Figure 8–52 engineered genes can be turned on and off with small molecules. Here, the DNA-binding portion of a bacterial protein (the tetracycline, Tet, repressor) has been fused to a portion of a mammalian transcriptional activator and expressed in cultured mammalian cells. The engineered gene X, present in place of the normal gene, has its usual gene control region replaced by cis-regulatory sequences recognized by the tetracycline repressor. In the absence of doxycycline (a particularly stable version of tetracycline), the engineered gene is expressed; in the presence of doxycycline, the gene is turned off because the drug causes the tetracycline repressor to dissociate from the DNA. This strategy can also be used in mice by incorporating the engineered genes into the germ line. In many tissues, the gene can be turned on and off simply by adding or removing doxycycline from the animal’s water. If the tetracycline repressor construct is placed under the control of a tissue-specific gene control region, the engineered gene will be turned on and off only in that tissue.
animal. For example, a gene can be fused to the gene for a fluorescent protein. When this altered gene is introduced into the genome, the protein can be tracked in the living organism by monitoring its fluorescence.
Altered genes can be created in several ways. Perhaps the simplest is to chemically synthesize the DNA that makes up the gene. In this way, the investigator can specify any type of variant of the normal gene. It is also possible to construct altered genes using recombinant DNA technology, as described earlier in this chapter. Once obtained, altered genes can be introduced into cells in a variety of ways. DNA can be microinjected into mammalian cells with a glass micropipette or introduced by a virus that has been engineered to carry foreign genes. In plant cells, genes are frequently introduced by a technique called particle bombardment: DNA samples are painted onto tiny gold beads and then literally shot through the cell wall with a specially modified gun. Electroporation is the method of choice for introducing DNA into bacteria and some other cells. In this technique, a brief electric shock renders the cell membrane temporarily permeable, allowing foreign DNA to enter the cytoplasm.
To be most useful to experimenters, the altered gene, once it is introduced into a cell, must recombine with the cell’s genome so that the normal gene is replaced. In simple organisms such as bacteria and yeasts, this process occurs with high frequency using the cell’s own homologous recombination machinery, as described in Chapter 5. In more complex organisms that have elaborate developmental programs, the procedure is more complicated because the altered gene must be introduced into the germ line, as we next describe.
Animals and plants that have been genetically engineered by gene deletion or gene replacement are called transgenic organisms, and any foreign or modified genes that are added are called transgenes. We discuss transgenic plants later in this chapter and, for now, concentrate our discussion on transgenic mice, as enormous progress has been made in this area. If a DNA molecule carrying a mutated mouse gene is transferred into a mouse cell, it often inserts into the chromosomes at random, but methods have been developed to direct the mutant gene to replace the normal gene by homologous recombination. By exploiting these “gene targeting” events, any specific gene can be altered or inactivated in a mouse cell by a direct gene replacement. In the case in which both copies of the gene of interest are completely inactivated or deleted, the resulting animal is called a “knockout” mouse. The technique is summarized in Figure 8–53.