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Inbred mice has no severe phenotype outcome?

Inbred mice has no severe phenotype outcome?



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Why does 20 generation of inbred mouse have no particular strange phenotypes, but on the contrary, when on purposely inbreed dogs or tigers for specific phenotype cause severe deformation of the bone structure or cranial structure?


Some information on inbred strains of laboratory mice:

https://en.wikipedia.org/wiki/Inbred_strain

http://what-when-how.com/molecular-biology/pure-line-molecular-biology/

A relevant quote on the consequences of inbreeding:

Inbreeding in allogamic organisms bring the deleterious recessive alleles to homozygosity; the immediate consequence is an increase in the frequency of defective offspring, or, in another words, an increase in the genetic load of the population. This phenomenon is called inbreeding depression or inbreeding degeneration. As inbreeding continues, the deleterious alleles are selected out and eventually disappear. The original heterozygous populations are often more fit than the resulting pure lines because they profit from heterosis and balanced polymorphisms; the main advantage of pure lines is the quick production of many individuals with the same well-adapted genotype, while the allogamy continuously generates new genotypes.

In other words, inbreeding is harmful because it makes it more likely that offspring will have two copies of bad recessive alleles, meaning those alleles get expressed, meaning the organism gets the bad consequences that wouldn't show up if they had only one copy. This is what happens over a single generation however; over many generations the bad alleles are selected against, precisely because they are harmful to the organism so it reproduces less, and after enough generations of inbreeding you hit a point where all individuals are genetically identical and have all the "good" alleles (if they were lucky; otherwise they die out or stay stuck with some bad-but-not-fatal alleles). They still may be worse off than their more diverse ancestors, but they're not completely messed up like their unfortunate great-aunts and uncles who didn't make it either.

The big difference between inbred mice and dogs or tigers is the "for specific phenotype" aspect. Laboratory organisms are inbred so that you get a large pool of genetically-identical individuals, meaning they're much easier to experiment on. The aim is the inbreeding itself, not any particular phenotype. For example if you look at the page for the most popular strain of laboratory mice, C57BL/6, you can see it has many different properties and is used for many different things.

On the other hand, dogs aren't inbred for the purpose of inbreeding or of being genetically identical; the aim is to get desirable phenotypes, and inbreeding is just an efficient way of achieving that aim. It also isn't obvious that many of the problems purebred dogs have is inbreeding (i.e. lack of genetic diversity, high levels of homozygosy) per se, but the fact that the traits being bred for are just plain unhealthy for the dog, or part of a bell curve that include bad outcomes at the edges. For example, Syringomyelia in the Cavalier King Charles Spaniel:

Some researchers estimate that as many as 95% of CKCSs may have Chiari-like malformation (CM or CLM), the skull bone malformation believed to be a part of the cause of syringomyelia, and that more than 50% of cavaliers may have SM.* It is worldwide in scope and not limited to any country, breeding line, or kennel, and experts report that it is believed to be inherited in the cavalier King Charles spaniel. CM is so widespread in the cavalier that it may be an inherent part of the CKCS's breed standard.

(emphasis mine)

Same thing for that spine malformation that's related to selecting for corkscrew tails. The genes that make the tail corkscrew also mess with the spine.

In other words, the issue isn't inbreeding or not but whether the genes themselves are harmful. When organisms are selected for traits that are directly harmful in their extreme, or are associated with harmful genes that just happen to be next to those that are selected for in the chromosome, then the harmful consequences will spread through the population. Inbreeding is only a problem insofar as it allows the process go faster (more offspring per generation have the desired trait). On the other hand when you're just inbreeding with no specific focus on phenotype, or not phenotypes that have obvious harm associated (i.e. no lab would select for a frivolous trait that also causes harm. They're either selecting for the harmful trait on purpose, or they're selecting against it, because they'll want animals that are as healthy as possible except for the one variable they're interested in), then you'll end up with populations that are fairly normal except for some of the direct consequences of genetic uniformity.

It should be noted that most purebred dogs probably aren't inbred strains the same way many laboratory animals are; those are genetically identical, so the whole point is that their offspring will be like they are. So while they may be less fit than a non-inbred version of them might be, their offspring won't be any less fit than they are. And this is not what's observed with purebred animals like dogs and horses; individuals aren't identical, and looking at the page on Syringomyelia it seems the problems are getting worse.


Because we have selected particular inbred strains that seemed "normal", at least superficially, and mostly for strains that produced good litters and didn't often bite the researchers. However, inbred strains of mice and rats have many deleterious mutations. In some cases they probably survive only because they are held in cages with ample food and water, so they are not subject to the same selection pressures as wild mice would be.

For example, many common mouse strains have the rd1 mutation (see: Jackson labs). This mutation leads to complete degeneration of photoreceptors by about 1 month of age. Note that these are not typically albino mice, so this is different from the vision deficits of albinos. These are mice that appear mostly normal, but they are totally blind.

The most common mouse strain used in research is the C57BL/6 mouse, sometimes called "black 6". Again, see Jackson Labs for some of the phenotypes observed in this strain. I'll list just a few here: hearing loss, high preference for alcohol, dermatitis bad enough to occasionally result in self-mutilation and death, susceptible to obesity, sensitive to atherosclerosis, small kidneys, susceptible to several tumor types, high rate of developmental issues with the lens of the eye, and on and on.

Given that this is the strain often considered "most normal" I think you can see that it isn't at all correct to think of inbred mice as not having any severe phenotypes.


Influence of genetic background on ex vivo and in vivo cardiac function in several commonly used inbred mouse strains

Inbred mouse strains play a critical role in biomedical research. Genetic homogeneity within inbred strains and their general amenability to genetic manipulation have made them an ideal resource for dissecting the physiological function(s) of individual genes. However, the inbreeding that makes inbred mice so useful also results in genetic divergence between them. This genetic divergence is often unaccounted for but may be a confounding factor when comparing studies that have utilized distinct inbred strains. Here, we compared the cardiac function of C57BL/6J mice to seven other commonly used inbred mouse strains: FVB/NJ, DBA/2J, C3H/HeJ, BALB/cJ, 129X1/SvJ, C57BL/10SnJ, and 129S1/SvImJ. The assays used to compare cardiac function were the ex vivo isolated Langendorff heart preparation and in vivo real-time hemodynamic analysis using conductance micromanometry. We report significant strain-dependent differences in cardiac function between C57BL/6J and other commonly used inbred strains. C57BL/6J maintained better cardiac function than most inbred strains after ex vivo ischemia, particularly compared with 129S1/SvImJ, 129X1/SvJ, and C57BL/10SnJ strains. However, during in vivo acute hypoxia 129X1/SvJ and 129S1/SvImJ maintained relatively normal cardiac function, whereas C57BL/6J animals showed dramatic cardiac decompensation. Additionally, C3H/HeJ showed rapid and marked cardiac decompensation in response to esmolol infusion compared with effects of other strains. These findings demonstrate the complex effects of genetic divergence between inbred strains on cardiac function. These results may help inform analysis of gene ablation or transgenic studies and further demonstrate specific quantitative traits that could be useful in discovery of genetic modifiers relevant to cardiac health and disease.

for decades biomedical research has relied heavily on the use of inbred mouse strains. The genetic homogeneity within these strains allows for well-controlled and highly reproducible studies. Over 450 distinct inbred strains have been described in the literature (3) and the Jackson Laboratory alone maintains over 180 inbred mouse strains (http://jaxmice.jax.org/list/cat481365.html). The most common strain of inbred mice used in research is the C57BL/6 mouse. This strain is the most commonly used background of genetically modified mouse strains maintained by the Jackson Laboratory (http://jaxmice.jax.org/literature/catalog/JAX_Mice_Catalog.pdf) and is currently the only inbred strain whose genome has been fully sequenced (57).

Though not as widely used, other inbred strains have found niches in biomedical research due to ease of genetic manipulation or differing propensities for developing specific pathologies. For instance, FVB/N mice are commonly used in conventional transgenesis (51), and 129 substrains have been used almost exclusively for the isolation of embryonic stem (ES) cells used for gene targeting (41, 56). Alternatively, other inbred strains have found use as disease models due to naturally occurring mutations in genes linked to human disease (5, 22, 38, 43). Even in the absence of engineered or known naturally occurring mutations, different inbred mouse strains can vary drastically in their susceptibility to clinically relevant diseases. Previous reports have taken advantage of this naturally occurring variation in susceptibility to a particular disease to identify quantitative trait loci (QTL) that modify disease pathogenesis (2, 6, 13, 48, 49).

Cardiovascular disease is the single leading cause of death in the United States, affecting 𢏁 in 3 Americans and causing 𾠀,000 deaths in 2006 (27). To effectively investigate the pathological processes associated with cardiovascular disease and devise new therapeutic strategies, the cardiovascular field has embraced genetically modified inbred mouse strains as tools to help understand the fundamental physiological processes governing function in the normal and diseased heart. A survey of the Jackson Labs website shows that they maintain 𾈥 genetically modified mouse lines used for cardiovascular research derived from inbred mouse strains. C57BL/6 is the most common genetic background ( Fig. 1A ). However, mutations are also maintained on other common inbred backgrounds as well. The majority of these mice were created using ES cells derived from a substrain of the 129 inbred strain, then often backcrossed and maintained on a C57BL/6 or mixed C57BL/6 × 129 background ( Fig. 1B ).

Summary of strain background of genetically modified mice and embryonic stem (ES) cells. A: genetic background of genetically modified mouse strains designed for cardiovascular research maintained at Jackson Labs. B: genetic background of ES cells used to create genetically modified mouse strains maintained at Jackson Labs.

Several studies have reported strain-dependent differences in cardiovascular function between inbred mouse strains by various in vitro and in vivo methods (4, 7, 23, 37, 40, 47). The majority of these strain-dependent differences in cardiac physiology have been carried out on a small subset of inbred strains in the absence of disease-related injury/stress. Previous reports have also demonstrated the potent effects of genetic background on modifying the cardiac phenotype of genetically modified mice (20, 21, 50). For instance, two distinct transgenic lines expressing hypertrophic cardiomyopathy-linked mutant tropomyosin E180G showed drastically different phenotypes, one displaying relatively mild diastolic dysfunction (29) and the other developing overt hypertrophic cardiomyopathy and heart failure (36). Excluding possible differences in animal care and housing, the main difference between these transgenic mice is their genetic background one mouse was created on the FVB/N background, the other on the C57BL/6 background. Although the precise mechanism underlying this effect is not understood, the phenotypic variation between these two transgenic lines contributes to the impetus for this study.

The purpose of this study was to compare the cardiac function of eight commonly used inbred mouse strains at rest and following physiologically relevant stress/injury. The rationale for conducting these studies is twofold. First, the large number of inbred strains used in medical research necessitates a consideration for the physiological differences that arise from genetic divergence between strains. Secondly, the fixed genetic divergence between inbred mouse strains serves as a potential substrate for the identification of reproducible quantitative traits that differ between inbred strains. Eight common inbred strains were used, six of which are listed on Jackson Laboratory's list of the most popular inbred mouse strains (http://jaxmice.jax.org/findmice/popular.html). The techniques used to measure cardiac function for this study are the Langendorff perfused isolated heart preparation and in vivo cardiac hemodynamic function as measured by catheter-based conductance micromanometry. The Langendorff preparation is a well-established technique for assessing function of a heart performing isovolumic contractions at a controlled end diastolic pressure (26, 45). In vivo hemodynamic analysis provides pressure and volume measurements of hearts in the intact animal where physiological loading and autonomic innervation remain intact (33). Additionally, we studied strain-dependent differences in cardiac function following physiologically relevant stresses. Specifically, isolated hearts were subjected to global ischemia and reperfusion. Mice instrumented for in vivo hemodynamic measurements were subjected to β-adrenergic blockade and an acute hypoxic challenge. Strain-dependent differences in cardiac function were found both at baseline and following physiologically relevant stress. Collectively, this study highlights strain-dependent differences between inbred mice that provide insight into the marked physiological divergence reported for cardiac transgenic mice generated on differing backgrounds. The identification of strain-dependent quantitative hemodynamic traits may form the foundation of future studies aimed at identifying QTL that modify cardiac function.


