Transfection, maximum efficiency

As is evident by the numerous choices available to the user, there are many ways to achieve the goal of transfection of DNA or siRNA into a mammalian cell. Depending on the cell line and other experimental conditions, especially keeping in view the downstream application of the transfection procedure, the researcher has to choose the optimal reagent and the appropriate methodology to achieve maximum efficiency. 

Two characteristic parameters are obtained when transfection is performed with a series of phage DNA concentrations (1) the dose response, relating the increase in biological activity to increasing DNA concentrations (2) the maximum efficiency of transfection. 

The maximum efficiency of transfection can be determined over a range where the transfection curves approach saturation, more precisely, at a point where the slope of the curve is unity. Table 2 lists the highest efficiencies of transfection reported. The values are not strictly comparable, since bacterial... 

Maximum efficiency of marker rescue requires that all cells be infected by the helper phage. In SP 82 G and 0105 transfections pronounced marker... 

TE of KL-1-14 without helper lipids was very low and reached only about twice the TE, which was found for the standard lipid DOTAP. Independent of the amount of DOPE incorporated in the lipoplexes (ratio of DOPE/KL-1-14 0.3, 0.5, 0.6, 0.7, 0.8, 0.8, 1.0, and 1.2), transfection behaviors (maximum transfection efficiencies and transfection profiles) of all mixtures were similar and comparable to the profile of KL-1-14/DOPE (1 1) as shown in Figure 2 (individual data for all mixtures are not shown). 

Most of the numerous commercially available transfection kits use cationic liposomes as transfection agent. The basic unit, Figure 2.4a, of such molecules mimics the natural lipids, which are also found as part of the cell membrane. The formulation and the exact chemistry of these molecirles is usually proprietary, hence it is difficult to postulate a mechanism or to suggest improvements of either the molecirle or the procedure. The protocols are simplified for routine use and do not present much information. Typical procedures state, for example add DNA to buffer A, add enhancer and mix with B, wait 5 minutes and add to cell culture, optimize the DNA concentration and the volume of the mix added to cells. In spite of the apparent simplicity, however, it is strongly advised to carry out the short optimization indicated by the protocol in order to obtain the maximum transfection efficiency. While kits are expensive, they require little previous experience and lead quickly to results. However, in our hands at least, these kits were not superior to calcium phosphate or PEl-mediated transfections. 

With respect to hybrid hyaluronic acid nanoparticles, the high efficiency seen in the transfection process was due to the interaction of hyaluronic acid with CD44 receptors, as illustrated in Figure 8.7. The transfection effect seemed to last at least 10 days, reaching its maximum level on the fourth day after transfection of human corneal cells (HCE) and human conjunctiva cells (lOBA-NHC) [13,133]. This was evidenced by blocking (using an antibody or excess HA) these receptors, which led to a dramatic decrease in the transfection efficiency. 

Cationic liposomes typically consist of a mixture of cationic and/or neutral lipids. They are highly efficient, of low cost, and widely available commercially. Stealth liposomes are cationic liposomes which have been PEGylated at the sirrface. Unless further modified with a targeting ligand they display poor transfection abilities. There is no maximum length of DNA/RNA that can be load into liposomes for transfection. 

Reference data on common chitosan salts are useful to assess the characteristics of complexes made by modified chitosans and DNA. Complexes formulated with several chitosan salts were assessed for complexing ability toward DNA, transfection efficiency in Chinese hamster ovary cells, and their effect on cell viability. Chitosan hydrochloride, lactate, acetate, aspartate and glutamate (chitosan MW 45 kDa) formed complexes with pcDNA3-CMV-Luc. Gel electrophoresis documented that complexes formed at N/P ratios above 2 with all chitosan salts. The transfection efficiency depended on the anion, on the chitosan MW, and on the N/P ratio maximum transfection efficiencies were at N/P ratio of 12, 12, 8, 6 and 6 for said salts, respectively. The MTT assay indicated that all chitosan/DNA complexes had low cytotoxicity (Weecharangsan et al., 2008). 

Similar studies were conducted in parallel on methylated N-(4-pyridinylmethyl) chitosan complexes of TM69Py62CS/DNA were formed at weight ratio above 1.1, whereas those of TM53Py40CS/DNA and TM52Pyl3CS/DNA were above 1.8 and 8, respectively. TM69Py-62CS showed superior transfection efficiency with a maximum at weight ratio 4, but the viability of the Huh-7 cells was adversely affected (Opanasopit et al., 20 08 127-134). 

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