Spontaneous autoimmunity in 129 and C57BL/6 mice-implications for autoimmunity described in gene-targeted mice

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disorder in which complex genetic factors play an important role. Several strains of gene-targeted mice have been reported to develop SLE, implicating the null genes in the causation of disease. However, hybrid strains between 129 and C57BL/6 mice, widely used in the generation of gene-targeted mice, develop spontaneous autoimmunity. Furthermore, the genetic background markedly influences the autoimmune phenotype of SLE in gene-targeted mice. This suggests an important role in the expression of autoimmunity of as-yet-uncharacterised background genes originating from these parental mouse strains. Using genome-wide linkage analysis, we identified several susceptibility loci, derived from 129 and C57BL/6 mice, mapped in the lupus-prone hybrid (129 × C57BL/6) model. By creating a C57BL/6 congenic strain carrying a 129-derived Chromosome 1 segment, we found that this 129 interval was sufficient to mediate the loss of tolerance to nuclear antigens, which had previously been attributed to a disrupted gene. These results demonstrate important epistatic modifiers of autoimmunity in 129 and C57BL/6 mouse strains, widely used in gene targeting. These background gene influences may account for some, or even all, of the autoimmune traits described in some gene-targeted models of SLE.     

Spontaneous Autoimmunity in 129 and C57BL/6 Mice—Implications for Autoimmunity Described in Gene-Targeted Mice

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disorder in which complex genetic factors play an important role. Several strains of gene-targeted mice have been reported to develop SLE, implicating the null genes in the causation of disease. However, hybrid strains between 129 and C57BL/6 mice, widely used in the generation of gene-targeted mice, develop spontaneous autoimmunity. Furthermore, the genetic background markedly influences the autoimmune phenotype of SLE in gene-targeted mice. This suggests an important role in the expression of autoimmunity of as-yet-uncharacterised background genes originating from these parental mouse strains. Using genome-wide linkage analysis, we identified several susceptibility loci, derived from 129 and C57BL/6 mice, mapped in the lupus-prone hybrid (129 × C57BL/6) model. By creating a C57BL/6 congenic strain carrying a 129-derived Chromosome 1 segment, we found that this 129 interval was sufficient to mediate the loss of tolerance to nuclear antigens, which had previously been attributed to a disrupted gene. These results demonstrate important epistatic modifiers of autoimmunity in 129 and C57BL/6 mouse strains, widely used in gene targeting. These background gene influences may account for some, or even all, of the autoimmune traits described in some gene-targeted models of SLE.     

ROSA26 mice carry a modifier of Min-induced mammary and intestinal tumor development

B6.129S7-Gtrosa26 (B6.R26) mice carry a LacZ-neoR insertion on Chromosome (Chr) 6, made by promoter trapping with 129 ES cells. Female C57BL/6J Apc ^Min /+ (B6Min/+) mice are highly susceptible to intestinal tumors and to the induction of mammary tumors after treatment with ethylnitrosourea (ENU). However, B6.R26/+Min/+ females develop fewer mammary and intestinal tumors after ENU treatment than do B6 Min/+ mice. B6.R26/+ mice from two independently derived congenic lines show this modifier effect. Each of these congenic lines carries approximately 20 cM of 129-derived DNA flanking the insertion, raising the possibility that the resistance is due to a linked modifier locus. To further map the modifier locus, we have generated several lines of mice carrying different regions of the congenic interval. We have found that resistance to mammary and intestinal tumors in ENU-treated Min/+ mice maps to a minimum 4-cM interval that includes the ROSA26 LacZ-neoR insertion. Therefore, the resistance to tumor development is due to either the ROSA26 insertion or a very tightly linked modifier locus. Received: 10 May 2000 / Accepted: 25 July 2000     

Confirmation and high resolution mapping of an atherosclerosis susceptibility gene in mice on Chromosome 1