Introduction

Owing to its metabolic and biotransformative capacity, the mammalian gut microbiota (GM) is now frequently regarded as a quasi-organ (Clarke et al. 2014 O’Hara and Shanahan 2006), with a collective metagenome dwarfing the host genome in terms of complexity and diversity. In addition to its profound influence on developmental processes and digestion and assimilation of nutrients, the GM also harvests carbon from xenobiotic compounds in the gut lumen, often changing the half-life and activity of parent compounds (Koppel et al. 2017), and is similarly responsible for other catabolic processes in the gut lumen. It follows that differences between individuals in the composition of their GM might, at least partially, explain differences in disease susceptibility or response to treatment (Gopalakrishnan et al. 2018 Routy et al. 2018 Matson et al. 2018). Accordingly, the biomedical research community has invested tremendous time and resources in endeavors like the Human Microbiome Project, the goal of which is to characterize the healthy human microbiota of various anatomic sites, and hundreds of other related studies examining deviations from the norm in various disease settings. Comparative studies using animal models have been critical to test the causality of associations found in human patients, and to define mechanisms underlying those associations.

Accordingly, there is a growing realization that the GM of laboratory mouse models must be considered in the context of biomedical research as a whole. For a researcher to not know the specific strain of mouse used in their experiments would be laughable, yet many researchers have minimal information regarding the GM of the mice in their research colonies and how it might be influencing the phenotype of their model. Indeed, multiple studies using mouse models have recapitulated or predicted relationships between the GM and host health in humans (Ivanov et al. 2009 Chen et al. 2018 Rosshart et al. 2019 Shin et al. 2014 Cuesta-Zuluaga et al. 2017), reflecting the utility of translational research in this field. With this in mind, the influences of the GM are implicated in three major facets of biomedical and translational research—reproducibility, translatability, and discovery. Here, we present a broad overview of these considerations, including current knowledge and best practices, with the goal of enhancing all three components.


“Autoinflammatory psoriasis”—genetics and biology of pustular psoriasis

Psoriasis is a chronic inflammatory skin condition that has a fairly wide range of clinical presentations. Plaque psoriasis, which is the most common manifestation of psoriasis, is located on one end of the spectrum, dominated by adaptive immune responses, whereas the rarer pustular psoriasis lies on the opposite end, dominated by innate and autoinflammatory immune responses. In recent years, genetic studies have identified six genetic variants that predispose to pustular psoriasis, and these have highlighted the role of IL-36 cytokines as central to pustular psoriasis pathogenesis. In this review, we discuss the presentation and clinical subtypes of pustular psoriasis, contribution of genetic predisposing variants, critical role of the IL-36 family of cytokines in disease pathophysiology, and treatment perspectives for pustular psoriasis. We further outline the application of appropriate mouse models for the study of pustular psoriasis and address the outstanding questions and issues related to our understanding of the mechanisms involved in pustular psoriasis.


Results

Characterization of P. berghei ANKA infection in 32 different mouse strains

To identify genes involved in resistance or susceptibility to P. berghei infections we analyzed body temperature, schizont-load in organs, and survival in eight mice (four females and four males) from each of 32 inbred mouse strains. A list of the mouse strains with their group association and statistical analyses for all quantified phenotypes are summarized in Table S1.

Survival.

In our setup, the average survival of all mice was 9 (S.D. 3.1), however, 46% of all mice died before day seven. Mice that lived longer than seven days showed a normal distribution with a median of 11 days. Differences in survival between the 32 strains varied significantly. In order to simplify the analysis, we grouped the different strains into three clusters using a one-way ANOVA analysis followed by Tukey-Kramer posttest with alpha = 0.05. Two of these (susceptible and resistant) showed statistically significant differences (Figure 1A) and the remaining strains were placed in an intermediate cluster. The susceptible cluster (mean survival in the cluster 6.7) included I/LnJ, RIIIS/J, CZECHII/EiJ, C3H/HeJ, BUB/BnJ, FVB/NJ, LG/J, MA/MyJ, SJL/J, SM/J, C57BL/6J, CE/J, PL/J, A/J, NZW/LacJ and CBA/J. The resistant cluster (mean survival of the cluster 13.0) included 129S1/1vlmJ, AKR/J, SWR/J, BTBR_T+_tf/J, DBA/2J, C58/J, NZO/HILtJ and WSB/EiJ. The intermediate cluster (mean survival of the cluster 9.6) included P/J, BALB/cByJ, MRL/MPJ, KK/HIJ, NOD/LtJ, PERA/EiJ, NON/LtJ, and C57BR/cdJ). Three strains showed statistical differences between males and females from the same strain (Table S1).

Four males and four females from 32 different mouse strains were infected with P. berghei and survival (A) terminal body temperature (B) and total luciferase counts from dissected organs (C) were compared. Phenotypes are presented as box and whiskers plots with whiskers showing min to max. The name of the strain is given on the x-axis and the strains are listed for increasing average survival. The number of mice analyzed for each strain and the statistical relationship between different strains are shown in Table S1. The strains were grouped into three clusters according to their survival phenotype: susceptible (blue), intermediate (yellow) and resistant (red).

To test if members of individual mouse strains grouped by ancestry, as shown in Table S1, are predominantly found in either the most susceptible, the intermediate or the most resistant cluster, the hypergeometric probability distribution for each group and cluster was calculated. Swiss mice and Castle's mice were significantly enriched in the most susceptible cluster (p = 1×10 −5 and p = 3×10 −5 , respectively) while significantly excluded from the most resistant cluster (p = 0.002 and p = 0.004, respectively). The Japanese and New Zealand strains were significantly excluded from the most susceptible cluster and enriched in the intermediate survival cluster (p = 0.0001 and p = 0.0006, respectively). Bagg albino derivates mice were highly significantly excluded from the most resistant cluster (p = 7×10 −7 ). Members of the three remaining mouse groups showed no preference for any cluster.

Body temperature.

In contrast to humans where a malaria infection is characterized by high fever, mice show a decrease in body temperature (hypothermia) upon infection with malaria parasites. Body temperature dropped significantly over the course of infection in all mouse strains (paired students t-test, p<0.04). With few exceptions, different mouse strains showed no significant differences in terminal body temperatures (for details see Figure 1B and Table S1). In four strains, female mice had significantly lower terminal body temperature than male mice from the same strain (unpaired students t-test p<0.03, for details see Table S1). In contrast there were significant differences in the course of hypothermia between the susceptible, intermediate and resistant clusters. Mice of the susceptible cluster had significantly lower body temperatures than mice from the resistant cluster from day three on (Figure 2B, one-way ANOVA followed by Tukey-Kramer post-test with alpha = 0.05). Over the course of infection the difference became more pronounced and mice of all three clusters showed significantly different body temperatures on day seven. Terminal body temperature of eight mouse strains correlated well with survival (for details see Table S1) but there was no correlation between terminal temperature and survival for all strains combined (R 2 = 0.001).

Survival and body temperature were recorded for mice infected with P. berghei. Mice were grouped into susceptible (blue dashed line, infected N = 122, dissected organs N = 42), intermediate (yellow solid line, infected N = 64, dissected N = 23) or resistant (red dotted line, infected N = 64, dissected N = 35) clusters according to their survival phenotype. A) Survival curve for the three clusters. Clusters were significantly different by a Log rank (Mantel Cox) test with p<0.0001. Average and STE for body temperature (B) over the course of infection with x-axis indicating time post infection. Statistical differences by one-way ANOVA followed by Tukey's posttest between clusters: a: susceptible versus resistant, b: susceptible versus resistant and intermediate, c: resistant versus susceptible and intermediate, and d: all three clusters differ. Luciferase expression of parasites in non-perfused organs was measured after dissection of organs from moribund mice. The luciferase expression per organ was expressed as the percentage of the total luciferase expression of all organs. C) Box and whisker plot with whiskers showing min to max relative luciferase expression for each organ. * indicates statistical significance of p<0.05 by one-way ANOVA analyses followed by Tukey's post test, ** indicates p between 0.01 and 0.001, *** p≤0.001.

Schizont distribution in organs.

Sequestration of infected erythrocytes in the microvasculature of organs is associated with malaria pathology in humans and mice [26]. To test for differences in sequestration patterns between various mouse strains a transgenic P. berghei line (pbGFP-LUCsch) was used that expresses a GFP-luciferase fusion protein under the control of the schizont-specific promoter from the ama1 gene of P. berghei [27]. Because the invading merozoite imports some of the fluorescence into the erythrocyte, young rings also show some luciferase expression. Fluorescent imaging of the whole body of a living mouse or dissected organs detects therefore schizonts, the sequestering parasite forms, as well as early ring stages. Over the course of infection whole body luciferase counts in individual mice increased as expected (Figure S1). Expression of luciferase in brain, lungs, spleen and liver was analyzed in non-perfused, dissected organs from mice that were moribund. Only about 40% of the mice were sacrificed and the number of analyzed mice varied therefore for each strain and we did not analyze gender differences.

The sums of the luciferase counts of all organs from single mice were averaged for each strain and compared between strains (Figure 1c). Only C58/J and C57BR/cdJ had significantly higher total luciferase counts than any other strain (For details see Table S2). The resistant cluster had significantly higher luciferase counts than the susceptible one (one-way ANOVA followed by Tukey-Kramer post-test with alpha = 0.05). Total luciferase counts of the organs are an indicator of total parasite load in the host.

Photon counts from intact organs were also expressed as the percentage of the total photon counts of all organs per mouse (relative luminescence, Figure 2c). There were statistically significant differences in luciferase expression per organ for the different survival clusters (Figure 2C) while only a few individual strains showed significant differences from the other strains (Table S2). Significantly higher levels of luciferase expression in the spleen were associated with susceptible strains while significantly higher expression in the liver was associated with resistance. For statistical details of the individual strains see Table S2. Overall the luciferase expression was lowest in the brain, with intermediate mice displaying higher luciferase expression than susceptible mice, and highest in the lungs with no statistical differences between the clusters. In agreement with the findings above, there was a moderate negative correlation between survival and relative luciferase expression in the spleen and a positive correlation between survival and relative luciferase expression in the liver (R 2 = 0.14 and R 2 = 0.18, respectively). Correlation for relative luciferase expression in the remaining organs compared to survival, and all organs compared to terminal body temperature was low (R 2 >0.06).

In comparing the outcome of all the different phenotypic analyses, survival clearly showed the most robust discrimination between the different strains and pedigree groups (Figure 1). Therefore, survival was used as trait in the following analyses of the underlying genotype.

