129X1/SvJ mouse strain has a novel defect in inflammatory cell recruitment.

Vitamin D-binding protein (DBP) has been reported to contribute to innate immunity. To verify prior in vitro and cell-based observations supporting this role, we assessed the ability of a recently developed DBP-null mouse line to recruit neutrophils and macrophages to a site of chemical inflammation. The interrupted DBP allele had been generated by homologous recombination in 129X1/SvJ embryonic stem cells and these cells were subsequently used to generate a line of DBP(-/-) (null) mice. Initial studies revealed a marked defect in the ability of these DBP(-/-) mice to recruit cells to the peritoneum after localized thioglycolate injection. However, progressive outcrossing of the DBP(-/-) mice to the C57BL/6J strain, conducted to provide a uniform genetic background for comparison of DBP-null and control mice, resulted in a progressive increase in cell recruitment by the DBP(-/-) mice and a loss in their apparent recruitment defect when compared with the DPB wild-type controls. These data suggested that the observed recruitment phenotype initially attributed to the absence of DBP was not linked to the DBP locus, but instead reflected the underlying genetic composition of the 129X1/SvJ ES cells used for the initial DBP gene disruption. A profound cell recruitment defect was confirmed in the 129X1/SvJ mice by direct analysis. Each of three commonly used inbred lines was discovered to have a distinct level of cell recruitment to a uniform stimulus (C57BL/6J > BALB/c > CD1 > 129X1/SvJ). Thus, this study failed to support a unique role for DBP in cellular recruitment during a model inflammatory response. Instead, the data revealed a novel and profound defect of cell recruitment in 129X1/SvJ mice, the strain most commonly used for gene deletion studies.     

Genetic factors influence cataract formation in alpha 3 connexin knockout mice

Connexin alpha 3 (Cx46 or Gja3) gene targeted null mice developed lens nuclear cataracts shortly after birth. A large variance in the cataracts was observed in alpha 3 null sibs on a mixed 129SvJae X C57BL/6J F3 background. This suggested that the genetic background might influence the cataract phenotype. Therefore, we placed the alpha 3 null mutation into a 129SvJae background, and also backcrossed the mutation for six generations into 129SvJ and C57BL/6J backgrounds. While alpha 3 nulls on the two 129 backgrounds contained severe cataracts associated with gamma crystallin cleavage, alpha 3 nulls on the C57B16 background had far milder cataracts with no detectable gamma crystallin cleavage. These findings suggest that a genetic modifier exists that influences gamma crystallin stability, and that gamma crystallin breakdown is associated with severe nuclear cataracts.     

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.     

Loss of PKD1 and loss of Bcl-2 elicit polycystic kidney disease through distinct mechanisms

We have recently demonstrated that ablation of one or both alleles of the proapoptotic gene Bim prevents the polycystic kidney disease (PKD) that develops in mice deficient for the prosurvival protein Bcl-2. The aim of the present study was to investigate whether loss of Bim or Bcl-2 could influence the disease in the PKD1del34/del34 mutant mice, a model of autosomal dominant PKD. PKD1del34/del34 mice were intercrossed with Bim-deficient mice and Bcl-2+/- mice to generate double mutants. Loss of Bim does not prevent the development of PKD in PKD1del34/del34 mice. On the C57BL/6 genetic background, most older PKD1del34/+ mice do not develop PKD, but present with liver cysts. Surprisingly, loss of Bim completely prevented liver cysts formation in PKD1del34/+ mice. Loss of one Bcl-2 allele did not influence the PKD1del34 phenotype significantly. We conclude that loss of PKD1 and loss of Bcl-2 elicit PKD through distinct mechanisms.     

