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).     

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.     

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.     

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.     

Impaired Pavlovian fear extinction is a common phenotype across genetic lineages of the 129 inbred mouse strain

Fear extinction is impaired in psychiatric disorders such as post-traumatic stress disorder and schizophrenia, which have a major genetic component. However, the genetic factors underlying individual variability in fear extinction remain to be determined. By comparing a panel of inbred mouse strains, we recently identified a strain, 129S1/SvImJ (129S1), that exhibits a profound and selective deficit in Pavlovian fear extinction, and associated abnormalities in functional activation of a key prefrontal-amygdala circuit, as compared with C57BL/6J. The first aim of the present study was to assess fear extinction across multiple 129 substrains representing the strain's four different genetic lineages (parental, steel, teratoma and contaminated). Results showed that 129P1/ReJ, 129P3/J, 129T2/SvEmsJ and 129X1/SvJ exhibited poor fear extinction, relative to C57BL/6J, while 129S1 showed evidence of fear incubation. On the basis of these results, the second aim was to further characterize the nature and specificity of the extinction phenotype in 129S1, as an exemplar of the 129 substrains. Results showed that the extinction deficit in 129S1 was neither the result of a failure to habituate to a sensitized fear response nor an artifact of a fear response to (unconditioned) tone per se . A stronger conditioning protocol (i.e. five × higher intensity shocks) produced an increase in fear expression in 129S1, relative to C57BL/6J, due to rapid rise in freezing during tone presentation. Taken together, these data show that impaired fear extinction is a phenotypic feature common across 129 substrains, and provide preliminary evidence that impaired fear extinction in 129S1 may reflect a pro-fear incubation-like process.     

Influence of test duration on the sensitivity of the two-bottle choice test

The long-term two-bottle choice test is commonly used as a simple screen to examine the acceptance of taste solutions by rodents. As part of an investigation of factors influencing the sensitivity of the two-bottle choice test, we determined the extent to which test duration influenced test sensitivity. C57BL6/J and 129X1/SvJ mice received four series of eight two-bottle tests, with each test lasting 1, 2, 4 or 6 days. Each series involved sequential tests with water, 2 mM saccharin, 5 and 50 mM citric acid, 30 and 300 micro M quinine hydrochloride, 75 mM NaCl and 10% ethanol. There were significant differences between the strains in intake of saccharin, 5 and 50 mM citric acid, NaCl and ethanol in 4 and 6 day tests, but only saccharin and ethanol in 2 day tests, and 5 mM citric acid and ethanol in 1 day tests. To compare the sensitivity of the tests, we developed an analytical approach based on the comparison of deviations of individual 129X1/SvJ mice from the C57BL6/J strain mean. Our results suggest that to discriminate between strains or treatments when using 'standard' laboratory conditions and methods, 1 day tests are generally inadequate and 2 day tests are useful only if large effects are anticipated. Tests lasting 4 or 6 days are more sensitive, but conducting 6 day tests provides little additional benefit and sometimes is detrimental relative to conducting 4 day tests.     

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.     

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.     

Differential profiles of genes expressed in neonatal brain of 129X1/SvJ and C57BL/6J mice: A database to aid in analyzing DNA microarrays using nonisogenic gene-targeted mice.

Strain-specific differences in gene expression have been observed among various inbred mouse strains. Two strains that are commonly used in gene-targeting research today are the 129 substrains, which are used to produce ES cell lines, and C57BL/6J, which is used for the extensive backcrosses required to produce isogenic knockout mice. When F2 nonisogenic littermates are assessed using DNA microarrays, one must determine whether the expression profiles obtained resulted either from specific alteration(s) induced by the targeted gene mutation or from gene expression differences related to the genetic background of the parent mouse strains. In the present study, we report the differential expression profile of genes expressed in neonatal brains and adult spleen and liver of 129X1/SvJ and C57BL/6J strains of mice. These comprehensive profiles were assessed using two types of Agilent Mouse Oligo Microarrays (development and standard) and were compiled into a publicly available database. Researchers can use this database to determine whether their microarray findings represent strain-specific differences in gene expression by comparing their data with those cataloged in our database. This database is useful for effectively analyzing DNA microarray data from nonisogenic littermates, and would help researchers avoid time-consuming backcrosses and confirmatory experiments requiring the use of many mice.     

