The breadth of genetic and phenotypic variation among inbred strains is often underappreciated because assessments include only a limited number of strains. Evaluation of a larger collection of inbred strains provides not only a greater understanding of this variation but collectively mimics much of the variation observed in human populations. We used a high-throughput phenotyping protocol to measure females and males of 43 inbred strains for body composition (weight, fat, lean tissue mass, and bone mineral density), plasma triglycerides, high-density lipoprotein and total cholesterol, glucose, insulin, and leptin levels while mice consumed a high-fat, high-cholesterol diet. Mice were fed a chow diet until they were 6–8 wk old and then fed the high-fat diet for an additional 18 wk. As expected, broad phenotypic diversity was observed among these strains. Significant variation between the sexes was also observed for most traits measured. Additionally, the response to the high-fat diet differed considerably among many strains. By the testing of such a large set of inbred strains for many traits, multiple phenotypes can be considered simultaneously and thereby aid in the selection of certain inbred strains as models for complex human diseases. These data are publicly available in the web-accessible Mouse Phenome Database (http://www.jax.org/phenome), an effort established to promote systematic characterization of biochemical and behavioral phenotypes of commonly used and genetically diverse inbred mouse strains. Data generated by this effort builds on the value of inbred mouse strains as a powerful tool for biomedical research.
- Mouse Phenome Database
- high-throughput phenotyping
- complex traits
- body composition
- mouse models
- high-density lipoprotein cholesterol
- metabolic syndrome
inbred strains and genetically engineered rodent models continue to provide invaluable tools for research aimed at improving human health through translation into prevention and treatment strategies. With the current availability of annotated human, mouse, and rat genomes (8, 16, 31, 33) comes a renewed interest in generating and accessing phenotypic data for maximal mining of genomic resources to understand both normal and disease processes. What and where are the genomic perturbations that lead to disease in mammals? How do genes interact to describe networks underlying these biological processes and pathways? Providing comprehensive phenotype data sets for a large number of inbred mouse strains will enable use of a multidisciplinary approach to uncover these phenomena and to develop and test relevant animal models. In addition to reporting the availability of this data set to the scientific community, this paper summarizes some important phenotypic differences both among a large set of inbred strains and between sexes toward identifying mouse models of human disease.
The Jackson Laboratory Heart, Lung, Blood, and Sleep Program for Genomic Applications [JAX-PGA; http://pga.jax.org (29)] uses high-throughput phenotyping to characterize both chemically induced mutant mice and the large set of inbred strains prioritized by the Mouse Phenome Project, an international collaborative effort established to guide the characterization of biochemical and behavioral phenotypes of commonly used and genetically diverse inbred mouse strains in a systematic manner. Results contributed by investigators worldwide are deposited in the web-accessible Mouse Phenome Database (MPD; http://www.jax.org/phenome). The inception and development of the Mouse Phenome Project have been described previously (2, 9, 25). All phenotyping criteria and detailed protocols accompany each submitted project, and the MPD website offers online analysis tools. The MPD currently contains more than 600 measurements for phenotypes relevant to human health, including atherosclerosis; gallstones; hypertension; obesity; osteoporosis; airway hyperactivity; pain response; hematology and clotting; neurological, behavioral, and sensory disorders; alcoholism; and toxicity to environmental pollutants. The MPD is continually updated with data from new studies.
This paper summarizes a large amount of the phenotypic data collected from a set of 43 inbred strains of mice by the JAX-PGA and contributed to the MPD. Numerous statistical and analytical tools are available as part of the MPD, and data sets held there are downloadable for further user-based analyses. We have analyzed measurements for body weight, lean and fat tissue mass, bone mineral density and plasma glucose, lipids, insulin, and leptin from a study to assess these parameters before and after mice consume a high-fat and high-cholesterol diet.
