Oligonucleotidedirected mutagenesis is used to study gene function when appropriate restriction sites are not available

a fertilized mouse egg contains two pronuclei, one from the sperm and one from the egg; these pronuclei later fuse to form the nucleus of the embryo. Mechanical devices can manipulate extremely fine, hollow glass needles to inject DNA directly into one of the pronuclei of a fertilized egg (I Figure 18.23). Typically, a few hundred copies of cloned, linear DNA are injected into a pronucleus, and, in a few of the injected eggs, copies of the cloned DNA integrate randomly into one of the chromosomes through a process called nonhomologous recombination. After injection, the embryos are implanted in a pseudopregnant female — a surrogate mother that has been physiologically prepared for pregnancy by mating with a vasectomized male.

Only about 10% to 30% of the eggs survive and, of those that do survive, only a few have a copy of the cloned DNA stablely integrated into a chromosome. Nevertheless, if several hundred embryos are injected and implanted, there is a good chance that one or more mice whose chromosomes contain the foreign DNA will be born. Moreover, because the DNA was injected at the one-cell stage of the embryo, these mice usually carry the cloned DNA in every cell of their bodies, including their reproductive cells, and will therefore pass the foreign DNA on to their progeny. Through interbreeding, a strain of mice that is homozygous for the foreign gene can be created. Animals that have been permanently altered in this way are said to be transgenic, and the foreign DNA that they carry is called a transgene.

Transgenic mice have proved useful in the study of gene function. For example, proof that the SRY gene (see p. 000 in Chapter 4) is the male-determining gene in mice was obtained by injecting a copy of the SRY gene into XX embryos and observing that these mice developed as males. In addition, a number of transgenic mouse strains that serve as experimental models for human genetic diseases have been created by injecting mutated copies of genes into mouse embryos.

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118.23 Transgenic animals have genomes that have been permanently altered through recombinant DNA technology. In this photograph a mouse embryo (red and blue) is being injected with DNA. (Photo: Jon Gordon/Phototake)

Knockout mice A particularly useful variant of the transgenic approach is to produce mice in which a normal gene has been disabled. The phenotypes of these animals, called knockout mice, help geneticists to determine the function of a gene. The creation of these knockout mice begins when a normal gene is cloned in bacteria and then "knocked out", or disabled. There are a number of ways to disable a gene, but a common method is to insert a gene called neo, which confers resistance to the antibiotic G418, into the middle of the target gene (I Figure 18.24). The insertion of neo both disrupts (knocks out) the target gene and provides a convenient marker for finding copies of the disabled gene. In addition, a second gene, usually the herpes simplex viral thymidine kinase (tk) gene, is cloned adjacent to the disrupted gene. The disabled gene is then transferred to cultured embryonic mouse cells, where it may exchange places with the normal chromosomal copy through homologous recombination.

After the disabled gene has been transferred to the embryonic cells, the cells are screened by adding the antibiotic G418 to the medium. Only cells with the disabled gene containing the neo insert will survive. Because the frequency of nonhomologous recombination is higher than that of homologous recombination and because the intact target gene is replaced by the disabled copy only through homologous recombination, a means to select for the rarer homologous recombinants is required. The presence of the viral tk gene makes the cells sensitive to gancy-clovir. Thus, transfected cells that grow on medium containing G418 and gancyclovir will contain the neo gene (disabled target gene) but not the adjacent tk gene. These cells contain the desired homologous recombinants. The nonhomologous recombinants (random insertions) will contain both the neo and the tk genes, and these trans-fected cells will die on the selection medium owing to the presence of gancyclovir. The surviving cells are injected into an early-stage mouse embryo, which is then implanted into a pseudopregnant mouse. Cells in the embryo carrying the disabled gene and normal embryonic cells carrying the wild-type gene will develop together, producing a chimera—a mouse that is a genetic mixture of the two cell types. The chimeric mice can be identified easily if the injected embryonic cells came from a black mouse and the embryos into which they are injected came from a white mouse; the resulting chimeras will have variegated black and white fur. The chimeras can then be interbred to produce some progeny that are homozygous for the knockout gene. The effects of disabling a particular gene can be observed in these homozygous mice.

Although they are a recent innovation, knockout mice have become important subjects for research in a number of fields. They have been used to study genes implicated in immune function, development, ethology, and human behavior.

Target gene

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Transferred sequence

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A normal gene is disabled by inserting the neo+ gene. The tk + gene is cloned adjacent to the target gene, and...

... the disabled gene is transferred to embryonic mouse stem cells.

neo+

A normal gene is disabled by inserting the neo+ gene. The tk + gene is cloned adjacent to the target gene, and...

... the disabled gene is transferred to embryonic mouse stem cells.

neo+

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