A transgenic animal is produced by introducing a foreign gene into the nucleus of a fertilized egg (Fig. 7.16a). The egg is then implanted into a foster mother and the offspring are tested to determine whether they carry the foreign gene. If they do, a transgenic animal has been produced. The first transgenic mice ever made were used to identify an enhancer sequence that activates the metallothionein gene when an animal is exposed to metal ions in its diet. The 5' flanking sequence of the metallothionein gene was fused to the rat growth hormone gene (Fig. 7.16ft). This DNA construct, the transgene, was injected into fertilized eggs. When the mice were a few weeks old, they were given drinking water containing zinc. Mice carrying the transgene grew to twice the size of their litter mates because the metallothionein enhancer sequence, stimulated by zinc, had increased growth hormone production.
Transgenic farm animals—such as sheep synthesizing human factor VIII in their milk— have been created. This is an alternative to producing human proteins in bacteria.
Transgenic mice are increasingly being used to prove a protein's function. To do this the gene for the protein is knocked out. This is done by either inserting a piece of foreign DNA into the gene, or by deleting the gene from the mouse genome. The consequences of the protein's absence are then established. Figure 7.17 describes the method called insertional mutagenesis for knocking out a gene's function. The first step is to isolate a genomic clone containing the gene to be knocked-out. A marker gene, such as the drug resistance gene neo, is then inserted into the genomic clone, usually in exon 2 of the gene. This means that the normal, functional product of the gene cannot be synthesized. This construct is the transgene, which is then introduced into embryonic stem (ES) cells. These are cells derived from the inner mass of a blastocyst—that is, a very early embryo—of a white mouse. Homologous recombination inside the embryonic stem cells will replace the normal gene with the transgene. Cells in which this rare event happens will survive when grown on neomycin while embryonic stem cells not containing the transgene will die. The genetically modified embryonic stem cells are inserted into the blastocyst cavity of a black mouse and the blastocyst implanted into a foster mother. Transgenic mice will be chimeric and have a mixed color coat because the cells derived from the genetically modified embryonic stem cells will give a white-color coat while the cells from the blastocyst will give a black-color coat. Subsequent breeding will produce a pure white mouse that is homozygous for the knocked-out gene. The effect of knocking-out the gene can then be analyzed.
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