G1 Mice Research Articles

Paternal exposure to low-dose lead acetate: effect on implantation rate,pregnancy outcome, and sex ratio in mice.

It has been reported from in vivo exposed experimental animals that paternal lead exposure reduces birth rate; however, there is limited evidence to suggest a decrease in the proportion of male births. This study investigated the role of paternal exposure to lower lead acetate doses on early embryonic development (implantation) and the sex ratio of their offspring. In total 180 Swiss Webster mice were used (60 male and 120 female). The males were divided into 3 groups: G1 (untreated group), G2 (treated daily with 50 µg/kg BW lead acetate), and G3 (treated daily with 100 µg/kg BW lead acetate). The implantation success rate, pregnancy outcome, and sex ratio were measured. The results showed a highly significant reduction in both the percentage of implantation rate and the number of offspring in the G3 mice, but there was no signification difference for the G2 mice. There was a slight insignificant reduction in the number of newborn males compared with females for both G2 and G3 mice. The findings of this study suggest lead exposure in experimental animals reduces implantation rate with paternal BLL of 28 µg/dL and the sex ratio for offspring showed a slight insignificant reduction with both paternal BLLs of 23.5 µg/dL and 28 µg/dL.

Eµ-TCL1mTerc-/- Mouse Model for Telomere Dysfunction in Chronic Lymphocytic Leukemia

Telomeres are nucleo-protein complexes at the ends of the chromosomes that play a key role in protection of the ends from being recognized as DNA damage and to prevent fusion of the chromosomes. The telomeric DNA shortens with each cell division in the absence of telomerase, due to end replication problem. In chronic lymphocytic leukemia (CLL), short telomeres were found to be associated with poor prognostic factors and poor survival in various univariable and multivariable analyses. Short telomeres in CLL are known to be frequently associated with increased DNA damage response and to undergo fusion events, conferring genomic instability. But the contribution of telomere dysfunction to CLL pathogenesis and disease progression has never been studied in vivo using mouse models.Here, we hypothesized that genomic instability resulting from telomere dysfunction could drive acquisition of genetic lesions, contributing to CLL pathogenesis, progression and disease evolution. Thus, the CLL mouse model with telomere dysfunction was generated by crossing the Eµ-TCL1 (TCL1+) mouse with mTerc-/- mouse. The first generation TCL1+ mTerc-/- (G1) mice were inter-crossed to obtain generations G2 and G3, as telomeres are known to shorten with subsequent generations. The TCL1+ mTerc-/- mice from the generations G1 (N=14), G2 (N=33) and G3 (N=26), including TCL1+ (N=34), wildtype (WT, N=18) and mTerc-/- G1 (N=4), G2 (N=5) and G3 (N=13) as controls were initially analyzed for disease burden in peripheral blood (PB) by bleeding at an interval of 4 weeks, starting from 12 weeks and the percentage of CD19+ CD5+ cells was estimated by FACS. No difference in disease onset or progression was observed between the TCL1+ mTerc-/- G1, G2 and G3 in comparison toTCL1+ mice (Fig. 1a). Similarly, analysis of survival showed no significant difference between the TCL1+ mTerc-/- G1 (N=14), G2 (N=33) and G3 (N=26) mice, compared to TCL1+ (N=34) (median: 53, 55, 52 weeks vs. 50.5 weeks, Fig. 1b).Spleen and liver weights in the TCL1+ mTerc-/- G1 (N=12), G2 (N=33) and G3 (N=26) mice were highly variable (spleen: 0.1g to 3.5g, liver: 0.1g to 8.0g) as in the TCL1+ (N=27, spleen: 0.3g to 5.0g, liver: 1.7g to 7.4g) mice but no significant difference in spleen (Fig. 1d) and liver weights was observed between the subgroups. Interestingly, spleen weights were associated with survival only in the TCL1+ mice, with larger spleens associated with worse survival (48.5 vs. 57.5 weeks, P=0.091). Since no difference in disease characteristics was observed, it was verified using Q-PCR, if telomere lengths vary in the tumors from the different subgroups. Telomere lengths of CLL cells from the spleen were significantly shorter (Fig. 1c) in the G1 (median: 20.5kb, P=0.0002), G2 (median: 18.5kb, P=0.0016) and G3 (median: 13.2kb, P<0.0001) compared to TCL1+ (median: 28.7kb). The absence of correlation of telomere length with survival in the murine CLL models with telomere dysfunction may indicate that a critical telomere length in the tumor is yet to be reached to elicit genetic alterations and clonal selection.Additionally, the G3 mTerc-/- microenvironment is known to restrict B and T lymphopoiesis and thus might influence CLL cell proliferation, masking disease aggressiveness in the TCL1+ mTerc-/- G3 mice. To overcome the influence of mTerc-/- microenvironment, CLL cells obtained from spleens of TCL1+ and TCL1+ mTerc-/- G3 mice were transferred into syngeneic C57Bl6 mice. Briefly, 20 million cells were intravenously injected into the tail vein and disease was monitored by analysis of CD19+ CD5+ cells in PB, once every 4 weeks. Early follow up of 8 weeks clearly show a trend towards increase in CLL cells in PB of mice transferred with TCL1+ mTerc-/- G3 tumors compared to those with TCL1+ tumors (median tumor load: 15.75% vs. 6.1%, P=0.0553). Longer follow up of the experiment is ongoing.In summary, the TCL1+ mTerc-/- mice across the generations G1, G2 and G3 showed no difference in disease onset, progression, disease burden and survival in comparison to TCL1+ mice. The absence of increased disease manifestation in the TCL1+ mTerc-/- may be attributed to the microenvironmental influence on lymphopoiesis, as syngeneic transfer of CLL from TCL1+ mTerc-/- G3 mice showed an increase in tumor load compared to that of TCL1+ tumors, indicating a contribution of telomere shortening to disease aggressiveness in CLL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Effect of low-level laser therapy (GaAlAs - λ660 nm) on muscle function

