Immunology Today, rot. 3, No. 8, 1982

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H o w pure are inbred strains of mice ? Donald W. Bailey The Jackson Laboratory, Bar Harbor, ME 04609, U.S.A.

As usejhI as immunologists find the inbred mouse, they cannot help but become disenchanted when they encounter genetically disparate mice in a purportedly inbred strain. Genetic impurity ( allogenicity) can arise from a number of sources: incomplete inbreeding, mutation, inadvertent outcrossing, mislabeling, and epistatic and heterozygote selection. In this article D. W. Bailey discusses these vitiating sources and the appropriateprecautions against them. Immunology was the first field of biology to exploit the genetic properties of the inbred mouse a n d in so doing came up with a major discovery. T h e muhiple-gene model that defines the genetic basis for the rejection and survival of tissue transplants was formulated by Little a n d Tyzzer 1 from data on crosses involving the J a p a n e s e Waltzing Mouse, an inadvertently inbred strain. T h a t model holds with validity to this date, as demonstrated so well by Snell 2, Moreover, the immunological basis of transplant rejection, as it was conceived at that time 3, was not far from our understanding of it today. However, outside of a few tumor transplant studies immunologists seemed to have parted company with the inbred mouse for about 30 years. Needless to say, the inbred mouse has since gained great popularity in immunological research due to the currently strong interests in identifying and studying the functions of tissue alloantigens a n d i m m u n e response genes; for these studies a homogeneous genome is obligatory. Most of our trust in the properties of the inbred mouse comes from our personal experiences or from published observations on the properties of inbred strains in experiments, and those observations are reinforced by well-developed genetic theory. This trust remains until the genetically exceptional mouse is encountered. T h e n disappointment and disenchantment ensue. The inbred mouse genome may be genetically homogeneous on paper, but how homogeneous is it in reality? Without sequencing the nucleotides of the D N A no one can know for sure. However, one can get a fair idea by typing a sample of loci that are commonly polymorphic in laboratory strains. Actually such sampling occurs routinely in m a n y research laboratories where typing of such loci is done in experiments. Such allogenicity indeed has been encountered as genes segregating either within an inbred strain, or as allelic differences existing between substrains of a n inbred strain. I m m u n o l o g i s t s seem to have provided more examples of unexpected genetic variants t h a n have' scientists in other fields, perhaps because the traits they investigate are usually at the molecular level and their techniques are more sensitive to small alterations © Elsevier Biomedical Piess 1982 0167-4919/82/0{)00-0000/$1 0(I

in molecular structure. T h e r e are m a n y examples of surprise encounters. Histocompatibility variants have been found in the strains: A, AKR, BALB/c, CBA, C57BL, DBA, W G 4"15. O t h e r m e m b r a n e alloantigenic v a r i a n t s have been found in the strains: A, AKR, BALB/c, CBA, C3H, C57BL, a n d C57L 16"2°. Variant genes affecting i m m u n e response have been found in the strains: C3H and C57BL 2>24. Encounters with genetically exceptional mice will become more frequent as more a n d more investigations depend on genetically defined mice and as the n u m b e r of m o u s e colonies, a n d m o u s e strains proliferate, and generations of m a i n t e n a n c e and inbreeding grow. I d y l l i c effects of i n b r e e d i n g T h r o u g h o u t this article I shall use the term isogenic in describing the genotypic status of a locus at which all individuals of an inbreeding generation are homozygous for the same allele (by descent). The allele has become genetically fixed a n d thereby invariable except by mutation. Isogenicity is not the same as homozygosity, in which case the status for the locus is that all individuals are homozygous, but not necessarily homozygous for the same allele. I shall use the term allogenic in describing the status of all loci that are not isogenic. Moreover, I shall express the degree of allogenicity of the genome in terms of the proportion of allogenic loci that existed in the foundation population (.35), which is then translated into the n u m b e r of allogenic protein-coding loci (.35 x 30,000 = 10,500 loci). T h e arguments for this conversion are presented elsewhere2L This is to enable us to compare the relative importance of various sources of allogenicity on a c o m m o n basis and to judge the relative probabilities of encountering allelic differences at the loci in which each of us might be interested: I shall consider only the protein-coding loci, but it should be remembered that the DNA of these genes consists only of a little more t h a n 1% of the total cellular DNA. According to theory, inbreeding will reduce genetic variability (allogenicity) asymptotically to zero. Therefore, mice taken from the strain early in the inbreeding regimen are expected still to carry some allogenic loci, the n u m b e r of which is reduced each