Fibrinolysis in LPS-induced chronic airway disease

To examine the role of the fibrinolytic system in LPS-induced airway disease, we compared the effect of a chronic LPS challenge in plasminogen activator inhibitor-deficient (C57BL/6JPAI-1-/-) mice and wild-type (WT) C57BL/6J mice. Physiological and biological assessments were performed, immediately after, and 4 wk after an 8-wk exposure to LPS or saline. Immediately after the LPS exposure, WT mice had increased estimates of airway reactivity to methacholine compared with C57BL/6JPAI-1-/- mice; however, airway inflammation was similar in both LPS-exposed groups. Significant increases in both active transforming growth factor (TGF)-beta1 and active matrix metalloproteinase (MMP)-9 was detected after LPS exposure in WT but not C57BL/6JPAI-1-/- mice. C57BL/6JPAI-1-/- mice showed significantly less TGF-beta1 in the lavage and higher MMP-9 in the lung tissue than WT mice at the end of exposure and 4 wk later. After LPS exposure, both WT and C57BL/6JPAI-1-/- mice had substantial expansion of the subepithelial area of the medium [diameter (d) = 90-129 microm]- and large (d > 129 microm)-size airways when compared with saline-exposed mice. Subepithelial fibrin deposition was prevalent in WT mice but diminished in C57BL/6JPAI-1-/-. PAI-1 expression by nonciliated bronchial epithelial cells was enhanced in LPS-exposed WT mice compared with the saline-exposed group. Four weeks after LPS inhalation, airway hyperreactivity and the expansion of the subepithelial area in the medium and large airways persisted in WT but not C57BL/6JPAI-1-/- mice. We conclude that an active fibrinolytic system can substantially alter the development and resolution of the postinflammatory airway remodeling observed after chronic LPS inhalation.     

Altered sensitivity of cerebellar granule cells to glutamate receptor overactivation in the Cln3(Δex7/8)-knock-in mouse model of juvenile neuronal ceroid lipofuscinosis

The juvenile onset form of neuronal ceroid lipofuscinoses (JNCL) is a recessively inherited lysosomal storage disorder characterized by progressive neurodegeneration. JNCL results from mutations in the CLN3 gene that encodes a lysosomal membrane protein with unknown function. Utilizing a Cln3-knock-out mouse model of JNCL that was created on the 129S6/SvEv genetic background, we have previously demonstrated that CLN3-deficient cerebellar granule cells (CGCs) have a selectively increased sensitivity to AMPA-type glutamate receptor-mediated toxicity. Our recent findings that CGCs from 129S6/SvEv and C57BL/6J wild type (WT) mice have significant differences in glutamate receptor expression and in excitotoxic vulnerability indicated that the genetic background possibly have a strong influence on how glutamate receptor function is dysregulated in CLN3-deficient neurons. Indeed, here we show that in the Cln3(Δex7/8)-knock-in mouse model, that is on the C57BL/6J genetic background, mimics the most frequent mutation observed in JNCL patients and considered a null mutant, the sensitivity of CGCs to both AMPA- and NMDA-type glutamate receptor overactivations is altered. Cultured wild type and Cln3(Δex7/8) CGCs were equally sensitive to AMPA toxicity after 2 or 3 weeks in vitro, whereas the subunit-selective AMPA receptor agonist, CPW-399, induced significantly more cell death in mature, 3-week-old Cln3(Δex7/8) cultures. NMDA receptor-mediated toxicity changed during in vitro development: Cln3(Δex7/8) CGCs were less sensitive to high concentration of NMDA after 2 weeks in culture but became more vulnerable than their WT counterparts after 3 weeks in vitro. Abnormally altered glutamate receptor function in the cerebellum may result in motor deficits, and we confirmed that 7-week-old Cln3(Δex7/8) mice, similarly to Cln3-knock-out mice, have a motor coordination deficit as measured by an accelerating rotarod. Our results demonstrate altered glutamate receptor function in Cln3(Δex7/8) neurons and suggest that both AMPA and NMDA receptors are potential therapeutic targets in JNCL.     

MARCO mediates silica uptake and toxicity in alveolar macrophages from C57BL/6 mice

Scavenger receptors (SR), on the surface of the macrophage, appear to be responsible for silica uptake and cell death signaling in the macrophage. The purpose of this study was to isolate which SRs (macrophage receptor with collagenous structure (MARCO), CD204, or CD36) were involved using a variety of SR single and double null mice. The findings indicated that MARCO was the critical SR involved in silica uptake and cytotoxicity in the primary alveolar macrophages (AM) from C57BL/6 mice, as there was no particle uptake or cell death in the absence of this SR. The level of MARCO expression on AM changed significantly with the absence of other SR, and silica uptake was proportional to cell surface MARCO expression. In addition, silica uptake and cytotoxicity were completely blocked by an anti-mouse MARCO antibody. Transfection of Chinese hamster ovary cells with human MARCO supported these conclusions, as silica particles bound to and initiated apoptosis in the MARCO-transfected cells. Strain differences with regard to SR distribution were also examined. There was a differential expression of these SR on AM from each strain, with MARCO dominant for C57BL/6, CD36 dominant on BALB/c, and all three SR expressed on 129/SvJ mice. Similar to the results with C57BL/6 AM, MARCO was involved with silica-induced cell death in the 129/SvJ strain. In contrast, BALB/c AM used an unidentified mechanism for silica uptake because the SR antibodies failed to block particle internalization. Taken together, these results indicate MARCO is the primary AM receptor interacting with silica, depending on mouse strain and level of constitutive expression.     