MATERIALS AND METHODS
Mice and Diets
Mouse strains were obtained from The Jackson Laboratory, Bar Harbor, ME; names and abbreviations are shown in Table 1. The sample size goal was 10 females and 10 males of each strain, but actual sample sizes varied from 4 to 26. Mice were housed in specific pathogen-free (SPF) barrier rooms with a 12:12-h light-dark cycle (lights on from 6:00 am to 6:00 pm) in pressurized individually ventilated (PIV) cages (Maxi-Miser PIV; Thoren Caging Systems, Hazelton, PA) covered by snap-on filter tops (Reemay 2033, Thoren Caging Systems, Hazelton, PA) and bedded with pine shavings (Crobb Box, Ellsworth, ME). Mice had ad libitum access to food and acidified water. Standard laboratory rodent chow (LabDiet 5K52, LabDiet, Scott Distributing, Hudson, NH) was fed from weaning until mice were 6–8 wk old, and an atherogenic diet containing (by weight) 15% dairy fat (30% caloric content), 50% sucrose, 0.5% cholic acid, and 1.0% cholesterol (20) was fed for the next 18 wk until they were 24–26 wk old.
In general, the same mice were tested for multiple phenotypes. In cases when mice did not survive the 18-wk protocol, additional mice were used to achieve a minimum number of four mice of each sex per strain for each measurement.
The phenotyping protocol, developed as part of the JAX-PGA, is based on high-throughput, noninvasive strategies. Table 2 summarizes the order in which measurements were obtained and the age of mice and duration of high-fat diet consumption at the time of each measurement. Data presented here include measurements pertinent to obesity, the metabolic syndrome, and osteoporosis. The complete protocol is available from the JAX-PGA website, but details relevant to this report are as follows. Mice were weaned when they were 3 wk old and fed standard rodent chow containing 6% fat. At 6–8 wk old, following a 4-h fast (7:00 am to 11:00 am), blood was withdrawn from the retroorbital plexus of each mouse through a heparin-coated hematocrit tube into a 1.5-ml Eppendorf tube containing 7.5 μl of sodium heparin (1,000 U/ml) and placed on ice. Samples were centrifuged at 14,000 rpm for 5 min, and plasma was removed for measurement of total cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides, and glucose. Mice were then fed the atherogenic diet until they were 16 wk old, at which time their weight was measured and they were examined for bone mineral density and body composition by dual-energy x-ray absorptiometry (DEXA). Mice continued consuming the atherogenic diet for nine more weeks. At age 24–26 wk, having consumed the atherogenic diet for 18 wk, mice were weighed and fasted, and blood samples were collected as described above for analysis of plasma total cholesterol, HDL cholesterol, triglycerides, and glucose. One week later, while mice continued to eat the atherogenic diet, blood samples (nonfasted) were collected for plasma leptin and insulin analyses, and mice were euthanized by CO2 asphyxiation. All bleeds for leptin and insulin determinations were obtained between the hours of 1 and 2 pm. The Animal Care and Use Committee at The Jackson Laboratory approved all animal protocols.
Phenotyping methods are briefly summarized here (details at http://pga.jax.org/protocols). Total plasma cholesterol, HDL cholesterol, triglycerides, and glucose were measured using a Beckman Coulter Synchron CX5 Delta Chemistry Analyzer (Beckman Coulter, Fullerton, CA) according to the manufacturer's instructions. Plasma insulin and leptin levels were determined by ELISA (ALPCO Diagnostics, Windham, NH; CrystalChem, Downers Grove, IL). Mice were weighed with an Ohaus Navigator scale set at “averaging weight” over a 15-s interval to correct for the constant motion of the mice. Bone mineral density, percent body fat, and lean tissue mass were determined using a Lunar PIXImus DEXA machine (Lunar, Madison, WI). For DEXA analysis, mice were anesthetized with tribromoethanol at a dose of 0.02 ml/g body wt. Because the skull is so bone dense, the head was excluded from DEXA analyses.
Data for all phenotypic measurements performed in this study on the strains listed in Table 1 are available for online evaluation or downloading from the MPD (19, 23). Before submission to the MPD, data were reviewed for completeness using a minimum requirement of four animals per sex per strain for each measurement. Separate analyses were performed for each sex. Individual values exceeding 2 SDs from the group mean were eliminated. This resulted in the loss of some strains for certain phenotypes. The missing strains and phenotypes are listed in Table 3. Strain CE was used only in leptin and insulin tests, and for many tests only one of the BALB strains (CBy or C) was used. Data were obtained for at least 36 strains for every phenotype measured. To describe differences in phenotypes between sexes within each strain, the MPD analysis tool “Sex differences” was used. In this analysis, a normalized sex difference is calculated for each strain as the average male value minus the average female value for a strain, divided by the average of male and female values.