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Fig. 1. The number of allogenic loci remaining after successive generations of full-sib mating. Curve A represents the allogcnicity expected according to theory; curve B, the allogenicity if one locus must remain heterozygous; and curve C, the mutational load, the floor which allogenicity approaches asymptotically. succeeding generation of full-sib mating. This effect is illustrated by curve A in Fig. 1, although instead of zero, the asymptote depicted is the mutational load to be discussed later. At generation F20, the arbitrary generation when a mouse strain can be pronounced 'inbred', the n u m b e r of allogenic loci remaining is estimated from curve A to be nearly 273 out of a postulated 10,500 polymorphic protein-coding loci originally allogenic in the base population 25. The likelihood of one of these loci being of specific interest to the investigator is small (2.6°70). However, if he has an interest in more t h a n one of them, the probability increases dramatically. For example, if he is interested in the block of histocompatibility (H) genes consisting of 30 loci 26, assumed to be unlinked inter se a n d allogenic in the original non-inbred population, then the probability of encountering at least one H gene segregating in an inbred strain at F20 would be: 1 - (1 - 0 . 0 2 6 ) 30 = 0 . 5 5 or a little better t h a n half of a collection of independently derived inbred strains would be carrying at least one segregating H gene in this generation.

Heterozygote selection Heterozygotes can have a reproductive advantage over homozygotes and thereby provide the more successful matings and slow down the progress of inbreeding. Because of this, curve A in Fig. 1 can be very misleading if followed too rigorously. T o illustrate the effect of one type of heterozygote selection, I have assumed that at one specific locus it is necessary that in each generation one of the parents must be heterozygous and the other must be homozygous for one of the alleles, a situation considered by Fisher 27. (The n u m b e r of such critical loci can not be m u c h greater t h a n one, or the incipient strain would soon become extinct due to the scarcity of fertile indivi-

duals.) The chromosomal segment bearing the locus in question a n d other linked loci will remain allogenic over a longer period of time t h a n the rest of the genome and will slow down the achievement of isogenicity as indicated in curve B of Fig. l. About 40 more genes remain segregating during the generations shown t h a n in the case without such selection. T h e effect of selection can be even more devastating .if during the development of a n inbred strain a n u m b e r of sublines have been formed. In such cases it is the practice to select the healthier, better breeding line for perpetuating the strain, a n d these are apt to be more heterozygous. T h e effect of this sort of subline selection can counter the effects of inbreeding progress much more than that from heterozygote selection within a single line, a subject discussed by Falconer 2s. A real life example of the trials a n d tribulations engendered by heterozygote selection during the inbreeding of the rabbit has been described by Chai 29.

Mutation Even if inbreeding were allowed to proceed to a generation when expected residual allogenicity is nil (say beyond F40) a n d if no heterozygote selection has occurred, the inbred strain would still be expected to show allogenicity due to mutation. M u t a t i o n is an ever recurring event in all strains; entropy is inescapable. No strain or substrain can be considered free of it, and therefore no substrain can truly be used as a s t a n d a r d genotype for the strain. W i t h i n an u n b r a n c h i n g inbred strain, or a single substrain, the n u m b e r of newly mutated proteincoded genes that accumulate to the point where the mutation and genetic-fixation rates are in equilibrium, is approximately four at any given generation 2~. This mutational load is represented by curve C in Fig. 1. Thus, residual allogenicity does not asymptotically approach zero as inbreeding theory would have it, but rather approaches the floor of mutational load. Curves A and B in Fig. 1 have been drawn accordingly. Inbreeding theoretically would reduce residual allogenicity to an amount equivalent to the mutational load by generation F40, as depicted in Fig. 1. Contamination The most insidious and devastating source of allogenicity can be the c o n t a m i n a t i o n that has occurred unbeknown to the investigator. T h e resulting allogenicity can vary in degree from a single gene (as a cross or substitution of a co-isogenic strain with its partner strain), to perhaps full allogenicity of the species (as a cross or substitution of two unrelated strains). Errors can arise at all levels: while transferring mice between pens, in labelling cage tags or when making entries in mating records; it can occur in the animal room, in the laboratory or' in transit to a new laboratory. The mere dropping of a substrain symbol from the strain designation can result in significant 'contamination'. M a n y researchers still refer to the

