Genetic analysis of low lambda 1 chain production in mice

Several commonly used strains of laboratory mice from Charles River Laboratories were found to produce extremely low or undetectable levels of serum immunoglobulins bearing lambda 1 light chain (lambda 1 Ig). Individual CF-1, CD-1, and CFW random-bred mice were tested for serum lambda 1 levels, lambda 1-specific anti-NP responses, and genomic polymorphisms at the lambda 1 locus. In all cases, a complete correlation among these parameters was observed. The results indicated that nearly all CFW, greater than 70% of CD-1 but none of the CF-1 mice produced low levels of lambda 1 light chain. The low lambda 1 Ig production is due to a genetic defect either similar or identical to that observed in SJL mice. The data suggest that the lambda 1 locus of CD-1, CFW, and SJL mice are derived from a common ancestor. We also surveyed lambda 1 Ig production in a series of wild mice. Mice producing low lambda 1 Ig were frequently observed. The wild mice with low lambda 1 Ig levels were captured in diverse geographic areas, including Europe, Middle East, Asia, and South America. Preliminary study suggests that the defect in the wild mice is different from that of SJL, CD-1, or CFW mice and implies that other mechanisms regulate lambda 1 Ig production in wild mice.     

Polymorphic microsatellite markers in the outbred CFW and ICR stocks for the generation of speed congenic mice on C57BL/6 background

Reliable definition of the phenotype of particular alleles is carried out in the genetic background of inbred strains. Appearance of mutations in outbred mice therefore requires the generation of congenic mice. The aim of this study was the establishment of a list of polymorphic microsatellite markers which can be used in a polymerase chain reaction (PCR)-based marker-assisted selection protocol (MASP) to allow the use of the two common outbred stocks, CFW and ICR, as donor animals for the fast generation of congenic C57BL/6 mice. The selection of informative microsatellite markers was carried out to provide a simple evaluation of the PCR products by conventional agarose gel electrophoresis. Outbred mice from three suppliers were examined. In total, 153 microsatellite loci were analysed. Here we present 76 and 70 microsatellite markers polymorphic for the outbred ICR and CFW stocks compared to C57BL/6. At least three microsatellite loci per chromosome were chosen as informative markers for the autosomal genome, giving rise to a maximum marker distance of 58 cM. Thus, additional individual markers have to be selected for the respective outbred mouse which is chosen as a donor animal.     

Spontaneous ophthalmic lesions in young Swiss mice.

BACKGROUND AND PURPOSE: Outbred mice are frequently used in toxicity evaluation. Due to their small size, ophthalmologic examination of such animals is difficult with regard to restraint and use of instruments designed for human medicine. The clinical appearance and incidence of spontaneous ophthalmic lesions should be helpful in selecting mice for toxicity studies and allow distinction between intercurrent spontaneous ocular changes and those attributable to drugs or chemicals. METHODS: Pretest ophthalmologic examinations of about 3,000 4- to 5-week-old Swiss mice, Crl:CD1 (ICR)BR, conducted in 1995 and 1996, provided information about spontaneous ocular changes and their incidence. Eye evaluations were performed after pupil dilatation (0.5% tropicamide instillation), using indirect ophthalmoscopy, and when indicated, a portable slit lamp. RESULTS: Lenticular opacities and heterogeneity/prominence were the most common findings (up to 19%) in the anterior segment. Abnormalities of the cornea and iris were detected in up to 4% of mice. Hyaloid artery remnant, as well as isolated cases of floating bodies or hemorrhage, was observed in the vitreous of 12 to 17% of mice. Approximately 2 to 4% of mice had colobomatous fundus, retinal fold, or retinal atrophy. A few mice had chorioretinal atrophy, hemorrhage, or abnormal pattern of the retinal vasculature. Remaining findings consisted of incomplete palpebral fissure, microphthalmia, exophthalmia, ophthalmic hemorrhage, and scleral mass. CONCLUSIONS: Due to severity of the condition or interference with ocular examination, affected mice should be eliminated from experimental studies. Hence, pretest ocular examinations of mice are indicated in safety-assessment toxicity studies.     