Lens density tracking in mice by Scheimpflug imaging

Scheimpflug imaging has recently been established for in vivo imaging of the anterior eye segment and quantitative determination of lens transparency in the mouse. This enables more effective investigations of cataract formation with the mouse model, including longitudinal studies. In order to enable recognition of disease-associated irregularities, we performed Scheimpflug measurements with the common laboratory inbred lines C57BL/6J, C3HeB/FeJ, FVB/NCrl, BALB/cByJ, and 129/SvJ in a period between 2 and 12 months of age. C57BL/6J mice showed lowest mean lens densities during the test period. Progressive cortical lens opacification was generally observed, with the earliest onset in C57BBL/6J, C3HeB/FeJ, and 129/SvJ, between 2 and 6 months after birth. Moreover, lenses of these inbred lines developed nuclear opacities. Calculated mean lens density significantly increased between 6 and 12 months of age in all inbred strains except 129/SvJ. Lens densities (and the corresponding standard deviations) of FVB/NCrl and 129/SvJ increased most likely because of differences in the genetic background. Albinism as confounder might be excluded since the albino Balb/cByJ mice are more similar to the C57BL/6J or C3Heb/FeJ mice. We further identified strain-specific anterior lens opacities (C57BL/6J) and cloudy corneal lesions (C57BL/6J, FVB/NCrl, and BALB/cByJ) at later stages. In conclusion, our results indicate that there are lifelong opacification processes in the mouse lens. The highest lens transparency and a dark coat color, which prevents interference from light reflections, make mice with the C57BL/6J background most suitable for cataract research by Scheimpflug imaging. We show that lens densitometry by Scheimpflug imaging in mouse eyes can resolve differences of less than 1 %, making it possible to detect differences in cataract development in different mouse strains, even if they are small.     

Amiloride inhibition on NaCl responses of the chorda tympani nerve in two 129 substrains of mice, 129P3/J and 129X1/SvJ

Amiloride, a sodium channel blocker, is known to suppress NaCl responses of the chorda tympani (CT) nerve in various mammalian species. In mice, the NaCl suppressing effect of amiloride is reported to differ among strains. In C57BL mice, amiloride inhibits NaCl responses to about 50% of control, whereas no such clear suppression was evident in prior studies with 129 mice. However, evidence from behavioral studies is not entirely consistent with this. Recently, it has been found that genetic backgrounds of 129 mice differ within substrains. 129X1/SvJ (formerly 129/SvJ) mice differ from the 129P3/J (formerly 129/J) strain by 25% of sequence length polymorphisms. Therefore, we examined possible substrain difference between 129P3/J and 129X1/SvJ mice in the amiloride sensitivity of electrophysiologically recorded NaCl responses. Amiloride significantly suppressed CT responses to NaCl without affecting responses to KCl both in 129P3/J and 129X1/SvJ mice. However, the magnitude of the amiloride inhibition was significantly larger (approximately 50% of control in response to 0.01-1.0 M NaCl by 100 microM amiloride) in 129X1/SvJ than in 129P3/J mice (approximately 20% of control in response to 0.03-0.3 M NaCl by 100 microM amiloride). Threshold amiloride concentration for suppression of responses to 0.3 M NaCl was 30 microM in 129P3/J mice, which was higher than that in 129X1/SvJ mice (10 microM). In 129X1/SvJ mice, the threshold amiloride concentration eliciting inhibition of NaCl responses and the magnitude of the inhibition were comparable with those in C57BL/6 mice. These results suggest that amiloride sensitivity of NaCl responses differs even among the 129 substrains, 129P3/J and 129 X1/SvJ, and the substrain difference of 129 mice in amiloride sensitivity is as large as that between two inbred strains (129P3/J and C57BL/6).     

A simple PCR-based method for the rapid genotyping of inherited fifth complement component (C5)-deficient mice

The fifth component of complement (C5) is considered to be the center of complement activation and function. However, there are no genetically engineered knockout mice for this gene, and the only commercially available inherited C5-deficient mice, in which a “TA” nucleotide deletion in the coding frame was previously identified, are in theC57BL/10Sn genetic background rather than the commonly used backgrounds C57BL/6 and BALB/c. Therefore, these mice must be backcrossed into the desired genetic background. Here, we developed an ARMS (amplification refractory mutation system) PCR method using a specific primer pair that was able to discriminate between the genotypes when the resulting product was analyzed by agarose gel electrophoresis. These results were supported by quantitative RT-PCR and semi-quantitative PCR and were consistent with the results from sequencing each backcrossed generation. Using ARMS-PCR method, we generated C5-deficient mice in the C57BL/6 background over 9 backcrossed generations and further verified the phenotype using complement-mediated hemolytic assays. In this study, we describe a simple, rapid and reliable PCR-based method for genotyping inherited C5-deficient mice that may be used to backcross C57BL/10Sn mice into other genetic backgrounds.     

