Lipofection is a lipid-based transfection technology that belongs to biochemical methods also including polymers, DEAE dextran and calcium phosphate.

The principle of lipofection is to associate nucleic acids with cationic lipids. The resulting molecular complexes, known as lipoplexes, are then taken up by the cells.

The main advantages of lipofection are its high efficiency, its ability to transfect all types of nucleic acids in a wide range of cell types, its ease of use, reproducibility, and low toxicity. In addition, this method is suitable for all transfection applications (transient, stable, co-transfection, reverse, sequential or multiple transfections…), high throughput screening assays, and has also shown good efficiency in some in vivo models.

Lipofection mechanism of action

The lipid-based reagents used for lipofection are generally composed of synthetic cationic lipids that are often mixed with helper lipids such as DOPE (1,2-dioleoyl-phosphatidyl-ethanolamine) or cholesterol. These lipid mixtures form liposomes or micelles with an overall positive charge at physiological pH and are able to form complexes (lipoplexes) with negatively charged nucleic acids through electrostatic interactions. The association of the lipid-based transfection reagent with nucleic acids results in tight compaction and protection of the nucleic acids.

Lipoplexes are mainly internalized by endocytosis. Once inside the cell, two mechanisms have been described for the release of the nucleic acids into the cytoplasm. One relies on the buffering capacity of endosomes for polycationic residues (called the “proton sponge effect”). The other describes the ability of cellular negatively charged lipids to neutralize the cationic residues of the transfection reagent leading to destabilization of endosomal membranes.

Finally, the cellular and molecular events leading to gene expression after the nuclear uptake of DNA (not required for siRNA) remain highly speculative. However, the significance of cell division on transfection efficiency favours the assumption that nuclear membrane disruption during the mitosis process promotes DNA nuclear uptake. Nonetheless, transfection of primary cells (non-dividing) and in vivo is also achievable with lipofection demonstrating that DNA can make its way to the nucleus where gene expression takes place.

Lipofection made easy

There are multiple parameters critical to achieving high transgene expression level (or high gene silencing) with minimized cytotoxicity. Those factors include cell type, plasmid DNA characteristics (size, promoter, reporter gene, purity), siRNA sequence & purity, cell culture conditions (medium with or without serum, cell number, absence of contaminations…), amount of nucleic acids and reagents, transgene assays to name a few.

Consequently, transfection reagents need to be specifically designed according to the nucleic acids to be delivered (DNA, siRNA, mRNA, ODN, shRNA etc.) and the cell types used in order to achieve optimal efficiency. In this context, OZ Biosciences has developed several outstanding transfection reagents:

• DreamFect™ Gold Transfection reagent: for all nucleic acids, achieving superior transgene expression level
• DreamFect™ Transfection reagent: for all nucleic acids, for all cells including suspension cell lines
• Lullaby™ Transfection reagent: for siRNA applications
• VeroFect™ Transfection reagent: for Vero cells transfection
• FlyFectin™ Transfection reagent: for insect cell transfection
• EcoTransfect™ Transfection reagent: for popular cell lines and routine transfection at low cost
• COSFect Transfection reagent: for COS cell transfection
• HeLaFect Transfection reagent: for HeLa cell transfection
• Ab-DeliverIN™ Transfection reagent: for intracellular antibody delivery
• Pro-DeliverIN™ Transfection reagent: for intracellular protein delivery

The lipofection method is especially suitable for immortalized cells. Please contact us for the list of cells successfully tested.

All these reagents are based on the TEE-Technology.

Triggered Endosomal Escape (TEE)-TECHNOLOGY

Cationic lipids (lipoplexes) and their polymers (polyplexes) are the most employed non-viral gene delivery systems. The TEE-Technology (Triggered Endosomal Escape) combines and exploits the properties of both lipoplexes and polyplexes to achieve extremely efficient nucleic acid delivery into cells. Indeed, this new generation of lipopolyamines contains a lipophilic part, such as lipids, and a charged polyamine moiety, such as cationic polymers. These moieties act in synergy to ensure the tight compaction and protection of nucleic acids and a very efficient destabilization of the endosomal membrane which allows the release of large nucleic acids amounts in the cytosol as well as DNA nuclear uptake. Furthermore, a particular focus on the synthesis of fully biodegradable entities was integrated into this technology. In this way, the transfection reagents do not interfere with cellular mechanisms, high cell viability is maintained in every experiment and any potential secondary effects are avoided.

Hence, the major TEE-Technology advantages are:

• Compaction of DNA in nanoparticles efficiently internalized by cells
• Protection of nucleic acids against nucleases degradation
• Efficient membrane destabilization and DNA delivery
• Highly efficient even with low amounts of nucleic acids
• Biodegradability

If you would like to know more about how these products can help with your research, do not hesitate to contact us.