A2B adenosine receptor blockade enhances macrophage-mediated bacterial phagocytosis and improves polymicrobial sepsis survival in mice

Antimicrobial treatment strategies must improve to reduce the high mortality rates in septic patients. In noninfectious models of acute inflammation, activation of A2B adenosine receptors (A2BR) in extracellular adenosine-rich microenvironments causes immunosuppression. We examined A2BR in antibacterial responses in the cecal ligation and puncture (CLP) model of sepsis. Antagonism of A2BR significantly increased survival, enhanced bacterial phagocytosis, and decreased IL-6 and MIP-2 (a CXC chemokine) levels after CLP in outbred (ICR/CD-1) mice. During the CLP-induced septic response in A2BR knockout mice, hemodynamic parameters were improved compared with wild-type mice in addition to better survival and decreased plasma IL-6 levels. A2BR deficiency resulted in a dramatic 4-log reduction in peritoneal bacteria. The mechanism of these improvements was due to enhanced macrophage phagocytic activity without augmenting neutrophil phagocytosis of bacteria. Following ex vivo LPS stimulation, septic macrophages from A2BR knockout mice had increased IL-6 and TNF-α secretion compared with wild-type mice. A therapeutic intervention with A2BR blockade was studied by using a plasma biomarker to direct therapy to those mice predicted to die. Pharmacological blockade of A2BR even 32 h after the onset of sepsis increased survival by 65% in those mice predicted to die. Thus, even the late treatment with an A2BR antagonist significantly improved survival of mice (ICR/CD-1) that were otherwise determined to die according to plasma IL-6 levels. Our findings of enhanced bacterial clearance and host survival suggest that antagonism of A2BRs offers a therapeutic target to improve macrophage function in a late treatment protocol that improves sepsis survival.     

In vitro development of one-cell embryos from outbred mice: Influence of culture medium composition

Summary One-cell embryos from outbred mice (CF1, CD-1, and Dub:ICR) were cultured in various modifications of egg culture medium (ECM). The best development was observed in medium in which inorganic salts of modified T6 medium (mT6) replaced those of ECM. In this modification (TE), 66% of one-cell CF1 embryos developed into blastocysts, comared to 46 and 43% for ECM and mT6, respectively. Moreover, the cell numbers of blastocysts developing in TE (74.9±3.3) were higher than the cell numbers of those developing in ECM (55.1±2.4). The culture requirements of embryos varied between different stocks of mice: Fewer CF1 embryos developed to the blastocyst stage than either Dub:ICR embryos (90%) or CD-1 embryos (84%). Lowering the osmolarity of the medium from 300 to 280 mOsm, increasing the concentration of KC1 from 1.42 to 25 mM, or omitting lactate from the medium during Day 1 of culture did not further improve development of embryos, in contrast to previous reports. However, the time at which embryos were transferred to outgrowth medium influenced their postblastocyst development. The best development was observed when embryos were transferred on Day 4 of culture at the late morula-early blastocyst stage. This work was supported by the Office of Health and Environmental Research, U.S. Department of Energy, Washington, DC, contract DE-AC03-76-SF01012.     

Beneficial effects of taurine on mouse zygotes developing in protein-free culture medium

The objectives of this study were to determine if mouse zygotes from outbred mice can develop in simple culture medium in the absence of bovine serum albumin (BSA), and if taurine can be used as a medium supplement to improve development. Zygotes from 2 stocks of outbred mice (CD-1 and CF-1) were cultured in simple embryo culture medium (TE medium) lacking BSA and with or without taurine (24 mM), or in regular TE medium with BSA. The presence of BSA had little or no effect on development, but development to post-blastocyst endpoints was enhanced when CD-1 zygotes were cultured in medium containing taurine. In addition, when CD-1 blastocysts were transferred to pseudopregnant animals, embryos cultured in the presence of taurine developed into fetuses more often than those cultured in medium without taurine, and their weights were higher than those of embryos cultured in regular TE medium with BSA. These beneficial effects of taurine do not appear to be the nonspecific effects of a fixed nitrogen source, because the addition of glycine to BSA-free TE medium did not have similar beneficial effects. It was concluded that mouse zygotes from outbred mice do not require BSA for their preimplantation development in culture and that the presence of taurine in preimplantation culture medium is beneficial not only for preimplantation development of the zygotes, but also for their post-blastocyst development.     