Genome wide analyses identify a locus linked to host survival on chromosome 6

To link the survival phenotypes of the different inbred mouse strains to their genotype, two different genome wide analysis methods were used. The haplotype associated mapping (HAM) algorithm uses ANOVA to calculate the strength of genetic associations between an input phenotype and the ancestral haplotype structure (as inferred using a local window of three adjacent single nucleotide polymorphisms (SNPs) across the genome). A weighted bootstrap method is used to detect association peaks conditional on the population structure in the mouse diversity panel. At each genetic locus, the association score is represented as the negative log10-transformed P value. A score of –Log10P = 6 is a maximal score resultant from 10 6 permutations performed at each locus. HAM analysis was performed for the survival phenotype across 32 strains using 297,674 informative SNPs. A locus with a –Log10P score of 4.77 was identified on chromosome 6 (115458884–115531474bp, NCBI M36) (Figure 3). Even though this –Log10P score is very good for this locus, it does not reach genome wide significance due to the conservative algorithm applied and the large number of SNPs tested. This locus contained the 3′ untranslated region (UTR) of peroxisome proliferator-activated receptor γ (Ppar-γ, GeneID: 19016, MGI: 97747) and the 5′ UTR of tRNA splicing endonuclease 2 homolog (S. cerevisiae) (Tsen2, GeneID: 381802, MGI: 2141599). In agreement with the nomenclature of previously identified QTLs [17], [18], we termed this newly identified locus on chromosome 6 berghei resistance loci 6 (berr6).

A) A genome wide scan with the haplotype associated mapping (HAM) algorithm using survival as trait of mice from 32 different mouse strains infected with P. berghei identified a locus with -Log10P score of 4.77 on chromosome 6 (arrow). The chromosomes numbers are indicated on the x-axis and -Log10P scores on the y-axis. B) Magnification of the locus on chromosome 6 identified by HAM analysis shows the –Log10P scores (red line) and the underlying genes and their chromosome positions. Three different haplotypes consisting of a combination of three SNPs were detected at the locus with the maximal –Log10P value of 4.77 (See also Figure 4a). The average survival of each strain carrying haplotype 1, 2 or 3 (yellow, blue and pink, respectively) are indicated in C). The efficient mixed-model for association (EMMA) method confirmed the locus on chromosome 6 identified with the HAM analysis (D). The –Log10P score of 9.87 with a false discovery rate of q = 3.5×10 −5 is genome wide significant.

The locus identified with the HAM analysis was confirmed with the efficient mixed-model for association (EMMA) mapping model [25]. This modeling method uses a different algorithm for association of phenotype to genotype and essentially uses a single SNP association rather than an inferred haplotype structure. It also corrects for confounding factors like genetic relatedness and population structures by estimating the pair-wise relatedness between all individuals and fitting these to the vector of the phenotype thereby decreasing false positives and false negatives. The locus identified through EMMA at position 115494951 (–log10P = 9.87), overlapped with the locus identified with the HAM analysis. This p value showed genome wide significance with a q value of 3.5×10 −5 . The q value is an estimation of the false discovery rate and was calculated as previously described [28]. For the mouse strains used in our study, this locus accounts for 53% of the variance in survival observed in females and 62% in males. While we tested other phenotypes as well, only body temperature on day 5 of male mice reached genome wide significance with a peak on chromosome 4 (123836655bp, -Log10P = 12.39).

While not much in known of the function of Tsen2, Ppar-γ forms a nuclear receptor heterodimer together with γ–retinoic X receptor (reviewed in [29]). It regulates transcription of a number of genes involved in adipose differentiation and metabolism, insulin sensitivity, bodyweight regulation, atherosclerosis and inflammation (reviewed in [30]). Ppar-γ has already been linked to malaria infection. An agonist of Ppar-γ enhanced the clearance of iRBCs of P. falciparum in vitro and improved the survival rate of mice infected with P. berghei and reduced parasitemia in the P. chabaudi chabaudi model in a CD36 dependent manner [31].

We compared the available expression data for uninfected mouse strains from the BioGPS [32], [33] portal of Tsen2 and Ppar-γ from the spleen, liver, adipose tissue and hypothalamus with the haplotypes of the various mouse strains at the berr6 locus. Tsen2 was significantly higher expressed in adipose tissue and the spleen of mouse strains sharing haplotype three while haplotype one showed significantly lower expression than two in the liver. Ppar-γ showed only significant expression differences in the liver where haplotype three had significantly lower expression than haplotype two (Figure 4). There were no differences in the expression levels in the hypothalamus (data not shown).

The HAM analysis is based on the formation of haplotypes consisting of three consecutive SNPs. The compositions of haplotypes at different positions at the berr6 locus are indicated for each mouse strain (A). Each haplotype is indicated by a number and labeled in a different color. The mouse strains are listed according to their survival. –Log10P values for each position are indicated and the haplotypes with the highest score are framed. B) Expression patterns of Tsen2 (upper panel) and Ppar-γ (lower panel) from different mouse strains were compared in adipose tissue, spleen and liver. The relative expression levels (y-axis) for each mouse strain were grouped and colored according to their haplotype at the locus with the highest –Log10P value (A). Significantly different expression levels were detected in all tissues for Tsen2 but only in the liver for Ppar-γ. Scatter dot plots with the mean and the standard error for the expression of Tsen2 and Ppar-γ haplotypes. * indicates statistical significance of p<0.05 by one-way ANOVA analyses followed by Tukey's post test, ** indicates p between 0.01 and 0.001, *** p≤0.001.


Summary and conclusions

Despite their significant clinical relevance, cardiac manifestations have so far rarely been the focus of experimental studies into autoimmune disease. The available literature is sparse and often limited to the first description and basic phenotypic characterisation of the respective mouse model. This means that, for most models, despite being well-established for immunological research, the mechanisms underlying cardiac involvement are not yet understood. More basic research is needed to allow direct comparison between cardiac disease mechanisms in human patients and the respective model. However, due to significant progress in our understanding of the immuno-cardio crosstalk, access to this information is becoming important to allow researchers to choose suitable models. Besides the phenotypic information provided above, we recommend that researchers also consider the following general challenges that are involved in attempting to model human disease using mice.

Complex pathophysiology of systemic autoimmune disease

Faithful modelling of diseases with aetiologies and phenotypic manifestations as diverse as those observed in systemic autoimmunity is immensely challenging. Pathological pathways underlying organ damage in systemic autoimmunity are many-fold and interconnected, and identification of a single triggering event or molecular culprit has not been possible so far. SLE has in fact been suggested to encompass several different disease groups (Toro-Domínguez et al., 2018). This opens the possibility for patient stratification and targeted investigation of aetiology and mechanisms in specific patient groups. In the meantime, researchers commonly resort to models that phenocopy selected manifestations of disease, and the use of several different models in parallel is highly recommended.

Environmental factors

A common concern with mouse models is that laboratory mice are born and maintained in controlled conditions with little correspondence to the human environment (Sundberg and Schofield, 2018). This is particularly relevant for immunological studies, which are known to show strong sensitivity to changes in diet, the microbiota, or the absence or presence of environmental microbes (Mu et al., 2017 Johnson et al., 2015). Careful experimental design and confirmation experiments in separate animal facilities, which differ in standard diet and repertoire of environmental microbes, may help to avoid spurious results and limit this concern.

Genetic diversity

Current studies in mouse models rely heavily on inbred mouse strains however, these cannot mimic the genetically diverse human population. Yet, variable susceptibility based on genetic variation has emerged as a critical factor determining the risk of developing autoimmunity as well as determining the main target organ in systemic autoimmunity (Almlöf et al., 2017 Lanata et al., 2018). Importantly, mouse genetic diversity panels have been generated to overcome this limitation. For example, the Collaborative Cross mouse diversity panel has been obtained from systematically crossing eight inbred founder strains (A/J, C57BL/6J, 129S1/SvImJ, NOD/ShiLtJ, NZO/H1LtJ, CAST/EiJ, PWK/PhJ and WSB/EiJ) (Iraqi et al., 2012). They provide the opportunity to identify genetic influences on manifestations of autoimmunity.

Inter-species differences in basic physiology

While cardiovascular system proteins, including myosin and troponin, are highly conserved, the immune system is under high evolutionary pressure from pathogens (Fumagalli et al., 2011). Therefore, mouse and human immunological factors may differ, which may affect direct translatability from model to patients. However, while individual molecules may have changed throughout evolution, the overall function of cells and networks often remains the same (Monaco et al., 2015). Thus, a specific target identified in mouse models needs to be carefully validated in humans, but it is likely that the functional pathways involved are similar.


Substrains of inbred mice differ in their physical activity as a behavior.

Exercise adaptations result from a coordinated response of multiple organ systems, including cardiovascular, pulmonary, endocrine-metabolic, immunologic, and skeletal muscle, recently reviewed by Boveris andNavarro [1], by Freidenreich and Volek [2], and by Perrino et al. [3]. Exercise training has been suggested as a promising countermeasure to prevent several disease states and as a rehabilitation tool aimed to restore both muscle strength and endurance, depending on the type of exercise [4]. Regular resistance exercise combined with adequate protein intake to maintain muscle mass is proposed to counteract sarcopenic obesity in an aging global population, a major public health challenge [5]. For all the above, rodent models of caloric intake and exercise are widely used [6] and novel molecular mechanisms underlying the effects of physical activity have been recently brought to light [7, 8]. Nonetheless, the anatomy and physiology of rodents differ significantly from those of humans. While it appears clear that Homo sapiens has evolved to support the svelte phenotype of an endurance runner [9], a better understanding of similarities and differences between human and animal models is becoming of paramount importance for translating discoveries in preclinical models to clinical settings.

The two main types of contractile activity that are classified as low muscular tension development over an extended duration, or high-tension generation of limited duration, are characteristic of endurance and resistance exercise, respectively. The aforementioned adaptive responses at the whole body and cellular and molecular levels depend on the mode of exercise performed [10]. For instance, increased strength [1113], power [14], muscle cross-sectional area [15-17], RNA, and protein content [18] typically occur following resistance exercise training. Aerobic, endurance exercise training has been shown to enhance exercise capacity [19], augment maximal oxygen consumption [20], increase oxidative enzymes [21], and elevate mitochondrial content [22].

Several protocols of exercise training were developed for rodent models to mimic either resistance or endurance exercise. For instance, to climb a vertical ladder as a mode of progressive resistance exercise has been used for rats [23]. Recently, a very interesting equipment and system of resistance exercise, based on squat-type exercise for rodents, with control of training variables, has been validated [24]. The latter is based on a conditioning system composed of sound, light, and feeding devices, thus being not necessary to impose fasting or electric shock for the animal to perform the task proposed. Endurance exercise is based on more standardized protocols, basically running. The intensity-controlled treadmill exercise represents a well-characterized model of endurance exercise [25]. Slope and velocity of treadmill can be regulated and the animals are hosted in an enclosed chamber with a shock grid for motivating mice to run. One of its major advantages is the possibility of increasing time-wise exercise intensity, thus allowing the researcher to submit rodents to specific training programs. One of the drawbacks of treadmill is the fact that it may induce stress in the mice due to environmental, nonphysiological conditions. On the contrary, spontaneous exercise is often the favored type of exercise for experimental purposes since it is physiologic: it is performed at will, mostly during the nighttime it mimics natural behavior, such as intermittent locomotion, typical of wildtype rodents finally, it has been shown that such a voluntary activity is repeatable and stable within individual mice [26]. Hosting the mice in wheel-equipped cages, in which they exercise at will, classically induces such a spontaneous physical activity. A drawback of this approach is a certain degree of inter and intrapopulation variability, which makes absolutely necessary to individually monitor running activity by tachometers.

