BOX 2.1 Knockout and Knockin Technology
In order to study the relationship between proteins and gene function, scientists now can prevent the manufacturing of a protein by a specific gene. By disabling a gene from a test organism, and then producing descendants that contain two copies of the disabled gene, it is possible to observe the descendants’ development in the absence of a particular protein. This practice, referred to as knockout technology, is an attempt to shut down or turn off a particular gene. Thus far, the mouse has been the mammal in which knockout technology has been most generally applied (University of Guelph, 2001). In essence, a “knockout” organism (e.g., the mouse) is created when an embryo cell (an embryonic stem cell—or ESC—which is a cell that has yet to divide into different tissue cells; NRC, 2002b) is genetically engineered, and then inserted into a developing embryo. The embryo then is inserted surgically into the womb of a host (e.g., a female mouse). Once the embryo has matured, a portion of its stem cells will produce egg and sperm with the knocked-out gene.
A gene also can be altered in function, in contrast to being deleted. When a gene is altered but not shut down, a “targeted mutation” effect is created. This practice is referred to as knockin technology, whereby a life form has an altered gene “knocked” into it (MGD, 2002).
Gene knockout/knockin technology is well established as an experimental tool in mice due to the availability of ES cell lines. The principleis to take advantage of a rather rare event that occurs after introduction of DNA into cells—homologous recombination between identical sequences in the genome and the transfecting DNA (Bronson and Smithies, 1994). In the most common protocol, a selectable marker (such as the neomycin resistance gene) is inserted within a piece of DNA corresponding to a portion of a gene of interest. After transfection of cells by this construct and selection for the marker (by growth in a medium containing the neomycin-related antibiotic G418, in this example), the selected cells are screened to identify the small fraction that has one copy of the gene of interest disrupted by the marker. Progeny animals derived from the cells will be heterozygous for the “knocked-out” or “knocked-in” gene; breeding to obtain homozygous animals is straightforward. Because the process is so inefficient, very large numbers of transfected cells must be screened, making the use of cultured cells essential, since it would be impracticable to screen large numbers of progeny from microinjected eggs. The galactosyl transferase-knockout pigs discussed above were generated from cultured fetal fibroblasts manipulated in this way. Nuclei from these cells then were transferred into oocytes as described in the next section.