In 2001, Martin Evans of the United Kingdom and Mario Capecchi and Oliver Smithies of the United States were honored for developing "gene targeting,'' a technology that allows the creation of ''knockout'' and other designer strains of mice in which almost any gene can be disabled and its function probed.6,33-35 Two principal conceptual advances in the 1980s, comparable in inventiveness and impact to other revolutionary biological innovations including recombinant DNA, DNA sequencing, polymerase chain reaction, and monoclonal antibodies, led to this breakthrough. The first was the development of stem cell methods to culture embryonic stem (ES) cells by Evans and the second was the development of the method of homologous recombination by Capecchi and Smithies independently. Homologous recombination occurs between a native target chromosomal gene and exogenous DNA to modify a specific target locus. The combination of the Evans technique with the Capecchi-Smithies technique led to construction of the first ''knockout'' mice in 1989, an advance that provided an efficient means of producing laboratory models of human disease in a predictable manner by making it possible to evaluate the function of almost any single gene. By 2001, well over 7000 genes of approximately 30,000 mouse genes had been analyzed with gene targeting.35
The gene-targeting protocol for generating chimeric mice from embryonic stem cells containing a targeted mutation, outlined in Figure 9.7, as it is now performed is as follows:35
The desired sequence modification is introduced into a cloned copy of the chosen gene by standard recombinant DNA technology. Then, the modification is transferred, by means of homologous recombination, the cognate genomic locus in ES cells and the ES cell lines carrying the desired alteration are selected. Finally, ES cells containing the altered genetic
locus are injected into mouse blastocysts, which are in turn brought to term by surgical transfer to foster mothers, generating chimeric mice that are capable of transmitting the modified genetic locus to their offspring.
Embryonic stem cells that contain the targeted mutation are enriched (if necessary) by a procedure involving positive selection for cells that have incorporated the targeting construct anywhere within the stem cell genome, followed by negative selection against the cells that have integrated the construct randomly into their genome. Homologous recombination between the targeting vector and chromosomal copy of the targeted gene results in disruption of one copy of the target gene and loss of the HSV-tk sequence. The vector is designed so that transfer of the HSV-tk gene does not accompany replacement of the endogenous gene. HSV-tk is excluded because it represents a discontinuity between homology and nonhomology with the target sequence. The genotype of the embryonic stem cells in which targeting has occurred will be Gene X+, neor+, HSV-tkwhereas the genotype of the cells in which random integration of the vector has occurred should be Gene X+, neo , HSV-tk+. At the concentration used for negative selection, gancyclovir is not toxic to parental embryonic stem cells but selectively kills cells containing the viral thymidine kinase gene. By selecting for cells containing a functional neor gene with G418, and against cells containing a functional HSV-tk gene with gancyclovir, the net effect is to enrich cells containing the targeted mutation.
The replacement vector used in gene targeting typically contains 10-15 kb of DNA homologous to the target gene, say Gene X, followed by a neomycin-resistant gene (neor) and a herpes simplex thymidine kinase gene (HSV-tk) adjacent to the target homology (Figure 9.8). The neor gene disrupts the coding sequence of the target gene and acts as a marker conferring resistance to a neomycin-like drug (G418) that is used for selecting cells that contain a copy of the recombinant vector. The pMC1 NEO neor vector is used in these constructs because it maximized the expression efficiency of the DNA integrated into the stem cells. The HSV-tk gene is used to negatively select for nonhomologous events. Nonhomologous events will lead to the insertion of tk and the resultant clone can be selected against with gancyclovir. In the more recent protocol, a nucleoside analog, FIAU (that specifically kills cells with functional HSV-tk genes, but is not toxic to cells with only cellular Tk), replaces gancyclovir.
Gene targeting has been used to generate animals with null ("knockout") alleles, overexpressed alleles, and "humanized" transgenic animals, that is, mice that have been modified by replacing a native mouse gene with its human counterpart. In fact, this technique can be used to modify the pattern of any property of a given gene including its transcription, mRNA, development, or the capacity of its gene products to interact with the products of other genes. The possibility of engineering large-scale changes such as chromosomal translocation or deletions into the mouse germ line by gene targeting has been demonstrated, which would facilitate creation of mouse models with chromosomal rearrangements that are associated with human cancers (see, for example, Appendix A). Even though transgenesis and gene targeting are often directed toward different ends, the former toward the gain of new functions and the latter toward augmenting or generating a loss of existing functions, the technique of gene targeting is applicable to the creation of animals of either type. Additional strategies have made possible the creation of mice with mutations that can be targeted to specific cells and tissues36 and timed to specific developmental stages.37
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