Small genetic differences may have a great influence on behavioral phenotypes [27]. Thus, the genetic background of different substrains should be carefully chosen, equated, and considered in the interpretation of mutant behavioral phenotypes. To this purpose, Knab et al. assessed the repeatability of a commonly used maximal exercise endurance treadmill test as well as voluntary physical activity measured by wheel running in mice: they found no significant differences in exercise endurance between different cohorts of BALB/c J and DBA/2 J mice indicating strains overall generally test the same [26]. Both strains are inbred mice that is, populations that are nearly identical to each other in genotype due to long inbreeding. The usual procedure is mating of brother-sister pairs for 20 generations, which will result in lines that are roughly 98% genetically identical. Indeed, inbred strains of animals are frequently used in laboratories for experiments where for reproducibility of conclusions, all the test animals should be as similar as possible.

BALB/c are an inbred strain of mice distributed globally and are among the most widely used inbred strains. The founding animals of the strain (the "Bagg albino") were obtained by Halsey J. Bagg of Memorial Hospital, NY, from a mouse dealer in Ohio in 1913. By 1935, the animals were in the possession of Muller's student, George Davis Snell, who moved them to The Jackson Laboratory. This stock provided the basis of all the BALB/c substrains that are now in use around the world. BALB/c ByJ (Jackson mice, donated to Jackson labs by Bailey J., in 1974) was separated from the BALB/c J strain in 1935. BALB/c ByJ mice have the advantage of better reproductive performance and less aggressiveness than the BALB/c J substrain and pose many other differences with the J substrain. Between the fifties and seventies, a third substrain got separated from the above-mentioned first two substrains, that is, the J and the ByJ: the Charles River AnNCrl (to Andervont in 1935 to NIH in 1951 from Andervont at F72 to Charles River in 1974 from NIH).

The three BALB/c substrains have been kept separated over decades and could have diverged to such an extent to develop sufficient genetic differences to account for behavioral differences among substrains, while remaining homogeneous within the same population. Mice may significantly differ for what concerns their physical activity as a behavior, which is of pivotal importance for the reproducibility and significance of studies exploiting exercise models. These differences may appear easily accountable when dealing with animals of different sexes or strains. However, we wondered whether even very fine differences (such as those distinguishing murine substrains of a single inbred strain) are able to determine significant behavioral differences. For this reason, we compared the physical activity behavior of the AnNCrl, the ByJ, and the J BALB/c mice and found striking differences concerning their willingness to run when hosted in wheel-equipped cages. Our findings have important experimental consequences with relevant economical and scientific fallouts.

2.1. Mice. Mice were generously provided by Janvier (Le Genest Saint Isle, St Berthevin Cedex, France). Throughout the study we used 7-week-old BALB/c mice of the following substrains: AnNCrl, ByJ, and J. We used a total of 21, 12, and 12 female mice of the substrains AnNCrl, ByJ, and J, respectively. Mice were allowed to settle in the animal facility for one day and then transferred to wheel equipped cages. Cachexia was induced by subcutaneous grafting, using a trocar of a 0.5 [mm.sup.3] fragment of colon carcinoma (C26, obtained from the National Cancer Institute) in the dorsal region as previously described [28]. Mice were hosted in standard conditions with day/night cycles of 12 hours and food ad libitum. Mice were treated in strict accordance to the guidelines of the Institutional Animal Care and Use Committee and to national and European legislation, throughout the experiments.

2.2. Cages. Cages were purchased from Animal Care System (Centennal, CO). The wheels, structured as a circular open ladder with a diameter of 15 cm, were purchased as wheels for rodents in general customer pet shops. The tachometers, either model DC-4 or DC-9, were purchased from Decathlon. Readings were recorded every morning, before 10 am. Sporadic events of day running activitywere observed. A Kleenex was introduced into the cage as material for the construction of a nest, with the aim to reduce stress due to being isolated (one animal per cage).

2.3. Tissue Immunohistochemical Analysis. NADH transferase staining was performed as described previously [28]. Morphometric analysis was performed on type IIB (low NADH transferase activity, glycolytic), type IIA/X (medium NADH transferase activity, intermediate), and type I (high NADH transferase activity, oxidative) fibers separately. For each muscle, the whole muscle cross-section was analyzed to calculate the percentage of each fiber type by using ImageJ 1.41 (freeware developed by Dr. W. Rasband at NIH, and available at http://rsb.info.nih.gov/ij/). The fibers with a medium and a high content in mitochondria were pooled and collectively considered as NADH transferase+ fibers, that is, oxidative. Photomicrographs were obtained by means of an Axioskop 2 plus system (Zeiss, Oberkochen, GE) or a Leica Leitz DMRB microscope fitted with a DFC300FX camera (Leica, Wetzlar, Germany).

2.4. Statistical Analysis. One-way or two-way analysis of variance (ANOVA) was used for one or two variate analysis, respectively. Either the Tukey LSD test or Student's f-test was used for the post hoc comparisons between specific groups, as indicated. The significance levels for these tests were set at a P < 0.05 or P < 0.01, as specified. Point and interval were estimated at the 95% confidence level. Statistical analyses were performed by using VassarStats, the website for statistical computation freely available at http://vassarstats.net/.

Mice of three BALB/csubstrains, that is, the AnNCrl, the ByJ, and the J, were obtained by the same vendor and hosted at the same time in the same animal facility. Each wheel-equipped cage was used for a single mouse, whose spontaneous wheel running activity was recorded by a commercial tachometer. The distance run over a period of four days was recorded daily the average Km/day over such period of time was considered to minimize daily variations. Mice clearly divide into two populations of runners and nonrunners, the latter totally ignoring the wheel as such and showing no interest in wheel running at all. The threshold to define a runner was set to 1 Km of distance run on the wheel over the 4-day period of time. Several rounds of independent experiments, involving 6 mice for each of the three substrains, were repeated and the percentage of runners for each substrain in each experiment was assessed. In this way, an average percentage of runners in a given experiment and its associated SEM were calculated as a function of the substrain. We found that the runners were 85.1 [+ or -] 3.4%, 72.0 [+ or -] 3.0%, and 38.2 [+ or -] 7.7% of the AnNCrl, the ByJ, and the J, respectively (Figure 1(a)). The 95% confidence intervals were found to be 76.9-93.2%, 59.1-84.9%, and 18.5-57%, respectively. One-way ANOVA (F = 21.61 P < 0.0001) demonstrated a significant dependence of the percentage of runners on the substrain, with the BALB/c J running showing a significantly lower number of runners as compared to the other two substrains by Tukey's HSD post hoc test. Considering only the population of runners, we then assessed the distance run daily on the wheel by representatives of the three substrains. We found that the mice run 5.0 [+ or -] 0.3 Km/d, 4.7 [+ or -] 1.4 Km/d, and 3.7 [+ or -] 0.6 Km/d for the AnNCrl, the ByJ, and the J substrain, respectively (Figure 1(b)). The 95% confidence intervals were found to be 4.1-5.8 Km/d, 3.3--6.1 Km/d, and 2.2--5.0 Km/d, respectively. While one-way ANOVA (F = 1.88 P = 0.167) failed to demonstrate a dependence of the observed trend in the daily run distance on the basis of the substrain, we tentatively used the Student's f-test to statistically explore the difference shown by the J substrain and found that is significantly lower as compared to the AnNCrl substrain. In summary, we observed that the majority of the BALB/c J mice do not spontaneously run on a wheel, differently from the BALB/c AnNCrl and the BALB/c ByJ, the vast majority of which is willing to run. Moreover, even when the BALB/c J do run, they cover on average a smaller distance as compared to the BALB/cAnNCrl and the BALB/c ByJ mice. We concluded that the BALB/c AnNCrl is the best substrain for studies involving spontaneous physical activity, such as wheel running. For this reason, this substrain has been used throughout the rest of the study.

With the aim to assess whether the observed wheel running activity displayed features of exercise training and whether the 5 Km/day represented the upper limit of physical activity for BALB/c mice, we recorded the kinetics of mouse wheel running over almost three weeks. For this set of experiments, we decided to use the AnNCrl substrain of BALB/c mice, since these behaved as the most active mice. We remarked that mouse running behavior is biphasic, with a first week spent to familiarize with the wheel, with an outstanding (for the size of the animals), yet moderate, daily distance if compared to the second and third week of activity, in which the daily distance covered by the mice reaches a plateau that it is more than twice the initial Km/d (corresponding to more than 11 Km/days, Figure 2). In this context, we also wondered whether mice were able to perform voluntary physical activity in pathological conditions, such as cancer-induced cachexia [29]. Thus, we recorded the daily running activity of C26 colon carcinoma-bearing mice, which develop a progressive and severe form of muscle wasting associated to weakness and fatigue [28]. C26-bearing mice run about the same Km/d as controls in the first week of activity, when the tumor size is still negligible however, they do not show the same progressive increase in the distance run on the wheel as the controls in the second week of activity. With the disease progression and overt cachexia, they keep running for about 7 Km/d, which is a striking amount of exercise, considering that tumor-bearing mice lose about 25% of the body weight in three weeks. Two-way ANOVA calculated over the last four days of the recordings (i.e., from day 15 to day 18) shows that only the presence of the tumor significantly affects the running behavior, with no interference with time (F = 19.74 P < 0.0001 Tukey's HSD P < 0.01 for control versus C26 bearing). These data indicate that the BALB/c AnNCrl mice not only spontaneously run several Km per day but also have the tendency to progressively increase the distance covered on the wheel. Mice bearing a tumor, a condition which deeply affects muscle mass and function, are still capable of performing a significant amount of physical activity, albeit to a lesser extent than healthy mice. This indicated that wheel running could be exploited as a model for endurance exercise intervention in pathological settings in rodents.

Endurance training activates metabolic pathways and remodeling in skeletal muscle. A prominent feature of this type of exercise is the stimulation of Krebs' cycle and the mitochondriogenesis in muscle fibers. Since in BALB/c AnNCrl mice we observed a spontaneous, yet significant, increase in physical activity during the second week of permanence in wheel-equipped cages, we hypothesized that the increased performance was associated to metabolic changes making the musculature adapted to endurance exercise. Thus, we performed a histochemical analysis of the skeletal muscle of BALB/c AnNCrl mice following two different periods of wheel running, that is, five and nineteen days, to monitor phenomena of fiber conversion to a more oxidative phenotype upon exercise. By NADH transferase staining on the Tibialis anterior (TA) muscle (Figures 3(a) and 3(b)), we highlighted the fibers rich in mitochondria (oxidative and intermediate fibers, typically corresponding to type I and IIA or X). The TA was chosen for its mixed population of fiber types (all types are represented), which appeared particularly suitable for studying shifts in fiber type. While we observed an increase in the number of NADH transferase+ fibers from 64 [+ or -] 5% to 72 [+ or -] 3% following nineteen days of exercise, one-way ANOVA (F = 2.39 P = 0.162) showed the lack of a statistically significant effect by exercise on this parameter (Figure 3(c)).

