Lipid reagents, transfection

Most adherent cell types can be efficiently transfected with lipid reagents. Even notoriously hard to transfect cells such as neurons, certain primary cells, and embryonic stem cells can be successfully transfected under optimized conditions with the appropriate reagents, presumably because siRNAs only need to be delivered to the cytoplasm (unlike DNA plasmids that must enter the nucleus for expression). For reasons that are not yet clear, suspension cells tend to be refractory to lipid-based transfection and typically require electroporation or viral-based methods for delivery of siRNA. While electroporation efficiently delivers naked siRNA directly to the cytosol for access to RISC, thereby bypassing the endosomal compartment, the extensive cell death that results and large amounts of siRNA required are prohibitive for broader-range applications. 

Various techniques for neuronal transfection have been reported including viral transfer, DNA/calcium-phosphate coprecipitation, electroporation, microinjection, biolistics, and liposomes. Although the ideal transfection method has not been developed, we describe a neuronal transfection method with LF2000, a commercially available cationic lipid reagent. This method yields consistent transfection efficiency without any time consumption, experienced culture technique, or cell toxicity. 

Due to the predominantly hydrophilic nature of PNAs they do not readily cross lipid membranes [93] and enter living cells [94]. Therefore in order to explore the ex vivo and in vivo potential of PNA as an antisense and/or antigene reagent, a number of different transfection protocols have been devised over recent years. 

Figure 1 The principles and variant parameters of lipofection. (i) Preparation of a lipofection reagent cationic liposomes were prepared from cationic lipids and helper (if required), (ii) Formation of positively charged lipoplexes by addition of DNA (e.g., reporter plasmid carrying the firefly luciferase gene) to the cationic liposomes, (iii) Transfection (lipofection) by incubation cells with the preformed lipoplexes. The efficiency of gene transfer (lipofection efficiency) can be determined from reporter gene amount or activity (e.g., luciferase activity). Most of the steps of a lipofection experiment can be varied and optimized (grey spots).

The transfection profiles of the most effective lipids of this group are similar, showing a peak (highest TE) at lipid/DNA ratios from 2 to 5. For the most effective lipids, the viability of the cells at maximum TE usually decreased to roughly 50%o. An exception is the lipid 14, which shows the highest TE as well as only a minor toxicity of about 70%o viability. We chose lipid KL-1-14 for further development of a versatile transfection reagent. 

Poly-L-lysine was one of the original reagents used to condense DNA into a transfecting particle (21) and numerous studies have been widely reported with these reagents. Oligolysine peptides as short as (K)i6 will also package pDNA, alone (polyplex) or in combination with lipids (lipopolyplex) (22-26). In both lipopolyplex and polyplex formulations, the DNA is condensed... 

Fig. 1. Fligti-ttiroughput RNAi screening using siRNA libraries. Assay plates containing library siRNA are treated with (1) diluted transfection reagent to complex the siRNA and lipid. (2) Cells are added to initiate the transfections. Assay plates are incubated for 72-96 h then (3) readout reagent is added. Assay plates are read and the data is analyzed (4-5).

Dilute Lipofectamine RNAiMAX in OptiMEM to a concentration of 3 pL lipid per mL of OptiMEM (see Note 6). Add diluted transfection reagent at a final volume of 20 pL/well to all plates (see Note 7). Allow transfection reagent to complex with siRNA at room temperature for at least 30 min. 

Since the introduction of the transfection reagent Lipofectin , a cationic liposome composed of 1 1 (w/w) mixture of the quaternary ammonium cationic lipid jV[1-(2, 3-dioleyloxy)propyl]-Ar, N, N-trimethylammonium chloride (DOTMA) and a colipid dioleoylphosphatidylethanolamine (DOPE) (Feigner et al., 1987), an increasing number of cationic lipids have been developed. To date, cationic lipids can be grouped into seven different categories ... 

Fig. 15 Transfection activity of cationic aminoglycerol-diamine conjugate lipids (up-left) employing (3-galactosidase (0.1 pg/well). The lipoplexes were used at DNA/lipids w/w ratios of 1 5 and 1 20. The transfection efficiencies of the lipids were compared to that of commercially available reagent, Effectene, which was calculated as 100% transfection efficiency [56]...

It is important to maintain low cytotoxicity, since transfection reagent-mediated toxicity could potentially mask the true phenotype of a target gene being studied. This chapter will attempt to describe the use of several cationic lipid/polymer-based transfection reagents for the in vitro delivery of siRNA and DNA. Emphasis will be laid on the key parameters that affect transfection efficacy. 

List of Some of the Cationic Lipid/Polymer-Based siRNA Transfection Reagents Currently Available... 

Brazas, R.M., and Hagstrom, J.E. (2005) Delivery of small interfering RNA to mammalian cells in culture by using cationic lipid/polymer-based transfection reagents. Methods Enzymol. 392 112-124. 

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