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J Mating Systems for Mutants There are many systems for breeding rodents, but not all of them can be used for the maintenance and propagation of mutants. The two major systems to be considered are inbreeding, which can be done either by brother x sister matings or by transferring a mutation to an inbred background, and propagation without inbreeding. Some mutants can be inbred easily. Generally, this is the case when the mutation has no immediate deleterious effects and the affected animals of both sexes are fertile. A more involved process is necessary when the mutation reduces viability or fertility. Those systems of breeding used for most of the immunologic mutants discussed in this report are presented, starting with the simplest maintenance scheme and progressing to the most complex. Mouse mutants are used to illustrate each method; however, the mating systems are applicable to all rodents. INBREEDING Brother x Sister Inbreeding Inbreeding of brother x sister littermates is the easiest system to use. At least 20 or more generations of consecutive brother x sister matings con- stitute an inbred strain. However, when expanding a strain beyond the F20 generation, all future matings must come from a common ancestor of at least the F20 generation, and, as inbreeding continues, the common ancestor should be kept as close as possible to the most advanced generation. The 155

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156 IMMUNODEFICIENT RODENTS greatest hazard of inbreeding is the inbreeding depression (decreased fertility) that can occur. Brother x Sister Inbreeding with Homozygosity This type of inbreeding is done with recessive mutations when both male and female homozygotes are viable and fertile. This method is most useful for animals with mutations that cannot be visually distinguished at either pre- or postweaning ages. For example, lymphoproliferation (Ipr' and generalized lymphoproliferative disease (gl]) are not apparent until 8 and 12 weeks, respectively. Other immunologic mutations that cannot be visually distin- guished include disease resistance and tolerance genes (Beg, Ity, Lsh, and Tol-l), mitogen responsiveness (Lps4), and immunodeficiency and comple- ment genes (scid, xid, and Hc). These are all maintained in inbred strains that are homozygous for the specific immunologic gene. Brother x Sister Inbreeding with Forced Heterozygosity Heterozygosity (the condition of having one or more pairs of dissimilar alleles) can be forced upon a locus either by backcrossing or by intercrossing. This method can be used to produce an inbred background that is selected for expression of the specific mutation or to maintain a mutation on an already inbred background when it is desirable to have a nonmutant sibling as a control. Backcrosses can be used if the gene is recessive and viable (i.e., r/ + x r/rJ or if the gene is dominant (i.e., Dl + x + / + ). Intercrosses can be used if the gene is recessive and lethal or sterile (i.e., r/ + x r/ + ~ or if the gene is dominant or semidominant and lethal (i.e., Dl+ x Dl+) (E. L. Green, 19661. The mutations discussed in this report that are maintained in this manner are hairless (hr), beige (bg), and dwarf (dw). Strain HRS/J is an example of an inbred strain produced by brother x sister matings of a haired (hrl+) female with a hairless (hrlhr) male. Strain DW/J is an example of an inbred strain produced by brother x sister matings of het- erozygotes (awl + ). The untreated homozygote (dwldw) is sterile. However, because heterozygotes are phenotypically indistinguishable from homozygous normal animals, each pair of normal-appearing siblings must produce dwldw offspring before that pair is placed in the breeding colony. Inbreeding a Balanced Stock Closely linked mutant genes can be used to distinguish heterozygotes of lethal or sterile recessive mutants. Because these lethal or sterile mutants must be bred from heterozygotes, there is a considerable saving of cage space

