Plasmids transfection efficiency

Dendrimers are three-dimensional hyperbranched, globular macromolecules that are capable of condensing DNA into small complexes, and thereby increase plasmid transfection efficiency (14). Dendrimers are typically stable in serum and not temperature sensitive, but are also non-biodegradable and cause significant cytotoxicity. 

Fig. 3. Comparison of transfection efficiencies obtained using PolyFect Reagent, a dendrimer-based transfection reagent, and a calcium phosphate-mediated procedure. COS-7 and HeLa cells were transfected in srx-weU plates with a /3-galactosidase expression plasmid using the appropriate protocol. For the calcium phosphate-mediated transfection, 6 pg of plasmid DNA was used and the medium was changed after 5 h incubation. Transfections were performed in triplicate, and transfection efficiency was measured by monitoring the /3-galactosidase activity of extracts obtained from the transfected cells. The amoimt of /3-galactosidase activity in the extracts correlates with the transfection efficiency. Cells were harvested 48 h post-trans-fection...

Once in the cytoplasm, a proportion of plasmid molecules are likely degraded by cytoplasmic nucleases, effectively further reducing transfection efficiencies. There are two potential routes by which plasmid DNA could reach the nucleus ... 

They used different generations of intact dendrimers to transfect plasmid DNA in a variety of cells (Table 18.2) using luciferase, CAT (chloramphenicol acetyltransferase) and /i-galactosidasc reporter genes to quantify transfection efficiency. 

Gregoriadis G, Saffle R, Hart SL. High yield incorporation of plasmid DNA within liposomes effect on DNA integrity and transfection efficiency. J Drug Target 1996 3 469 75. 

Figure 6 Lipofection results (lipofection profiles) of lipoplexes from the R-configu-rated cationic lipids KL-1-1 to KL-1-17 (Table 1) in a mixture with equimolar amounts of l,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) (counterion chloride) and the pCMVluc-plasmid. Each bar represents the mean ( S.D.) of three wells of a 96-well microtiter plate. T-axis (left) represents the transfection efficiencies expressed in relative light units (RLU) (lu/pg protein). X-axis (right) represents the viability of the cells compared to nontreated control cells. F-axis represents the different cationic lipid/plasmid DNA-charge ratios from 1 to 15.

It has been reported that the plasmid vector is unable to translocate to the nucleus unless complexed in the cytoplasm with nuclear proteins possessing NLS. NLS are short karyophilic peptides on proteins that bind to specific transporter molecules in the cytoplasm, mediating their passage through the pore complexes to the nucleus. Examples of these peptides will be given later in this section. DNA can also be presented to cells in culture as a complex with polycations such as polylysine, or basic proteins such as protamine, total histones or specific histone fractions (110), cationized albumin, and others. These molecules increase the transfection efficiency. In addition histone HI is identified as transfection-enhancing protein in cell culture (111). 

Aral, C., Akbuga, J. (2003). Preparation and in vitro transfection efficiency of chitosan microspheres, containing plasmid DNA poly (L-lysine) complexes. J. Pharm. Phantn. Sci., 6(3), 321-326. 

Chemg, J.Y., Van de Wetering, R, Talsma, H., Crommelin, D.J.A., Hennink, W.E. (1996). Effect of size and serum proteins on transfection efficiency of poly ((2-dime-thylamino)ethyl methacrylate)-plasmid nanoparticles. Pharm. Res., 13, 1038-1042. 

Cherng, J.Y., Schuurmans-Nieuwenbroek, N.M., Jiskoot, W., Talsma, H., et al. (1999). Effect of DNA topology on the transfection efficiency of poly((2-dimethy-lamino)ethyl methacrylate)-plasmid complexes. J. Control Release, 60, 343-353. 