Introduction:Low-level laser therapy (LLLT) is effective in preventing fatigue and in stimulating the microcirculation and cellular activity. In this study, we examined the effect of LLLT on injured tibial muscle in vivo by assessing muscle function during fatigue.MethodsTwenty-four male mice were used. Each mouse received an injection of sterile 0.9% saline solution (50 µL) in the right tibialis anterior muscle, after which the tendon of the muscle was exposed, connected to an isometric transducer and subjected to a resting tension of 1 g. A bipolar electrode was attached to the tibial nerve for electrical stimulation. The mice were randomly allocated to one of two groups: G1 (control: 3 h – n=8 and 9 h – n=5) and G2 (treated with GaAlAs laser, λ660 nm, 35 mW, 0.6 J, 17 s: 3 h – n=6 and 9 h – n=5).ResultsIn G1 mice, the amplitude of the tetanic contracture in response to induced fatigue remained unchanged during six consecutive tetani. The amplitude of the tetanic contractions in response to electrical stimulation (4-8 mV) was also unchanged. These results indicated muscle intactness in response to the load imposed by tetanus. In G2 mice, there was an increase in the amplitude of contraction after 3 h and 9 h when compared to G1 at 83% tetanus.ConclusionThese results indicate that exposure of muscle to LLLT enhanced the contractile force and increased the resistance to muscle fatigue without causing morphological damage to cellular structures.

An ENU Mutagenesis Screen of FLT3-ITD Knock-in Mice Identifies Novel Gene Mutations That Lead to an Exacerbated Myeloproliferative Neoplasm