Effects of genetic background on thermoregulation and fatty acid-induced uncoupling of mitochondria in UCP1-deficient mice

An interaction between free fatty acids and UCP1 (uncoupling protein-1) leading to de-energization of mitochondria was assumed to be a key event for triggering heat production in brown fat. Recently, Matthias et al., finding indistinguishable de-energization of isolated brown fat mitochondria by fatty acids in UCP1-deficient mice and control mice, challenged this assumption (Matthias, A., Jacobsson, A., Cannon, B., and Nedergaard, J. (1999) J. Biol. Chem. 274, 28150-28160). Since their results were obtained using UCP1-deficient and control mice on an undefined genetic background, we wanted to determine unambiguously the phenotype of UCP1 deficiency with the targeted Ucp1 allele on congenic C57BL/6J and 129/SvImJ backgrounds. UCP1-deficient congenic mice have a very pronounced cold-sensitive phenotype; however, deficient mice on the F1 hybrid background were resistant to cold. We propose that heterosis provides a mechanism to compensate for UCP1 deficiency. Contrary to the results of Matthias et al., we found a significant loss of fatty acid-induced de-energization, as reflected by membrane potential and oxygen consumption, in brown fat mitochondria from UCP1-deficient mice. Unlike cold sensitivity, fatty acid-induced uncoupling of mitochondria was independent of the genetic background of UCP1-deficient mice. We propose that intracellular free fatty acids directly regulate uncoupling activity of UCP1 in a manner consistent with models described in the literature.     

Severely impaired B lymphocyte proliferation, survival, and induction of the c-Myc:Cullin 1 ubiquitin ligase pathway resulting from CD22 deficiency on the C57BL/6 genetic background

Understanding the molecular mechanisms through which CD22 regulates B lymphocyte homeostasis, signal transduction, and tolerance is critical to defining normal B cell function and understanding the role of CD22 in autoimmunity. Therefore, CD22 function was examined in vivo and in vitro using B cells from CD22-deficient (CD22(-/-)) mice. Backcrossing of founder CD22(-/-) mice onto the C57BL/6 (B6) genetic background from a B6/129 mixed background resulted in a dramatically reduced B cell proliferative response following IgM ligation, characterized by a paucity of lymphoblasts and augmented apoptosis. Also, the phenotype of splenic B6 CD22(-/-) B cells was uniquely HSA(high) and IgD(low)/CD21(low) with intermediate levels of CD5 expression, although the percentages of mature and transitional B cells were normal. That B6 CD22(-/-) B cells predominantly underwent apoptosis following IgM ligation correlated with this unique tolerant phenotype, as well as defective induction of the c-Myc:Cullin 1 (CUL1) ubiquitin ligase pathway that is necessary for progression to the S phase of cell cycle. CD40 ligation compensated for CD22 deficiency by restoring lymphoblast development, proliferation, c-Myc and CUL1 expression, and protein ubiquitination/degradation in IgM-stimulated B6 CD22(-/-) B cell cultures. Thereby, this study expands our current understanding of the complex role of CD22 during B cell homeostasis and Ag responsiveness, and reveals that the impact of CD22 deficiency is dictated by the genetic background on which it is rendered. Moreover, this study defines CD22 and CD40 as the first examples of lymphocyte coreceptors that influence induction of the c-Myc:CUL1 ubiquitin ligase pathway.     

