Lipofectamine 2000

Hela cells are transfected with either pcDNA-Ren-HCV-FF or pGL3-Ren/CrPV/FF in 10-cm plates using Lipofectamine-plus reagent as described by the manufacturer (Invitrogen). Twenty-four hours posttransfection, the cells are trypsinized and re-plated in 24-well plates at 5 x10s cells/well. 

HeLa cells were transfected using 1 /d lipofectamine (Invitrogen, Breda, The Netherlands), 0.5 fig plasmid DNA and 50 /d Opti-MEM per 35 mm dish holding a 24 mm 0 1 coverslip. Samples... 

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. 

For each transfection, dilute 1 to 2 ug of DNA in 100p,l of serum-free medium. In a separate tube, dilute 8p,l of lipofectamine in 100 xl of serum-free medium. 

Add 0.8 ml of serum-free medium to the tube of DNA and lipofectamine. Mix and overlay the mixture onto the rinsed cells. Do not use antibacterial agents to the media during transfection. 

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

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. 

Various approaches based on guanidinium moieties as cationic headgroups have been explored. Some approaches introduced both polyamines and guanidinium groups. Product RPR115335 in Figure 15.18, displayed enhanced transfection activity as compared to LipofectAMINE in NIH3T3, rabbit SMC, 3LL... 

Other cationic lipids combine several elements of different families, for example DOSPA, which is composed of a quaternary ammonium salt and polyamine spermine. The formulation of DOSPA with DOPE results in the well-known commercial Lipofectamine (Hawley-Nelson et al., 1993). Although this lipid displayed high transfection activity, its cytotoxicity prevented its further development. 

Byk, T., Haddada, H., Vainchenker, W. and Louache, F. (1998d) Lipofectamine and related cationic lipids strongly improve adenoviral infection efficiency of primitive human hematopoietic cells. Hum. Gene Then, 9, 2493-2502. 

As an extension of the HA film approach, Yun and coworkers [32] synthesized hyaluronan microspheres using the chemistry described above, but the synthesis was completed in emulsion in one step, yielding 5- to 20-pm microspheres. These microspheres were found to be biodegradable and released three times more pDNA when incubated with hyaluronidase in PBS (phosphate buffered saline) solution (vs enzyme-free PBS). As in the case of films, DNA release from the microspheres was dependent on the DNA loading. DNA-HA microspheres were not directly used for transfection instead, DNA obtained from release experiments was used in transfection of Chinese hamster ovary (CHO) cells using Lipofectamine. The relative levels of transfection over time had the same trend as DNA release from the DNA-HA microspheres and confirmed that released DNA is bioactive. 

Dodds, E., Dunckley, M. G., Naujoks, K, et al. Lipofection of cultured mouse muscle cells A direct comparison of Lipofectamine and DOSPER. Gene Ther. 5 542—551,1998. 

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