We have shown that three substrains of the same inbred mice, namely, the BALB/c AnNCrl, ByJ, and J, display striking differences in their behavior concerning spontaneous physical activity. This phenomenon was observed in spite of the fact that the three substrains display remarkable genetic similarities, exemplified by the absence of histocompatibility barriers. The fact that the vast majority of the AnNCrl and, to a lesser extent, of the ByJ mice run for several Km per day distinguishes these two substrains from the J mice. To our knowledge, this is the first paper on significant behavioral differences among substrains of the same inbred mouse strain. Researchers planning to perform experiments requiring wheel running should be aware of these unexpected findings. In fact, the selection of the J substrain, which is less prone to wheel running, would determine a very little amount of exercise performed by a limited number of mice. Interestingly, the J substrain is the cheapest (at least with the vendor used for this study) however, if a relevant number of animals are excluded from a study since they do not exercise, the costs must be recalculated. Researchers that, for an experimental reason, need to use the J substrain and that absolutely require that they exercise by wheel running may consider selecting the mice in preliminary experiments according to their running behavior to sort the runners from the nonrunners in advance. Otherwise, researchers interested in using BALB/c mice for exercise-related experiments may simply want to choose either the AnNCrl or the ByJ substrain.

The idea that different mice strain behave differently about wheel running is not new, but, again, our study shows that even very fine differences (such as those distinguishing substrains) are able to determine significant behavioral differences. Several studies associated genetic influence with physical activity, but animal studies were often conducted with only one sex or a limited number of strains, thus reducing the genomic coverage and generality of the result that Lightfoot et al. clearly showed that physical activity as a behavior has a genetic basis [30]. Their results suggest that potential genetic mechanisms arising from traditional noncoding regions of the genome may be involved in regulation of physical activity [30]. Of course, other studies clearly show that mouse substrains differ for several features other than physical activity as a behavior. For instance, it has been shown that the genetic background of the four different mouse substrains affects their vulnerability to cope with environmental challenges, such as exposure to novelty the authors consistently suggest considering substrain-specific guidelines and protocols, taking the substrain-specific adaptive capabilities into account [31]. We totally agree with the authors of this study. Another intriguing study showed that two substrains of BALB/c mice, the BALB/cByJ and the BALB/cAnNCr, are resistant and susceptible, respectively, to Theiler's murine encephalomyelitis, a virus-induced demyelinating disease [32]. The fact that the two substrains are histocompatible makes them a nice model for studying mechanisms of virus infection, since they permit the transfer of cells between naturally resistant and naturally susceptible mice in the absence of immunodepression. Similarly, one could foresee the possibility of satellite cell grafts among different BALB/c substrains to verify whether fine genetic differences are responsible for differential muscle stem cell features, such as their capability of being engrafted into regenerating muscles one could even wonder whether satellite cells from spontaneous runners could donate to the derived muscle fibers intrinsic mechanical or contractile properties more suitable for running.

Our study is in agreement with previous results showing that mice spontaneously run for 5 to 10 Km per day [30]. This incredible level of voluntary activity is an important fact to keep in mind and represents an outstanding feat for such a diminutive species. It has been stated that "such distances covered daily by us much larger humans would probably cure most of the epidemic diseases facing the world, including obesity and type 2 diabetes" [33].

Running is associated with distinct metabolic adaptations of the skeletal muscle [34, 35]. In particular, it has been reported that voluntary running exercise induces a steady increase in the percentage of NADH transferase-positive fibers in the TA muscle, which was significant after 4 weeks of voluntary exercise [36]. We obtained very similar results in terms of exercise effect on the percentage of NADH transferase+ fibers, with the exception that we failed to demonstrate the significancy of such an effect. This may be due to several differences between the two studies: the mouse strain (BALB/c, C57/Bl6), the sex (female, male), the distance (5 Km/day, 6.8 Km/day on average), and the time frame (3 weeks, 4 weeks) each of these differences could be sufficient to explain why we could not observe a statistically significant increase in the oxidative fibers of exercise mice. The observed trend is consistent with an increased demand of muscle oxidative capacity suggesting that endurance exercise invariably affects muscle metabolism by favoring the oxidative muscle fiber phenotype.

Finally, it should be noted that the therapeutics and ergogenic effects of controlled exercised as opposed to spontaneous exercise may differ significantly in rodents. Intriguingly, the two types of exercise may have very different outputs depending on the targeted organ. For example, voluntary activity causes a more evident plastic changes in the hippocampal formation of rat than that one induced by forced exercise [37].

Recognizing the proven benefits of exercise training on health outcomes and the trend towards increasing inactivity at the population level has made recommending exercise a directive of paramount importance. In parallel, studies on organismal and muscle-specific adaptations to increased physical activity steadily increase over time, as shown by the trend in PubMed citations with the keywords exercise and endurance/resistance. With such a proliferation of animal and experimental models dedicated to exercise, it is important to clearly define the major features of the experimental models used in a given study and to be very formal in assessing to which extent generalization of the results can be driven. We report here that three substrains of the same inbred mouse strain, the BALB/c, display significant differences in physical activity as a behavior. We propose that not only the strain of mice used but also the substrain must be clearly specified and chosen consciously, since the differences in spontaneous physical activity between substrains can impact exercise-induced muscle adaptations.

D. Coletti is supported by UPMC Emergence 2011 and by AFM 2012. Z. Li is supported by ANR and by AFM. PRIN 2009 (Project no. 2009WBFZYM_001) and Italian Space Agency (ASI), OSMA project, grant to S. Adamo are also acknowledged. The authors are indebted to Carla Ramina for her precious technical assistance. They gratefully thank Richard Lowry, Ph.D., Professor of Psychology Emeritus at the Vassar College for his web-based, user-friendly tool for performing statistical computation, VassarStats, which They used for statistical analysis. The mice used throughout this study were a generous gift of Janvier SAS (Le Genest St Isle, St Berthevin Cedex, France).

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Dario Coletti, (1,2,3) Emanuele Berardi, (4) Paola Aulino, (1,2,3) Eleonora Rossi, (2,3) Viviana Moresi, (2,3) Zhenlin Li, (1) and Sergio Adamo (2,3)

(1) UR4 Aging, Stress, Inflammation, University Pierre et Marie Curie Paris 6, 7 Quai Saint Bernard, 75005 Paris, France

(2) Department of Anatomical, Histological, Forensic & Orthopaedic Sciences, Section of Histology & Medical Embryology, Sapienza University of Rome, Via Scarpa 16, 00161 Rome, Italy

(3) Interuniversity Institute of Myology, 00161 Rome, Italy

(4) Laboratory of Translational Cardiomyology, Department of Development and Regeneration, Katholieke Universiteit Leuven, 3000 Leuven, Belgium

Correspondence should be addressed to Dario Coletti [email protected]

Received 30 December 2012 Accepted 4 February 2013

Academic Editors: L. Guimaraes-Ferreira, H. Nicastro, J. Wilson, and N. E. Zanchi


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Rodents (1996)

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Criteria for Selecting Experimental Animals SPECIES AND STOCKS Choosing a Species for Study For a scientific investigation to have the best chance of yielding useful results, all aspects of the experimental protocol should be carefully planned. If animal models will be used, an important part of the process is to con- sider whether nonanimal approaches exist. If, after careful deliberation and review of the existing literature, the investigator is satisfied that there are no suitable alternatives to the use of live animals for the study in question, the next question that should be addressed is what species would be most appropriate to use. In choosing a species for study, it is important to weigh a variety of scientific and operational factors, including the following: . In which species is the physiologic, metabolic, behavioral, or dis- ease process to be studied most similar to that of humans or other animals to which the results of the studies will be applied? · Do other species possess biologic or behavioral characteristics that make them more suitable for the planned studies (e.g., generation time and availability'? · Does a critical review of the scientific literature indicate which species has provided the best, most applicable historical data? 16

CRITERIA FOR SELECTING EXPERIMENTAL ANIMALS 17 · Do any features of a particular species or strain including ana- tomic, physiologic, immunologic, or metabolic characteristics render it in- appropriate for the proposed study? · In light of the methods to be used in the study, would any physical or behavioral characteristics of a particular species make the required physical manipulation or sampling procedures impossible, subject to unpredictable failure, or difficult to apply? . Does the proposed study require animals that are highly standard- ized either genetically or microbiologically? Those and other considerations often lead to the selection of a laboratory rodent species as the most appropriate model for a biomedical research protocol. Rodents are generally easy to obtain and relatively inexpensive to acquire and maintain. Other advantages of laboratory rodents as research models include small size, short generation time, and availability of micro- biologically and genetically defined animals, historical control data, and well-documented information on ohvsiolo

Pathologic, and metabolic ,,_^,

7 rip processes. The order Rodentia encompasses many species. The most commonly used rodents are laboratory micel, laboratory rats (Rattus norvegicus), guinea pigs (Cavia porcellus), Syrian hamsters (Mesocricetus auratus), and gerbils (Meriones unguiculatus). All those rodents have been extensively studied in the laboratory, and information about them can be found in the peer- reviewed literature and in a number of texts (e.g., Altman and Katz, 1979a,b Baker et al., 1979-1980 Foster et al., 1981-1983 Fox et al., 1984 Gill et al., 1989 Darkness and Wagner, 1989 Van Hoosier and McPherson, 1987 Wagner and Manning, 19761. Rodent Stocks The same factors used in selecting a species for study can be used in selecting a rodent stock. Rodents have been maintained in the laboratory environment for more than 100 years. Some, such as the mouse, have been very well characterized genetically and have undergone genetic manipula- tion to produce animals with uniformly heritable phenotypes. A hallmark of good scientific method is reproducibility, which is accomplished by minimizing and controlling extraneous variables that can alter research results. In stud- ies that are mechanistic, genetic uniformity is highly desirable. In contrast, genetic uniformity might be undesirable in studies that explore the diversity 1 laboratory mice are neither pure Mus domesticus nor pure Mus musculus therefore, geneticists have determined that there is no appropriate scientific name (International Commit- tee on Standardized Genetic Nomenclature for Mice, 1994a).

