study involving transgenic cattle, seven out of eight transgenic founder males produced by pronuclear DNA injection were mosaics (Eyestone, 1999). Mosaic founder animals might not pass the transgene to their offspring at all, or they might transmit it at a normal or reduced rate.
In mice and pigs, the inefficiency associated with microinjection can be compensated for to a great extent by implanting recipient females with multiple embryos. In cattle, however, this can result in difficult births as well as masculinization of the female offspring (freemartinism) if both a male and a female embryo are transferred. For this reason, embryos usually are cultured temporarily in vitro or in recipient cow, sheep, or rabbit oviducts until the stage at which longer-term viability can be established (Eyestone, 1994). If cows are used, these developed embryos need to be recovered and then transferred to the recipient animals. Although this technique requires the use of additional animals for the “culturing” stage, it can reduce the number of recipient cows needed by up to 90 percent.
Because inserted DNA can insert itself into the middle of a functional gene, insertional mutations that alter or prevent the expression of that functional gene inadvertently might be generated (Chapter 2). Meisler (1992) estimates that 5 to 10 percent of established transgenic mice lines produced by microinjection have such mutations, and it is likely that similar rates would be found in microinjected livestock. Most (about 75 percent) of these are lethal prenatally, but those that are not are responsible for an array of defects in mice, including severe muscle weakness, missing kidneys, seizures, behavioral changes, sterility, disruptions of brain structure, neuronal degeneration, inner ear deformities, and limb deformities. Individuals with such mutations can vary enormously with respect to the degree and type of impairment shown. And because many insertional mutations are recessive, their effects do not become obvious until the animals are bred to transgenic relatives (Chapter 2). For example, although mice engineered with a transgene for herpesvirus thymidine kinase were normal, their offspring that were homozygous for the transgene had truncated hind limbs, forelimbs lacking anterior structures and digits, brain defects, congenital facial malformations in the form of clefts, and a greatly shortened life expectancy (McNeish et al., 1988).
Many of the problems associated with random-site integration, including insertional mutagenesis, could be circumvented by gene targeting (Chapter 2), which allows for the controlled integration of transgenes into predetermined loci within the genome. In addition to site-specific transgene insertions, gene targeting also permits the removal (knockout) and replacement of existing genes. However, problems with the expression of inserted genes still can arise,