The first and best-understood step of transfection is therefore the necessity for “compaction” of the large, negatively charged DNA molecule. A suitable compacting agent is a positively charged molecule able to interact with the DNA and to neutralize or even overcompensate the negative charges. During compaction, the DNA forms stable complexes with the compaction agent, which either stay in solution or form a precipitate. In a typical transfection experiment, the complexes are formed in a reaction mixture containing the given amounts of purified DNA as well as the compaction agent under defined pH and salt conditions. The complex formation occurs spontaneously upon mixing. Within the next 30 minutes the complexes are added to the target cells. Usually, cells are exposed for several hours to the complexed DNA. Subsequently, the medium is exchanged in order to minimize possible toxic effects.
Two groups of molecules are currently investigated as compaction agents: cationic lipids and cationic polymers. Protonated amino groups provide the required positive charges in both cases. Amino groups are also found in some of the naturally occurring compaction agents such as spermine and spermidine. They are clearly the group of choice, since they allow the generation of a positive charge at physiological (neutral) pH. In addition, eukaryotic cells developed over eons of evolution special proteins (nucleosomes) with a high affinity to DNA, which also can complex DNA. The structure of these nucleosomes may in the future inspire the design of novel compaction agents. Prominent representatives are histones or protamines, naturally occurring ubiquitous DNA binding (compacting) proteins.
Cationic lipids are usually fairly small molecules, which mimic the structure of the cell’s plasma membrane and hence facilitate the passage of DNA into the cell by increasing the solubility of the DNA in the plasma membrane. These molecules consist of a hydrophobic (hydrocarbon) tail and a positively charged head-group. The hydrophobic tail promotes in aqueous solutions self-aggregation into larger structures (micelles, double layers) capable of interaction or even fusion with the cellular membrane.
The cationic polymers (such as polyethyleneimine, polyvinyl pyrrolidone) commonly used for transfection are fairly large molecules (up to 1,000,000 g/mol). They are soluble in water at neutral pH due to their positive charges. Linear as well as branched molecules are employed for transfection. In contrast to the cationic lipids, which usually were developed as dedicated transfection reagents, most cationic polymers have been developed for other applications and purposes. They are therefore available from several suppliers in a wide variety of purity and chemical homogeneity.
Tags: Bio Technology, Bio Genetics, Gene Compaction
