Chromosomal insertion of foreign DNA.

The main route and, in most species, the only reliable route to the generation of transgenic animals is by microinjecting DNA into an early embryo, generally one of the pronuclei of a newly fertilized egg (a one-cell embryo). In most cases, a small number (perhaps 100) of identical cloned DNA molecules is introduced in this way. The weight of evidence supports the view that this DNA forms extrachromosomal concatemers (arrays), mainly of monomers orientated in the same direction, by rounds of homologous recombination. Since this occurs when a population of identical linear molecules is introduced, productive recombination can only take place after a population of circularly permuted monomers has been generated by circularization and random cleavage. Extrachromosomal recombination is known to occur by a nonconservative process in transfected mammalian cells in culture. Concatemeric molecules integrate into the chromosomes, more or less at random, by illegitimate recombination. This may occur during DNA replication, consistent with the very high observed frequency of transgenic founder animals that are mosaics of transgenic and nontransgenic cells. Foreign genes integrated in this way are frequently liable to chromosomal position effects, which can adversely affect expression. In the commercial arena this often necessitates the production of a large number of transgenic founders in the hope of obtaining one with a high expression level. Ways of approaching this practical problem are explored.