Previously, we demonstrated that Ath1 is a quantitative trait locus for aortic fatty streak formation, located on Chromosome (chr) 1, with susceptibility in C57BL/6J mice and resistance in C3H/HeJ and BALB/cJ mice fed an atherogenic diet. In this study, we find an atherosclerosis susceptibility locus in the same region of Chr 1 by constructing two congenic strains with the resistance phenotype transferred from different resistant strains, PERA/EiJ or SPRETUS/EiJ. By backcrossing one congenic strain to C57BL/6J and testing recombinant animals, we reduced the distance of the atherosclerosis susceptibility region to 2.3 cM between D1Mit14 and D1Mit10. Further testing of nine recombinant animals showed that eight of the nine were consistent with a further narrowing between D1Mit159 and D1Mit398 a distance of 0.66 cM. This region encompasses a number of potential candidate genes including the thiol-specific antioxidant gene Aop2, also known as peroxiredoxin 5 (Prdx5). AOP2 is capable of reducing hydroperoxides and lipid peroxides in the cell. To investigate Aop2 as a potential candidate, we mapped Aop2 in our backcross and localized it to the atherosclerosis susceptibility interval. We determined that Aop2 is highly expressed in atherosclerosis-related tissues including liver and heart. We also found an inverse correlation between Aop2 mRNA in liver and atherosclerosis phenotype for strains C57BL/6 and the resistant congenic derived from SPRETUS/EiJ. Since LDL oxidation has been implicated in the pathogenesis of this disease, and AOP2 possesses antioxidant activity, we suggest the role of Aop2 in atherosclerosis susceptibility needs to be further explored.     

The inhibiting Fc receptor for IgG, FcγRIIB, is a modifier of autoimmune susceptibility

FcγRIIB-deficient mice generated in 129 background (FcγRIIB(129)(-/-)) if back-crossed into C57BL/6 background exhibit a hyperactive phenotype and develop lethal lupus. Both in mice and humans, the Fcγr2b gene is located within a genomic interval on chromosome 1 associated with lupus susceptibility. In mice, the 129-derived haplotype of this interval, named Sle16, causes loss of self-tolerance in the context of the B6 genome, hampering the analysis of the specific contribution of FcγRIIB deficiency to the development of lupus in FcγRIIB(129)(-/-) mice. Moreover, in humans genetic linkage studies revealed contradictory results regarding the association of "loss of function" mutations in the Fcγr2b gene and susceptibility to systemic lupus erythematosis. In this study, we demonstrate that FcγRIIB(-/-) mice generated by gene targeting in B6-derived ES cells (FcγRIIB(B6)(-/-)), lacking the 129-derived flanking Sle16 region, exhibit a hyperactive phenotype but fail to develop lupus indicating that in FcγRIIB(129)(-/-) mice, not FcγRIIB deficiency but epistatic interactions between the C57BL/6 genome and the 129-derived Fcγr2b flanking region cause loss of tolerance. The contribution to the development of autoimmune disease by the resulting autoreactive B cells is amplified by the absence of FcγRIIB, culminating in lethal lupus. In the presence of the Yaa lupus-susceptibility locus, FcγRIIB(B6)(-/-) mice do develop lethal lupus, confirming that FcγRIIB deficiency only amplifies spontaneous autoimmunity determined by other loci.     

Microsatellite-directed selection of breeders for the next backcross generation by using a minimal number of loci

To establish a minimal number of markers for direct selection of candidate mice used for the next mating to produce congenic mice, recombination frequencies of 53 microsatellite loci on chromosomes (chr.) 1 and 19 were examined using 41 N2 mice: the donor strain was BALB/c, and recipient strain was C57BL/6J (B6J) or C57BL/6N (B6N). These markers were spaced at 0.1 to 24.2 centimorgans (cM). Among the 41 mice, B6/B6 homozygosity ranged from 18 to 24 animals (mean, 20; 2 standard deviations, 1.36) for a given locus. There was no difference in recombination frequency between chr. 1 and 19. The recombination frequency of B6J was higher than that of B6N (P < 0.05). Various densities of markers, 10 (5 markers/chr.), 8 (4 markers/chr.), and 6 (3 markers/chr.) spaced at 12.0 to 29.3, 9.0 to 45.0, and 24.5 to 53.0 cM, respectively, were selected from the 53 markers, and homozygosity was compared in each mouse. In mice with decreased homozygosity when tested using 53 markers, homozygosity differed depending on the density of the markers. The results suggested that 3 markers/chr. are sufficient for selection of the highest percentages of homozygosity but are not suitable to define mice with lower percentages of homozygosity.     

