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2005 Nature Publishing Group http://www.nature.com/naturegenetics

The origins and uses of mouse outbred stocks


Ruth Chia1, Francesca Achilli1, Michael F W Festing2 & Elizabeth M C Fisher1
Outbred mouse stocks, often used in genetics, toxicology and pharmacology research, have been generated in rather haphazard ways. Understanding the characteristics of these stocks and their advantages and disadvantages is important for experimental design. In many studies these mice are used inappropriately, wasting animals lives and resources on suboptimal experiments. Recently, however, researchers from the field of complex trait analysis have capitalized on the genetics of outbred stocks to refine the identification of quantitative trait loci. Here we assess the most widely used outbred stocks of mice and present guidelines for their use. Outbred stocks of rats and mice are commonly used in academic and industrial research. We estimate that from January 2002 to July 2005 33% of all mouse papers and 85% of all rat papers in the PubMed bibliographic database involved outbred stocks. These percentages do not take into account the unpublished commercial studies that use these stocks. Because the potential and limitations of rodent outbred stocks (outbred colonies are usually referred to as stocks, whereas inbred ones are referred to as strains or lines) are often not recognized by the researchers who use them, here, in a companion piece to our paper on the genealogies of mouse inbred strains1, we describe the origins and characteristics of some mouse outbred stocks, and when it is appropriate to use them. Whats in a name? The international standardized nomenclature for outbred stocks of both mice and rats2 takes the following form: the company or laboratory designation followed by a colon, the stock designation, and then a hyphen followed by the mutation designation (if a mutation is known to be present). For example, Hsd:NIHS refers to Harlan (Hsd) National Institutes of Health (NIH) Swiss outbred mice (http://www.harlan. com/); Crl:CF-1-Abcb1amds is the Charles River Laboratories (Crl) CF-1 outbred mouse stock carrying a mutation in the Abcb1a transporter called multiple drug sensitive (http://www.criver.com); and Tac:SW is the Taconic (Tac) Animal Models, Swiss Webster outbred mouse stock (http://www.taconic.com). This nomenclature has replaced an older system used only in the US (and still in use) that includes the remote ancestry, which is thought not to be of great importance in the international rules, and broad disease status. For example, Crl:CFW(SW)BR
1Department of Neurodegenerative Disease, Institute of Neurology, Queen Square, London WC1N 3BG, UK. 2c/o MRC Toxicology Unit, Hodgkin Building, University of Leicester, PO Box 138, Lancaster Road, Leicester LE1 9HN, UK. Correspondence should be addressed to E.M.C.F. (e.fisher@prion.ucl.ac.uk).

is a stock of Carworth Farms Swiss Webster (CFW) mice with Swiss ancestry (SW), sold by Charles River Laboratories and maintained in barrier conditions (BR). Outbred stock nomenclature guidelines are available in the Mouse Genome Informatics database (http://www. informatics.jax.org/nomen/strains.shtml). Because the names of the stocks are often given trademarks by individual companies, the name of the same stock can change when it is sold by a different company. Thus, the LACA outbred stock is a renamed version of CFW (Table 1). As a result, there is often no substantial difference between stocks with different names from distinct companies3,4. Conversely, because of inbreeding, genetic drift, directional selection and genetic contamination, stocks with the same name from different breeders35, or even distinct cohorts from the same breeder6, may vary genetically. Sometimes, the same designation is used both for an outbred stock and for an inbred strain derived from it (e.g., ABH, ABL, NMRI and NIH; Table 1 and ref. 1). In these situations, it is impossible to repeat experiments with the same mice supplied by different breeders. The Institute for Laboratory Animal Research has a tool on its website (http://dels.nas.edu/ilar_n/ilarhome/) that searches the websites of suppliers of laboratory animals for named strains and stocks. It also lists separately the websites of most suppliers (albeit with a strong emphasis on the US). The International Mouse Strain Resource (http://www. imsr.org/) is a dynamic and current database of strains and stocks that are available worldwide and has mandatory updates from contributing repositories7. Currently, 19 repositories worldwide contribute strain listings to this resource, and this number continues to grow (J. Eppig, personal communication). Breeding outbred stocks Like inbred mouse strains, outbred mouse stocks have an official definition: a closed population (for at least four generations) of genetically variable animals that is bred to maintain maximum heterozygosity2. The goal in maintaining an outbred stock is usually to minimize genetic change. The coefficient of inbreeding, F (the probability that two alleles at a locus are identical by descent8), should increase by less than 1% per generation9. With random mating, the increase in F per generation is given by the formula F = 1/8Nm + 1/8Nf, where F is the increase in F, Nm is the number of males and Nf is the number of females per generation8. According to this formula, the increase in F per generation will be less than 1% provided that the colony is maintained by at least 25 breeding pairs. The rate of inbreeding depends on the numbers of the least numerous sex. The rate of inbreeding per generation can be halved by ensuring that each breeding female contributes one female and each breeding male contributes one male to the next generation8. In practice, this can be achieved by using a rotational breeding system8,1012.