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Fig. 2. The number of fixed gene differences that are expected to develop between substralns of an inbred strain. Curve A represents the level reached if the substrains were separated at F20; curve B, at F25; curve C, at F30; curve D, at F35; and curve E the accumulation of fixed mutated-gene differences which could be added to each of the above curves to give a combined effect of the two sources, mutation and residual allogenieity.

' C 5 7 B L ' or to the ' B A L B / c ' strain in spite of the published differences between substrains as listed above. C o n t a m i n a t i o n can be avoided. T h e consequences of wasted research make precautionary measures well worthwhile, but few researchers adopt them. A few measures would be: to check the reliability of the source of breeding stock, to hire knowledgeable lab technicians, to conduct routine genetic tests (e.g. skin graft tests), a n d to label strain and substrain designations fully. Genetic drifting of substrains The sources of allogenicity that are effective within strains or substrains can also contribute to differences between substrains of an inbred strain. Differences occurring between substrains are probably more important t h a n those occurring within substrains, for the'latter, m u c h like that caused by poorly controlled environmental conditions 3°, will tend only to add 'noise' (error variance) to an experiment and perhaps therein waste the effort of the one laboratory, whereas substrain differences can cause two or more laboratories to come to contradictory conclusions a n d waste the efforts of those laboratories a n d many others in reconciling the source of the conflicting results. O n the other hand, once a substrain difference is identified, it can become quite useful in its own right. It is likely to contribute to the very scientific field with which it originally interfered, as has been the case for m a n y of the encountered differences listed above. T h e n u m b e r of differences expected to arise from the differential fixation of genes that were left segregating (residual allogenicity) at the time of sub-

strain separation have been calculated a n d illustrated in Fig. 2. They have been treated in greater detail elsewhere 4,25. For this example, I have considered two substrains separated at generation F20, F25, F30 or F35, then inbred for an additional 30 generations, or so, in order that all such loci have become fixed. From curve A in Fig. 2 it is seen that substrains separated at F20 will eventually differ in regard to fixed alleles at 117 loci. However, substrains derived from the more established strains will not be so troubled, and the more recently separated substrains will contain very little of this component. However, new mutations will contribute fixed differences between substrains at a rate proportionate to the total n u m b e r of generations each substrain has been independently maintained 2s. T h e numbers of gene differences expected to arise by m u t a t i o n are illustrated by curve E in Fig. 2. Fixed mutated gene differences between two substrains would be expected to have accumulated in 80 years (i.e. about 200 generations maintenance for each substrain), to a n u m b e r comparable to that which would have originated from residual allogenicity if the two substrains had separated at generation F20. This is illustrated in Fig. 2 by the junction of curves A and E at full-sib generation 200. Congenic strains These strains are even more misunderstood and with potential misuse t h a n are other inbred strains. Congenic strains are constructed by introducing a foreign marker (differential) gene into a n inbred strain genome by repeated crossing of the marker gene carrier, which is selected each generation, back to a m e m b e r of the inbred (background) strain. Thus, congenic strains are subject not only to the same vitiating forces confronting other inbred strains but additional ones generated by the process of incorporating the foreign chromosomal segment. In the process of constructing a congenic strain, the differential gene does not come alone; both linked and unlinked genes are introduced in the initial cross. According to theory, the unlinked genes are reduced each succeeding backcross generation by a doubling dilution while linked genes are lost more slowly. Leslie 3~ has presented the theoretical mathematical basis for calculating the linked a n d unlinked allogenicity expected after repeated backcrossing. Unlinked genes