Strain difference in expression of the adult-type polycystic kidney disease gene, pcy, in the mouse

DBA/2FG-pcy and C57BL/6FG-pcy congenic strains were established by transferring the polycystic kidney disease gene, pcy, to DBA/2 and C57BL/6 mice. We carried out pathological and hematological examinations of these strains at 4, 8, 16 and 30 weeks of age. In DBA/2FG-pcy mice more than 8 weeks of age, macroscopic renal cysts were observed on the surface of both kidneys. Their kidneys weight was significantly greater than in DBA/2 mice at all ages examined. Microscopic renal cysts were evenly distributed at 4, 8 and 16 weeks of age. At 30 weeks of age, the kidneys were filled with numerous polycysts. In C57BL/6FG-pcy mice, no macroscopic renal cysts were found until the animals were 30 weeks old, and the weight of their kidneys was greater than in B6 mice of the same age. From 8 weeks of age on, a limited number of microscopic renal cysts was observed, and many renal cysts were found adjacent to the enlarged Bowman's capsules. With age, the red blood cell count and hematocrit level decreased while the platelet count increased in both strains, with greater changes occurring in DBA/2FG-pcy mice than in C57 BL/6FG-pcy mice. These findings demonstrate that polycystic kidney disease exhibits strain differences in animals with a DBA/2 and C57BL/6 background. Our results suggest that phenotypic expression of the pcy gene in the mouse depends on genetic background, and that variations in the severity of human polycystic kidney disease may be explained, at least in part, by individual differences in genetic background.     

Derivation and comparison of C57BL/6 embryonic stem cells to a widely used 129 embryonic stem cell line

Typically, embryonic stem (ES) cells derived from 129 mouse substrains are used to generate genetically altered mouse models. Resulting chimeric mice were then usually converted to a C57BL/6 background, which takes at least a year, even in the case of speed congenics. In recent years, embryonic stem cells have been derived from various mouse strains. However, 129 ES cells are still widely used partially due to poor germline transmission of ES cells derived from other strains. Availability of highly germline-competent C57BL/6 ES cells would enormously facilitate generation of genetically altered mice in a pure C57BL/6 genetic background by eliminating backcrossing time, and thus significantly reducing associated costs and efforts. Here, we describe establishment of a C57BL/6 ES cell line (LK1) and compare its efficacy to a widely used 129SvJ ES cell line (GSI-1) in generating germline chimeras. In contrast to earlier studies, our data shows that highly germline-competent C57BL/6 ES cell lines can be derived using a simple approach, and thus support broader use of C57BL/6 ES cell lines for genetically engineered mouse models. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

129/SvJ mice have mutated CD23 and hyper IgE

CD23, the low affinity IgE receptor, is hypothesized to function as a negative regulator of IgE production. Upon discovering reduced CD23 surface levels in 129/SvJ inbred mice, we sought to further investigate 129/SvJ CD23 and to examine its influence on IgE levels. Five amino acid substitutions were found in 129/SvJ CD23. Identical mutations were also observed in CD23 from New Zealand Black and 129P1/ReJ mice. 129/SvJ B cells proliferated more rapidly than those from BALB/c after stimulation with IL-4 and CD40 ligand trimer. However, in vitro IgE levels in supernatants from stimulated 129/SvJ B cells were significantly reduced. Contrary to the in vitro findings, the 129/SvJ CD23 mutations correlated with a hyper IgE phenotype in vivo and 129/SvJ were able to clear Nippostrongylus brasiliensis infection more rapidly than either BALB/c or C57BL/6. Overall, this study further suggests that CD23 is an important regulatory factor for IgE production.     

