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Suspension cell lines have traditionally proved laborious to transfect. The common cause of this is the reduced attachment of the transfection complex (including liposome-based transfection reagents containing the target gene) to the cell surface, resulting in decreased uptake of target DNA. Importantly, suspension cells are not easy to culture and die easily, which also causes some difficulties for cell transfection.
Polyethylenimine (PEI) is one of the most utilized transfection agents for small-scale to large-scale protein expression because it is simple, efficient, and cost-effective. PEI is a cationic polymer that binds DNA and condenses it into small cationic nanoparticles, thereby promoting DNA cell uptake, most likely through their interaction with cell surface heparin sulfate proteoglycans. Linear and branched 25 kDa PEI are the most widely used polymers, and linear PEI is more effective and potentially less toxic for HEK293 cells grown in the suspension cultures. Current transfection procedures involve the formation of PEI-DNA complexes (poly complexes) before the addition of cell cultures (indirect method). Typically, PEI and DNA are mixed and incubated at room temperature for 10-15 minutes to allow complexation before they are added to cells. This incubation time allowed the complex to grow to the optimum size for maximum transfection efficiency, which was significantly reduced when incubation time exceeded 30 min. PEI has successfully been applied to transient transfection of suspension cell CHO-S and HEK 293 GnTi-, as well as other suspension cells.
In electroporation, nucleic acids and cells are suspended in a conductive solution, sandwiched between the anode and cathode, and electrical pulses are applied continuously to create transient holes in the cell membrane through which charged extracellular nucleic acids such as DNA and RNA are injected into the membrane. The physical method has the advantages of high introduction efficiency and being easier than chemical and biological methods, but it also has the disadvantages of cell death due to electrical impulses, such as the failure of pores formed by electrical impulses, and the need for expensive special equipment.
Diethylaminoethyl-dextran (DEAE-dextran) is a cationic polymer that forms complexes with negatively charged DNA. It is used as a complexing agent for nucleic acids (DNA and RNA) and also as a coating of liposomes. The complexation of DNA and DEAE-glucan through electrostatic interaction resulted in the formation of a positively charged complex that adhered to the negatively charged plasma membrane. This complex may enter the cytoplasm through dimethyl sulfoxide (DMSO) or glycerol-induced osmotic shock, or it may cross the plasma membrane through nonspecific endocytosis. In addition, chloroquine can be added to the medium to be transfected in this way, since chloroquine is thought to enhance transfection efficiencies by neutralizing lysosomal hydrolases, which can degrade the DNA. The method is inexpensive and easy to perform and can be applied to the transfection of lymphocyte cell lines and other suspended cell lines. However, due to its toxicity, it is only suitable for transient transfection and not for stable transfection.