Taste solution preferences of C57BL/6J and 129X1/SvJ mice: influence of age, sex, and diet

To examine whether age influences taste solution preferences, we measured taste preferences of C57BL/6J and 129X1/SvJ mice given a series of 48-h 2-bottle tests with a choice between water and one of the following taste solutions: 2 mM saccharin, 5 mM citric acid, 30 microM quinine hydrochloride, 75 mM sodium chloride (NaCl), 10 mM inosine monophosphate (IMP), 50 mM calcium chloride (CaCl(2)), and 10% ethanol. We tested separate groups of male mice fed Teklad 8604 chow at ages 4, 6, 9, 12, 15, 20, 25, 30, 40, and 50 weeks and retested some of these mice at 54, 75, and 100 weeks and again at 125 weeks. Female mice fed chow were tested at ages 4, 12, 25, and 50 weeks and retested at 54, 75, 100, and 125 weeks. Male mice fed AIN-93G semisynthetic diet were tested at ages 4, 12, 25, and 50 weeks and retested at 54, 75, and 100 weeks. Concentration-response functions for each taste solution were collected from male and female mice fed chow aged 8 or 125 weeks. In general, the results showed that age had little effect on taste preferences. Exceptions included 1) a small increase in quinine hydrochloride preference between 54 and 125 weeks in mice of both strains and sexes, 2) a marked increase in NaCl preference between 4 and 12 weeks in female B6 mice, 3) a gradual decrease in IMP preference between 4 and 125 weeks in male and female 129 mice, 4) a marked decrease in CaCl(2) preference between 54 and 125 weeks in male and female 129 mice, and 5) a marked reduction in ethanol preference between 4 and 12 weeks in male B6 mice fed AIN-93G diet but not chow. These results show that over a wide range and with the exceptions noted, age contributes little to the variation in taste preferences observed in C57BL/6J and 129X1/SvJ mice.     

Mitotic arrest in teratoma susceptible fetal male germ cells

Formation of germ cell derived teratomas occurs in mice of the 129/SvJ strain, but not in C57Bl/6 inbred or CD1 outbred mice. Despite this, there have been few comparative studies aimed at determining the similarities and differences between teratoma susceptible and non-susceptible mouse strains. This study examines the entry of fetal germ cells into the male pathway and mitotic arrest in 129T2/SvJ mice. We find that although the entry of fetal germ cells into mitotic arrest is similar between 129T2/SvJ, C57Bl/6 and CD1 mice, there were significant differences in the size and germ cell content of the testis cords in these strains. In 129T2/SvJ mice germ cell mitotic arrest involves upregulation of p27(KIP1), p15(INK4B), activation of RB, the expression of male germ cell differentiation markers NANOS2, DNMT3L and MILI and repression of the pluripotency network. The germ-line markers DPPA2 and DPPA4 show reciprocal repression and upregulation, respectively, while FGFR3 is substantially enriched in the nucleus of differentiating male germ cells. Further understanding of fetal male germ cell differentiation promises to provide insight into disorders of the testis and germ cell lineage, such as testis tumour formation and infertility.     

The impact of genetic background and Bid on the phenotype of Bcl-2-deficiency in mice

How a central apoptosis mechanism could be modulated during a specific developmental or homeostatic process to comply with the specific needs of a particular tissue is poorly understood. Bcl-2 is a key anti-apoptosis regulator and its deletion resulted in multiple defects in mice, indicating its broad involvement in development and homeostasis of various tissues. We found that the severity and extensiveness of the defects could be greatly influenced by the genetic background of the mice. Hence, Bcl-2-deficient mice predominantly on C57BL/6 background had the most severe presentation with increased embryonic lethality, whereas Bcl-2-deficient mice predominantly on 129/SvJ background had a significantly minor phenotype. In particular, the 129/SvJ background could almost completely rescue the polycystic kidney disease phenotype of the Bcl-2 deficiency, resulting in normal renal functions. These observations would be consistent with the assumption that the C57BL/6 background is more pro-death while the 129/SvJ background is more pro-survival. Concurrent deletion of Bid, a BH3-only molecule, in either genetic background, could significantly increase the birth rate of the Bcl-2 deficient progenies and lessen lymphocytopenia, although the double knockout mice still developed the polycystic kidney diseases. Overall, our work indicates that the phenotype of Bcl-2 deficiency can be affected by multiple genetic elements, resulting in tissue-specific modulations of the cell death program during development and cellular homeostasis.