Rodent brain and heart catecholamine levels are altered by different models of copper deficiency

Limiting dopamine beta-monooxygenase results in lower norepinephrine (NE) and higher dopamine (DA) concentrations in copper-deficient Cu- tissues compared to copper-adequate Cu+ tissues. Mice and rat offspring were compared to determine the effect of differences in dietary copper Cu deficiency started during gestation or lactation on catecholamine, NE and DA, content in brain and heart. Holtzman rat and Hsd:ICR (CD-1) outbred albino mouse dams were fed a Cu- diet and drank deionized water or Cu supplemented water. Offspring were sampled at time points between postnatal ages 12 and 27. For both rat and mouse Cu- tissue, NE and DA changes were greater at later ages. Though Cu restriction began earlier in rats than mice in the gestational model, brain NE reduction was more severe in Cu- mice than Cu- rats. Cardiac NE reduction was similar in Cu- rodents in the gestation models. In the lactation model, mouse catecholamines were altered more than rat catecholamines. Furthermore, following lactational Cu deficiency Cu- mice were anemic and exhibited cardiac hypertrophy, Cu- rats displayed neither phenotype. Within a species, changes were more severe and proportional to the length of Cu deprivation. Lactational Cu deficiency in mice had greater consequences than in rats.     

Spontaneous wasting disease in nude mice associated with Pneumocystis carinii infection

Several cases of wasting disease and dyspnea occurred in outbred Crl: CDI(ICR) nu/nu mice obtained recently from a commercial supplier. Gross necropsy revealed pulmonary consolidation, and histologically there was alveolar-wall thickening and filling of alveoli with macrophages and honey-combed eosinophilic material. Electron microscopic examination of lung tissue confirmed infection with Pneumocystis carinii.     

Catheter colonization and abscess formation due to Staphylococcus epidermidis with normal and small-colony-variant phenotype is mouse strain dependent

Coagulase-negative staphylococci (CoNS) form a thick, multilayered biofilm on foreign bodies and are a major cause of nosocomial implant-associated infections. Although foreign body infection models are well-established, limited in vivo data are available for CoNS with small-colony-variant (SCV) phenotype described as causative agents in implant-associated infections. Therefore, we investigated the impact of the Staphylococcus epidermidis phenotype on colonization of implanted PVC catheters and abscess formation in three different mouse strains. Following introduction of a catheter subcutaneously in each flank of 8- to 12-week-old inbred C57BL/6JCrl (B6J), outbred Crl:CD1(ICR) (CD-1), and inbred BALB/cAnNCrl (BALB/c) male mice, doses of S. epidermidis O-47 wild type, its hemB mutant with stable SCV phenotype, or its complemented mutant at concentrations of 10(6) to 10(9) colony forming units (CFUs) were gently spread onto each catheter. On day 7, mice were sacrificed and the size of the abscesses as well as bacterial colonization was determined. A total of 11,500 CFUs of the complemented mutant adhered to the catheter in BALB/c followed by 9,960 CFUs and 9,900 CFUs from S. epidermidis wild type in BALB/c and CD-1, respectively. SCV colonization was highest in CD-1 with 9,500 CFUs, whereas SCVs were not detected in B6J. The minimum dose that led to colonization or abscess formation in all mouse strains was 10(7) or 10(8) CFUs of the normal phenotype, respectively. A minimum dose of 10(8) or 10(9) CFU of the hemB mutant with stable SCV phenotype led to colonization only or abscess formation, respectively. The largest abscesses were detected in BALB/c inoculated with wild type bacteria or SCV (64 mm(2) vs. 28 mm(2)). Our results indicate that colonization and abscess formation by different phenotypes of S. epidermidis in a foreign body infection model is most effective in inbred BALB/c followed by outbred CD-1 and inbred B6J mice.     