BACKGROUND: Activating mutations of the FLT3 gene are common in acute myeloid leukemia (AML), with approximately 25-30% of cases containing an internal tandem duplication (ITD) in the juxtamembrane domain. The presence of a FLT3-ITD mutation is associated with a poor prognosis, the severity of which, can be modulated by the combination of co-occuring mutations. In animal models, expression of Flt3-ITD by transgenesis, bone marrow transplantation or gene knock-in does not lead to an acute leukemia but a myeloproliferative disease, resembling CMML, suggesting a requirement for additional co-operating mutations (Lee et al, Cancer Cell. 2007, 12: 367). This is supported by in vivo models which demonstrate that the combination of the Flt3-ITD mutation with other genetic lesions leads to the development of an acute leukemia in mice (Chen et al, Genes Dev. 2013, 27: 1974).AIMS: To identify and characterise novel genes that alter Flt3-ITD induced MPN by using an N-ethyl-N-Nitroso-urea (ENU) mutagenesis strategy in mice with an Flt3-ITD homozygous knock-in background.METHODS: An autosomal dominant screen for Flt3-ENU co-operating mutations was carried out at the Australian Phenomics Facility by mating ENU-mutagenised male mice homozygous for the Flt3-ITD knock-in allele to homozygous Flt3-ITD females. G1 mice were screened for changes in blood cell parameters indicative of an altered disease state (compared to that induced by Flt3-ITD alone). Mice with blood cell parameters outside two standard deviations of the relevant G1 mean were identified as potential mutation carriers and bred to Flt3-ITD homozygous knock-in mice to test for heritability of the phenotype. Where pedigrees were generated demonstrating heritable phenotypes, multiple affected and unaffected littermates were subject to exome sequencing and analysis to identify a list of candidate gene mutations segregating with the disease phenotype.RESULTS: 150 G1 mice were screened, leading to the identification of four pedigrees with heritable phenotypes marked by an exacerbated MPN. Exome sequencing has identified a short list of 3 genes for one pedigree (pedigree 37) that includes a mutation in Neurofibromatosis 1 (Nf1), a gene known to be involved in the induction of juvenile myelomonocytic leukemia and frequently lost in AML (Parkin et al,Clin Cancer Res 2010, 16:4135). In another pedigree (pedigree 24) we identified a mutation in Ndufa10 as the single candidate segregating with the phenotype (Figure 1A-B). Ndufa10 encodes a subunit of the mitochondrial respiratory complex I, which is the first and largest complex in the mitochondrial electron transport chain. Importantly, germline mutations in this gene lead to a complex I deficiency syndrome in humans, indicating that it is a critical subunit of this complex. We hypothesise that mutation of Ndufa10 leads to altered cellular metabolism in hematopoietic stem and progenitor cells which contributes to exacerbation of the MPN, possibly through an alteration in production of reactive oxygen species and a shift in the balance between glycolysis and oxidative phosphorylation. Breeding of the Ndufa10 mutation onto a non Flt3-ITD background shows that action of the mutation is not dependent on the presence of Flt3-ITD, as these mice also have altered blood counts, including increased WBC (Figure 1C).CONCLUSIONS: It is possible to identify mutations that exacerbate Flt3-ITD induced MPN through mutagenesis and an efficient blood screening strategy. In addition, using this strategy, we have identified novel mutations that act independently of Flt3-ITD to induce changes in the haematological compartment. Translation of these findings to human AML may indicate pathways that will be targets for new and complementary treatments in AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Effects of background mutations and single nucleotide polymorphisms (SNPs) on the Disc1 L100P behavioral phenotype associated with schizophrenia in mice.

BackgroundDisrupted-in-schizophrenia 1 (DISC1) is a promising candidate susceptibility gene for psychiatric disorders, including schizophrenia, bipolar disorder and major depression. Several previous studies reported that mice with N-ethyl-N-nitrosourea (ENU)-induced L100P mutation in Disc1 showed some schizophrenia-related behavioral phenotypes. This line originally carried several thousands of ENU-induced point mutations in the C57BL/6 J strain and single nucleotide polymorphisms (SNPs) from the DBA/2 J inbred strain.MethodsTo investigate the effect of Disc1 L100P, background mutations and SNPs on phenotypic characterization, we performed behavioral analyses to better understand phenotypes of Disc1 L100P mice and comprehensive genetic analyses using whole-exome resequencing and SNP panels to map ENU-induced mutations and strain-specific SNPs, respectively.ResultsWe found no differences in spontaneous or methamphetamine-induced locomotor activity, sociability or social novelty preference among Disc1 L100P/L100P, L100P/+ mutants and wild-type littermates. Whole-exome resequencing of the original G1 mouse identified 117 ENU-induced variants, including Disc1 L100P per se. Two females and three males from the congenic L100P strain after backcrossing to C57BL/6 J were deposited to RIKEN BioResource Center in 2008. We genotyped them with DBA/2 J × C57BL/6 J SNPs and found a number of the checked SNPs still remained.ConclusionThese results suggest that causal attribution of the discrepancy in behavioral phenotypes to the Disc1 L100P mutant mouse line existing among different research groups needs to be cautiously investigated in further study by taking into account the effect(s) of other ENU-induced mutations and/or SNPs from DBA/2 J.Electronic supplementary materialThe online version of this article (doi:10.1186/1744-9081-10-45) contains supplementary material, which is available to authorized users.

