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6.1.1 The specific locus test

Genetic variation — the existence of at least two forms — is the essential ingredient present in all genetic experiments. Phenotypic variation, in particular, is used as a means for uncovering the normal function of a wild-type allele. As discussed in the first chapter of this book, it was the availability of many variant phenotypes within the fancy mouse trade that made the house mouse such an ideal organism for studies by early geneticists. In a sense though, the house mouse won by default because in the absence of domestication and artificial selection, variation in traits visible to the eye is extremely rare, and thus, other small mammals were genetically intractable. Although the fancy mouse variants provided material for a host of early genetic studies, the number of different variants was still limited, and the rate at which new ones arose spontaneously in experimental colonies was exceedingly low: it is now known that, on average, only one gamete in 100,000 is likely to carry a detectable mutation at a particular locus.

During the 1920s, several investigators began investigating the effects of X-rays on reproduction and development. In two laboratories, at least, new mutant alleles were recovered in the offspring of irradiated parents, but the investigators failed to make any connection between irradiation and the induction of these mutations (Little and Bagg, 1924; Dobrovolskaia-Zavadskaia, 1927). The connection was finally made by Muller who, in 1927, published his classic paper explaining the induction of heritable mutations by X-rays (Muller, 1927). Since that time, geneticists who study all of the major experimental organisms — from bacteria to mice — have used both ionizing irradiation and various chemicals as agents of mutagenesis to uncover novel alleles as tools for understanding gene function.

Large-scale mouse mutagenesis experiments were first begun at two government-based "atomic energy" laboratories: the Oak Ridge National Laboratory in Oak Ridge, Tennessee, in the U.S. and the MRC Radiobiological Research Unit first at Edinburgh, Scotland, and then at Harwell, England, in the U.K. Both of these experimental programs were begun initially after World War II as a means for quantifying the effects of various forms of radiation on mice and, by extrapolation, humans, to better understand the consequences of detonating nuclear weapons. The U.S. effort was directed by W. L. Russell and the British effort was directed by T. C. Carter (Green and Roderick, 1966). Scientists at both laboratories quickly realized the potential of their newly created resource of mutant animals, and both laboratories have since gone on to generate mutations by chemical agents as well. The very large-scale studies conducted at Oak Ridge and Harwell — where 10,000 to 60,000 first generation animals were typically analyzed in an experimental protocol — have provided most of the empirical data currently available on the mechanisms and rates at which mutations are caused by all well-characterized mutagenic agents in the mouse.

The experiments performed by Russell and Carter, and other colleagues who followed in their footsteps, were designed to obtain discrete values for the mutagenic potential of different radiation protocols. Rather than attempt to examine all animals for all effects of a particular irradiation protocol (as was common in earlier experiments), these mouse geneticists chose instead to look only at the small fraction of animals that were mutated at a small set of well-defined "specific" loci. The rationale for the "specific locus test" was that effects on individual loci could be more easily quantitated and that the limited results obtained could still be extrapolated for an estimate of whole genome effects. Russell decided that mutation rates should be followed simultaneously at a sufficient number of loci to distinguish and avoid problems that might be caused by locus-to-locus variations in sensitivity to particular mutagens. He decided further that the same set of loci should be examined in each experiment performed. The seven loci chosen to be followed in the specific locus test were defined by recessive mutations with visible homozygous phenotypes that were easily distinguished in isolation from each other, and had no effect on viability or fertility. The seven loci are agouti (a is the recessive non-agouti allele), brown (b), albino (c), dilute (d), short-ear (se), pink-eyed dilution (p), and piebald (s). A special "marker strain" was constructed that was homozygous for all seven loci.

In its simplest form, the specific locus test is carried out by mating females from the special marker strain to completely wild-type males that have been previously exposed to a potential mutagen. In the absence of any mutations, offspring from this cross will not express any of the seven phenotypes visible in the marker strain mother. However, if the mutagen has induced a mutation at one of the specific loci, the associated mutant phenotype will be uncovered. This test is very efficient because it only requires a single generation of breeding and visual examination is all that is required to score each animal.