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MATING SYSTEMS FOR MUTANTS 157 if the heterozygote can be recognized at least 70 percent of the time. To make a balanced stock, a carrier of the recessive mutation in question (m/ + ~ is crossed to a mouse that is homozygous or heterozygous for a linked marker gene (gig or g/ + ). The offspring are then intercrossed, and, if both mutant phenotypes (m/m and gig) are among their progeny, the intercrossed parents are double heterozygotes (m + / + g) in the repulsion phase; that is, the two recessive mutant alleles are on different members of the homologous chromosome pair. From double heterozygote repulsion matings, three classes of offspring can be distinguished: homozygotes of the gene in question; homozygotes of the closely linked marker gene; and normal-appearing mice, which are ex- pected at least 70 percent of the time to be carriers of both genes if the genes are linked as close as 7 cM (M. C. Green, 19661. Only normal-appearing offspring from parents that have produced both mutants should be used to propagate the next generation (for more details regarding this type of mating see M. C. Green, 19664. Examples of stocks that are maintained in this manner are gray-lethal (al) and diabetes (db). The gray-lethal mutation in strain GL/Le is balanced with downless-J idled, which is located approxi- mately 7 cM away from gl on chromosome 10. Diabetes (db) on C57BL/Ks is maintained balanced with misty (m), which is about 1 cM away on chro mosome 4. Closely linked marker genes can also be used in coupling (both mutant alleles are on the same chromosome) to help identify mutants before the effect of the gene can be detected. Transferring a Mutation to an Inbred Background One of the standard inbred strains (e.g., C57BL/6J or C3H/HeJ) is used to provide the background for this system of breeding. The mutant gene is crossed into the selected strain in one of the following ways, depending on whether the mutation is dominant, recessive, or recessive and lethal or sterile. Bachcross Matings Backcross matings are generally used for a dominant gene such as viable dominant spotting (WV). The heterozygote (Wt/ + ~ is repeatedly backcrossed to a member of the selected inbred strain ~ + / + ). This backcrossing continues for several generations. The letter "N" is used to denote the number of times a mutation has been crossed to the inbred background. Between N7 and N10, nearly all alleles that are not closely linked to the W locus will have come from the selected inbred strain (E. L. Green, 19661.

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158 IMMUNODEFICIENT RODENTS Cross-Intercross Matings Using Homozygotes Cross-intercross matings, using animals homozygous for the recessive gene, are generally used when the homozygote is viable and fertile. In this type of mating, the homozygous recessive mutant (r/r) is crossed to the selected inbred strain i+/+. The offspring are mated brother x sister (intercrossed = r/+ x r/+), and the homozygote (r/r) is recovered and crossed back again to the inbred strain ~ + / + ). This pattern of cross-inter- cross matings is continued until at least 7-10 crosses (N7 to N10) back to the inbred strain have been completed. At this point, crossing into the inbred strain can continue or brother x sister matings can be made using either backcross- or intercross-type matings. However, if the brother x sister method is used, it is wise to occasionally cross back to the inbred strain to prevent subline divergence. The mutation Ipr has been put on several different inbred backgrounds to at least the N10 generation by this method and then maintained by brother x sister matings of homozygotes. Cross-Intercross Matings Using Heterozygotes Cross-intercross matings using heterozygotes are made when the recessive gene is lethal or sterile. A known heterozygote (r/ + ~ is crossed to the selected inbred strain (+/+ ). Only one out of two of the offspring, or one out of four pairs, is expected to be a carrier; therefore, as many as 12 brother x sister pairs might have to be made up from the progeny to ensure that two carriers are mated. Carriers are identified by the production of mutant off- spring. Once identified, the heterozygote (r/ + ~ is crossed to the inbred strain (+/+ ), and the progeny are again intercrossed. This breeding pattern can be continued indefinitely, or after the mutation has been placed on the inbred background with at least seven crosses (N7) to ensure histocompatibility, the ovarian transplantation technique can be used. Ovarian transplantation is a more efficient method of maintenance because all intercross pairs are known carriers, and, consequently, fewer pairs are needed to maintain the stock. Cross-lntercross Matings Using Ovarian Transplantation Cross-intercross matings using ovarian transplantation are used to maintain lethal or sterile recessive mutations. The ovaries of the homozygous mutant are removed at any time from 14 days to several weeks of age and are transplanted into the empty ovarian capsule of a histocompatible female host. The host must be of such a genotype (generally a coat color is used) that the appearance of the offspring will determine whether they were produced from eggs from the transplanted ovary or from residual tissue in the host ovary. The host female is then crossed to a male of the selected inbred strain. For