The detailed data for each mouse is stored in MPD, which also has analysis tools and the ability to download data, so we encourage the investigator to visit the MPD website. As expected, broad diversity in phenotypes among strains was observed. Strain averages as well as values for strains showing extreme high or low phenotypes are given in Table 4. Significant sexual dimorphism was observed for most traits measured; hence the data are presented and analyzed separately for each sex. The following paragraphs briefly describe sex and strain trends for each phenotype under high-fat-diet conditions.
Measurements obtained from the DEXA scan, including body weight, percent fat, lean tissue mass, and bone mineral density, were obtained from 16-wk-old mice fed the atherogenic diet for 8 wk and are shown in Fig. 1.
Considerable variation in body weight among the strains occurs with a 3.0-fold (females) and a 2.8-fold (males) difference between the smallest and largest strains. Although most strains fall within 1 SD of the overall mean (22.4 g for females; 27.9 g for males), six strains are more than 1 SD above the mean (AK, BTBR, NZW, both sexes; NON females; CBA, KK males), and seven strains are more than 1 SD below the mean (CAST, CZECH2, MOLF, MSM, WSB, both sexes; PL, females; SPRET, males). It is noteworthy that all but one strain (PL) with low body weight are recently derived from the wild. Males weighed more than females except in strains SPRET and NON, for which females weighed 15% and 6% more than their male counterparts, respectively.
The overall strain mean of 23.3% fat is identical for females and males. The difference between the leanest and fattest strains is 2.7-fold among females and 3.7-fold among males. Nine strains are more than 1 SD above the mean (AKR, CB, both sexes; JF1, NON, NZW, PERA, females; 129S1, KK, LP, males), and seven strains are more than 1 SD below the mean (C58, MOLF, SW, both sexes; CZECH2, WSB, females; B6, SPRET, males). Females have higher percent fat than male counterparts in 21 strains; the largest differences occur in strains SPRET and FVB (54% and 38% greater, respectively). Males have higher percent fat in 14 strains; the largest differences occur in strains KK, BR, and 129S1 (56%, 35%, and 32% greater, respectively). No sex difference is seen for this trait in MOLF.
LEAN TISSUE MASS.
Lean tissue mass varies 2.6-fold in females and 2.5-fold in males. Seven strains are greater than 1 SD above the mean (AKR, BTBR, NZW, both sexes; BUB, KK, NON, females; NZB males), and seven strains are greater than 1 SD below the mean (CAST, CZECH2, MOLF, MSM, WSB, both sexes; PL females; SPRET males). Males have greater lean tissue mass than females in all strains except SPRET, which shows no sex difference. The greatest sex differences are seen in SM and FVB (males 41% and 38% greater than females, respectively). The wild-derived strains appear as low value outliers.
BONE MINERAL DENSITY.
Bone mineral density was the least variable of the body composition phenotypes measured. The difference between the strains with the most and least dense bones is 1.4-fold for females and 1.5-fold for males. Strain means are not significantly different between females and males (0.0495 ± 0.005 for females; 0.0497 ± 0.005 for males). Sex differences do not exceed 14% for any strain. Five strains are more than 1 SD above the mean (AKR, LP, NZW, both sexes; NZB, KK, males), and seven strains are more than 1 SD below the mean (CAST, CZECH2, MOLF, MSM, WSB, both sexes; SM, PWK, females). Except for strain SM, the strains with the lowest bone density are wild derived.
Plasma lipids and glucose.
Plasma lipids (total and HDL cholesterol, triglycerides) and glucose were measured in fasted 25-wk-old animals fed the atherogenic diet for 17 wk and are shown in Fig. 2. Total cholesterol (Fig. 2A) and HDL cholesterol (Fig. 2B) are measured directly. An estimate of non-HDL- cholesterol, which in the mouse consists of low-density lipoprotein and very low density lipoprotein cholesterol, may be obtained by subtracting HDL cholesterol from total cholesterol. The distribution of non-HDL cholesterol is available on MPD.