Backcrossing is equivalent to making doubling dilutions of the foreign genome for all but the chromosomal segment carrying the differential marker gene. Thus, the expected reduction in allogenicity of untinked genes ideally would follow the doubling dilution curve A in Fig. 3. Only 10 unlinked polymorphic structural genes are expected to be present above the mutational load floor after 10 backcross generations and less t h a n 1 after 15 generations as based on

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Immunology Today, vol. 3, No. 8, 1982

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A chromosomal segment containing the differential marker gene grows shorter each backcross generation. T h e length can be estimated as 2 0 0 / n centimorgans (cM), where n is the n u m b e r of generations of crossing back to the background strain beyond the F131. The allogenicity that comes along in the chromosomal segment bearing the marker gene is rather large and is illustrated in Fig. 3 by the increment that curve B has over curve A. T h e segment length is expected to vary considerably from the theoretical m e a n due to the r a n d o m determination of the points of recombination. Therefore, the n u m b e r of differing passenger genes can be much greater t h a n the theoretical m e a n n u m b e r as illustrated by the upper 95°70 confidence limits of the estimate, curve C in Fig. 3, as calculated by the equations developed by J o h n s o n 32. This emphasizes the potential allogenicity that can still be present in a congenic strain merely by chance alone. Congenic strains are subject to genetic drift like other substrains. Thus, it would be p r u d e n t for the holder of a congenic strain periodically (say every 10 years) to ]~urge it of accumulated fixed mutational differences by a series of backcrosses to the inbred background (partner) strain.

C o n c o m i t a n t counter s e l e c t i o n W h e n the marker-bearing segment is selected during congenic strain construction, other genes that interact epistatically with the differential marker gene or with any of the passenger genes will also be selected if the interaction results in a more viable or reproductively more fit individual. There are two serious consequences. T h e effect of the epistatic gene will likely be mistakenly attributed to the differential marker gene, a n d the attainment of reduced allogenicity will be retarded. For an example, I take the case where the foreign genes at one locus, unlinked to the marker, are necessary for viability a n d continuation of the strain. The retardation in reducing allogenicity is illustrated by curve D in Fig. 3. After 10 generations (often taken as the generation when the strain can be termed 'congenic') there would be 287 genes still segregating within such a strain compared with the 10 expected by theory. Out of 10,500 allogenic loci at the start 2s, the chance of one of these 287 being the one the researcher is interested in is 2.7%; but if he is interested in a block of them, say 16, then the probability of encounter goes up considerably: 1 - (1 - 0 . 0 2 7 ) 16= 0 . 3 5 C o n c l u d i n g remarks With all of the possible sources of genetic heterogeneity, it is surprising that inbred strains can still in

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Fig. 3. The number of allogenic loci theoretically expected to be remaining after successive generations of crossing back to an inbred strain, the procedure used to construct a congenic strain. Curve A represents the number of allogenic loci unlinked to the differential marker locus. Curve B represents the allogenic loci, both linked and unlinked, expected to remain during the construction of a congenic strain for which a marker has been selected. Curve C represents the upper 95% confidence limit for the number of allogenic loci carried in the congenic strain, curve D represents the number of allogenic loci remaining if a second gene, unlinked to the marker, is carried along because it interacts epistatically with the marker to provide a trait essential for reproductive fitness, and curve E represents the mutational load. practice be accepted as being genetically homogeneous. As a matter of fact, only the allogenicity originating from m u t a t i o n is inescapable, although even that can likely be reduced by employing frozenembryo storage 25. Such storage is expected to reduce genetic drift essentially by reducing the n u m b e r of generations per year. The allogenicity originating from heterozygote selection, a n d epistatic-gene selection, when they are present, as well as allogenicity of the introduced segment in congenic strains, can be reduced to insignificant amounts over a long time span by a continued, persistent full-sib mating or backcrossing regimen 28. The investigator can avoid the other sources. To do so the investigator should: (1) whenever practicable, obtain mice from a reliable breeding source in lieu of maintaining a separate subline; (2) avoid comparing results from substrains that arose early in the strains inbreeding regimen or that have been long separated; (3) be wary of using inbred strains that have not yet reached F30, or preferably F40, a n d of using congenic strains that have not yet reached their 15th, or preferably 20th backcross generation; (4) take precautions (listed above) against contamination a n d mislabeling; a n d (5) use substrain symbols in all publications a n d correspondence. Impurity of inbred strains cannot be completely eliminated but it can be reduced to levels that make

lmmunology Today, vol. 3, Nv. 8, 1982

214 e n c o u n t e r s of genetically e x c e p t i o n a l m i c e e x t r e m e l y unlikely.