18 RODENTS: LABORATORY ANIMAL MANAGEMENT of application of a phenomenon over a range of phenotypes, such as prod- uct-registration studies, including safety evaluation of compounds that have therapeutic potential. In many such studies, a varied genetic background might be appropriate, as long as the range of variation can be characterized and is to some degree reproducible (Gill, 19801. Genetically Defined Stocks Inbred Strains. The mating of any related animals will result in inbreed- ing, but the most common and efficacious method for establishing and main- taining an inbred strain is brother x sister (i.e., full-sib) mating in each genera- tion. Full-sib inbreeding for 20 generations will result in more than 98 percent genetic homogeneity, at which point the members of the stock are isogenic, and the stock is considered an inbred strain. Many inbred strains of mice and rats have been developed (Festing, 1989 Festing and Greenhouse, 1992), and they are widely used in biomedical research. Many of the commonly used strains have been inbred for over 200 generations. A few inbred strains of guinea pigs, Syrian hamsters, and gerbils have also been developed (Altman and Katz, 1979b Festing, 1993 Hansen et al., 1981

. The isogeneity of the members of an inbred strain provides a powerful research tool. Although some genes might remain heterogeneous, most metabolic or physiologic processes, as well as their phenotypic expression, will be identical among individuals of an inbred strain, thereby eliminating a source of experimental variation. Isogeneity also allows exchange of tissue between individuals of an inbred strain without rejection. F1 Hybrids. F1 hybrid animals are the first filial generation (the F1 generation) of a cross between two inbred strains. They are often more hardy than animals from either of the parental strains, having what is called hybrid vigor. F1 hybrids are heterozygous at all genetic loci at which the parental strains differ nevertheless, they are uniformly heterozygous. Be- cause of the heterogeneity, F1 hybrids will not breed true to produce them one must always cross animals of the parental inbred strains. Reciprocal hybrids are developed by reversing the strains from which the dam and the sire are taken. Reciprocal male hybrids will have Y-chromosome differ- ences. Reciprocal female hybrids will have identical genotypes but might have differences caused by inherited maternal effects. F1 hybrids will ac- cept tissue from either parental strain, except in the case of a Y-chromo- some incompatibility (e.g., a skin graft from a male of either parental strain will be rejected by a female F1 hybrid). Special Genetic Stocks. The effects of specific genes or chromosomal regions can be studied by using various breeding or gene manipulation

CRITERIA FOR SELECTING EXPERIMENTAL ANIMALS 19 methods to create a new strain that differs from the original strain by as little as a single gene. · A segregating inbred strain is an inbred strain maintained by full- sib matings however, male-female pairs are selected for mating so that one pair of genes will remain heterozygous from generation to generation. This method of mating permits well-controlled experiments because a single sibship contains both carriers and noncarriers of the gene of interest, and all the animals are essentially identical except for that gene. · A coisogenic strain is an inbred strain in which a single-gene muta- tion has occurred and has been preserved it is otherwise identical with the nonmutant parental strain. If the mutation is not deleterious when homozy- gous, the strain can be maintained by simple full-sib matings. If the muta- tion adversely affects breeding performance, the coisogenic strain can be maintained by one of several special breeding systems (Green, 1981 NRC, 1989

. To avoid subline divergence between the coisogenic strain and the nonmutant parental inbred strain, periodic back-cro$sing (see next para- graph) with the parental strain is recommended. · A congenic strain is a close approximation to a coisogenic strain. It is created by mating an individual that carries a gene of interest, called the differential gene, with an individual of a standard inbred strain. An offspring that carries the differential gene is mated to another individual of the same inbred strain. This type of mating, called back-crossing, is contin- ued for at least 10 generations to produce a congenic strain. Back-crossing for 10 generations minimizes the number of introduced genes other than the differential gene and its closely linked genes. Details on developing congenic strains have been published (Bailey, 1981 Green, 1981

. Both coisogenic and congenic strains can be maintained by full-sib matings if the differen- tial gene is homozygous however, to avoid subline divergence between the congenic strain and the standard inbred strain, periodic back-crossing with the standard strain is recommended. . A transgenic strain is similar to a coisogenic or congenic strain in that it carries a segment of genetic information not native to the strain or individual (Hogan et al., 1986 Merlino, 1991

. The introduced genetic mate rial can be from the same or another species. Transgenic animals are de scribed in more detail in Chapter 8. . Recombinant inbred (RI) strains are sets of inbred strains produced primarily to study genetic linkage. Each RI strain is derived from a cross between two standard inbred strains. Animals from the F1 generation are then bred to produce the second filial generation (the F2 generation), mem- bers of which are randomly selected and mated to produce a series of RI lines. Members of the F2 generation are used to found RI lines because, unlike the F1 generation, they are not isogenic. The mice derived from any parental pair will be genetically homogeneous when inbreeding is complete

20 RODENTS: LABORATORY ANIMAL MANAGEMENT however, each line in a set will be homozygous for a given combination of alleles originating from the two parental inbred strains. Alleles that are linked in the parental strains will tend to remain together in the RI lines this is the basis for their use in genetic-mapping studies. · Recombinant congenic strains are like recombinant inbred strains except that each strain of a series has been derived from a back-cross in- stead of an F2 cross (Demant, 1986

. The number of back-crosses made before full-sib inbreeding is started determines the proportion of genes from each of the parental inbred strains. Series of recombinant congenic strains are particularly useful in the genetic analysis of multiple-gene systems, such as that responsible for cancer susceptibility. Nongenetically Defined Stocks The terms noninbred, random-mated, and outbred are all used to refer to populations of animals in which, theoretically, there is no genetic uniformity between individuals. Nongenetically defined stocks make up the majority of rodents used in biomedical research and testing, and they are generally less expensive and more readily available than genetically defined stocks. Noninbred refers to a population of animals in which no purposeful inbreeding system has been established. Random-mated refers to a group of animals in which the selection of breeding animals is random. It assumes an almost infinite population with no external selection pressures. In prac- tice, such a colony probably does not exist. Outbred refers to a colony in which breeding is accomplished by a purposeful scheme that minimizes or eliminates inbreeding. Animals produced by these breeding systems have varied genotypes, and characterizing the range and distribution of pheno- types requires a large sample of the population. The degree of heterozygosity in any nongenetically defined stock is continuously varying, so two populations developed from the same parental stock will show differing degrees of heterozygosity at any loci at any time. Spontaneous mutations can occur and become fixed because no purposeful selection is imposed on the population to eliminate the mutant genes. Out- bred populations are always evolving and therefore are more variable than inbred strains. For that reason, large sample numbers are needed to account for phenotypic variation that could have an impact on the charactersitics being studied. If outbred animals are used, treatment and control groups in a study will not necessarily be identical, nor will the population of animals necessarily be identical if the study is repeated. The genetic variation in outbred stocks, which can be magnified by sampling error, can make results from different laboratories difficult to compare. Background data on stock characteristics will vary over time, so concurrent controls are needed to allow useful interpretation of data.

CRITERIA FOR SELECTING EXPERIMENTAL ANIMALS STANDARDIZED NOMENCLATURE FOR RODENTS 21 Standardized nomenclature allows scientists to communicate briefly and precisely the genetics of their research animals. The International Commit- tee on Standardized Genetic Nomenclature for Mice and the International Rat Genetic Nomenclature Committee, which are affiliated with the Interna- tional Council for Laboratory Animal Science, are responsible for maintain- ing the nomenclatures for genetically defined mice and rats, respectively, and modifying them as necessary. The sections below briefly describe the nomenclature for inbred, mutant, and outbred mice and rats. The complete rules for mice can be found in the third edition of Genetic Variants and Strains of the Laboratory Mouse (Lyon and Searle, in press). Those rules are regularly updated, and updates are published in Mouse Genome (for- merly called Mouse News Letter Oxford University Press) and are available on-line in MOD, the Mouse Genome Database. Information on MOD can be obtained from the Mouse Genome Informatics Group, The Jackson Laboratory, Bar Harbor, ME 04609 (telephone, 207-288-3371 fax, 207-288-5079 Internet, [email protected]). The rules for rats have been published as an appendix to the report Definition, Nomenclature, and Conservation of Rat Strains (NRC, 1992a), and updates will be published in Rat Genome, Heinz W. Kunz, Ph.D., editor, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261. Investigators using other labo- ratory rodents should follow the rules for mice or rats. Inbred Strains An inbred strain is designated by capital letters (e.g., mouse strains AKR and CBA and rat strains BN and LEW). The mouse rules, but not the rat rules, allow the use of a combination of letters and numbers, beginning with a letter (e.g., C3H), although this type of symbol is considered less desirable. Brief symbols (generally one to four letters) are preferred. Ex- ceptions are allowed for strains that are already widely known by designa- tions that do not conform (e.g., mouse strains I01 and 129 and rat strains F344 and DONRYU). Substrains An established strain is considered to have divided into substrains when genetic differences are known or suspected to have become established in separate branches. These differences can arise either from residual het- erozygosity at the time of branching or from new mutations. A substrain is designated by the full strain designation of the parent strain followed by a slanted line (slash) and an appropriate substrain symbol, as follows:

22 RODENTS: LABORATORYANIMAL MANAGEMENT · Mice. The substrain symbol can be a number (e.g., DBA/1 and DBA/ 2

a laboratory code, which is defined below (e.g., C3H/lIe, where He is the laboratory code for Walter E. Heston) or, when one investigator or laboratory originates more than one substrain, a combination of a number and a laboratory code, beginning with a number (e.g., C57BL/6J and C57BL/ lOJ, where J is the laboratory code for the Jackson Laboratory, Bar Harbor, Maine). Exceptions, such as lower-case letters, are allowed for already well-known substrains (e.g., BALB/c and C57BR/cd). · Rats. The substrain symbol is always a number when genetic differ- ences have been demonstrated. The founding strain is considered the first substrain, and the use of /1 for it is optional (e.g., KGH or KGH/11. A laboratory code (e.g., Pit for the University of Pittsburgh Department of Pathology and N for the NIH Genetic Resource) is used to designate a substrain when genetic differences are probable but not demonstrated (e.g., BN/Pit and BN/N). Laboratory Codes Each laboratory or institution that breeds rodents should have a laboratory code. The registry of laboratory codes is maintained by ILAR, National Re- search Council, 2101 Constitution Avenue, Washington, DC 20418 (telephone, 202-334-2590 fax, 202-334-1687 URL:http://www2.nas.edulilarhome/

. The laboratory code, which can be used for all laboratory rodents, consists of either a single roman capital letter or an initial roman capital letter and one to three lower-case letters. . Mice. A particular colony is indicated by appending an [email protected]" sign and the laboratory code to the end of the strain or substrain symbol (e.g., [email protected], the colony of strain SJL mice bred at the Jackson Laboratory C3H/ [email protected], the He substrain of strain C3H bred at the NIH Genetic Resource and CBA/[email protected], the Ca substrain of strain CBA carrying the se mutation and bred at the Jackson Laboratory). If the substrain symbol and laboratory code are the same, the @ symbol and the laboratory code can be dropped for simplicity (e.g., SJL/[email protected] becomes SJL/J). The laboratory code is al- ways the last symbol used and is meant to indicate that the environmental conditions and previous history of a colony are unique. When a strain is transferred to a new laboratory, the laboratory code of the originating labo ratory is dropped, and the code of the recipient is appended laboratory codes are not accumulated. . Rats. Normally, a rat strain is designated by the strain name, a slash, the substrain designation (if any), and the laboratory code (e.g., BN/ [Pit). When a strain is established in another laboratory, the new labora- tory code is appended (e.g., BN/lPitN). In general, more than two labora- tory codes are not accumulated. Intermediate codes are dropped to avoid excessively long designations.