Published online 27 October 2005; doi:10.1038/ng1665

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Table 1 Mouse outbred stocks
Symbol
ABH ABL AP CD-1 CF-1

Names, origins and current status


Biozzi mousehigh responder. Selected from a Swiss population for the production of hemagglutinins, the H stock being high producers57. Also susceptible to experimentally induced allergic encephalomyelitis47,58. Biozzi mouselow responder. Selected from a Swiss population for the production of hemagglutinins, the L stock being low responders57. Has a markedly lower susceptibility to experimentally induced allergic encephalomyelitis than has ABH47,58. An outbred stock maintained at Alderley Park, Cheshire, UK, by AstraZeneca. Possibly of Swiss outbred stock origin59. Derived from Lynchs Swiss mice, by Roswell Park Memorial Institute, Buffalo, New York, and the Institute of Cancer Research, Fox Chase, Philadelphia, Pennsylvania, by Charles River Laboratories, USA, in 1959 (ref. 16). Carworth Farms No. 1. Obtained by Carworth Farms, UK, from a Missouri laboratory and thought not be descended from Swiss mice. Founded in 1935 (ref. 60) and inbred by Carworth Farms for more than 20 generations, before being reduced to a single pair from which progeny were outbred to make a new CF-1 stock, which went to Charles River Laboratories in 1974. CF-1 maintained by Carworth Farms (subsequently called Anglia Laboratory Animals Limited). Stock name was changed to CFLP by Lane-Petter; this company later went out of business. Carworth Farms White. Swiss Webster stock from the Rockefeller Institute, New York. Inbred for 21 generations by Carnochan and outbred as a closed colony from then on; the stock went to Charles River Laboratories in 1974. General Purpose stock. Derived from Swiss mice from the Rockefeller Institute and given to the NIH in 1935 (ref. 16). Heterogeneous stock currently at G24. Maintained to maximize heterozygosity and created from four different inbred lines: BALB/c, C57BL/6, DBA/2 and LP (R. Hitzemann, personal communication). Heterogeneous Stock Collaborative Cross, currently at G4. Maintained to maximize heterozygosity and created from eight different inbred lines: 129Sv/Im, A, C57BL/6, CAST/Ei, NOD/Lt, NZO/HILt, PWK/Ph and WSB/Ei (R. Hitzemann, personal communication). Center for Developmental and Health Genetics. HSIBG stock (see below) was imported from G55 to Pennsylvania State University, University Park, Pennsylvania, in 1996 and rederived into a barrier facility; currently held at G73 (Supplementary Note online and G. McClearn, personal communication). Boulder Heterogeneous Stock Institute for Behavioral Genetics. An outbred stock created at the Behavioral Genetics Laboratory at the University of California, Berkeley, and transferred to the Institute for Behavioral Genetics at the University of Colorado, Boulder (ref. 50 and J. Stitzel, personal communication); maintained to maximize heterozygosity and created from eight different inbred lines maintained at the Cancer Research Genetics Laboratory, University of California, Berkeley: A/J, AKR, BALB/c, C3H, C57BL/6, DBA/2, Is/Bi and RIII. Northport Heterogeneous Stock. An outbred stock maintained to maximize heterozygosity and created from eight different inbred lines: A/J, AKR/J, BALB/cJ, C3H/HeJ, C57BL/6J, CBA/J, DBA/2J and LP/J at the Northport Veterans Affairs Medical Center, New York43. Institute of Cancer Research. Produced in 1947 by Hauschka at the Institute of Cancer Research, Fox Chase. Derived from Swiss mice of the Rockefeller Institute and now widely distributed (Taconic and refs. 16,61). Originally from outbred ddN mice of the Central Laboratory of Experimental Animals, Kawasaki, Japan, in 1960. Selectively bred for regular estrus cycles (Jackson Laboratory)62 and now also inbred1. Laboratory Animal Centre Albino. Derived from a Swiss Webster stock with 20 generations of brother-sister mating at the Rockefeller Institute; went first to Carworth Farms in 1937, where it became CFW (see above), and then to the Laboratory Animals Centre, Carshalton, Surrey, UK, where the name was changed