Semen quality parameters and embryo lethality in mice deficient for Trp53 protein

Trp53 is a protein which is able to control semen parameters in mice, but the extent of that control depends on the genetic background of the mouse strain. Males from C57BL/6Kw, 129/Sv, C57BL×129 -p53+/+ (wild type controls) and C57BL×129-p53-/- (mutants) strains were used in the study, and histology and light microscopy were applied to evaluate the influence of genetic background and Trp53 (p53) genotype on testes morphology and semen quality in male mice. We showed that sperm head morphology, maturity and tail membrane integrity were controlled only by the genetic background of C57BL/6Kw and 129/Sv males, while testes weight and sperm concentration depended on both the genetic background and p53 genotype. Cell accumulation in seminiferous tubules may be responsible for heavier testes of p53-deficient males. In addition, to examine the effect of sex and p53 genotype on embryo lethality, pairs of control (C57BL×129-p53+/+) and heterozygous (C57BL×129-p53+/-) mice were examined. Before day 7 post coitum (dpc), female and male embryos were equally resorbed in both crosses types. After 7 dpc, preferential female embryo lethality in the heterozygote pairs was responsible for the skewed sex ratio in their progeny. Also, mutant female and male newborns were underrepresented in the litters of the heterozygous breeding pairs.     

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.     

Behavioral differences among fourteen inbred mouse strains commonly used as disease models

We compared the behavior of 14 inbred mouse strains and an F1 hybrid commonly used in transgenic and knockout production. These strains were 129P3/J, 129S1/SvImJ, 129S6/SvEvTac, 129T2/SvEmsJ, 129X1/SvJ (formerly 129/J, 129/Sv-p+Tyr+Kitl+/J, 129/SvEvTac, 129SvEmsJ, and 129/SvJ, respectively), A/JCrTac, BALB/cAnNTac, C3H/HeNTac, C57BL/6J, C57BL/6NTac, DBA/2NTac, FVB/NTac, NOD/MrkTac, SJL/JCrNTac, and the hybrid B6129S6F1Tac. Performance in three behavioral tests (rotorod, open-field activity-habituation, and contextual and cued fear conditioning) was determined. On the rotorod assay, SJL/JCrNTac mice had the shortest latencies to fall on the first day of testing, and DBA/2NTac mice showed impaired motor learning. Open-field behavior was analyzed using the parameters total distance, center distance, velocity, and vertical activity. 129T2/EvEmsJ and A/JCrTac were least active in the open field, whereas NOD/MrkTac mice were most active. Contrary to earlier studies, we found that all strains habituated to the open field in at least one of these parameters. In contextual and cued fear conditioning, all strains displayed activity suppression. However, FVB/NTac mice reacted less strongly to both context and cue than did most of the other strains. There were no significant behavioral differences between C57BL/6J and C57BL/6NTac, except for higher open-field activity in C57BL/6J female mice. These findings illustrate the importance of the appropriate selection of background strain for transgenic, gene targeting, or drug research.     

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 modifiers of the Kv beta2-null phenotype in mice

Shaker-type potassium (K+) channels are composed of pore-forming alpha subunits associated with cytoplasmic beta subunits. Kv beta2 is the predominant Kv beta subunit in the mammalian nervous system, but its functions in vivo are not clear. Kv beta2-null mice have been previously characterized in our laboratory as having reduced lifespans, cold swim-induced tremors and occasional seizures, but no apparent defect in Kv alpha-subunit trafficking. To test whether strain differences might influence the severity of this phenotype, we analyzed Kv beta2-null mice in different strain backgrounds: 129/SvEv (129), C57BL/6J (B6) and two mixed B6/129 backgrounds. We found that strain differences significantly affected survival, body weight and thermoregulation in Kv beta2-null mice. B6 nulls had a more severe phenotype than 129 nulls in these measures; this dramatic difference did not reflect alterations in seizure thresholds but may relate to strain differences we observed in cerebellar Kv1.2 expression. To specifically test whether Kv beta1 is a genetic modifier of the Kv beta2-null phenotype, we generated Kv beta1.1-deficient mice by gene targeting and bred them to Kv beta2-null mice. Kv beta1.1/Kv beta2 double knockouts had significantly increased mortality compared with either single knockout but still maintained surface expression of Kv1.2, indicating that trafficking of this alpha subunit does not require either Kv beta subunit. Our results suggest that genetic differences between 129/SvEv and C57Bl/6J are key determinants of the severity of defects seen in Kv beta2-null mice and that Kv beta1.1 is a specific although not strain-dependent modifier.