References 1 Little, C. C. and Tyzzer, E. E. (1916) J. Med. Res. 33,393 2 Snell, G. D. (1978) in Origins oflnbred Mice (H. C. Morse III, ed.) p. 119, Academic Press, New York. 3 Tyzzer, E. E. (1916) J. CancerRes. 1,125 4 Bailey, D. W. (1977) in The Freeziug of Mammalian Embryos, Ciba Foundation Symposium 52 (Elliott, R. and Whelan, J., eds) pp. 291-299, Elsevier/North Holland Amsterdam 5 Billingham, R. E., Brent, L., Medawar, P. B. and Sparrow, E. M. (1954) ProcRoy. Soc. London Ser. B 143, 43 6 Bittner, J. J. (1930) Pap. Mich. Acad. Sci 11,349 7 Dux, A., Corduvener, D. and Muhlbock, O. (1971) in Irrzmunogenetics of the H-2 System (Lengerova, A. and Vojtiskova, M., eds) pp. 163-165, Karger, Basel 8 Egorov, I. K. and Blandova, Z. K. (1968) Genetika 4 (12), 63 9 Graft, R. J., Valeriote, F. and Medoff, G. (1973) J. Natl Cancer Inst. 55, 1015 10 Kindred, B. (1963) Aust. J. Biol. Sci. 16,863-868 11 Liebelt, A. G. (1978) in Origins of lnbred Mice (H. C. Morse IIi, ed.) p. 475, Academic Press, New York 12 Linder, A. E. A. (1963) Transplantation 1, 58-60 13 Silvers, W. K. and Gasser, D. L. (1973) Genetics75,671 14 Snell, G. D. (1960)J. Nail CancerInst. 25, 1191 15 Zatz, M. M. (1978) in Origins oflnbred Mice (H. C. Morse llI, ed.) p. 467, Academic Press, New York 16 Acton, R. T., Blankenhorn, E. P., Douglas, T. C., Owen,

R. D., Hilgers, J., Hoffman, H. A. and Boyse, E. A. (1973) Nature New Biol. 245, 8-11 17 Cherry, M., Bailey, D. W. and Snell, G. D. (1978) in Origins of Inbred Mice (H. C. Morse III, ed.) p. 481, Academic Press, New York 18 Flaherty, L. (1978) in Origins of Inbred Mice (H. C. Morse lII, ed.) p. 409, Academic Press, New York 19 Morse, H. C. llI (1978) in Orzgins of Inbred Mice (H. C. Morse lII, ed.) p. 441, Academic Press, New York 20 Mosier, D. E. (1978) in Origins of Inbred Mice (H. C. Morse llI, ed.) p. 471, Academic Press, New York 21 Maurer, P. H., Merryman, C. F. and Jones, J. (1974) lrnmunogeneti~' 1,398 22 Rosenstreich, D. L. (1978) in Origins of Inbred Mice (H. C. Morse III, ed.) p. 457, Academic Press, New York 23 Tagliabue, A., Ruco, L., McCoy, J. L., Meltzer, M. S. and Herberman, R. B. in Origins of Inbred Mice (H. C. Morse III, ed.) p. 461, Academic Press, New York 24 Taniguchi, M., Tada, T. and Tohuhisa, T. (1976) J. Exp. Med. 144, 20 25 Bailey, D. W. (1978) in Origins of Inbred Mice (H. C. Morse III, ed.) p. 197, Academic Press, New York 26 Bailey, D. W. and Mobraaten, L. E. (1969) Transplantation 7, 394 27 Fisher, R. A. (1949) The Theory of Inbreeding, Oliver and Boyd, Edinburgh 28 Falconer, D. S. (1960) Introduction to Quantitative Genetics, Oliver and Boyd, Edinburgh 29 Chai, C. K. (1969) J. IIered. 60, 64 30 Riley, V. (1981)Science212, i100 31 Leslie, J. F. (1981) Genet. Res. 37, 239 32 Johnson, L. L. (1981) J. Hered. 72, 27