Strain MOLF exhibited extremely high total plasma cholesterol (>2 SD above strain means) in both females (1,235 ± 188 mg/dl) and males (2,143 ± 392 mg/dl) after consuming the high-fat diet. Eight other strains have levels greater than 300 mg/dl, a level at which humans are treated with cholesterol-lowering drugs (A, C58, CAST, MA, NOD, NZB, both sexes; BR, females; JF1, males). To more accurately compare cholesterol values among strains after consuming the high-fat diet, we removed MOLF and used average values of 243 ± 107 mg/dl for females and 244 ± 92 for males. Seven strains fall greater than 1 SD above the recalculated mean (A, C58, CAST, NOD, both sexes; MA, NZB, females; JF1, males), and six strains are greater than 1 SD below the strain mean (D1, CZECH2, MSM, both sexes; CB, D2, SJL, females). Males show higher total plasma cholesterol levels than females in 20 strains with the greatest differences in strains JF1, MOLF, SJL, D2, and CBA (58%, 53%, 50%, 42%, and 40%, respectively; Fig. 2A). In 13 strains, females have higher levels than males with the greatest differences in strains C58, R3, BUB, and MA (40%, 38%, 35%, and 32%, respectively). No sex differences were found in strains CAST and SW.
The average HDL cholesterol among all strains differs by only 10 mg/dl between the sexes (83.0 for females; 92.5 for males) although values are highly variable among sexes, with a difference in females of 4.9-fold and in males a remarkable 14.5-fold difference (Fig. 2B). Five strains are more than 1 SD from the mean for all strains (MA, NOD, NZB, both sexes; PERA, females; 129S1, males), and three strains are greater than 1 SD below the mean (D1, CAST, I, females). Males of 35 strains have higher HDL on the high-fat diet than female counterparts with differences exceeding 50% in strains CB, SEA, C3, and D1. In strain MOLF, females have a remarkable 136% higher HDL than males, reflecting the notable decrease in HDL levels observed in males. Levels of HDL are higher in females than males in 10 strains, with C58, PERA, and NZB showing the greatest differences (39%, 23%, and 18%, respectively) after MOLF.
SPRET females have extremely high triglycerides after consuming the high-fat diet (534 ± 409 mg/dl), and for comparisons we recalculated the strain mean for females after excluding this value and used 85 ± 50 mg/dl. Eight strains are greater than 1 SD above this recalculated mean (CAST, MOLF, MSM, both sexes; KK, PL, RF, females; NOD, SPRET, males), and only one strain, B6 males, is more than 1 SD below the mean. Five strains, all wild derived, have values above 190 mg/dl, which is considered high in humans (MSM, both sexes; CAST, MOLF, SPRET, males). In 21 strains, males have higher triglyceride levels than their female counterparts, and differences exceeded 30% in nine of them (Fig. 2C) with strain NOD exhibiting the greatest difference (107%). In 14 strains, females show higher values than males with strain SPRET showing an 87% higher value while other differences were <30%.
In strain NOD, a model of type I autoimmune diabetes, males have plasma glucose levels of 348 ± 237 mg/dl, exceeding the range for other strains, as expected (Fig. 2D). With this value excluded, the recalculated strain mean for males is 159 ± 33.4 mg/dl. Seven strains have values greater than 1 SD above the recalculated mean (B6, CB, SPRET, both sexes; B10, females; NON, SM, males), and seven strains have levels greater than 1 SD below this mean (A, C, C58, MOLF, both sexes; 129S1, females; PERA, WSB, males). Plasma glucose levels for males in 27 strains other that NOD exceed that of females, with NON and SM showing the greatest differences (63% and 32%, respectively) after NOD. In the 11 strains for which female values exceeded male values, differences are 20% or less.
RESPONSE TO HIGH-FAT DIET.
Baseline values for lipids and glucose, measured before mice began the high-fat diet, are available for all strains from the MPD (19, 24). Figure 3, A–D, summarizes strain responses to the high-fat diet for plasma total cholesterol, HDL cholesterol, triglycerides, and glucose, respectively. Response is expressed as fold increase in values compared with those measured in animals consuming standard chow. Since mice consumed the diet for 18 wks, this would include any change induced by age as well as the response to diet. All strains show an increase in plasma total cholesterol for both sexes (average female increase = 3.5-fold; average male increase = 2.7-fold), ranging from 1.3-fold for D2 males to 24.3-fold in MOLF males. HDL cholesterol values differ by 1.5-fold in females and by 1.3-fold in males, with some strains showing a slight decrease in values. Of note, however, is the remarkable decrease in HDL cholesterol levels for MOLF males to only 20% of chow values. Triglycerides are generally lower after the high-fat diet, although in five strains values increase by 1.5- to 5-fold. Glucose levels remain close to those on chow for both sexes in most strains, with fold increases ranging from 0.5 to 1.7.
