As discussed above, introduction of DNA into a cell—whether somatic or germline—is not a well-controlled process and can lead to a number of undesired genetic consequences.

Unintended Genetic Side Effects

Introduction of DNA into random sites in the germline is a mutagenic event that will affect any gene that happens to be at or near the site of integration. The most obvious effect is the disruption of the integrity of a gene into which the insertion occurs. Since a large fraction of the mammalian genome is noncoding DNA derived from various kinds of silenced transposable elements, not all integration events will lead to gene inactivation; however a fraction of animals selected for the presence of a transgene has been found to carry associated genetic lesions. In mice, for example, it has been estimated that about 5 percent of MLV proviruses integrated into the germline have led to mutations o this sort (Boeke and Stoye, 1997). Direct DNA introduction can lead to numbers of integrated copies at multiple sites, leading to a risk of creating animals with a variety of genetic defects, which should be carefully screened for in the course of subsequent breeding. For example, one of the very first transgenic mouse lines generated, intended to contain an inserted active oncogene, also suffered a lesion that caused a severe recessive developmental limb defect (Woychik et al., 1985). A number of other examples of insertional inactivation by transgenes introduced into mice are known, and this approach has been proposed as a useful technique for mutagenesis (Woychik and Alagramam, 1998). Two additional points should be noted. First, the successfully transfected embryo might have inserted DNA sequences other than those that express the transgene, so the point of damage can be at a location different from the active transgene. Second, damage of this sort is often (but not always) recessive, so that it can only be detected by inbreeding to derive animals homozygous at the site(s) of the inserted DNA, adding to the complexity of the screening process.

A related effect is the activation of gene expression in the vicinity of the transgene through the action of the introduced promoter elements. This sort of inappropriate activation of expression is the mechanism of cancer induction in animals infected by a variety of retroviruses, and it has been well-studied as a model for oncogenesis. There are a number of mechanisms by which the expression of genes adjacent to (or even at some distance from) the integration site can be activated, including promoter and enhancer insertion, as well as gene fusion and introduction of elements that stabilize messenger RNA (Rosenberg and Jolicoeur, 1997). Indeed, alteration of expression of genes at genome sites far removed from a transgene has been reported in cell lines, apparently due to altered methylation (Muller et al., 2001). Whether this effect



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