Targeting the Transgene to a Specific Location

TARGETING THE TRANSGENE TO A SPECIFIC LOCATION

Targeting the incoming transgene to a particular location on the host chromosome requires homologous recombination, as opposed to the random integration that usually happens with injected DNA. Inserting a transgene in a specific location may be desirable for several reasons. First, chromosomal location often affects the expression of a transgene (see earlier discussion). Second, the transgene is not necessarily a novel gene. Sometimes the objective of genetic engineering is to replace the original version of a particular gene by an altered version. In this case it is obviously preferable to insert the incoming gene in the same location, and under the same regulation, as the gene it is replacing.

Gene targeting relies on homologous recombination, and special targeting vectors are designed to direct the integration. The DNA to be inserted is flanked by sequences that are homologous to those at the target location. Targeting vectors may be subdivided into those designed to insert novel DNA and those that replace DNA (Fig. 15.9). Targeting vectors are often linearized just before transforming the DNA into the cell because this promotes more efficient recombination. Integration of the required DNA segment may be selected by antibiotics or by some other positive selection.