Functional interplay between intrinsic B and T cell defects leads to amplification of autoimmune disease in New Zealand black chromosome 1 congenic mice

Genetic loci on New Zealand Black (NZB) chromosome 1 play an important role in the development of lupus-like autoimmune disease. We have shown previously that C57BL/6 mice with an introgressed NZB chromosome 1 interval extending from approximately 35 to 106 cM have significantly more severe autoimmunity than mice with a shorter interval extending from approximately 82 to 106 cM. Comparison of the cellular phenotype in these mice revealed that both mouse strains had evidence of increased T cell activation; however, activation was more pronounced in mice with the longer interval. Mice with the longer interval also had increased B cell activation, leading us to hypothesize that there were at least two independent lupus susceptibility loci on chromosome 1. In this study, we have used mixed hemopoietic radiation chimeras to demonstrate that autoimmunity in these mice arises from intrinsic B and T cell functional defects. We further show that a T cell defect, localized to the shorter interval, leads to spontaneous activation of T cells specific for nucleosome histone components. Despite activation of self-reactive T cells in mixed chimeric mice, only chromosome 1 congenic B cells produce anti-nuclear Abs and undergo class switching, indicating impaired B cell tolerance mechanisms. In mice with the longer chromosome 1 interval, an additional susceptibility locus exacerbates autoimmune disease by producing a positive feedback loop between T and B cell activation. Thus, T and B cell defects act in concert to produce and amplify the autoimmune phenotype.     

Deletion of p21 (WAF-1/Cip1) does not induce systemic autoimmunity in female BXSB mice

Cell cycle, apoptosis, and replicative senescence are all influenced by the cyclin-dependent kinase inhibitor, p21. It was previously reported that deletion of p21 in 129/Sv x C57BL/6 mixed genetic background mice induced a severe lupus-like disease, almost exclusively in females. However, we did not confirm this finding in an independently derived stock of 129/Sv x C57BL/6 p21(-/-) mice. To further address this discrepancy, we examined the effects of p21 deletion in BXSB female mice that develop late-life, mild lupus-like disease. Survival, polyclonal Igs, anti-chromatin Abs, and kidney histopathology in these mice were unremarkable and identical to wild-type littermates for up to 14 mo of age. We conclude that p21 deficiency does not promote autoimmunity even in females of a predisposed strain. The findings indicate that the use of mixed background 129/Sv x C57BL/6 mice to study effects of gene deletions in systemic autoimmunity may be confounded by the genetic heterogeneity of this cross. We suggest that studies addressing gene deletion effects in systemic autoimmunity should use sufficiently backcrossed mice to attain genetic homogeneity, include wild-type littermate controls, and preferentially use congenic inbred strains with late-life lupus predisposition to emulate the polygenic nature of this disease.     

Fine mapping of Ahl3 affecting both age-related and noise-induced hearing loss

A region in the vicinity of D17Mit119 on mouse chromosome 17 harbors a susceptibility gene, designated as Ahl3, to age-related hearing loss (AHL). We produced congenic lines of C57BL/6 background that substituted regions around D17Mit119 with MSM-derived ones, and examined auditory brainstem response (ABR) thresholds for their hearing capacity at 6 and 12months of age. Three congenic lines carrying the approximately 14-Mb region between D17Mit274 and D17Mit183 retained normal hearing at 12months of age whereas two congenic lines not carrying this region tended to lose hearing at that age. We also investigated noise-induced hearing loss (NIHL) in congenic lines at 1, 7 and 14days after exposure to the noise of 100dB for 1h. Most congenic mice carrying the 14-Mb region did not exhibit permanent threshold shift (PTS) whereas mice not carrying this region exhibited a strong tendency of PTS, indicating the role of Ahl3 in susceptibility to NIHL. These results indicate that Ahl3 exists within the 14-Mb region and affects not only AHL but also NIHL.     

Quantitative assessment and characterization of visceral nociception and hyperalgesia in mice

Colorectal distension (CRD) is a well-characterized model of visceral nociception, which we adapted to the mouse. CRD reproducibly evoked contractions of the abdominal musculature [visceromotor response (VMR)], which was graded to stimulus intensity. The magnitude of VMR was greater in male C57BL6 and female 129S6 mice than in male 129S6 and B6.129 mice. In 129S6, C57BL6, and B6.129 mice strains, VMR was reduced dose dependently by morphine (1-10 mg/kg) and by the kappa-opioid agonist U-69593 (0.2-2 mg/kg), although U-69593 was significantly less potent in C57BL6 mice. In additional experiments, the VMR was recorded from adult male 129S6 mice before and after intracolonic administration of various irritants. Only 30% ethanol significantly enhanced responses to CRD. The colon hyperalgesia persisted for 14 days and was associated with a significant shift of the morphine dose-response function to the left. We believe this will be a useful model for study of visceral nociception and hyperalgesia, including studies of transgenic mice with mutations relevant to pain.     