Copper deficient rats and mice both develop anemia but only rats have lower plasma and brain iron levels

Iron homeostasis depends on adequate dietary copper but the mechanisms are unknown. Mice (Mus musculus) and rat (Rattus norvegicus) offspring were compared to determine the effect of dietary copper deficiency (Cu-) on iron status of plasma, liver, brain and intestine. Holtzman rat and Hsd:ICR (CD-1) outbred albino mouse dams were fed a Cu- diet and drank deionized water or Cu supplemented water. Offspring were sampled at time points between postnatal ages 13 and 32. Cu- rat and mouse pups were both anemic, but only rat pups had lower plasma and brain iron levels. Plasma iron was lower throughout the suckling period in Cu- rats but not Cu- mice. Cu- mice derived from dams restricted of Cu only during lactation were also severely anemic without hypoferremia. Intestinal metal analysis confirmed that Cu- pups had major reductions in intestinal concentration of Cu, increased Fe, and normal Zn. However, whole mouse (less the intestine) analysis demonstrated normal content of Fe indicating that the limitation in iron transport by intestinal hephaestin had no consequence to total iron reserves of the mouse. Further research will be needed to determine the reason Cu- mice were anemic since the "ferroxidase" hypothesis does not explain this phenotype.     

Comparison of tetraploid blastocyst microinjection of outbred Crl:CD1(ICR), hybrid B6D2F1/Tac, and inbred C57BL/6NTac embryos for generation of mice derived from embryonic stem cells

Embryo electrofusion and tetraploid blastocyst microinjection is a modification of the traditional embryonic stem cell (ES cell)-based method to generate targeted mutant mice. Viability of tetraploid embryos is reportedly lower than with diploid embryos, with considerable interstrain variation. Here we assessed fetus and pup viability after ES cell microinjection of tetraploid blastocysts derived from outbred, hybrid, and inbred mice. Two-cell mouse embryos (C57BL/6NTac [B6], n = 788; B6D2F1/Tac [BDF1], n = 1871; Crl:CD1(ICR) [CD1], n = 1308) were electrofused; most resultant tetraploid blastocysts were injected with ES cells and surgically transferred into pseudopregnant recipient mice. Reproductive tracts were examined at midgestation for embryologic studies using B6 and BDF1 blastocysts; implantation sites and viable fetuses were counted. Pregnancies were carried to term for studies of targeted mutant mice using BDF1 and CD1 blastocysts, and pup yield was evaluated. Electrofusion rates of 2-cell embryos did not differ among B6, BDF1, and CD1 mice (overall mean, 92.8% +/- 5.4%). For embryologic studies, 244 B6 blastocysts were surgically transferred and 1 fetus was viable (0.41%), compared with 644 BDF1 blastocysts surgically transferred and 88 viable fetuses (13.7%). For targeted mutant mouse studies, 259 BDF1 blastocysts were surgically transferred yielding 10 pups (3.9%); 569 CD1 blastocysts yielded 44 pups (7.7%).     

Etonitazene-induced antinociception in μ1 opioid receptor deficient CXBK mice: Evidence for a role for μ2 receptors in supraspinal antinociception

The prevailing view is that supraspinal μ opioid-mediated antinociception in mice is mediated via the μ₁ subtype. The purpose of the present study was to determine if the highly μ-selective compound etonitazene could produce supraspinal (intracerebroventricular; i.c.v.) antinociception in CXBK mice, which are deficient in brain μ₁, but not μ₂, opioid receptors. CXBK or normal Crl:CD-1 ^®(ICR)BR mice were administered graded doses of etonitazene i.c.v. and 15 min later antinociception was assessed by a standard radiant-heat or 55°C water tail-flick test. Etonitazene produced dose-related antinociception that was blocked by naloxone and by β-FNA (demonstrating a μ opioid mechanism), but not by either ICI-174,864 or naltrindole (demonstrating the lack of involvement of δ opioid receptors). These findings suggest that μ₂ opioid receptors are important contributors to opioid-induced supraspinal antinociception in mice.     

Rapid induction of thymic lymphomas by isopropyl methanesulfonate: a preliminary report

The direct-acting SN1 alkylating agent isopropyl methanesulfonate (IMS) was carcinogenic by subcutaneous injection in female Hsd:(ICR)BR mice, causing thymic lymphoid neoplasms within 7 months in at least 20 of 32 treated mice. No such neoplasms were observed in mice treated with the direct-acting SN2 methyl homolog, methyl methanesulfonate (MMS). Both the IMS-treated mice and the MMS-treated mice initially received 20 mumole of the respective compounds by sc injection once weekly; however, because of toxic effects the dose of IMS was reduced to 10 mumole per injection on the 63rd day and further reduced to 5 mumole per injection on the 120th day, after which this dose was maintained until the 202nd day when the last surviving IMS-treated mouse became moribund and was sacrificed. In 2 of the MMS-treated mice, 93% of which were alive at 288 days, tumors were observed at the site of injection, one being a papilloma and the other a subcutaneous sarcoma. IMS has not previously been implicated as a carcinogen, to our knowledge. Its induction of thymic lymphomas may conceivably be related to its ability to alkylate exocyclic oxygen atoms in the DNA of hemopoietic cells.     