to LACA in the 1970s. Long sleep time (also see SS below). Selected with avoidance of inbreeding from the Boulder Heterogeneous Stock for long sleep time under alcohol anesthetic46. The LACA stock (renamed CFW) was sent to OLAC (Oxfordshire Laboratory Animal Colonies, UK) before 1970, where it was crossed with CS-1 mice (possibly a renamed CF-1 stock) from Scientific Products Farm, Manston, Kent, UK63. Mouse Outbred Roche Only. An albino stock bred by Roche in Basel and then transferred to Fllinsdorf in 1959. Originally a Swiss mouse strain (M. Josten, personal communication). Descended from Swiss Webster stock rederived by the University of Notre Dame, Indiana (Harlan). NIH Swiss. Derived from an NIH GP colony (see GP above) in 1936. From 1936 to the 1960s, the stock was bred by a multiple branched inbreeding system described as brother-sister mating without any systematic plan16. In the early 1960s, a strict random mating system was adopted and an SPF colony was rederived by hysterectomy and foster-nursing in 1972 (ref. 16). Naval Medical Research Institute. Originated from Lynchs Swiss stock and obtained at the NIH in 1937 by Poiley, who bred and named the stock NIH/PI. From Poiley it went to the NMRI, USA. Was subsequently randomly bred at the Zentralinstitut fr Versuchstierzucht, Hannover, Germany, and was also sent to the Danish mouse breeding company M&B A/S (now Taconic) in 1961 and 1985, where it is maintained as an outbred stock. Non Swiss Albino. Descended from animals bought from a breeder in the 1930s. Passed to Carworth Farms where it was renamed CF-1 (see above; Harlan). Originated in 1930 from a single pair of albino mice of unknown origin from a breeder in Albany, New York64. Maintained by the New York State Department of Health, Albany. The Nya:NYLAR mouse colony has been divided into two portions since 1962: a larger, outbred stock (Nya:NYLAR); and a smaller, inbred strain (NYLR/Nya; Harlan and ref. 65). Oncine France 1. Derived from the CF-1 outbred stock (see above) and introduced into France by Charles River Laboratories in 1966 (ref. 60). Australian Quackenbush outbred stock. Maintained as frozen embryos currently at University of Sydney, Australia. Originally bred at the Department of Veterinary Physiology, University of Sydney, in 1996 from Swiss outbred stock colonies that had high fecundity. Extremely robust mice that are larger than average Swiss mice5 (The University of New South Wales; J.D. Kirton, F.W. Nicholas, I.C.A. Martin & C. Moran, unpublished data; and Supplementary Note online). An Israeli outbred stock, bred by the Authority for Animal Facilities of the Hebrew University, Jerusalem, Israel. Currently maintained by Harlan Israel. Origins seem to be unknown (R. Kalman, personal communication). Sensitive to skin carcinogenesis. Derived by Boutwell and Baird in the 1960s and early 1970s by selective breeding of Rockland AP with CD-1 mice to produce progeny sensitive to papillomas induced by an initiation-promotion protocol. The most widely used skin tumorsensitive mouse. Several inbred strains are now available66,67. SKH1-hr is an uncharacterized hairless mouse stock acquired by Temple University, Pennsylvania, from a small commercial supplier in New York City. The stock went to Charles River Laboratories from the Skin and Cancer Hospital, Temple University, in 1986 (ref. 68). Short sleep. Selected for short sleeping time under alcohol anesthetic (see LS above). Swiss Webster. Different Swiss Webster outbred stocks are sold by different laboratory animal suppliers. All of these mice originated from the Swiss mice of Lynch, which were passed on to Webster in 1932. The different stocks are therefore related; for example, see Charles River Laboratories and Simonsen Laboratories Inc. Thylers original. Thought to be derived from Swiss Webster stock, possibly in the 1920s or 1930s (ref. 28). Thyler worked on infectious diseases at the Rockefeller Institute. TO also refers to an inbred strain69 and the outbred Tuck ordinary mouse (see below). Tuck ordinary from Trevor Tuck, mouse breeder, UK. Origin seems to be unknown.

