Basic principles

Perhaps the most powerful application of recombinant DNA technologies is the ability to precisely manipulate the genotype and phenotype of a cell. This is accomplished by the intracellular delivery of DNA molecules of defined sequence which have the capacity to function in the recipient cell. This technology provides the opportunity to evaluate the biological information and function specified by the nucleotide sequence of a gene and can frequently distinguish the function of normal and mutant gene products. In addition, the regulatory properties of specific nucleotide sequences can also be assessed. While these methods have led to the development of highly sophisticated strategies to change the genotype of whole oiganisms (either by addition or deletion of genetic material), the following discussion will target specific issues relating to gene transfer methodologies in cultured mononuclear phagocytes.

Gene transfer strategies generally fall into two categories: those which involve short-term or transient expression, and those in which the transferred gene is permanently integrated in the genome of the recipient cell. Each of these approaches has distinct advantages and disadvantages both in general terms and with respect to mononuclear phagocytes. The primary advantage of transient transfection is its potential application in the largest selection of cell types. It has been difficult to obtain stable integration of the transgene material in many differentiated cell types including macrophages. In many cases, however, these cells can be transiently transfected. Furthermore, the transient gene transfer assays can be conducted with much greater speed because selection and expansion of stably transfected clones is not required. Although the focus in the following section is on methods for the transient transfection of macrophage populations, each of these methods can be used to generate stable transfectants, assuming the plasmid DNA carries an appropriate selectable marker.

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