Polygenic determination of quinine aversion among mice

There are substantial differences among inbred mouse strainsin avoidance of quinine solutions in two-bottle preference tests.x A Mendelian cross-breeding experiment was conducted to testthe hypothesis that a single locus Qui has a major influenceon quinine aversion. Inbred strains C57B1/6J (B6, avoider) andC3HeB/FeJ (C3, indifferent) were progenitors of two segregatinggenerations. Phenotypic ratios for 100 µM and 30 µMquinine sulfate (QSO4) in these generations were not consistentwith ratios expected for a single gene. Frequency distributionsfor individual preference ratios were more characteristic ofa polygenic trait. An outbred strain (CFW/Crl) which displayssegregation for the Soa locus was tested for both QSO4 and sucroseocta-acetate (SOA) avoidance. Correlated avoidance patternsfor the two bitter compounds were found in these mice. A Soaeffect might not have been seen in the C3.B6 cross because bothstrains are relatively poor SOA avoiders. A second Mendeliancross was made between strains C3 and SWR/J (SW, SOA and QSO4avoider). One segregating generation was tested with both compounds.In these mice, as in the CFW population, QSO4 aversion was correlatedwith SOA aversion. These results suggest that quinine aversionis polygenic, that there is a relationship between SOA sensitivityand quinine sensitivity, and that this association may be theresult of variation at the Soa locus.     

Sleep-time variation for ethanol and the hypnotic drugs tribromoethanol, urethane, pentobarbital, and propofol within outbred ICR mice.

To evaluate the phenotypic variation within a commercial outbred mouse stock, we examined sleep-time (or duration of loss of righting reflex) of outbred ICR mice after i.p. injection of ethanol (4.0 g/kg of body weight), urethane (1.3 g), tribromoethanol (250 mg), and pentobarbital (60 mg), and after i.v. injection of propofol (30 mg). We observed high-grade individual differences in sleep-time that ranged from 0 to 179 min, 83.1 +/- 4.3 (mean and SEM of 100 mice) for ethanol; 0 to 169 min, 64.5 +/- 3.1 for pentobarbital; 0 to 160 min, 36.6 +/- 3.6 for urethane; 0 to 120 min, 21.5 +/- 2.2 for tribromoethanol; and 3 to 20.5 min, 7.1 +/- 0.3 for propofol. This extensive phenotypic variance within the outbred stock was as great as the variation reported among inbred strains or selected lines, and the varied susceptibility within the colony was inherited by Jcl:ICR-derived inbred strains IAI, ICT, IPI, and IQI. The range of sleep-time variance for ethanol, pentobarbital, urethane, tribromoethanol, and propofol within four-way cross hybrid Jcl:MCH(ICR) mice was 86.6%, 63.3%, 124%, 61.0%, and 53.1% that of outbred Jcl:ICR mice, respectively. The present study indicates that phenotypic variance within an outbred Jcl:ICR stock was at high risk for susceptibility to the drugs that depress the central nervous system and that Jcl:ICR-derived inbreds may be an excellent source of animal models for studying the anesthesia gene.     

Development of mouse embryos in vitro is affected by strain and culture medium

One-cell and two-cell embryos from three random-bred strains of mice–CF1, Dub:(ICR), and CFW (Swiss-Webster)–were cultured to the blastocyst stage in Spindle's, Earle's, Ham's F10, Whittingham's T6, or Hoppe and Pitts' medium. CFW embryos were more successful than CF1 and Dub:(ICR) embryos in developing to the blastocyst stage in all five media. Dub:(ICR) and CFW two-cell embryos showed the best development in Spindle's, Whittingham's T6, and Hoppe and Pitts', whereas CF1 two-cell embryos were most successful in developing in Hoppe and Pitts' medium. Similar results were obtained with one-cell embryos, although fewer developed to the blastocyst stage, and T6 rather than Hoppe and Pitts' medium sustained the best development of CF1 one-cell embryos. For all strains, the least successful development was in Ham's F10, but CFW embryos did show good development in this medium. In addition to the effects of various media on mouse embryo development, our results indicate that the strain of mouse used for the bioassay of media is of critical importance. Random-bred CFW (Swiss-Webster) mice are as suitable as a hybrid strain for this purpose.     