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CFLP CFW GP HS4 HSCC HSCDHG HSIBG

HSNPT ICR IVCS LACA

LS MF1 MORO ND4 NIH Swiss NMRI

NSA NYLAR

OF1 QS

SABRA SENCAR SKH SS SW TO TO, TUCK

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How outbred is outbred? The degree of genetic heterogeneity in an outbred colony depends on the previous history of the stock and can range from almost zero, when it has been maintained in small numbers for many generations, to very extensive, when a stock has been outcrossed relatively recently. Several studies have been carried out in various outbred stocks to survey several loci and to determine the proportion that are polymorphic in each individual stock2,5,10,1317. For example, an assay of ten polymorphic loci in five different Australian outbred stocks of Swiss-derived mice (sampling 1015 mice from each stock) showed that the proportion of polymorphic loci ranged from 0.3 three of ten loci were polymorphic) to 0.8 with a mean of 0.52 in these stocks, and the average expected heterozygosities ranged from 0.08 to 0.37 with a mean of 0.21 (ref. 6); comparable figures have been found for three different American outbred stocks of Swissderived mice6,16. Genetic quality control, the extent to which an outbred colony changes over a period of time, is largely unaddressed but is an important issue. Currently, larger commercial breeders monitor at least some of their stocks with microsatellites and possibly other types of marker, but the methods that they use are often not published. Institutional colonies are largely uncharacterized. Some theoretical work on appropriate sample sizes and the number of loci that should be studied is urgently needed25,10,1316,1821. The genetic make-up of many colonies has been affected by a poor understanding of the breeding schemes for outbred stocks22 and by historical events such as directional selection and bottlenecks, which lead to reduced variation, and genetic drift, mutation and genetic contamination, which result in genomic differences over time in individual colonies. Directional selection can be minimized by choosing future breeding stocks strictly at random, without regard to their phenotypic characteristics. But the genetic characteristics of some outbred stocks have been affected by directional selection over many years. Laboratory animal technicians tend to cull small mice as runts and, as a result, mice from most outbred stocks usually weigh more at a given age than those from inbred strains. For example, the mean weight at 70 d of age in males in a collection of 17 inbred strains was found to average 25.4 g (range 20.030.8 g), whereas that in 3 outbred stocks was 34.7 g (range 31.737.7 g); similar differences were seen in females (M.F.W.F., unpublished data). This difference cannot be attributed solely to heterozygosity; the mean body weight at 9 weeks of age in males of seven mouse F1 hybrids was only 4.0 g greater than that of the inbred strains used to produced them (Jackson Laboratory Mouse Catalog 2001). In addition, there may be a positive genetic correlation between body weight and litter size. Thus, the CD-1 mouse stock is larger and tends to produce more pups per litter than any inbred mouse strain (Mouse Genome Informatics (http://www.jax.org/) and refs. 23,24). Both classical outbred stocks and inbred strains are much larger, more prolific and, in many other ways, genetically distinct from wild mice. Thus, they do not represent anything that exists in nature. An example of a population bottleneck is provided by the CFW outbred mouse stock (Table 1). This stock was reduced to a single pair and progeny were outbred from that point onwards (Charles River Laboratories); thus, there has been a bottleneck in the population and probably genetic drift from the original Swiss colony16. Genetic drift has been also seen in a CFLP outbred mouse stock. Over 6 years the frequency of the glucose phosphate isomerase isozyme a allele fell from 0.96 to 0.59, statistically a highly significant change, and mandible shape also changed, indicating that the stock had drifted over this period25, although the possibility that these changes are due to genetic contamination cannot be ruled out. Genetic drift is minimized by minimizing the