Although recessive mutations at all loci other than the specific seven will go undetected in the first generation offspring from this cross, it is possible to detect a dominant mutation at any locus so long as it is viable and produces a gross alteration in heterozygous phenotype such as a skeletal or coat color change. One should realize that the most common effect of any undirected mutagen will be to "knock-out" a gene and, in the vast majority of cases, the resulting null allele will be recessive to the wild-type. There is, however, a very small class of loci at which null alleles will act in a dominant or semidominant fashion to wild-type. These "haplo-insufficient" phenotypes are presumably caused by a developmental sensitivity to gene product dosage. Among the best characterized of the dominant-null mutations are the numerous ones uncovered at the T locus — which result in a short tail — and the W locus — which result in white spotting on the coat.

6.1.2 Mutagenic agents

Mutations can be induced by both physical and chemical means. The physical means is through the exposure of the whole animal to ionizing radiation of one of three classes — X-rays, gamma rays, or neutrons (Green and Roderick, 1966). The chemical means is to inject a mutagenic reagent into the animal such that it passes directly into the gonads and into differentiating germ cells. The specific locus test has provided an estimate of the relative efficiency with which each reagent induces mutations. Under different protocols of exposure, X-irradiation was found to induce mutations at a rate of 13 - 50 X 10-5 per locus, which is a 20 to 100 fold increase over the spontaneous frequency, but still not high enough to be used by any but the largest facilities as a routine means for creating mutations (Rinchik, 1991). The mutations created by irradiation are often large deletions or other gross lesions such as translocations or complex rearrangements.

The class of known chemical agents that can induce mutations (known as mutagens) is very large and expanding all the time. However, two chemicals in particular — ethylnitrosourea (ENU) and chlorambucil (CHL) — have been found to be extremely mutagenic in mouse spermatogenic cells (Russell et al., 1979; Russell et al., 1989). Both of these chemicals produce much higher yields of mutations than any form of radiation treatment tested to date (Russell et al., 1989). Optimal doses of either ENU or CHL can induce mutations at an average per locus frequency which is greater than one in a thousand — 150 X 10-5 with ENU and 127 X 10-5 per locus with CHL (Russell et al., 1982; Russell et al., 1989). Although the rates at which ENU and CHL induce mutations are very similar, the types of mutations that are induced are quite different. In general, ENU causes discrete lesions which are often point mutations (Popp et al., 1983), whereas CHL causes large lesions which are often multi-locus deletions (Rinchik et al., 1990a). Originally, it was thought that the basis for mutational differences of this type was the chemical nature of the mutagen itself (Green and Roderick, 1966), but this no longer appears to be the case. Rather, it now appears that the germ-cell stage in which the mutation arises is the major determinant of the lesion type (Russell, 1990). The correlation observed between chemical and lesion type is a result of the fact that different mutagens are active at different stages of spermatogenesis. Thus, ENU acts upon premeiotic spermatogonia where mutations are likely to be of the discrete type, and CHL acts upon postmeiotic round spermatids where mutations are likely to be of the large lesion type (Russell et al., 1990).

ENU was the first chemical to be identified that was sufficiently mutagenic to be used by smaller laboratories in screens for mutations at particular loci or chromosomal regions of interest (Bode, 1984; Shedlovsky et al., 1988). ENU has also been used in screens for non-locus-specific phenotypic variants that could serve as models for various human diseases (McDonald et al., 1990). Several laboratories are beginning to use ENU for saturation mutagenesis of small chromosomal regions defined by deletions, as one tool (among several complementary ones) for obtaining a complete physical and genetic description of such a region (Shedlovsky et al., 1988; Rinchik et al., 1990b; Rinchik, 1991). The major limitation to the global use of this approach is the very small number of genomic regions in the mouse at which large deletions have been characterized.