CRITERIA FOR SELECTING EXPERIMENTAL ANIMALS 23 For both mice and rats, a strain's holder is responsible for maintaining a strain history. F1 Hybrids An F1 hybrid is designated by the full strain designation of the female parent, a multiplication sign, the full strain designation of the male parent, and F1 (e.g., the hybrid mouse C57BL/6J x DBA/2J Fl and the hybrid rat F344/NNia x BN/RijNia Fig. If there is any chance of confusion, parenthe- ses should be used to enclose the parental strain names te.g., (C57BL/6J x DBA/21)F1 and (F344/NNia x BN/RijNia)Fl]. The correct formal name should be given the first time the hybrid is mentioned in a publication an abbreviated name can be used subsequently [e.g., C57BL/6J x DBA/2J F1 (hereafter called B6D2F1) and F344/NNia x BN/RijNia F1 (hereafter called FBNF1

. Coisogenic, Congenic, and Segregating Inbred Strains In mice, a coisogenic strain is designated by the strain symbol, the substrain symbol (if any), a hyphen, and the gene symbol in italics (e.g., CBA/H-kd). When the mutant or introduced gene is maintained in the heterozygous condition, this is indicated by including a slash and a plus sign in the symbol (e.g., CBA/H-kdl+

. A congenic strain is designated by the full or abbreviated symbol of the background strain, a period, an abbre- viated symbol of the donor strain, a hyphen, and the symbol of the differen- tial locus and allele (e.g., B10.129-H12b). Segregating inbred strains are designated like coisogenic strains however, indication of the segregating locus is optional when it is part of the standard genotype of the strain (e.g., 129/J and 129/J-CCh/C mean the same thing, and either can be used). In rats, a coisogenic strain (except for alloantigenic systems see NRC, 1992a) is designated like a coisogenic strain in mice, except that the labora- tory code follows the substrain symbol and the gene symbol is not italicized (e.g., RCS/SidN-rdy). A con

enic rat strain (except for alloantigenic sys _ _

ke a coisogenic strain (e.g., LEW/N-rnu). For segre gating inbred strains developed by inbreeding with forced heterozygosis, indication of the segregating locus is optional. Recombinant Inbred (RI) Strains The symbol of an RI strain should consist of an abbreviation of both parental-strain symbols separated by a capital X with no intervening spaces (e.g., CXB for an RI strain developed from a cross of BALB/c and C57BL mouse strains and LXB for an RI strain developed from a cross of LEW and BN rat strains). Different RI strains in a series should be distinguished by numbers (e.g., CXB1 and CXB2 in mice and LXB1 and LXB2 in rats).

24 RODENTS: LABORATORYANIMAL MANAGEMENT Genes The rules for gene nomenclature are very complicated because they apply not only to mutant genes, but also to gene complexes, biochemical variants, and other special classes of genes (e.g., transgenes). This descrip- tion will cover only a small portion of the gene nomenclature. The full rules can be found in the references given previously. The symbols for loci are brief and are chosen to convey as accurately as possible the characteristic by which the gene is usually recognized (e.g., coat color, a morphologic effect, a change in an enzyme or other protein, or resemblance to a human disease). Symbols for loci are typically two- to four-letter abbreviations of the name. For mice, the symbols are written in italics for rats, they are not. For convenience in alphabetical lists, the initial letter of the name is usually the same as the initial letter of the symbol. Arabic numbers are included for proteins in which a number is part of the recognized name or abbreviation (e.g., in mice, C4 and C6, the fourth and sixth components of complement, respectively in rats, C4 and C63. Except in the case of loci discovered because of a recessive mutation, the initial letter of the locus symbol is capitalized and all other letters are lower-case. Hyphens are used in gene symbols only to separate characters that together might be confusing. This rule was adopted for mice in 1993, and hyphens should be deleted from all gene symbols except where they are necessary to avoid confusion. Gene designations are appended to the desig- nation of the parental strain, and they are separated by a hyphen. Loci That Are Members of a Series A locus that is a member of a series whose members specify similar proteins or other characteristics is designated by the same letter symbol and a distinguishing number (e.g., Esl, Es2, and Es3 in mice and Esl, Es2, and Es3 in rats). For morphologic or "visible" loci with similar effects (e.g., genes that cause hairlessness', distinctive names are given because the gene actions and gene products can ultimately prove to be different (e.g., hr and nu in mice and fz and mu in rats). Alleles An allele is designated by the locus symbol with an added superscript. For mice, the superscript is written in italics for rats, it is not. An allele superscript is typically one or two lower-case letters that convey additional information about the allele. For mutant genes, no superscript is used for the first discovered allele. When further alleles are found, the first is still designated without a superscript (e.g., nu for nude and nuStr for streaker in

CRITERIA FOR SELECTING EXPERIMENTAL ANIMALS - 25 mice and fa for fatty and faCP for corpulent in rats). If the information is too complex to be conveyed conveniently in the symbol, the allele is given a superscript (e.g., Esla and Eslb in mice and Esla and Eslb in rats), and the information is otherwise conveyed. Indistinguishable alleles of independent origin (e.g., recurrences) are designated by the gene symbol with a series symbol, consisting of an Arabic number corresponding to the serial number of the recurring allele plus the laboratory code, appended as a superscript in italics. To avoid confusing the number "1" and the lower-case letter "1," the first discovered allele is left unnumbered, and the second recurring allele is numbered 2 (e.g., bg, beige bgJ, a recurrence of the mouse muta- tion bg at the Jackson Laboratory and bg27, a second recurrence of the mutation bg at the Jackson Laboratory). A mutation or other variation that occurs in a known allele (except for alloantigenic systems in the rat) is designated by a superscript m and an appropriate series symbol, which consists of a number corresponding to the serial number of the mutant allele in the laboratory of origin plus the labo- ratory code. The symbol is separated from the original allele symbol by a hyphen (e.g., Mupla-m7

for the first mutant allele of mouse Mupla found by the Jackson Laboratory). For a known deletion of all or part of an allele, the superscript m may be replaced with the superscript dl. This nomencla- ture is used for naming targeted mutations (often called "knockout" muta- tions), as well as spontaneously occurring ones. 7 _ Transgenes Nomenclature for transgenes was developed by the ILAR Committee on Transgenic Nomenclature (NRC, 1992b). A transgene symbol consists of three parts, all in reman type, as follows: TgX(YYYYYY)#####Zzz, where TgX is the mode, (YYYYYY) is the insert designation, and #####Zzz represents the laboratory-assigned number (#####) and laboratory code (Zzz). The mode designates the transgene and always consists of the letters Tg (for "transgene") and a letter designating the mode of insertion of the DNA: N for nonhomologous recombination, R for insertion via infection with a retroviral vector, and H for homologous recombination. The purpose of this designation is to identify it as a symbol for a transgene and to distinguish between the three fundamentally different organizations of the introduced sequence relative to the host genome. When a targeted mutation introduced by homologous recombination does not involve the insertion of a novel functional sequence, the new mutant allele (the knockout mutation) is des- ignated in accordance with the guidelines for gene nomenclature for each species. The gene nomenclature is also used when the process of homolo

26 RODENTS: LABORATORYANIMAL MANAGEMENT gous recombination results in integration of a novel functional sequence, if that sequence is a functional drug-resistance gene. For example, Mbpm

Dn would be used to denote the first targeted mutation of the myelin basic protein (Mbp) in the mouse made by Muriel T. Davisson (Dn). In this example, the transgenic insertion, even if it contains a functional neomycin- resistance gene, is incidental to "knocking out" or mutating the targeted locus (see also International Committee on Standardized Genetic Nomen- clature for Mice, 1994b). The insert designation is a symbol for the salient features of the transgene, as determined by the investigator. It is always in parentheses and consists of no more than eight characters: letters (capitals or capitals and lower-case letters) or a combination of letters and numbers. Italics, superscripts, sub- scripts, internal spaces, and punctuation should not be used. Short symbols (six or fewer characters) are preferred. The total number of characters in the insert designation plus the laboratory-assigned number may not exceed 11 (see below) therefore, if seven or eight characters are used, the number of digits in the laboratory-assigned number will be limited to four or three, respectively. The third part of the symbol is a number and letter combination that uniquely identifies each independently inserted sequence. It is formed of two components. The laboratory-assigned number is a unique number that is assigned by the laboratory to each stably transmitted insertion when germline transmission is confirmed. As many as five characters (numbers as high as 99,999) may be used however, the total number of characters in the insert designation plus the laboratory-assigned number may not exceed 11. No two lines generated within one laboratory should have the same assigned number. Unique numbers should be given even to separate lines with the same insert integrated at different positions. The number can have some intralaboratory meaning or simply be a number in a series of transgenes produced by the laboratory. The second component is the laboratory code. Thus, the complete designation identifies the inserted site, provides a sym- bol for ease of communication, and supplies a unique identifier to distin- guish it from all other insertions [e.g., C57BL/6J-TgN(CD8Ge)23Jwg for the human CD8 genomic clone inserted into C57BL/6 mice from the Jack- son Laboratory (J' and the 23rd mouse screened in a series of microinjec- tions done in the laboratory of Jon W. Gordon (Jwg)

. The complete rules for naming transgenes have been published (NRC, 1992b). TBASE, a database developed at Oak Ridge National Laboratory, Oak Ridge, Tennessee, as a registry of transgenic strains, is maintained at the Johns Hopkins University, Baltimore, Maryland. Information on TBASE can be obtained from the Genome Database and Applied Research Labora- tory, The Johns Hopkins University, 2024 East Monument Street, Balti- more, MD 21205 (telephone, 410-955-1704 fax, 410-614-04341.

CRITERIA FOR SELECTING EXPERIMENTAL ANIMALS Outbred Stocks 27 An outbred-stock designation consists of a laboratory code, a colon, and a stock symbol that consists of two to four capital letters (e.g., mouse stock Crl:ICR and rat stock Hsd:LE). The 'stock symbol must not be the same as that for an inbred 'strain of the same species. As an exception, a stock derived by outbreeding a formerly inbred strain may continue to use the original symbol in this case, the laboratory code preceding the stock symbol characterizes the stock as outbred. An outbred stock that contains a specified mutation is designated by the laboratory code, a colon, the stock symbol, a hyphen, and the gene symbol (e.g., Crl:ZUC-fa). The transfer of an outbred stock between breeders is indicated by list- ing the laboratory code of the new holder followed by the laboratory code of the holder the stock was obtained from (e.g., HsdBlu:LE for rats obtained by Harlan Sprague Dawley from Blue Spruce Farms). To avoid excessively long designations, only two laboratory codes should be used. QUALITY In selecting rodents for use in biomedical research, consideration should be given to the quality of the animals. Quality is most commonly character- ized in terms of microbiologic status and of the systems used in raising animals to ensure that a specific microbiologic status is maintained. How- ever, the genetics of an animal, as well as the genetic monitoring and breed- ing programs used to ensure genetic consistency, clearly also play an im- portant part in defining rodent quality. Microbiologic Quality Rodents can be infected with a variety of adventitious pathogenic and opportunistic organisms that under the appropriate circumstances can influ- ence research results at either the cellular or subcellular level. Some of those agents can persist in animals throughout their lives others cause tran- sient infections and are eliminated from the animals, leaving lasting sero- logic titers as the only 'indicators that ' the organisms were present. The types of organisms that can infect rodents include bacteria, protozoa, yeasts, fungi, viruses, rickettsia, mycoplasma, and such nonmicrobial agents as helminths and arthropods. Many of the common organisms that infect laboratory rodents have been studied extensively, and some of their research interactions have been characterized (see Bhatt et al., 1986NRC, 1991, for review). Unfortu- nately, information about the effects of many other organisms is incomplete or is not available. There is no general agreement on the importance of

28 RODENTS: LABORATORYANIMAL MANAGEMENT many organisms that latently infect rodents, especially opportunistic organ- isms that cause disease or alter research results only under narrowly defined conditions and even then usually affect only a very small proportion of the population. Any decision on the quality of rodents to be selected for a particular research project should include a realistic assessment of the or- ganisms that have a reasonable probability, as determined by documentation in the peer-reviewed literature, of producing confounding effects in the proposed study. It is commonly assumed that animals for which the most extensive health monitoring has been done and to which the most rigorous techniques for excluding microorganisms have been applied are the most appropriate for use in all studies. However, for both scientific and practical reasons, that assumption is not always valid. Rodents that are free of all microor- ganisms (axenic rodents, see definition below) or axenic rodents that have purposely been inoculated with a few kinds of nonpathogenic microorgan- isms (microbiologically associated rodents) can have altered physiologic and metabolic processes that make them inappropriate models for some studies. They can also rapidly become contaminated with common micro- organisms unless they are maintained with specialized housing and hus- bandry measures, which are expensive and can fail. The commercial avail- ability of such rodents is limited, and they are more expensive than rodents in which the microbial burden is not so restricted. For those reasons, the rodents most commonly used in research are ones that are free of a few specific rodent pathogens and some other microorganisms that are well known to have confounding effects on specific kinds of research. The quality of laboratory animals is generally related to the microbiologic exclusion methods used to breed and maintain them. There are three major types of maintenance: isolator-maintained, barrier-maintained, and no-con- tainment or conventionally maintained animals. An isolator is a sterilizable chamber that is usually constructed of metal, rigid plastic, vinyl, or polyure- thane. It usually has a sterilized air supply, a mechanism for introducing sterilized materials, and a series of built-in gloves to allow manipulation of the animals housed within. All materials moved into the isolator are sterilized, and animals raised within the isolator are generally maintained free from con- tamination by either all or specified microorganisms. Barrier-maintained animals are bred and kept in a dedicated space, called a barrier. For barrier facilities, personnel enter through a series of locks and are usually required to disrobe, shower, and use clean, disinfected clothing. All body surfaces that will potentially make contact with animals are cov- ered. All equipment, supplies, and conditioned air provided to the barrier facility are sterilized or disinfected. Barrier facilities can be of any size and can consist of one or more rooms. They are designed to exclude organisms for which rodents are the primary or preferred hosts but generally will not exclude organisms for which humans are hosts.