rate of inbreeding. The chance of genetic contamination (as a result of inadvertent mixing with stock of a different strain) can be minimized by keeping stocks of similar phenotype physically isolated from one another, although such contamination is not infrequent3,4,9. Like human populations, outbred stocks can carry recessive mutations that may affect experimental results. For example, the percentage of mice that are homozygous with respect to retinal degeneration, a mutation that causes blindness from 6 weeks of age, was found to range from 0 to 98% in a study of eight outbred stocks26. This variation may affect behavioral responses, particularly because a stock may contain a mixture of homozygous, heterozygous and wild-type mice. Although the same mutation is found in some inbred strains, all mice in an inbred strain will be genetically identical and thus will be affected equally. Little can be done to minimize the occurrence of mutations, but these are relatively rare events. Given the widespread use of outbred stocks, it may be helpful for academic and commercial interests to develop a single database of these stocks that records their origins (and therefore their relatedness), degree of inbreeding and genotypic and phenotypic characteristics. Because of the labile nature of outbred stocks, however, the first step probably should be to characterize each colony genetically. Such a characterization would not be trivial because large sample sizes and many loci need to be studied to estimate the allele frequency at each polymorphic locus25,10,1316,1821. Only then would it be possible to assess the genetic similarity of different colonies with a measure such as Neis genetic distance27. (High-density sets of markers for genotyping several outbred stocks, including MF1 and ICR, are available; http://www.well.ox.ac.uk/mouse/INBREDS/ and R. Mott, personal communication.) Phenotypic information for many of the outbred stocks can be gathered from the literature, but because this information will not have been related to any defined genotype and because most phenotypes also depend on environmental factors, its reliability will be doubtful3,4, even if the mice were supplied by the same breeder6. Thus, it is mostly up to each user to characterize the stock that he or she plans to use without relying too much on existing background information. Outbred stock origins We collated the origins of the main outbred mouse stocks (Table 1) and the principal Swiss mouse genealogies (Supplementary Fig. 1 online); these listing are not comprehensive but include all the main outbred stocks that are currently in use. Many of the mouse outbred stocks are derived from nine mice that were imported to the US by Clara J. Lynch28, a cancer researcher with an interest in genetics who was working at the Rockefeller Institute for Medical Research (now Rockefeller University) in New York. In 1926, Lynch imported two male and seven female mice from the noninbred albino stock of Andr de Coulon of the Centre Anticancereux Romand in Lausanne, Switzerland, and all mice derived from these mice are known as Swiss mice from the nickname used by Lynch and her laboratory28. Coulon had previously maintained the colony at the Institute of Hygiene in Strasbourg, France, and derived it from an outbred colony from the Pasteur Institute in Paris, which was almost certainly bred from a group of mice bought from a dealer some time before 1920. Thus, Swiss mice are not known to be derived from any of the current inbred lines28 (although inbred strains such as FVB, SJL and SWR have been subsequently derived from Swiss outbred stocks1). Almost all outbred mice and rats used in biomedical research are albino. This characteristic may give many research workers the spurious impression that they are genetically uniform when in fact they are genetically heterogeneous. Outbred stocks, like inbred strains1, are often related to each other. Lynch sent her Swiss mice to other researchers both inside and outside the Rockefeller Institute. In 1932, one such researcher was Leslie Webster, who was carrying out experiments on susceptibility to viral infection and who passed on the mice to academic and commercial breeders including Carworth Farms, UK, which then produced the Swiss