The availability to Drosophila geneticists of deletions (or deficiencies as the fly people call them) that span nearly every segment of the fly genome has played a critical role in the identification and characterization of large numbers of genes and the production of both gross functional maps and fine-structure point mutation maps by the very approach just described above. Clearly, a method to accumulate a similar library of deletions for the mouse would be well-received. The mutations induced by X-rays are often large-scale genomic alterations including translocations, inversions, and deletions. Indeed, most mouse deletion mutations maintained in contemporary stocks were derived in this manner. However, the overall yield of X-ray-induced deletions is quite low, and because of other problems inherent in this approach, it is not ideal for global use.

In 1989, CHL was reported to be an attractive alternative to X-rays as an agent for the high-yield induction of deletion mutations in the mouse (Russell et al., 1989). The per locus mutation rate was found to be on the order of one in 700 in germ cells of the early spermatid class, and of the eight mutations induced at this stage that were analyzed, all were deleted for DNA sequences around the specific locus marker (Rinchik et al., 1990a). This study also showed that CHL-induced mutations were often associated with reciprocal translocations. This last finding is unfortunate because translocations can reduce fertility with consequent negative effects on strain propagation.

There is hope that CHL can be used as a means for generating sets of overlapping deletions that span entire chromosomes (Rinchik and Russell, 1990). Projects of this type will require very large animal facilities and support resources and will consequently be confined to only a handful of labs. However, once mouse strains with deletions have been created and characterized, they can serve as a resource for the entire community.

6.1.3 Mouse mutant resources

An advantage to using the mouse as a genetic system is the strong sense of community that envelops most of the workers in the field, and it is in the context of this community that strains carrying many different mutations — both spontaneous and mutagen-induced — have been catalogued and preserved and are made available to all investigators. A catalog containing detailed descriptions of all mouse mutations characterized as of 1989 has been compiled by Margaret Green and is included as the centerpiece chapter in the Genetic Variants and Strains of the Laboratory Mouse edited by Mary Lyon and Tony Searle (Green, 1989). This catalog is now available in an electronic form that is updated regularly (see Appendix B). Of course, many more mutant animals are found and characterized with the passing of each year, and an updated list is published annually in the journal Mouse Genome. This list contains information on the individual investigators that one should contact to actually obtain the mutant mice.

The largest collection of mutant mouse strains is maintained at the Jackson Laboratory under the auspices of the "Mouse Mutant Resource" (MMR) which is currently maintained under the direction of Dr. Muriel Davisson (Davisson, 1990). In 1990, over 250 mutant genes were maintained in this resource, accounting for two-thirds of all known mouse mutants alive at the time (Davisson, 1990). Each year, animal caretakers identify an additional 75 to 80 "deviant" animals among the two million mice that are produced by the Jackson Laboratory's Animal Resources colonies (Davisson, 1993). Approximately 75% of the deviant phenotypes are found to have a genetic basis and breeding studies are conducted on these to determine whether or not they represent mutations at previously characterized loci. If they do, DNA samples are recovered and the lines are discarded or placed into the frozen embryo repository. If a mutation is novel, its mode of transmission (autosomal/X-linked, dominant/recessive) is determined, the phenotypic effect of the mutation is characterized, and it is mapped to a specific chromosomal location with the use of breeding protocols to be described in Section 9.4 (Davisson, 1990; Davisson, 1993). Descriptions of all newly characterized mutations are publicized, and mutant mouse strains are made available for purchase through the standard Jackson Laboratory catalog. In 1992, over 35,000 mice from the MMR were distributed to investigators throughout the world (Davisson, 1993).

Space limitations make it impossible for the MMR to maintain breeding stocks of mice that contain every known mutant gene, with the total number expanding each year. Fortunately, mutant stocks that are not currently in demand by investigators can be maintained (at minimal cost) in the form of frozen embryos. The importance of embryo freezing as a storage protocol cannot be over-emphasized. Time and time again, modern-day molecular researchers have reached back to use mutations described long-ago as critical tools in the analysis of newly cloned human and mouse loci.

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