The molecular mechanisms of the immunoglobulin class switch Tasuku Honjo Department of Genetics, Osaka University, Medical School, Osaka 530, Japan. R e c e n t m o l e c u l a r genetic s t u d i e s have b e g u n t h e e l u c i d a t i o n of several i n t e r e s t i n g i m m u n o b i o l o g i c a l p h e n o m e n a s u c h as variable region diversity, allelic e x c l u s i o n a n d t h e class s w i t c h ~ . T h e m o l e c u l a r m e c h a n i s m of t h e class switch h a s i n t e r e s t e d developm e n t a l biologists as well as i m m u n o l o g i s t s b e c a u s e one VI1 region a n d different CH regions are e x p r e s s e d as a single p o l y p e p t i d e c h a i n in t h e lineage of a given l y m p h o c y t e - a n a p p a r e n t c o n t r a d i c t i o n of t h e welle s t a b l i s h e d fact t h a t a single p o l y p e p t i d e c h a i n is e n c o d e d by a single gene. Deletion model Specific CH g e n e s are deleted in m o u s e m y e l o m a D N A s p r o d u c i n g different i m m u n o g l o b u l i n classes 6. T h e deletion profile first e s t a b l i s h e d w a s c o n s i s t e n t w i t h t h e C u gene o r d e r 5'--VH c l u s t e r - C , - C v 3 Cvt-Cy2b-Cv2a-Ca-3'. It was a s s u m e d t h a t t h e e x p r e s sion of a p a r t i c u l a r CH gene w a s a c c o m p a n i e d by t h e deletion of a n i n t e r v e n i n g D N A s e g m e n t b e t w e e n a V H a n d t h e CH gene. T h e deletion of CH genes a n d t h e © glscvicr Biomcdical Prcss 1982 0167 4919/82/0000-0000/$1.00

gene o r d e r were later c o n f i r m e d by e x t e n s i v e S o u t h e r n blot a n a l y s e s u s i n g cloned p r o b e s 7 12. Several g r o u p s have isolated h e a v y c h a i n g e n e s from g e n o m i c libraries of m y e l o m a a n d e m b r y o n i c (or n o n l y m p h a t i c cell) D N A s , a n d c o m p a r e d t h e s t r u c t u r e of t h e e x p r e s s e d a n d g e r m l i n e H c h a i n genes. S u c h s t u d i e s 13-15 have lead to t h e p r o p o s a l t h a t a c o m p l e t e H c h a i n gene is c r e a t e d by at least t w o t y p e s of D N A r e a r r a n g e m e n t as i l l u s t r a t e d in Fig. 1. T h e first, V - D - J r e c o m b i n a t i o n , .joins t h e t h r e e s e p a r a t e D N A s e g m e n t s VH, D a n d JH e a c h e n c o d i n g a p a r t of a V region, into one piece, r e s u l t i n g in t h e e x p r e s s i o n of t h e c o m p l e t e d VH gene as a g c h a i n b e c a u s e t h e C , gene is closest to t h e J u s e g m e n t . T h e s e c o n d D N A rea r r a n g e m e n t , S - S r e c o m b i n a t i o n , t a k e s place bet w e e n S regions w h i c h are located at t h e 5' side of e a c h CH gene. T h e S - S r e c o m b i n a t i o n b r i n g s t h e c o m pleted VH gene a d j a c e n t to a n o t h e r CH g e n e by t h e deletion of a n i n t e r v e n i n g D N A s e g m e n t . T h e m o d e l h a s p r e d i c t e d several s t r u c t u r a l c h a r a c teristics of t h e Igh loci: (1) T h e o r d e r of t h e CH gene is

How pure are inbred strains of mice?

As useful as immunologists find the inbred mouse, they cannot help but become disenchanted when they encounter genetically disparate mice in a purport...
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