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Figure 1 Comparison of the response to chloramphenicol in outbred and inbred mice. Shown is the reanalysis of a study of the effects of chloramphenicol given at five different doses and a control on eight hematological parameters (40 differences in total) in 47 CD-1 outbred mice (filled symbols) and 48 mice of four inbred strains (C57BL/6, BALB/c, C3H/He and CBA/Ca; open symbols)35. The response is the absolute difference between the mean value from the treated mice and that from the control mice in standard deviation units. Dashed line shows the number of differences likely to be observed with 25 mice per group in a two-sample t-test with a power of 90%, a significance level of 5% and a two-sided test assuming this mix of doses. Thus, for the CD-1 mice 10 comparisons would be judged to be statistically significant, whereas for the multistrain mice 24 comparisons would be significant. Ten significant comparisons in the multistrain study would be found with an effect size of 2.5 standard deviations, requiring a sample size of about five mice in each group (dotted lines). Thus, the multistrain study would give equivalent statistical power using one-fifth of the number of mice. The study with CD-1 mice did not detect any response in the white blood cell lineage, whereas such a response was seen in the multistrain study.

Effect size (standard deviations)

8 7 4 3 2 1 0 0 10 20 30 40

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Rank of effect

Webster outbred stock16,28. The CD-1 mouse outbred stock was first produced by Charles River Laboratories in 1959 (ref. 16). This stock originally came from researchers at the Roswell Park Memorial Institute, Buffalo, New York, who got them from a colony bred in the 1940s at the Institute of Cancer Research, Fox Chase, Philadelphia, Pennsylvania, that can be traced back directly to Websters Swiss mice from the Rockefeller Institute16. Thus, CD-1 and Swiss Webster outbred stocks are related but were separated in the 1930s. Research uses of outbred stocks Outbred stocks of mice and rats are widely used in toxicology, pharmacology and fundamental biomedical research, although in our opinion most such studies could be done much more effectively with inbred strains. Outbred stocks, however, have been used very successfully in genetic studies as a base population for selection to develop new or improved mouse models of human conditions and, recently, for genetic finemapping of quantitative trait loci (QTLs). They are also sometimes used in the development and maintenance of genetically modified mice, largely because they have a better breeding performance than inbred strains. Such use is best avoided, however, except as a last resort. The expression of most transgenes depends to some extent on the genetic background; thus, an undefined background that is subject to random genetic drift is not ideal. It is usually much better to backcross to an inbred genetic background before studying the effects of genetic modification in any detail. Most characters or outcomes of interest to toxicologists, pharmacologists and others doing fundamental or applied research have a polygenic mode of inheritance. For such characters, the phenotypic variance has both a genetic and an environmental component: VP = Vg + Ve, where Vp is the total phenotypic variance and Vg and Ve are the genetic and environmental variances, respectively. In an inbred strain there is no genetic variation because the mice are all identical, so Vg = 0 and VP = Ve; however, in an outbred stock there is genetic variation, so Vg > 0. Thus, the phenotypic variation is greater in an outbred stock than in an inbred strain, although the extent of this variation will depend on the heritability of the character and the degree of genetic variation in an individual colony8. Because statistical tests such as the Students t-test relate the mean difference between treatment groups to the within-group standard deviation, experiments using phenotypically variable outbred stocks either lack statistical power (i.e., they may fail to detect the effect of a treatment) or require larger sample sizes than do experiments using inbred strains. In a study of sleeping time under hexobarbital anesthetic, for example, the

