![]() 1995), and its role may be to release these complexes from endosomes as well as to facilitate fusion of the outer cell membrane with liposome/nucleic acid complexes. DOPE is considered a “fusogenic” lipid (Farhood et al. It is unclear how the nucleic acids are released from the endosomes and lysosomes and traverse the nuclear membrane. Following cellular internalization, the complexes appear in the endosomes and later in the nucleus. Entry of the liposome complex into the cell may occur by endocytosis or fusion with the plasma membrane via the lipid moieties of the liposome (Gao and Huang, 1995). For cultured cells, the overall net positive charge of the liposome/nucleic acid complex generally results in higher transfer efficiencies, presumably because it allows closer association of the complex with the negatively charged cell membrane. The cationic portion of the lipid molecule associates with negatively charged nucleic acids, resulting in compaction of the nucleic acid in a liposome/nucleic acid complex (Kabanov and Kabanov, 1995 Labat-Moleur et al. Often, the cationic lipid is mixed with a neutral lipid such as L‑dioleoyl phosphatidylethanolamine (DOPE Figure 3), which can enhance the gene transfer ability. 1995).Ī lipid with overall net positive charge at physiological pH is the most common component of liposomes developed for gene delivery (Figure 2). Unlike DEAE-dextran or calcium phosphate chemical methods, liposome-mediated nucleic acid delivery can be used for in vivo transfer of DNA and RNA into animals and humans (Felgner et al. Cells transfected by liposome techniques can be used for transient expression studies and long-term experiments that rely on integration of DNA into the chromosome or episomal maintenance. Liposome-mediated delivery offers advantages such as relatively high efficiency of gene transfer, the ability to transfect certain cell types that are resistant to calcium phosphate or DEAE-dextran, in vitro and in vivo applications, successful delivery of DNA of all sizes from oligonucleotides to yeast artificial chromosomes, delivery of RNA, and delivery of protein (reviewed in Kim and Eberwine, 2010 Stewart et al. The cationic head group of the lipid compound associates with negatively charged phosphates on the nucleic acid. The next advance in liposomal vehicles was the development of synthetic cationic lipids (Felgner et al. Artificial liposomes were first used to deliver DNA into cells in 1980 (Fraley et al. The term “liposome” refers to lipid bilayers that form colloidal particles in an aqueous medium (Sessa and Weissmann, 1968). In addition, small pH changes (± 0.1) can compromise transfection efficiency (Felgner, 1990).ĭue to their shortcomings, chemical reagents have been largely superseded by synthetic lipid-based reagents. However, calcium phosphate co-precipitation is prone to variability and is not suited for in vivo gene transfer into whole animals. ![]() Calcium phosphate transfection is routinely used for both transient and stable transfection of a variety of cell types. The precipitate is taken up by cells via endocytosis or phagocytosis. The controlled addition generates a precipitate that is dispersed onto the cultured cells. The protocol involves mixing DNA with calcium chloride, adding this mixture in a controlled manner to a buffered saline/phosphate solution and incubating the mixture at room temperature. The method is still popular because the components are easily available and inexpensive, the protocol is easy to use, and it is effective with many different types of cultured cells. 1996).Ĭalcium phosphate co-precipitation as a transfection method was introduced in the early 1970s (Graham and van der Eb, 1973). 1995) and dendrimers (Haensler and Szoka, 1993 Kukowska-Latallo et al. Other synthetic cationic polymers have been used to transfer DNA into cells, including polybrene (Kawai and Nishizawa, 1984), polyethylenimine (Boussif et al. However, DEAE-dextran is not generally useful for stable or long-term transfection studies that rely upon integration of transferred DNA into the chromosome. This method successfully delivers nucleic acids into cells for transient expression that is, for short-term expression studies of a few days. Uptake of the complex is presumably by endocytosis. An excess of positive charge, contributed by the polymer in the DNA:polymer complex, allows the complex to come into closer association with the negatively charged cell membrane. DEAE-dextran, a cationic polymer, was one of the first chemical reagents used to transfect cultured mammalian cells (Vaheri and Pagano, 1965 McCutchan and Pagano, 1968).
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