Transfection reagent

PAMAM dendrimers have the following characteristics which are important for their use as transfection reagents. They bind and form complexes with nucleic acids, allow transfer of the DNA-dendrimer complex into the cytoplasm of the... 

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...

The generation number, activation procedure, and core moiety of PolyFect Transfection Reagent have been developed specifically for transfection of certain cell lines, including HeLa and COS-7. Optimized amounts of DNA and den-drimer delivered transfection efficiencies in both cell lines significantly higher than those obtained using a standard transfection method. 

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. 

Polyethylenimine (PEI) stock The transfection reagent (Aldrich, catalogue number 40,872-7 25 kDa branched PEI ). Stock solution of PEI is prepared as follows prepare stock solution of 100 mg/ml PEI in water, mix and further dilute to 1 mg/ml, neutralize with HCI and filter sterilize store 5 ml aliquots frozen. 

Lipofectamine RNAiMAX Transfection reagent (Life Technologies, Carlsbad, CA). 

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. 

Serum-free cell media can be substituted for OptiMEM for dilution of the transfection reagent. Adjustments to the serum concentration of the media used in cell addition may be needed. 

FuGENE6 Transfection Reagent (Roche Diagnostics, Basel, Switzerland). 

DNA using FuGENE6 Transfection Reagent according to the manufacturer s instructions and cultured for 36 h in DMEM with 10% FBS. 

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. 1 (a) Synthesis of dextran-spermine conjugates, (b) Fluorescence micrographs of dextran-spermine compared to common transfection reagents in HEK293 and NIH3T3 cells. Adapted with permission from [38]. 2002 American Chemical Society... 

In this volume, we have brought together articles from various experts in the chemical transfection reagent field. Topics covered include descriptions of the chemistry of chemical transfection compounds and details of the fundamental parameters that influence transfection efficiencies. 

Several authors describe distinct classes of chemical transfection reagents. Fischer et al. summarize synthesis methods, features, and applications of hyper-branched polyamines as carriers for nucleic acids. 

A review from Sizovs et al. discusses carbohydrate-based polymeric transfection reagents, turning special attention to their potential applications in preclinical models and for the treatment of diseases. 

While several reviews collected in this volume describe the basic chemical composition of transfection reagent classes, Salcher and Wagner focus on the modification and functionalization of polymeric gene carriers to improve their transfection efficiencies for DNA or siRNA. Such modifications include the introduction of cell-specific ligands or pH-sensitive lytic residues. 

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