mean sleeping time among five inbred strains ranged from 18 to 48 min with a standard deviation of 3.2 min averaged across the strains29. The mean sleeping times in two outbred stocks of 43 and 48 min were within the same range, but the standard deviation averaged across the two strains was 13.5 min. Toxicologists sometimes use sleeping time under barbiturate anesthetics to assess whether a chemical is influencing drug metabolizing enzymes. Suppose the aim was to plan an experiment to detect a 4-min difference in sleeping time between two groups of mice consisting of a control group with an assumed mean sleeping time of 40 min and a group treated with a test chemical. Power analysis30 can be used to determine the sample size required (a free sample size calculator is available at Biomath, http://www.biomath.info/). Assuming that the mean sleeping times are to be compared with a two-sample t-test, using a 5% significance level, a two-sided test and a power of 80%, this experiment could be done using 12 inbred mice per group or 180 outbred mice per group. Thus, it would require 15 times more outbred than inbred mice. Alternatively, suppose the investigator decided to use 20 mice per group; the power of the experiment to detect the 4-min difference can then be estimated with the same assumptions. With an inbred strain the chance of detecting that difference (i.e., the power of the experiment) would be 97%. With one of these outbred stocks, however, the power would only be 14%; therefore, it would not be worthwhile doing the experiment because it would have a very low chance of detecting any treatment effect. Although this may be an extreme example, because sleeping time is highly heritable, the increased phenotypic standard deviation in outbred mice tends to obscure the effects of treatments, leading to low-powered experiments. As general research animals, outbred stocks have many other disadvantages as outlined above: they are genetically labile, nothing is known about the genotype of individual mice, the extent of genetic variation in an individual stock is usually unknown, and genetic quality control methods have not been established. Research workers are often reluctant to switch to using inbred strains, however, because each strain consists of only a single genotype that may not be typical of the species. In a toxicity study, for example, the chosen inbred strain may be genetically resistant to the test chemical and thereby give misleading results. Unfortunately, this problem cannot be overcome by using outbred stocks because the genetic variation simply obscures the effect of the treatment. But it can be largely overcome without increasing the total number of mice that are studied by using more than one inbred strain and a factorial experimental design31. Instead of using 20 outbred mice in both the control and treated groups in an experiment, for example, it would be better to use 5 mice from each