Leptin levels, shown in Fig. 2E, are highly variable, with differences between extreme high and low values of 13.7-fold in females and 33.6-fold in males. Four strains are greater than 1 SD above the strain mean (KK, RF, both sexes; CE, females; LP, males), and three strains are more than 1 SD below the mean (MOLF, both sexes; CZECH2, females; CBy, males). Strain C shows the greatest sex difference, with females having 128% higher levels than males. In 17 additional strains, female values are greater than male values, with notable differences for strains FVB, CBy, and PERA (95%, 93%, and 93%, respectively). In 23 strains, male values are greater than those of females, with the greatest differences in strains BR, PL, and LP (107%, 92%, 71%, respectively). Strain SEA shows no difference in leptin levels between sexes.
Insulin (Fig. 2F) varies 5.6-fold in females and 8.5-fold in males. Five strains are greater than 1 SD above the strain mean (D2, KK, both sexes; AK, CB, LP, males), and three strains are greater than 1 SD below the mean (A, PERA, MOLF, males). Males have greater values than females in all but six strains, with differences exceeding 100% in strains BTBR, LP, and 129S1. In 16 additional strains, male values exceed female values by >50%. In strains for which female values are greater, PERA females have an 88% higher level than males, while other differences are <30%.
Inbred Strains as Models
To summarize the overall phenotypic state of the inbred strains used in this study after 18 wk consuming the high-fat diet, strains with two or more average trait values lying above or below 1 SD unit from strain means per sex are shown in Table 5. In this summary, we have focused on phenotypes prevalent in the metabolic syndrome, namely percent fat and plasma levels of triglycerides, HDL cholesterol, and glucose, and have also included insulin levels. For some strains, only one sex exhibits extreme traits, but for many strains both sexes show extreme traits, although both sexes of a strain do not always share the same trait profile. For example, using risk factors for metabolic syndrome as defined by the American Heart Association (10) (e.g., elevated triglycerides, low HDL cholesterol, elevated glucose, obesity), three inbred strains from Table 5 can be readily identified as carrying two of these features: CAST females have high triglycerides and low HDL; CB females and males have increased fat with high glucose (CB males also have high insulin); MSM (both sexes) have high triglycerides and high glucose. Strains with only one extreme metabolic syndrome-defined trait but also having high insulin levels are also included in Table 5 (AK, males; KK, both sexes; LP, males). Table 5 aids in the practical consideration of inbred strains as models of complex human diseases such as the metabolic syndrome, and such a table could also be constructed from relevant phenotype data to summarize other complex traits such as diabetes, obesity, or cardiovascular disease. Such a list can also be generated for strains fed the standard chow diet. Other measurements available from MPD, such as systolic blood pressure (4, 7, 14, 30), can supplement this list, providing additional information when choosing strains for conducting genetic crosses to further investigate complex traits.
Laboratory mouse inbred strains are derived from a limited number of gene pools (27, 32), and the distribution of phenotypes across tens of inbred strains often shows a unimodal continuous variation, suggesting that we can treat them as a population. These distributions can then be interrogated for features that mimic human disease. The large amount of phenotype data presented here provides a basis for profiling inbred strains in the context of complex trait analysis and toward their development as experimental models.
As the genetic structure of inbred mouse strains is revealed (5, 13, 35, 36), phenotypic information becomes an increasingly important element for understanding how genes drive disease processes. Environmental factors are also important gene regulators, and in this study mice were exposed to a high-fat, high-cholesterol diet as an environmental perturbation to which susceptibility to disease-related traits can be evaluated. Broad phenotypic diversity was observed among these inbred strains both in baseline (prediet) and, especially, in response to the dietary challenge. Moreover, sexual dimorphism among strains was observed for most traits measured. This observation underscores the necessity to account for sex in evaluating phenotype data, and failure to do so may impact experimental conclusions (15). Many studies overcome this issue by using only one sex in an experimental design. However, in a large comprehensive study involving multiple phenotypic observations, a better understanding about pathways leading to disease is gained by testing both sexes.