CRITERIA FOR SELECTING EXPERIMENTAL ANIMALS 29 Barrier maintenance can also be achieved at the cage or rack level with equipment that can be sterilized or otherwise disinfected. This type of maintenance depends heavily on providing large volumes of filtered or ster- ilized air to the animal cages. Such systems can be used successfully to maintain animals with a highly defined microbiologic status the success of such systems depends on the techniques used and is difficult to monitor because microbiologic status might differ from cage to cage. No-containment, or conventionally maintained, animals are raised in areas that have no special impediments to the introduction of microorganisms. This method of maintaining animals cannot ensure stability of the microbiologic status, because unwanted organisms can be introduced at any time. Several classifications have been developed to define the microbiologic quality of laboratory animals, as follows (see also NRC, 1991

: · Axenic refers to animals that are derived by cesarean section or embryo transfer and reared and maintained in an isolator with aseptic tech- niques. It implies that the animals are demonstrably free of associated forms of life, including viruses, bacteria, fungi, protozoa, and other saprophytic or parasitic organisms. Animals of this quality require the most compre

. hensive and frequent monitoring of their microbiologic status and are the most difficult to obtain and maintain. . Microbiologically associated, definedflora, or gnotobiotic refers to axenic animals that have been intentionally inoculated with a well-defined mixture of microorganisms and maintained continuously in an isolator to prevent contamination by other agents. Generally, a small number (usually less than 15) of species of microorganisms are used in the inoculum, and it is implied that these organisms are nonpathogenic. · Pathogen-free implies that the animals are free of all demonstrable pathogens. It is often misused, in that there is no general agreement about which agents are pathogens, what tests should be used to demonstrate the lack of pathogens and with what frequency, and how the populations should be sampled. Use of this term should be avoided because of the lack of precision of its meaning. . Specific-pathogen-free (ape) Is applied to animals that show no evidence (usually by serology, culture, or histopathology) of the presence of particular microorganisms. In its strictest sense, the term should be related to a specific set of organisms and a specific set of tests or methods used to detect them. An animal can be classified as SPF if it is free of one or many pathogens. . Conventional is applied to animals in which the microbial burden is unknown, uncontrolled, or both. In addition, the term clean conventional is sometimes used to describe ani- mals that are maintained in a low-security barrier and are demonstrated to be free of selected pathogens. This term is even less precise than pathogen- free, and its use is discouraged (NRC, 1991

30 RODENTS: LABORATORY ANIMAL MANAGEMENT Commercial suppliers have coined various terms to indicate SPF status. All the terms are related to specific organisms of which the animals are stated to be free and for which they are regularly monitored. In some cases, the terms (e.g., virus-antibody-free and murine-pathogen-free) imply a quality of animals beyond the actual definitions of the terms. Virus-antibody-free animals, for example, are animals that are free of antibodies to specific rodent viruses. The term is a variation of SPF, in that it relates to specified viruses. The implied method of detection is serology. Animals might not be free of viruses other than those specified and might not be free of other . . microorganisms. Genetic Quality In spite of diligent maintenance practices that are required in any breeding colony to identify animals properly and house them securely, people can make mistakes. In addition, loose animals, including animals that escape their housing unnoticed and wild rodents, can enter cages, mate with the inhabitants, and produce genetically contaminated offspring. Good hus- bandry practices carried out by trained personnel, including keeping a pedi- gree and clearly identifying animals and cages, can help to reduce the oc- currence of such events. Nevertheless, to avoid devastating consequences of genetic contamination, a good program of genetic monitoring is war- ranted. Genetic monitoring consists of any method used to ensure that the genetic integrity of individuals of any particular strain has not been vio- lated. Several commercial sources provide genetic monitoring services for inbred mouse and rat strains. Personnel should be alert to phenotypic changes in the animals, such as unexpected coat colors or large changes in reproductive performance. In a pedigree-controlled foundation colony (see Chapter 4), it is important to monitor the breeding stock at least once every two generations so that a single erroneous mating can be detected quickly. Retired breeders or some of their progeny can be tested. In an expansion or production colony, in which it might not be cost-effective or practical to monitor so closely, sampling is recommended. The extent of such sampling can be as broad as resources and need permit. If genetic contamination occurs outside the foundation colony, contamination will eventually be purged by the infusion of breeders from the more rigorously controlled foundation colony. The extent of necessary testing depends on the number and genotypes of neighboring strains. A testing system should be capable of identifying the strain to which the individual belongs and differentiating it from other strains maintained nearby. Most strains can be identified with a small set of any genetic markers for which an assay is available. Newer DNA-typing methods that use multilocus probes, minisatellite markers, and "DNA-fin

CRITERIA FOR SELECTING EXPERIMENTAL ANIMALS 31 gerprinting" analysis are powerful tools for distinguishing strains, espe- cially strains that are closely related, but electrophoretic methods that type isoenzymes are generally more cost-effective for genetic monitoring (Hedrich, 1990 Nomura et al., 1984), in that such monitoring is most commonly done to detect mismatings. Immunologic methods are also used, and the ex- change of skin grafts between individuals of a strain is a particularly effec- tive method for screening a large number of loci in a single test. DNA from - representative breeders ot a stra

n can be stored for future use in identifying suspected genetic contaminations. Genetic monitoring is used orimarilY to verify the authenticity of a given strain new mutations are rarely detected by this means. It is impos- sible to monitor all loci for new mutations, given the large number of unknown loci and known loci that do not produce a visible phenotype. A good breeding-management program, as described in Chapter 4, will help to reduce unwanted genetic changes caused by mutations. SELECTED ASPECTS OF EXPERIMENTAL DESIGN An experiment in which laboratory animals are used should be designed carefully, so that it produces unequivocal information about the questions that it was designed to address. The two most important requirements of proper experimental design in that connection are as follows: . Animals in different groups should vary only in the treatment that the experiment is designed to evaluate, so that the experimental outcome will not be confounded by dissimilarities in the constitution of the groups or in how they are treated or measured. · Each treatment should be given to enough animals for the experi- mental outcome to be attributed confidently to treatment difference and not merely to chance. The best way to ensure that groups of experimental animals are compa- rable is to draw them from a single homogeneous pool and to assign them randomly to treatment groups. Choosing animals of the same age, sex, and inbred strain for all treatment groups and even assigning littermates ran- domly to different treatment groups can eliminate factors that might par- tially account for group-to-group differences in experimental outcome. once animals are assigned to groups, they should be handled identi- cally, except for the treatment differences that the experiment is designed to evaluate. Food, water, bedding, and other features of animal husbandry should be the same. For long-term experiments, cages should be rotated to minimize group differences caused by cage position. For invasive experi- mental treatments, sham or placebo procedures should be performed in compar

32 RODENTS: LABORATORY ANIMAL MANAGEMENT ison groups for example, animals given treatment by gavage should be compared with controls given the vehicle by Savage, animals treated surgi- cally should be compared with animals that undergo sham surgical opera- tions, and animals exposed to treatment by inhalation should be compared with animals placed in inhalation chambers that circulate only air. Follow- ing those precautions will ensure that differences in outcome between groups can be attributed to the experimental treatment itself and not to ancillary differences associated with the administration of the treatment. Finally, wherever possible, the outcome of interest should be measured by people who are unaware of which treatment each animal received, be- cause such knowledge can magnify or even create observed treatment dif- ferences. It is particularly important to carry out "blind" studies when the outcome is to be evaluated subjectively (e.g., by grading of disease sever- ityJ, rather than measured quantitatively (e.g., by measuring concentrations of serum constituents). The number of animals needed in each group will depend on many features of the experimental design, including the following: . . the goals of the study · the primary outcome measure that will be compared the number of groups that will be compared · the expected number of technical failures or usable end points the number and type of comparisons that will be made · the expected animal-to-animal and measurement variability in the outcome · the statistical design and analysis that will be used · the magnitude of the differences between control and treatment groups that it is desirable to detect · the projected losses and · the maximal tolerable chance of drawing erroneous conclusions. The more variable an outcome measure is, either because outcomes in identically treated animals vary substantially or because there is a high degree of measurement variability, the more animals will be needed in each group to distinguish between group differences caused by treatment and those caused by chance. How outcome measurement variability, treatment difference to be detected, and tolerable chance of drawing an erroneous conclusion affect the required sample size depends on the measurement to be made' the type of croup comparison to be made. and the statistical ,

analysis to be used. Tables and formulas for comparing proportions among two or more groups have been published (Gart et al., 1986), as has useful information for other types of outcomes (Mann et al., l991J. For most experiments, it is highly desirable to collaborate with a statistician through- out, beginning with the design stage, so that appropriately defined groups of sufficient size will be available for a proper statistical analysis.


Author information

These authors contributed equally: Thomas Naert and Dieter Tulkens.

Affiliations

Department of Biomedical Molecular Biology, Ghent University, Technologiepark 71, 9052, Ghent (Zwijnaarde), Belgium

Thomas Naert, Dieter Tulkens, Marjolein Carron, Suzan Demuynck & Kris Vleminckx

Cancer Research Institute Ghent, Ghent, Belgium

Thomas Naert, Dieter Tulkens, Suzan Demuynck & Kris Vleminckx

Division of Developmental Biology, Perinatal Institute, and Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati Children’s Hospital, Cincinnati, USA

Nicole A. Edwards & Aaron M. Zorn

Center for Medical Genetics, Department of Biomolecular Medicine, Ghent University, Ghent, Belgium

Marjolein Carron, Annekatrien Boel, Paul Coucke, Andy Willaert & Kris Vleminckx

National Xenopus Resource and Eugene Bell Center for Regenerative Biology and Tissue Engineering, Marine Biological Laboratory, Woods Hole, MA, 02543, USA


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