Although there are very few examples of dominantly inherited neoplastic disease in lower ani- mals, as indicated above, the ability to manipulate the genome of mammalian organisms has made enormous technological advances during the last decade. This has led to the production of transgenic animals bearing oncogene or related constructs as a component of their genome (Jae- nisch, 1988) and the production of animals with specific gene alterations resulting from gene targeting (knockout) methods (Bronson and Smithies, 1994). Figure 5.8 presents a diagram of the method used in producing transgenic animals. In this technique it is necessary to introduce a specific genetic construct by means of microinjection into the fertilized egg. This is then im- planted in the uterus of a pseudopregnant female, allowing embryonic development to proceed through the birth of the fetus (cf. Camper, 1995). Pups are tested for the presence of the trans- gene by securing samples of their DNA and analyzing it for the introduced genetic material. Usually the transgene is expressed in a dominant manner. In contrast, when gene targeting is employed, blastocysts (Chapter 2) are isolated from pregnant females, and the inner cell mass is cultured to produce embryonic stem (ES) cells (Chapter 14). The gene construct used to target the gene of interest is then transfected into the ES cells. Those cells exhibiting the altered gene structure are isolated and injected directly into blastocysts, which are implanted into a foster mother as with the microinjected fertilized eggs above. From this is produced a chimera which, if the targeted ES cell has populated the germline, will be heterozygous for the gene alteration. Subsequent breeding will produce homozygous progeny. This technique is outlined in Figure 5.9, along with a diagram depicting the results and mechanisms of the transfection step. Basi- cally this step depends on homologous recombination of the transfected gene into the position of
Figure 5.8 Diagram of method for the development of transgenic mice, beginning with injection of cloned DNA construct into the nucleus of a fertilized egg, implantation into pseudopregnant female, and monitoring of offspring for the presence of the transgene. With subsequent breeding of transgene-positive founder animals, inheritance of the transgene through the germline can be determined.
the normal gene, with subsequent recombination and replacement of one copy of the normal gene with the gene construct containing a selectable marker in place of one or more of the ge- netic components of the gene. Culture of ES cells having undergone this process of homologous recombination will allow enrichment and isolation of the ES cells. The selectable marker allows growth of cells containing such a gene in the presence of a drug which would be toxic to cells not having this selectable gene. Complications arise in that some random incorporation of the transfected gene occurs, and thus all clones must be tested to ensure that the one to be utilized contains the disrupted or “knocked out” gene.
By means of these two techniques, it thus becomes possible to develop animals having extra copies of a specific gene in their germline as well as animals missing one or both alleles of a critical gene, such as a tumor suppressor gene.
Figure 5.9 Diagram of the procedure for homologous recombination (a) and the scheme for the develop- ment of animals with a homozygous loss of the targeted gene (b). In the homologous recombination proce- dure there is deletion of exon 2 (1) and its replacement by a selectable gene (the neor gene). This construct is transfected into ES cells (3) in which homologous recombination occurs with replacement of the normal gene by the gene containing the selectable sequence. In the scheme on the right, ES cells are cultured and those containing the selectable genes isolated and grown in the presence of the drug (neo). Surviving cells are implanted into a foster mother, and progeny in which the germ cells have been populated by the tar-
geted ES cell are obtained by selective breeding. Such chimeras are then bred to produce homozygous off- spring exhibiting complete loss of the targeted gene. (From Yamamura and Wakasugi, 1991, with permission of the authors and publisher.)
Gene targeting (knockout) has been used in mice to study the effects of complete elimina- tion as well as heterozygosity of the RB and p53 tumor suppressor genes. Mice in which one allele of the Rb gene is mutated do not exhibit retinoblastomas, but some of the animals have pituitary neoplasms that arise from cells in which the wild-type RB allele is absent (Jacks et al., 1992). The elimination of both alleles results in death prior to the 16th day of embryonic life. Such animals exhibit multiple defects with abnormalities in the hematopoietic system and central nervous sys- tem (Lee et al., 1992a). On the other hand, mice with mutations in one or both of the p53 tumor suppressor genes appear normal during the first few months of life but then begin to develop a variety of mesenchymal and epithelial neoplasms (Donehower et al., 1992; Hooper, 1994).
In contrast, considerable work has been done with the transgenic approach, largely with viral and cellular oncogenes as the structural gene within the constructs. In addition, constructs in which growth factors, hormones, and even homeobox genes are the structural component of the construct have been utilized (Table 5.8). Table 5.8 shows some examples of transgenic car-
cinogenesis with various constructs following the technique depicted in Figure 5.8. The listing is only representative and by no means exhaustive. In addition, transgenic carcinogenesis has also been established in other rodent species including the rat (Hully et al., 1994) and the rabbit (Knight et al., 1988). One may assume that neoplasms resulting from transgenic carcinogenesis as exemplified in Table 5.8 are the result of dominant effects of the structural genes utilized to produce the animals, but some phenotypes resemble dominantly inherited neoplastic disease syndromes of the human. For example, mice made transgenic with the HTLV tat gene under the regulation of its LTR produce a syndrome very similar to neurofibromatosis I in the human (see above). In addition, mice made transgenic with the c-mos proto-oncogene under the regulation of the Moloney virus LTR develop a syndrome very similar to MEN II (Schalz et al., 1992). As shown in the table, in some instances the regulatory region (promoter/enhancer) of the gene con- struct targets the expression of the structural gene to specific tissues, with resultant neoplasms arising only in those tissues. An exception to this is the description by Schaffner et al. (1995) of the use of the promoter for prostate-specific antigen with a mutated Ha-ras gene in which no prostatic neoplasms developed, but rather carcinomas of the salivary gland and the gastrointesti- nal tract. Some regulatory components, especially the metallothionine promoter, have been uti- lized so that the expression of the structural gene may be regulated by external factors, in this instance zinc administration (Lee et al., 1992b; Dyer and Messing, 1989).
Although the induction of cancer by modern genetic techniques is, on the surface, quite different from carcinogenesis by chemical and physical agents, there are many similarities in the development of neoplasms from genetic, chemical, and physical carcinogens. Thus, the genetic changes that occur during the process of carcinogenesis initiated by any of these methods may have more similarities than differences, and both careful investigation and comparison of the development of neoplasia from several different carcinogenic mechanisms may prove to be ex- tremely fruitful in ultimately elucidating the critical mechanisms of cancer development.