Some of the phenotypic variation observed between sexes may be attributable to the atherogenic diet, as is shown by comparing lipid and glucose values before and after high-fat-diet feeding. It is known that inbred strains vary in their susceptibility to diet-induced obesity (28, 34) and atherosclerosis (20, 22). Most strains had lower plasma triglyceride levels after consuming the high-fat diet, and Rossmeisl and colleagues (26) have suggested a role for respiratory uncoupling protein in this response. The five strains that did not follow this trend but rather showed increases in triglyceride levels may be useful for further investigation of this mechanism. Insulin and leptin levels, measured in this study after mice consumed the high-fat diet for 18 wk, were among the most variable traits we measured. Insulin levels have been shown to vary according to diet (1, 3), and circulating leptin levels are also affected by a high-fat diet, especially in strains prone to diet-induced obesity (1, 6). Hence, it is plausible that much of the variation we observed is in response to the high-fat diet.
Nine strains have total plasma cholesterol levels exceeding 300 mg/dl after consuming the high-fat diet. One would expect that these strains would be susceptible to diet-induced atherosclerosis. However, only three of these strains (C58, both sexes; BR, females; A, males) develop significant aortic lesions in response to this diet (23); other strains appear to be resistant to lesion development. Interestingly, both females and males of strain A have total cholesterol levels above 500 mg/dl on the high-fat diet, while only males develop significant aortic lesions. Strain MOLF, with cholesterol levels above 1,000 mg/dl in both sexes, develops only modest lesions compared with more susceptible strains. Mice have high HDL cholesterol compared with humans, likely because of their inherent deficiency in cholesteryl ester transfer protein (12). Elevated HDL cholesterol may account for the high total cholesterol in three of the nine strains (MA, NOD, NZB) and may also contribute to their resistance to atherosclerosis. However, in the other strains with high cholesterol that are resistant to atherosclerosis, mechanisms independent of HDL level are likely to explain this protection.
Mouse strains more recently developed for laboratory use from the wild have been an important tool for mapping genes by providing both phenotypic and genotypic diversity to the repertoire of available strains (11, 18). Our data identify many of these strains as having extreme phenotypes, such as the recurrence of strains CAST, CZECH2, MOLF, MSM, and WSB as low outliers for body composition parameters (weight, lean tissue mass, bone mineral density) and CAST, MOLF, MSM, and SPRET comprising the high outliers for triglycerides.
The great range of phenotypic variation among 43 inbred strains for the phenotypes measured in this study restates that multiple genes in concert direct an interplay contributing to complex disorders. This variation can be used to further investigate underlying processes and identify genes affecting these phenotypes by following their segregation when strains are mixed. We have provided evidence supporting the use of certain inbred strains for modeling human diseases such as obesity, diabetes, osteoporosis, and the metabolic syndrome. Does this range capture the diversity observed in humans? How well will these models represent human disease? By comparing typical normal human reference values (17) to the traits measured in mice in this study under both normal chow and high-fat diet conditions, we have evaluated this assertion in Table 6. Values for mice on a normal chow diet were obtained from MPD (21). The mouse data encompass most of the normal or desirable human values and provide a realistic range of extreme values for the traits that we were able to compare. Mouse and human obesity are difficult to compare using weight data alone, and a better estimate would be provided by a comparison of percent body fat, a measurement not readily available for humans. The range of glucose levels for mice is considered normal. By demonstrating such a broad range of response to consuming a high-fat diet, we establish the utility of the laboratory mouse for dissecting important pathways controlling normal and disease processes implicated in the regulation of body composition, bone health, and lipid metabolism. Using rapidly emerging bioinformatics tools, investigators can begin to map these divergent phenotypes to genomic elements driving this diversity.
Program for Genomic Applications Award HL-66611 from the National Heart, Lung, and Blood Institute supported this work.
The authors thank Willson Roper, Sara Connolly, and Stacey Dannenberg for expert phenotyping and mouse care support.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2007 the American Physiological Society