Effect of N-ethyl-N-nitrosourea on the fatality, recovery of fertile period and breeding record in C57BL/6J mice

N-ethyl-N-nitrosourea (ENU) is a potent mutagen in a mouse model by inducing point mutation in a random manner and, in particular, causing heritable base substitutions in spermatogonia. In this study, systematic development of phenotype-driven mutant mice with large scale was carried out by using ENU. Nine-week-old male mice of C57BL/6J received intraperitoneal injection at three times with 100 mg/kg of ENU at weekly intervals for three weeks. After injections with ENU, the changes of body weight, fatality, recovery of fertile period, and breeding record were measured in these mice. Body weight lost as a result of ENU treatments was reversed after the last ENU injection. Live fertile male mice recovered from infertility from 104 to 165 days after ENU treatments were mated with C57BL/6J female mice for generation of G1 offspring. An average birth rate was 5.9 mice from 1 pair of paternal and maternal mice. All of 231 G1 offspring mice were analyzed by modified-SHIRPA with standard procedure at nine weeks of age. Among G1 mice, 166 mice were identified as mutagenic phenotypes in 20 test items. The changes in mutagenic phenotypes after ENU treatments, for instance, pattern in the region with a different color, touch escape, changes in head morphology, pupil, and teeth, and negative geotaxis etc., were found in these mice. Taken together, these results indicate that ENU may be a trans-generational mutagen in C57BL/6J mice.

A Sensitized Whole Genome ENU Mutagenesis Screen Identifies an Arp2 Missense Mutation As a Novel Suppressor of Lethal Thrombosis in the Factor V Leiden Mouse

Abstract 493Only ∼10% of individuals carrying the common risk factor, Factor V Leiden (FVL), will develop venous thrombosis. In order to identify potential FVL modifier genes, we performed a sensitized dominant ENU mutagenesis screen based on the perinatal synthetic lethal thrombosis observed in mice homozygous for FVL (FVQ/Q) and hemizygous for tissue factor pathway inhibitor deficiency (Tfpi+/−). The screen was performed by crossing ENU-treated male FVQ/Q mice with FVQ/+ Tfpi+/− females. Surviving G1 offspring were analyzed to identify survivors with the lethal FVQ/Q Tfpi+/− genotype. Analysis of 7,128 G1 offspring (∼2X genome coverage) identified 98 FVQ/Q Tfpi+/− mice that survived to weaning. Fourteen FVQ/Q Tfpi+/− G1 mice exhibited successful transmission of a putative suppressor mutation to two or more FVQ/Q Tfpi+/− G2 offspring. Whole exome sequencing was performed on a progeny tested member of 8 of the 14 lines using the Agilent SureSelect mouse whole exome capture kit resulting in ∼100 fold coverage. Variant analysis revealed a small number of high confidence novel heterozygous (dominant) single nucleotide variants (SNVs) in each sample. Sanger re-sequencing of all 11 SNVs in a cohort of mice from line 1 confirmed that the G to C mutation (chromosome 11 base 19,977,300) in the Actr2 gene (present in 15/16 re-sequenced progeny p<0.0001) is the dominant FVL modifier in this line. This mutation resulted in an R286G substitution in a highly conserved amino acid in the Arp2 protein, which is essential for the function of the Arp2/3 complex. Arp2/3 is responsible for intracellular actin branching and polymerization, which is critical for the regulation of cell shape. Analysis of 31 progeny from an Arp2 R/G × Arp2 R/G cross revealed only one live Arp2 G/G mouse (p<0.05), suggesting that R286G is a loss of function mutation and that homozygous deficiency is lethal. Complete blood counts (Advia 2120) performed on 14 Actr2 heterozygous and 10 wildtype littermates revealed no significant differences in platelet count, red and white blood cell counts, hematocrit or hemoglobin. However, measurements of platelet size and size distribution including mean platelet dry mass, platelet volume distribution width and the platelet component distribution width were significantly altered in Actr2 heterozygous mutant mice (p<0.05 for each measure). Thus, partial deficiency of Arp2 appears to alter platelet structure/function resulting in a shift in hemostatic balance facilitating survival of the otherwise lethal FVQ/Q Tfpi+/− phenotype. These results suggest that variation in Arp2 or related genes could potentially modify thrombosis risk in humans, and might also identify novel therapeutic targets for the treatment of this class of disorders. Disclosures:Ginsburg:Shire Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees; Portola Pharmaceuticals: Consultancy; Catalyst Biosciences: Consultancy; Baxter Pharmaceuticals: benefit from payments to Children’s Hosptial, Boston, and the University of Michigan Patents & Royalties; Merck Pharmaceuticals: Consultancy.