Epistatic suppression of systemic lupus erythematosus: fine mapping of Sles1 to less than 1 mb

Sle is a susceptibility locus for systemic autoimmunity derived from the lupus-prone NZM2410 mouse. The New Zealand White-derived suppressive modifier Sles1 was identified as a specific modifier of Sle1 and prevents the development of IgG anti-chromatin autoantibodies mediated by Sle1 on the C57BL/6 (B6) background. Fine mapping of Sles1 with truncated congenic intervals localizes it to a approximately 956-kb segment of mouse chromosome 17. Sles1 completely abrogates the development of activated T and B cell populations in B6.Sle1. Despite this suppression of the Sle1-mediated cell surface activation phenotypes, B6.Sle1 Sles1 splenic B cells still exhibit intrinsic ERK phosphorylation. Classic genetic complementation tests using the nonautoimmmune 129/SvJ mouse suggests that this strain possesses a Sles1 allele complementary to that of New Zealand White, as evidenced by the lack of glomerulonephritis, splenomegaly, and antinuclear autoantibody production seen in (129 x B6.Sle1 Sles1)F(1)s. These findings localize and characterize the suppressive properties of Sles1 and implicate 129 as a useful strain for aiding in the identification of this elusive epistatic modifier gene.     

Gene-environment interaction: a significant diet-dependent obesity locus demonstrated in a congenic segment on mouse Chromosome 7

We have previously reported suggestive evidence for a locus on Chromosome (Chr) 7 that affects adiposity in F₂ mice from a CAST/Ei × C57BL/6J intercross fed a high-fat diet. Here we characterize the effect of a high-fat (32.6 Kcal% fat) diet on male and female congenic mice with a C57BL/6J background and a CAST/Ei-derived segment on Chr 7. Adiposity index (AI) and weights of certain fat pads were approximately 50% lower in both male and female congenic mice than in control C57BL/6J mice, and carcass fat content was significantly reduced. The reduction of fat depot weights was not seen, however, in congenic animals fed a low-fat chow diet (12 Kcal% fat). The congenic segment is approximately 25 cM in length, extending from D7Mit213 to D7Mit41, and includes the tub, Ucp2, and Ucp3, genes, all of which are candidate genes for this effect. Some polymorphisms have been found on comparing c-DNA sequences of the Ucp2 gene from C57BL/6J and CAST/Ei mice. These results suggest that one or more genes present in the congenic segment modulate the susceptibility to fat deposition on feeding a high-fat diet. We were unable to show any significant difference between the energy intakes of the congenic and the control C57BL/6J mice on the high-fat diet. Also, measurements of energy expenditure in male mice at 6 weeks of age, during the first 2 weeks of exposure to the high-fat diet, failed to show any differences between control and congenic animals. Received: 30 September 1998 / Accepted: 22 December 1998     

The ROSA26 LacZ-neo R insertion confers resistance to mammary tumors in Apc Min/+ mice

B6.129S7-Gtrosa26 (ROSA26) mice carry a LacZ-neo ^R insertion on Chromosome (Chr) 6, made by promoter trapping with AB1 129 ES cells. Female C57BL/6J Apc ^Min /+ (B6 Min/+) mice are very susceptible to the induction of mammary tumors after treatment with ethylnitrosourea (ENU). However, ENU-treated B6 mice carrying both Apc ^Min and ROSA26 are resistant to mammary tumor formation. Thus, ROSA26 mice carry a modifier of Min-induced mammary tumor susceptibility. We have previously mapped the modifier to a 4-cM interval of 129-derived DNA that also contains the ROSA26 insertion. Here we report additional evidence for the effect of the ROSA26 insertion on mammary tumor formation. To test the hypothesis that the resistance was due to a linked modifier locus, we utilized two approaches. We have derived and tested two lines of mice that are congenic for 129-derived DNA within the minimal modifier interval and show that they are as susceptible to mammary tumors as are B6 mice. Additionally, we analyzed a backcross population segregating for the insertion and show that mice carrying the insertion are more resistant to mammary tumor development than are mice not carrying the insertion. Thus, the resistance is not due to a 129-derived modifier allele, but must be due to the ROSA26 insertion. In addition, the effect of the ROSA26 insertion can be detected in a backcross population segregating for other mammary modifiers. Received: 29 December 2000 / Accepted: 4 April 2001     

Mapping of jog locus to the region between D6Mit104 and D6Mit336 on mouse chromosome 6.

