The most promising new techniques for the study of in vivo mammalian mutagenesis make use of transgenenic mice carrying a recoverable vector. Mutation systems in mammals can be based on the selection of altered phenotypes among cells sampled from the whole animal, but they are then limited to the very few cell types in which the marker gene is expressed. Such systems require both in vivo and in vitro cell proliferation for expression and verification of the mutations. To avoid these complications, the study of mutations in most tissues must be based on the detection of genetic alterations in a vector that is independent of the phenotype of the mammalian cell. The vector is only a small portion of the mammalian genome, and may of the procedures for recovering the vector are inhibited by the host DNA. For this reason, partial purification is necessary. The purification is made possible by using vectors which are not cut by restriction enzymes that cut the host DNA to pieces of an average size considerably smaller than the vector. The efficiency for measuring mutation frequencies depends on the number of vectors which can be recovered from a certain amount of DNA and is affected by the number of vectors per mammalian genome and the transfection efficiency of the paritally-purified vector. In order to avoid selection against or for the spontaneous or induced mutations, the transfection efficiency of the vector from the transformed DNA and of the pure vector DNA should be of the same order of magnitude. Differences in the response to mutagens between the mammalian genome and the procaryotic vector may be expected due to the lack of unique mammalian topographical features in the vectors. Any mutation induction which depends preferentially on these unique features of the mammalian genome may not be detected in a shuttle vector system unless the vector has been engineered or specifically designed to include such topographical characters. The shortcoming of short-term tests that use mutagenicity for predicting human carcinogenicity is usually lack of correlation between mutagenesis in the short-term tests and the corresponding results in carcinogenesis bioassays in mammals. One factor which could contribute to the lack of correlation between the short-term test systems and the bioassays is that we are comparing mutations in totally different genes in different organisms. By using the ΦX174 shuttle system, one of the variables may be eliminated. In this system, we can compare mutation rates between different stages of the same target DNA ranging from in vivo in transgenic mice to naked single-stranded DNA. In other words, it may provide the opportunity to study how various organisms respond to the same initial mutagenic damage on a constant target. In order to mimic in vivo mammalian metabolism, liver microsomes are used in many in vitro tests for mutagenicity. In reality, there is no possible way to simulate the complex activation, detoxification and repair systems that occur in vivo. By using a shuttle vector system such as ΦX174 we can hope to elucidate the influence of these factors on mutagenesis. Using these systems, the mutation frequency and the cancer rate can be compared for the same tissue in the same strains of animals. This comparison may bring us closer to understanding the role of mutations in the development of cancer. Only 20–30 compounds have been tested for the induction of transmissible mutations in mammalian germinal tissue, and most of these compounds are strong alkylating agents (Ray et al., 1987). A shuttle vector system based on the detection of mutations in DNA from sperm of exposed animals may provide the method needed for expanding the data base on mammalian germ-cell mutagenesis. Unfortunately, due to the limited amount of DNA available from the female germinal tissue, none of the shuttle vector systems currently envisioned can be applied to enhance the study of mutagenesis in female germinal tissue. The mitotic indices of many mammalian tissues are very low or essentially zero. This is especially true of neurological tissue. It is reasonable to assume that DNA damage occurs in such tissues, is repaired and somtimes misrepaired, and that results in mutations. In order to study mutations in nonmitotic tissue, a shuttle vector system must be designed in such a way that the mutation frequency is determined directly among the DNA molecules isolated from the tissue and that mutation detection does not require expression of the mutant phenotype in mammalian cells in vivo. The ability to determine mutation frequencies in nonmitotic tissues such as the brain may promote a better understanding of the causative events in age-related degenerative diseases of the central nervous system.
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