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MATING SYSTEMS FOR MUTANTS 159 example, if a lethal mutant gene arose on the C57BL/6J strain, it will be nonagouti (ala) in color. The host might be a white-bellied agouti female from the coisogenic C57BL/6J-AW-~/Aw-~ strain or a hybrid of C57BL/6J- AW-~lAw-~ x any inbred strain that is homozygous agouti (A/A) in color. After ovarian transplantation, this host is crossed to a C57BL/6J male that is nonagouti (ala). If the offspring are of the correct color (ala), they will be intercrossed (brother x sister mated) and a homozygous mutant offspring will be selected as the ovarian donor for the next transplant. If the offspring are agouti in color, they are from the host ovary and are discarded. The new host with the nonagouti mutant ovary is then crossed to the inbred C57BL/6J strain. Each cross to the inbred strain represents another N generation. However, before ovarian trans- plantation to an inbred strain can be made, the lethal or sterile recessive mutation must be placed on the selected inbred strain to N7 by the cross-intercross system using the heterozygote to ensure histocompatibility (unless it arose on that strain by mutation and is, therefore, coisogenic and histocompatible). The mutations motheaten (me), viable motheaten (met), microphthalmia (mi), and obese (ob) are all maintained on inbred strains by ovarian transplantation. PROPAGATION WITHOUT INBREEDING Some mutant mice cannot be successfully inbred; consequently, the use of the hybrid mouse has become an invaluable means for both maintaining and producing these mutants. In general, the vigor of the hybrid results in hardier, faster-growing, and longer-lived mutants, as well as better repro- ductive performance in the breeders. Transferring a Mutation to a Hybrid Background A mutation can be transferred to a hybrid background in two ways. First, the mutation can be transferred onto two different standard inbred strains. Hybrid mutant mice can then be produced by crossing one mutant-bearing strain to the other. The mutant from this cross is a true hybrid or F1. However, this is an expensive' and space-consuming process, and, although hybrid mutants and controls with known genotypes are produced, the breeding stock is inbred and is often very difficult to maintain. The second method is to transfer the mutation to a hybrid background that is made from two standard inbred strains and that is also color coded to permit ovarian transplantation. For example, strain C57BL/6J is nonagouti (ala), and the coisogenic strain C57BL/6J-AW-~/Aw-7 is white-bellied agouti. Strain C3HeB/FeJ is agouti (A/A), and the congenic strain C3HeB/FeJLe- ala is nonagouti. From these four strains two sets of compatible hybrids can be made, a nonagouti, B6C3Fe-a/a Fit (C57BL/6J x C3HeB/FeJLe-a/a Fly, and an agouti, C3FeB6-A/AW~~ Fit (C3HeB/FeJ x C57BL/6J-AW-~/Aw-~F~.

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160 IMMUNODEFICIENT RODENTS The type of cross used to transfer a mutation onto a hybrid background depends on whether the mutation is dominant, recessive, or recessive and lethal or sterile. Backcross Matings Backcross matings to the hybrid can be made with a dominant gene just as they are made to an inbred strain. Dominant hemimelia (Dh) is maintained in this manner. Cross-Intercross Matings . . . ~ Cross-intercross matings can be made with recessive genes that are either homozygous or heterozygous to any hybrid in the same manner that a re- cessive gene is transferred to a standard inbred strain. Lethargic (Ih) is maintained on the B6C3-a/A Fit (C57BL/6J x C3H/HeSnJ Fly background . . . in this manner. Cross-Intercross Matings Using Ovarian Transplantation Cross-intercross matings using ovarian transplantation can be accom- plished using the compatible sets of hybrids just described. For example, the mutation osteopetrosis (op.) is recessive, and the homozygote does not breed. It is maintained by crossing to the nonagouti, B6C3Fe-a/a Fit hybrid via ovarian transplantation and intercrossing those progeny. However, before ovarian transplantation can be done, the mutant gene must first be transferred to this hybrid background by at least seven crosses (N7) using a heterozygote (opl + ). Once the oplop mouse is compatible with the B6C3Fe-a/a F1 back- ground, an a/a oplop ovary is transplanted into the ovarian capsule of the C3FeB6-A/AW~7 Fit female. This host is crossed to a B6C3Fe-a/a Fit male, and all nonagouti (a/a) offspring will be opl + and can be intercrossed. Any agouti offspring are from host tissue and can be discarded. This intercross is expected to produce one out of four affected (oplop) offspring, out of which a female that is a/a oplop is again selected for an ovarian transplant. Each cross to the B6C3Fe-a/a Fit male is another N generation. Osteopetrosis (op.), osteosclerosis (oc), and wasted (wst) are maintained in this manner. Because of the hybrid vigor, the mutants are generally husky and the breeders have large litters; therefore, many heterozygotes can be produced in a very limited space. This is the most efficient method of maintaining lethal and sterile mutations that cannot be successfully inbred.