal structure of the complexes can directly determine the mechanism of transfection [4, 23, 25]. We have found that for Lac CL-DNA complexes, the membrane charge density (aM) is a predictive parameter for transfection efficiency [21] (see Sect. 2), i.e., the data for monovalent and multivalent cationic lipids are described by a simple bell-curve. In contrast, for inverted hexagonal HnC CL-DNA complexes, TE is independent of aM, suggesting a distinctly different mechanism of transfection. Consistent with the TE data, confocal microscopy revealed distinctly different CL-DNA complex pathways and interactions with cells, which depended on both the structure (HnC vs Lac) and, for Lr/ complexes, on aM [25]. Thus, the mechanism of transfection by CL-DNA complexes is dependent both on their structure and, for a given structure, on chemical and physical parameters of the complexes. 

The mechanism of transfection by cationic lipids is not known, but cationic lipids probably form stable complexes with anionic DNA molecules to facilitate entry into the cell. Physical studies of DNA/DOTAP/DOPE complexes suggest that DNA molecules are arranged in a regular pattern between layers of lipid bilayer [54]. This multilamellar arrangement permits the charged lipid-head groups to interact with the charged DNA molecules. When mixtures... 

The picture emerging from the data discussed in the previous section reveals two properties, the correct interpretation of which is essential for an understanding of the mechanism of transfection 1. the existence of two types of transfection characterized by linear and multipowered dose responses, and 2. the low efficiency of transfection in comparison to infection. To analyze these observations, the fate of DNA after uptake into cells must be followed. 

Rutberg, L., Hoch, J. A., Spizizen, J. Mechanism of transfection with deox3n-ibo-nucleic acid from the temperate Bacillus bacteriophage 0IO5. J. Virol. 4, 50-57... 

The first idea to consider is the effect of receptor density on sensitivity of a functional system to agonists. Clearly, if quanta of stimulus are delivered to the stimulus-response mechanism of a cell per activated receptor the amount of the total stimulus will be directly proportional to the number of receptors activated. Figure 5.8 shows Gi-protein-mediated responses of melanophores transiently transfected with cDNA for human neuropeptide Y-l receptors. As can be seen from this figure, increasing receptor expression (transfection with increasing concentrations of receptor cDNA) causes an increased potency and maximal response to the neuropeptide Y agonist PYY. 

The transfection mechanism of plasmid-chitosan complexes as well as the relationship between transfection activity and cell uptake was analyzed by using fluorescein isothiocyanate-labeled plasmid and Texas-Red-labeled chitosan. Several factors affect transfection activity and cell uptake, for example the molecular mass of chitosan, stoichiometry of complex, seriun concentration and the pH of the transfection medium. The level of transfection with plasmid-chitosan complexes was found to be highest when the molecular mass of chitosan was 40 or 84 kDa, the ratio of chitosan nitrogen to DNA phosphate was 5, and serum at pH 7.0 was 10%. Plasmid-chitosan complexes most likely condense to form large aggregates (5-8 p,m), which absorb to the cell surface. After this, plasmid-chitosan complexes are endocytosed, and accumulate in the nucleus [97]. 

Besides this purported action on DAT, amphetamine has also been suggested to act upon the vesicular transporter as well. Pifl et al.87 examined COS cells transfected with cDNA for either DAT or the vesicular transporter, or both. A marked increase in DA release was noted in cells that expressed both DAT and the vesicular transporter when compared to the release from cells that express only DAT or the vesicular transporter. The mechanism of action for amphetamine was further defined with the work of Giros et al.59 In transgenic mice lacking the DAT, amphetamine did not produce hyperlocomotion or release DA. 

J. L. Lemoine, R. Farley, and L. Huang. Mechanism of efficient transfection on the nasal airway epithelium by hypotonic shock. Gene Ther 12 1275-1282 (2005). 

Xu Y, Szoka FC Jr. Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection. Biochemistry 1996 35(18) 5616-5623. 

Integrin receptor-binding peptides have been used to enhance liposome binding, uptake, and expression (25,47 9). The inclusion of an 0(5pi integrin-targeted peptide into a liposomal complex enhanced transfection efficiency four- to five-fold in Jurkat cells and 10- to 13-fold in TF-1 cells (48). Confocal and electron microscopy revealed that the mechanism of cell entry conferred by RGD peptides on liposomes is predominantly by clathrin-coated endocytosis rather than by phagocytosis (50). 

Therefore, evidence exists to support numerous possible mechanisms for the uptake promoted by TAT peptides. An alternative explanation is that TAT is a sticky opportunistic peptide that has the ability to bind to the cell surface and exploit multiple mechanisms in order to enter the cytoplasm (92). Clearly, the mechanism of internalization requires further study. Nevertheless, it has been observed that transfection with CPPs requires less lipid and therefore proves to be less cytotoxic to cells in vitro and in vivo, making it a promising vector system for future gene therapy (95,104). 

Transfection is the process of introducing DNA or RNA into eukaryotic ceils. The use of transfection is to study the role and regulation of proteins or to understand the mechanisms of a pathway. Transfection can be transient for rapid analysis or stable , mostly for induction of expression. There are various methods of transfection which include electroporation, viral vectors, DEAE-Dextran, calcium phosphate or Lipofectamine. The choice of transfection depends on the cell type used. The most desirable technique is the one which gives high efficiency of nucleic acid transfection with less interference to the cells physiology and high reproducibility. 

Packer M (1992) The neurohumoral hypothesis a theory to explain the mechanism of disease progression in heart failure. J Am Coll Cardiol 20 248-254 Pepperl DJ, Regan JW (1993) Selective coupling of a2-adrenergic receptor subtypes to cAMP-dependent reporter gene expression in transiendy transfected JEG-3 cells. Mol Pharmacol 44 802-809... 

Zhou, X., Huang, L. (1994). DNA transfection mediated by cationic liposomes containing lipopolysine characterization and mechanism of action. Biochim. Biophys. Acta, 1189, 195-203. 

Garcia, M., Sakamoto, K., Shigekawa, M., Nakanishi, S., Ito, S. Multiple mechanisms of arachidonic acid release in Chinese hamster ovary cells transfected with cDNA of substance P receptor, Biochem. Pharmacol. 1994, 48, 1735-1741. 

These experimental observations suggest a simple direct mechanism of DNA release from endosomal membranes into the cytoplasm that is consistent with the higher transfection efficiencies in mammalian cell cultures reported empirically in CL/DNA complexes containing DOPE compared to those containing DOPC, which are known to exhibit the structure. These findings unambiguously establish a correlation between... 

Coonrod, A., Li, F.Q. and Horwitz, M. (1997) On the mechanism of DNA transfection efficient gene transfer without viruses. Gene Ther., 4, 1313-1321. 

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