Gender influences infectivity in C57BL/6 mice exposed to mouse minute virus

Two natural outbreaks of mouse minute virus (MMV) are described. Observations during management of the naturally infected colonies led to a study in which 4-wk-old C57BL/6NCr and C57BL/6Tac mice were inoculated oronasally with an immunosuppressive variant of MMV (MMVi), as were adult C57BL/6NCr lactating dams or their pups (age, 10 d). By day 28 postinoculation, 100% of the 4-wk-old male C57BL/6NCr and C57BL/6Tac mice, 56.2% of 4-wk-old C57BL/6NCr female and 62.5% of 4-wk-old C57BL/6Tac female mice, 100% of adult lactating C57BL/6NCr dams, and 100% of inoculated pups (10 d) had seroconverted. Serologically positive nursing dams did not infect their nursing pups. In contrast, when nursing pups were inoculated, 100% of their dams seroconverted by 28 d postinoculation. Only 1 of 4 facility sentinels (Tac:SW female mice) seroconverted to MMVi and none of the 4 research sentinels (Tac:SW female mice) seroconverted under a once-weekly bedding transfer program. Consequently, 4 new research Tac:SW sentinels of each gender (n = 8) were placed in known-positive cages at cage-change; 100% of the male mice but 0% of the females seroconverted by day 48. Study results suggest gender influences both infectivity and the ability to detect subclinical infections of MMVi. Other factors that may influence detection of MMV include mouse strain or stock, short shedding period, and prolonged time between cage changes. In light of the data from both the natural infections and the experimental cases, cessation of breeding likely will be beneficial when trying to eradicate this virus.     

Heligmosomoides polygyrus (=Nematospiroides dubius): the development of self-cure and/or protection in several strains of mice.

Strains of outbred (ICR/CD1 and S--W) and inbred (BALB/C and C57BL/6) mice vaccinated subcutaneously (SQ) with 500, 1,000, or 2,000 exsheathed Heligmosomoides polygyrus larvae developed varying levels of protection upon subsequent oral challenge with larvae. In contrast, the inbred C3H/HEJ strain failed to develop protection at any dosage level tested. ICR/CD1 mice vaccinated intraperitoneally with exsheathed larvae developed a high level of resistance but exhibited extensive adhesions of the viscera. When ensheathed larvae were used for vaccination, ICR/CD1 mice developed a moderate level of protection; but 1% of the vaccine dose was recovered in the intestine as adult stages. Both the inbred and outbred strains given multiple oral infections developed a protection response similar to that strain's response following parenteral vaccination. The specificity of this protection was demonstrated using various complex foreign antigens. In contrast, the self-cure response was observed only in the S--W strain.     

Observations on soft tissue calcification in DBA/2NCrj mice in comparison with CRJ:CD-1 mice

Male and female DBA/2NCrj (DBA/2) mice 3, 4, 5 and 10 weeks old were examined biochemically and pathologically and the results obtained were compared with those for CRJ:CD-1 (ICR) mice of the same age. The plasma levels of glucose, triglyceride and total cholesterol tended to be lower in DBA/2 mice than in ICR mice but the levels of non-esterified fatty acid, calcium and inorganic phosphorus were almost the same in the two strains. The mean body weight of DBA/2 mice was significantly lower than that of ICR mice at each examination, and the relative weights of the hearts of male and female DBA/2 mice were significantly greater than those of male and female ICR mice. Cardiac calcinosis, tongue calcification and corneal degeneration occurred exclusively in DBA/2 mice with incidences of 30%-100%. The incidence and severity of these lesions increased with age but no sex differences were seen. It was difficult to relate differences in biochemical features of the two strains with pathological findings obtained in the DBA/2 mice. The numbers of cells secreting adrenocorticotropic hormone in the pituitary glands were significantly greater in male and female DBA/2 mice than in ICR mice, suggesting a higher secretion of corticosteroids in the former strain.