Gazing into a crystal ball at the behest of the Environmental Mutagen Society, I see three paths, all leading to Investigation of Spontaneous Mutation more sophisticated questions about heritable mutations, somatic mutations, cancer, and other diseases. Those fol-Studies with transgenic animals have revealed surprislowing the first path use the existing transgenic animals, ing things about spontaneous mutation. The spontaneous especially the MutaMouse and the Big Blue Mouse frequency of mutants is unexpectedly high and unexpect- [Gossen et al., 1989;Kohler et al., 1991]. Those following edly similar in all tissues, about 40 1 10 6 [for review, the second path combine these existing lacZ and lacI see Zhang et al., 1995]. The largest difference between reporter genes with other special strains of mice, espetissues seems to be male germ cells in the Big Blue cially knockout mice, as has been done for p53. This Mouse (lacI) where it is less than half that of the somatic route starts out beautifully but runs through increasingly cells [Kohler et al., 1991]. Regardless of the tissue, the difficult terrain because of Mendel's Laws. It is not too spontaneous mutant frequency is at the high end of the difficult to put two loci together, but combinations of spectrum for all of the loci examined so far, save the three, four, and more become ''exponentially'' more dif-fX174 mouse, in which a single basepair is the mutaficult, especially if the genes have to be homozygous. As tional target [Burkhart et al., 1992]. Skopek has sugeach new genetic character is added, the preexisting loci gested that the high spontaneous frequency of mutants are rendered heterozygous, as is the new one, and all will in the Big Blue Mouse is due to a high frequency of segregate in the next generation, requiring the breeding CpG sites in the lacI transgene, sites that have been and testing of a large number of mice. So, creation of selected against in mammalian codons during evolution mice that have multiple deficiencies, like many of the because of their high spontaneous mutation rate [ Skoinformative bacterial strains, is promising but not easy. pek et al., 1996 ]. The third path involves the generation of new kinds of Another explanation for the difference in mutant frereporters that will permit simpler and cheaper analyses quency between hprt and the transgenes is that hprt cells and will have characteristics that render them more like are selected against in the mouse, as they are in Leschendogenous genes. Certainly we need them, for the cost Nyhan heterozygotes [Strauss et al., 1980]. One of the of this work is definitely impeding the field and inducing difficulties is not knowing the somatic mutation rate of people to take shortcuts in their experiments. Since the endogenous genes, of which we have far too few for work of Piegorsch et al. [1994] encouraged us to make analysis, and whether or not selection against cells with our samples large enough-both of mice and of plaques, mutations in endogenous genes may be reducing the obthe most egregious of these shortcuts has been the failserved mutant frequency at these loci. The transgenes, ure of most researchers to consider manifestation time. being nonessential and unexpressed, are genetically neutral and simply accumulate mutations [Tao et al., 1993; This is the interval between treatment and sampling Shaver-Walker et al., 1995]. Each gene has, of course, [ Heddle, 1998 ]. The largest component of this interval its own characteristic mutation rate. The observed rate is is the turnover of cells in the tissue, which is why the term ''manifestation time'' is more appropriate than ''fixation time'' or ''expression time.'' If the interval between the last treatment and fixation is less than the Contract grant sponsors: Cancer Research Society, Inc.