The joggle mouse is a recessive ataxic mutant carrying an unknown mutation in a C3H/He (C3H)-derived chromosomal segment. Taking advantage of the mouse genome database, we selected 127 DNA microsatellite markers showing heterozygosity between C3H and C57BL/6J (B6) and a first round of screening for the joggle mutation was performed on B6-jog/+ partial congenic mice (N4). We identified 4 chromosomal regions in which 13 microsatellite markers show heterozygosity between C3H and B6. Then, we analyzed the genotype of these 4 chromosomal regions in mice that showed the joggle phenotype and mapped the jog locus between markers D6Mit104 (111.4 Mb) and D6Mit336 (125.1 Mb) (an interval of 13.7 Mb) on chromosome 6. By using a partial congenic strain together with the mouse genome database, we successfully mapped the chromosomal localization of the jog locus much more efficiently than by conventional linkage analysis.     

Strain-specific modification of lethality in fucose-deficient mice

The FX locus encodes an essential enzyme in the de novo pathway of GDP-fucose biosynthesis. Mice homozygous for a targeted mutation of the FX gene manifest a host of pleiotropic abnormalities including a lethal phenotype that is almost completely penetrant in heterozygous intercrosses on a mixed genetic background. Here we have investigated genetic suppression of FX-mediated lethality. Reduced recovery of heterozygous mice was observed while backcrossing the null FX allele to C57BL/6J (B6), but was less dramatic in an outcross to CASA/Rk and absent in an outcross to 129S1/SvImJ, indicating that genetic background modifies survival of FX+/- progeny. Substantial strain-specific differences in pre- and postnatal survival of FX-/- progeny were also detected in heterozygous crosses of C57BL/6J congenic, 129S1B6F1, and B6CASAF1 mice. Specifically, intrauterine survival of FX-/- mice was greatly increased during a heterozygous intercross on a uniform C57BL/6J genetic background compared with survival on a hybrid genetic background consisting of a mixture of C57BL/6J and 129S2/SvPas. In addition, statistically significant clustering of FX-/- progeny into litters and specific breeding cages was noted during a B6CASAF1 FX+/- intercross, suggesting a rare mechanism for modifier gene action in which parentally expressed genes define the phenotype, in this case the survival potential, of mutant offspring. Our results disclose that lethality in FX mutant mice is determined by one or more strain-specific modifier loci.     

Genetic analysis of alcohol intake in recombinant inbred and congenic strains derived from A/J and C57BL/6J progenitors

The objective of the present study was to map quantitative trait loci (QTL) for alcohol intake using A × B/B × A recombinant inbred (RI) and AcB/BcA recombinant congenic (RC) strains of mice that were independently derived from the A/J and C57BL/6J progenitors. Mice were screened for levels of alcohol consumption with four days of forced exposure to alcohol, followed by three weeks of free choice between water and a 10% alcohol solution. Alcohol consumption data previously collected for 27 A × B/B × A RI strains were reanalyzed using a larger marker set and composite interval mapping. The reanalysis found markers on Chromosome 2 (D2Mit74, 107 cM) (males and females) and on Chromosome 11 (Pmv22, 8 cM) (females only) that exceeded the threshold for significant loci, and found suggestive loci (in males) on Chromosomes 10 (D10 Mit126, 21 cM), 12 (D12Mit37, 1 cM), 15 (Pdgfb, 46.8 cM), and 16 (D16Mit125, 29 cM). An additional suggestive locus was identified in female RI mice on Chromosome 11 (D11Mit120, 47.5 cM). Composite interval mapping (CIM) analysis indicated that there was a significant association between loci at Pdgfb and D2Mit74 in both males and females. Analysis of the AcB/BcA RC strains identified 11 QTL on Chromosomes 2, 3, 5,6, 7, 8, 9, 10, 12, 13, and 15. QTL on Chromosomes 7, 10, 12, and 15 were identified in both the A × B/B × A RI and AcB/BcA RC strains of mice. Additional QTLs identified on Chromosomes 2, 3, 7, 11, and 15 overlap with those previously identified in the literature using strains of mice with a C57BL/6J progenitor.     