An ENU Mutagenesis Screen to Identify Genes Involved in the Induction of FLT3-ITD Positive Acute Myeloid Leukemia

Abstract 1400 BACKGROUND:Activating mutations of the FLT3 gene are common in AML, with approximately 25–30% of cases containing an internal tandem duplication (ITD) in the juxtamembrane domain. The presence of a FLT3-ITD mutation is associated with a poor prognosis which can be modulated by the combination of co-operating mutations. In animal models, expression of FLT3-ITD by transgenesis, bone marrow transplantation or gene knock-in does not lead to an acute leukemia but a myeloproliferative disease, resembling CMML, suggesting a requirement for additional co-operating mutations (Lee et al, Cancer Cell. 12: 367). This is supported by in vivo models which demonstrate that the combination of the FLT3-ITD mutation with other genetic lesions leads to an acute leukemia in mice. AIMS:To identify novel genes co-operating with FLT3-ITD to induce acute leukemia by using an N-ethyl-N-Nitroso-urea (ENU) mutagenesis strategy in mice with a FLT3-ITD background. METHODS:An autosomal dominant screen for FLT3-ENU co-operating mutations was carried out at the Australian Phenomics Facility by mating ENU mutagenised male mice homozygous for the FLT3-ITD knock-in allele to females homozygous for the FLT3-ITD knock-in allele. G1 mice were screened for changes in blood cell parameters indicative of an altered disease state. Peripheral blood differential analysis was performed at 8, 12 and 16 weeks of age as well as immunophenotyping for the myeloid markers Mac1 and Gr1 and the progenitor marker c-kit. Analysis of a non-mutagenised cohort of mice indicated a sex-specific differential effect of FLT3-ITD on multiple blood cell parameters, so male and female data were analysed separately. Mice with blood cell parameters outside two standard deviations of the relevant G1 mean were identified as potential mutation carriers (Figure 1) and bred to FLT3-ITD homozygous knock-in mice to test for heritability of the phenotype (Figure 2). [Display omitted] [Display omitted] RESULTS:To date 150 G1 mice have been screened, leading to the identification of four heritable phenotypes (Designated pedigrees 12, 21, 24 and 37, Figure 1). All pedigrees were characterised by increased WBC counts compared to ‘unaffected' mice (FLT3-ITD homozygous background) and by further expansion of the myeloid compartment. Preliminary analysis of the bone marrow from affected mice identified an increase in colony replating ability in response to GM-CSF, suggestive of a block in differentiation. Three out of the four pedigrees showed disease penetrance only in males, with several mice from pedigree 24 succumbing to disease (Figure 2). Whole exome sequencing is being undertaken to identify the sequence variants that segregate with the phenotype in each of the pedigrees. CONCLUSIONS:It is possible through mutagenesis to identify co-operating mutations with FLT3-ITD. Further characterisation of these pedigrees, the associated phenotype and identification of the associated mutation in each case, will provide important information regarding pathways that co-operate with FLT3-ITD in leukemogenesis. Translation of these findings to human AML will have potential implications for predicting the course of disease in AML patients and may indicate pathways that will be targets for new and complementary treatments in AML. Disclosures:No relevant conflicts of interest to declare.

Whole-Exome Sequencing to Identify Mutations in Thrombosis Modifier Genes Isolated From a Factor V Leiden-Dependent Sensitized ENU Suppressor Screen in the Mouse