While isolation of a specific inhibitor will be necessary to assess the definitive role of the cytosolic nuclease in the low transfection efficiency in vivo, circumstantial evidence suggests that the metabolic instability of plasmid DNA represents one of the cellular barriers to gene transfer. Microinjection of DNA complexes with PEI has augmented the transfection efficiency (Pollard et al., 1998). Although the stability of the PEI-complexed DNA has not been determined in vivo, it has been demonstrated that the nuclease resistance of plasmid DNA is dramatically increased upon complex formation in vitro (Cappaccioli et al., 1993 Chiou et al., 1994 Thierry et al., 1997). Therefore, it is conceivable that faster diffusional mobility and decreased nuclease susceptibility jointly lead to the enhanced nuclear targeting efficiency of the PEI-condensed plasmid DNA. 

Chloroquine drastically improves the transfection of cells when DNA/polylysine conjugates are used (Zenke et al., 1990 Midoux et al., 1993). So far, the mechanism of action of chloroquine has not been completely elucidated. Chloroquine is supposed to protect the internalized plasmid from intracellular degradation as a result of the neutralization of acidic compartments and the inhibition of endosome fusion with lysosomes. Furthermore, the swelling of vesicles can be induced when the concentration of chloroquine is high enough. However, there is no direct relationship between the neutralization of the acidic cell compartments and the transfection efficiency (Erbacher et al., 1996a). Once taken up by the cells, chloroquine accumulates inside acidic vesicles where it can reach a concentration more than 50 mM, based on the estimated volume of the vesicular compartments. At physiological pH, 82% of chloroquine is protonated and can bind to nucleic acids. Therefore, the interaction of chloroquine with DNA appears to be... 

Instead of poly lysine, commercially available oligolysine of dp 19, which is less polydisperse than poly lysine, has been used to condense pDNA. A minimal repeating lysine chain of 18 residues followed by a tryptophan and an alkylated cysteine (AlkCWK18) has been found to condense plasmid DNA into small particles of 78 nm that mediated efficient in vitro transfection (Wadhwa et al., 1997) (Table 16.4). Compared to a commercially available K19, AlkCWK18 induced a 40-fold reduction in particle size and a 1000-fold increase in transfection efficiency. 

The assembly of NLS in peptide-based gene delivery systems has been achieved by the non-covalent binding of plasmid to either free NLS embedded with polyplexes or to NLS linked to a cationic sequence, such as (PKKKRKV)4-K2o (Table 16.7), AKRARLSTSFNPVYPYEDES-K20 (Table 16.7) or H9-2 sequence (nls-H9-2) (Table 16.4). With nls-H9-2, the transfection efficiency with a formulation containing... 

In an original manner, one SV40 NLS sequence has been covalently linked to one end of a linear plasmid encoding Luciferase (Zanta et al., 1999). In combination with polyethylenimine, transfection efficiency was increased 10- to 1000-fold, depending on the cell types compared to the same construction containing a nonfunctional mutated NLS. 

Anti-deoxyribonucleic acid autoantibodies from human and mice suffering from Lupus erythematosus can penetrate into cells and accumulate in the cell nucleus. Based on the characteristics of a mi-ON A autoantibodies, VAYISRGGVSTYYSDTVKGRFTRQKYNKRA peptide (P3), which exhibits a-helix, has been used as a vector for the intracytoplasmic and intranuclear translocation of macromolecules (Table 16.7) (Avrameas et al., 1998, 1999). P3 shares similar capabilities with Antenapedia peptide (Derossi et al., 1994), but in contrast P3 operates only at 37 °C by an energy dependent mechanism. P3 linked to a 19 lysine residue sequence (K19-P3) forms complexes with plasmid DNA. Efficient transfection of mouse 3T3 cells and hamster lung CCL39 cells were obtained with these complexes. This transfection was not impaired by the presence of serum and did not require helper molecules such as chloroquine. These observations suggest that peptides from cell specific anti-DNA autoantibodies may represent a source of peptide-based gene delivery system with different specificities. 

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