Efficiency alleles of the Pctr1 modifier locus for plasmacytoma susceptibility

The susceptibility of BALB/c mice to pristane-induced plasmacytomas is a complex genetic trait involving multiple loci, while DBA/2 and C57BL/6 strains are genetically resistant to the plasmacytomagenic effects of pristane. In this model system for human B-cell neoplasia, one of the BALB/c susceptibility and modifier loci, Pctr1, was mapped to a 5.7-centimorgan (cM) chromosomal region that included Cdkn2a, which encodes p16(INK4a) and p19(ARF), and the coding sequences for the BALB/c p16(INK4a) and p19(ARF) alleles were found to be polymorphic with respect to their resistant Pctr1 counterparts in DBA/2 and C57BL/6 mice (45). In the present study, alleles of Pctr1, Cdkn2a, and D4Mit15 from a resistant strain (BALB/cDAG) carrying DBA/2 chromatin were introgressively backcrossed to the susceptible BALB/c strain. The resultant C.DAG-Pctr1 Cdkn2a D4Mit15 congenic was more resistant to plasmacytomagenesis than BALB/c, thus narrowing Pctr1 to a 1.5-cM interval. Concomitantly, resistant C57BL/6 mice, from which both gene products of the Cdkn2a gene have been eliminated, developed pristane-induced plasma cell tumors over a shorter latency period than the traditionally susceptible BALB/cAn strain. Biological assays of the p16(INK4a) and p19(ARF) alleles from BALB/c and DBA/2 indicated that the BALB/c p16(INK4a) allele was less active than its DBA/2 counterpart in inducing growth arrest of mouse plasmacytoma cell lines and preventing ras-induced transformation of NIH 3T3 cells, while the two p19(ARF) alleles displayed similar potencies in both assays. We propose that the BALB/c susceptibility/modifier locus, Pctr1, is an "efficiency" allele of the p16(INK4a) gene.     

Identification of 129S6/SvEvTac-specific polymorphisms on mouse chromosome 11

Polymorphisms such as single-nucleotide polymorphisms (SNPs) and insertions/deletions (Indels) can be associated with phenotypic traits and be used as markers for disease diagnosis. Identification of these genetic variations within laboratory mice is crucial to improve our understanding of the genetic background of the mice used for research. As part of a positional cloning project, we sequenced six genes (Mettl16, Evi2a, Psmd11, Cct6d, Rffl, and Ap2b1) within a 6.8-Mb domain of mmu chr 11 in the C57BL/6J and 129S6/SvEvTac inbred strains. Although 129S6/SvEvTac is widely used in the mouse community, there is very little current (or projected future) sequence information available for this strain. We identified 6 Indels and 21 novel SNPs and confirmed genotype information for 114 additional SNPs in these 6 genes. Mettl16 and Ap2b1 contained the largest numbers of variants between the C57BL/6J and 129S6/SvEvTac strains. In addition, we found five new SNPs between 129S6/SvEvTac and 129S1/SvImJ within the Ap2b1 locus. Although we did not detect differences between C57BL/6J and 129S6/SvEvTac within Evi2a, this locus contains a relatively high SNP density compared with the surrounding sequence. Our study highlights the genetic differences among three inbred mouse strains (C57BL/6J, 129S6/SvEvTac, and 129S1/SvImJ) and provides valuable sequence information that can be used to track alleles in genomics-based studies.     

Analysis of factors decreasing testis weight in MRL mice

MRL/MpJ (MRL) mouse testes have several unique characteristics, including the appearance of oocytes, the occurrence of metaphase-specific apoptosis of meiotic spermatocytes, and the presence of heat-shock-resistant spermatocytes. In the present study we used chromosomal mapping to determine the genomic background associated with small testis size in MRL mice. We prepared and analyzed C57BL/6-based congenic mice carrying MRL mouse loci. Quantitative trait loci (QTL) analysis revealed susceptibility loci for small testis size at 100 cM on chromosome (Chr) 1 and at around 80 cM on Chr 2. Analysis with B6.MRLc1 and B6.MRLc2 congenic mice and double-congenic mice confirmed the QTL data and showed that low testis weight in MRL mice was caused by germ cell apoptosis. Through histological examinations we found that B6.MRLc1 and B6.MRLc2 mice showed stage-specific apoptosis in their testes, the former at metaphase stage XII and the later at pachytene stage IV. Metaphase-specific apoptosis of spermatocytes occurs due to mutation of the exonuclease 1 (Exo1) gene located at 100 cM on Chr 1. Thus, the mutation of the Exo1 gene is also responsible for low testis weight caused by metaphase-specific apoptosis. In conclusion, testis weight is reduced in MRL mice due to apoptosis of germ cells caused by mutations in loci on Chrs 1 and 2.