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of four inbred strains in each group. This experimental design retains the statistical power of the inbred strains, shows whether the response is under genetic control by showing whether all strains respond in the same way, and obviates the problem of possible resistance associated with the use of a single inbred strain. Multistrain studies of this type would be particularly valuable in toxicological screening, such as carcinogenesis bioassays3234, but could be used more widely in numerous fundamental and applied studies (as, in fact, could recombinant inbred lines; R. Mott, personal communication). Figure 1 shows the response to chloramphenicol in 47 CD-1 outbred mice compared with the much better response in 48 mice from four inbred strains35. Not only was the multistrain study quantitatively more powerful, but it showed a white blood cell response not seen in the CD-1 stock and gave an estimate of the extent of genetic variation in response to chloramphenicol. Similarly, in a comparison of three experiments to study the toxic effects of gentamycin in the rat (H. Jacob, personal communication) using outbred Sprague-Dawley, inbred F344 or seven isogenic strains of rats with the same number of rats in each experiment, the multistrain study was substantially more powerful than the studies using a single outbred stock or a single inbred strain. Lack of power in the experiment using F344 rats was due to resistance in this strain rather than to increased phenotypic variability. Within-animal experimental designs are, however, likely to be equally powerful whether the animals are inbred or outbred, although such designs are relatively rare. Many of these points have been discussed in more detail (http://www.isogenic.info/). Use of outbred stocks for QTL mapping There can be no more excuses for using outbred stocks in most fundamental research or for testing effects of substances, but there are reasons for working with these mice in the genetic analysis of complex traits36. The outbred stocks accumulate many breakpoints over time that split chromosomes into fine-grained mosaics (as occurs in the outbred human population), facilitating the high-resolution mapping of complex traits. These breakpoints cannot be visualized in single inbred lines because the mice have no polymorphic loci. In the best examples, a QTL that has been localized by using crosses with inbred strains can be then mapped in an outbred stock cross to within 1 cM; thus, identification of the underlying nucleotide sequence becomes feasible3742. CD-1 and MF1 outbred mouse stocks have been used in these analyses to identify a susceptibility locus for pulmonary adenoma38 and one for a gene influencing anxiety42, respectively. Specially bred heterogeneous stocks are being also used to identify complex trait loci3941,4345. These mouse heterogeneous stocks have been bred from up to eight known founder inbred lines and have been carefully maintained to maximize diversity36. For example, the Boulder heterogeneous stock (HSIBG, Table 1) has been bred for more than 60 generations36 and was created from A/J, AKR, BALB/c, C3H, C57BL/6, DBA/2, I and RIII inbred lines44,45. Similarly, the Northport heterogeneous stock (HSNPT, Table 1) has been bred for more than 40 generations36 and was derived from A/J, AKR/J, BALB/cJ, C3H/HeJ, C57BL/6J, CBA/J, DBA/2J and LP/J inbred strains43. More recent heterogeneous stocks, such as the HSCC stock, are being constructed with increased diversity by the inclusion of wild-derived inbred lines such as CAST/Ei, PWK/Ph and WST/Ei (Table 1). The key to working with heterogeneous stocks for fine-mapping is the ability to make a probabilistic reconstruction of the genomic mosaic that makes up each chromosome39, and other outbred stocks potentially may be also used in this way42. The advantages and disadvantages of outbred stocks for complex trait mapping have been reviewed36. Use of outbred stocks as a base population for selection Outbred stocks have been used extensively as a base population for selective breeding for phenotypes of interest. The best known examples are the many strains of mice selected for various aspects of response to alcohol, of which the long- (LS) and short-sleep (SS) mice are the best known example46. Mice have been also selected for immune responses to sheep red blood cells47, high and low blood pressure48, sensitivity to skin carcinogenesis49, low antioxidant defences50, high activity51, prolificacy and weight52, obesity53 and various traits related to drug abuse54,55, among others. All these models have been used extensively to look for physiological, biochemical and genetic correlates with the traits of interest. A review of this work is beyond the scope of this perspective. If the eventual aim is to identify loci controlling the trait of interest and to detect genetically correlated traits, however, it is important to maintain an unselected control or to select in both directions and to avoid inbreeding so that the positive or negative alleles are enriched at loci controlling the trait, without fixing unrelated loci. The total response to selection depends on both the selection differential (difference between mean of selected group and mean of whole group) and the effective population size8. Sibling mating will therefore limit the response because some unfavorable loci will be fixed by chance. Such a differential fixation of neutral loci between the selected and control groups will also lead to spurious genetic correlations between traits. For example, all the rat strains selected for hypertension have been identified by a method involving simultaneous sibling mating. As a result, the hypertensive SHR strain differs from its control strain WKY at many loci, including several associated with characters that are unrelated to hypertension. A direct comparison of the two strains is therefore useless for identifying candidate QTLs, although these can still be identified by crossing the strains. Fortunately, most mouse selection experiments have not fallen into this trap. Most have started with a heterogeneous stock, often involving a cross between eight inbred strains, that is then subjected to random mating for a few generations before the selective breeding is started, and inbreeding has been avoided by maintaining large population sizes. Combining QTL mapping, selective breeding and gene microarrays (e.g., to detect changes in expression in genes that map to regions known to contain QTLs) may provide a powerful method of identifying candidate genes for traits of biomedical interest56. Conclusions The outbred stocks are a genetically ill-defined set of laboratory mice that are often used erroneously in toxicology, pharmacology and basic research. There is no system of genetic quality control for these stocks and even their names provide little information about their genetic or phenotypic characteristics. These stocks should not be used in situations where smaller numbers of mice from a range of inbred strains would give optimal results, for example, for determining sensitivities to substances or for examining physiological parameters. But outbred stocks, especially the experimentally designed and carefully monitored heterogeneous stocks, are helpful both in positional cloning of QTLs and as a phenotype and genotype pool from which to select specific traits of interest. Nevertheless, we think that most papers that are currently published on the back of outbred stocks should be using inbred lines and any experimental plans, including grant applications and papers, based on outbred stocks need to be fully justified to avoid wasting animals and funding.
Note: Supplementary information is available on the Nature Genetics website. ACKNOWLEDGMENTS We thank R.D. Mastrocco for information about Clara Lynch; J. Eppig, R. Hitzemann, H. Jacob, M. Josten, R. Kalman, I. Martin, G. McClearn, R. Mott, J. Stitzel and R. Williams for personal communications; R. Young for graphics; and the Brain Research Trust and the Motor Neuron Disease Association for funding. COMPETING INTERESTS STATEMENT The authors declare competing financial interests (see the Nature Genetics website for details).

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