Methods

Because the uptake of plasmids or oligonucleotides is cell type specific, transfection conditions need to be optimized. The number of hours for uptake, the dose of vehicle (preferably cationic liposomes such as Lipofectin ) needed to optimize introduction of the nucleic acid sequence, and the subsequent time after transfection to allow expression of the desired function require standardization for particular cell types. Protocols useful for established adherent cell lines may be too harsh for primary diploid cells or tissues, and lymphoid and other nonadherent cells require protocols unlike those used for adherent cells. In addition, agents that favor uptake of short or long nucleic acid sequences may also increase excessively the uptake of antibiotics such as penicillin, streptomycin, and neomycin, which are frequently used in tissue culture. Hence, the presence of antibiotics should be avoided when gene transfer is being attempted together with cationic agents such as positively charged liposomes or other agents such as calcium chloride, which are commonly used during transfection (6,10-12).

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