Vectors and Delivery

For gene transfer, it is necessary to incorporate the desired cDNA into various vectors, such as appropriate plasmids or replication-deficient viruses. Vectors used for transfection generally possess two essentially independent functions: 1) carrying genes to the target cell and 2) expressing the genes properly in the target cell. There are currently many commercial vectors with different features from which to choose, and the choice is dependent on the purpose of the experiments and on the characterizations of exogenous desired genes. In one form of transfection, stable transfection, cells expressing the gene of interest can be actively selected by a marker (e.g., neomycin resistance genes, Neo), and the cDNA or other type of foreign DNA is stably integrated into the DNA of a chromosome. In transient transfection, however, cells express the gene of interest for a few days. Cells for hosting the foreign DNA can be either established cell lines or primary cultured neuronal cells. The desired cDNA delivered into cells may be native genes, fragments of the genes, mutant genes, or chimeric genes.

The main barrier to the delivery of DNA into cells is getting the foreign DNA through the cellular membrane. Over the past decades, various methods have been developed to convey the foreign DNA molecules into mammalian cells. These include chemical or physical techniques, such as calcium phosphate coprecipitation of DNA, liposome fusion, electroporation, microinjection, ballistic injection, and viral infection. At present, methods dependent on incorporation of DNA into cationic liposomes (e.g., lipofectin) are used most widely. These methods of transfection are accessible to both cell lines and cultured primary neuronal cells. The ratio of DNA to liposomal suspension, cell density, and time duration of exposure to the DNA-liposomal complex must be optimized for each cell type in culture.

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