Abstract 1183Only ∼10% of individuals carrying the common venous thrombosis risk factor, Factor V Leiden (FVL) will develop venous thrombosis in their lifetime. In order to identify potential FVL modifier genes, we performed a sensitized dominant ENU mutagenesis screen, based on the perinatal synthetic lethal thrombus previously observed in mice homozygous for FVL (FVQ/Q) and hemizygous for tissue factor pathway inhibitor deficiency (Tfpi+/−). The genome-wide ENU mutagenesis screen was performed by crossing ENU-treated male FVQ/Q mice with FVQ/+ Tfpi+/− females. Surviving G1 offspring were analyzed to identify survivors with the otherwise lethal FVQ/Q Tfpi+/− genotype. As proof of concept, we demonstrated that reduced tissue factor (Tf+/−) suppresses the lethal FVQ/Q Tfpi+/− phenotype, suggesting that mutations at Tf should be among the suppressor genes identified by our screen. Analysis of 7,128 G1 offspring (∼2X genome coverage) identified 98 FVQ/Q Tfpi+/− mice that survived to weaning. Fourteen FVQ/Q Tfpi+/− G1 mice exhibited successful transmission of a putative suppressor mutation to two or more FVQ/Q Tfpi+/− G2 offspring. Extensive genotyping of mice from an expanded genetic cross from one of these lines mapped a candidate suppressor locus to a chromosome 3 region encompassing the TF gene (LOD=4.93). With continued improvements in next generation sequencing technologies, we have now applied whole exome sequencing to analysis of 8 of the remaining 13 lines. The entire DNA coding region (the “exome”, totaling 49.6 Mb of DNA sequence) from a progeny-tested member of each line was captured using the Agilent SureSelect mouse exome capture system. Whole-exome sequencing using the Illumina HiSeq high-throughput sequencer yielded 12–15 gigabases of sequence data per sample, corresponding to an average of ∼200 fold sequencing coverage for each nucleotide position. Variant analysis using the Gene Analysis Toolkit revealed the presence of a small number of high confidence novel heterozygous (dominant) variants in each sample. Each of these heterozygous variants is a candidate suppressor mutation and these are presently being tested in remaining FVQ/Q Tfpi+/− mice from each respective line. Based on previous studies where ENU-induced mutations in mice have been identified, we anticipate the identification of putative exomic mutations in approximately 80% of tested suppressor lines. Identification of these mutations should provide candidate modifier genes for FVL and other human hemostatic disorders. Disclosures:Ginsburg:Portola Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Catalyst Biosciences: Membership on an entity's Board of Directors or advisory committees; Shire Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Cytopenia induction by 5-fluorouracil identifies thrombopoietic mutants in sensitized ENU mutagenesis screens

The ability of random mutagenesis techniques to annotate the mammalian genome can be hampered due to genetic redundancy and compensatory pathways that mask heterozygous mutations under homeostatic conditions. The objective of this study was to devise a pharmacologically sensitized screen using the chemotherapeutic drug, 5-fluorouracil (5FU), to induce cytopenia. 5FU dose was optimized in the 129/SvImJ, C57BL/6J, BALB/cJ, and C3H/HeJ strains of laboratory mice. N-ethyl-N-nitrosourea (ENU) mutagenesis was performed on 129/SvImJ males and phenotypic variants were identified by backcrossing on to the C57BL/6J background. G1 animals were challenged with 100 μg/g 5FU and phenodeviants with altered platelet recovery were monitored. Of 546 G1 animals tested, 15 phenodeviants were identified that displayed increased baseline platelet number, a platelet overshoot, or delayed platelet recovery, thereby demonstrating the utility of this approach for uncovering mutations in megakaryocyte and platelet development. Four G1 mice were selected for further analysis. The phenotypes were heritable in all four strains and genetic mapping identified a chromosome location in two of the three G2 lines tested. In conclusion, our group has developed a sensitized random mutagenesis screen utilizing 5FU and has shown that the strain combination of 129/SvImJ × C57BL/6J is robust for identification of founder lines with defects in megakaryocyte and platelet development.

ENU Mutagenesis Screening for Dominant Behavioral Mutations Based on Normal Control Data Obtained in Home-Cage Activity, Open-Field, and Passive Avoidance Tests

To establish the cutoff values for screening ENU-induced behavioral mutations, normal variations in mouse behavioral data were examined in home-cage activity (HA), open-field (OF), and passive-avoidance (PA) tests. We defined the normal range as one that included more than 95% of the normal control values. The cutoffs were defined to identify outliers yielding values that deviated from the normal by less than 5% for C57BL/6J, DBA/2J, DBF(1), and N(2) (DXDB) progenies. Cutoff values for G1-phenodeviant (DBF(1)) identification were defined based on values over +/- 3.0 SD from the mean of DBF(1) for all parameters assessed in the HA and OF tests. For the PA test, the cutoff values were defined based on whether the mice met the learning criterion during the 2nd (at a shock intensity of 0.3 mA) or the 3rd (at a shock intensity of 0.15 mA) retention test. For several parameters, the lower outliers were undetectable as the calculated cutoffs were negative values. Based on the cutoff criteria, we identified 275 behavioral phenodeviants among 2,646 G1 progeny. Of these, 64 were crossed with wild-type DBA/2J individuals, and the phenotype transmission was examined in the G2 progeny using the cutoffs defined for N(2) mice. In the G2 mice, we identified 15 novel dominant mutants exhibiting behavioral abnormalities, including hyperactivity in the HA or OF tests, hypoactivity in the OF test, and PA deficits. Genetic and detailed behavioral analysis of these ENU-induced mutants will provide novel insights into the molecular mechanisms underlying behavior.

ENU-Based Gene-Driven Mutagenesis in the Mouse: A Next-Generation Gene-Targeting System

As a new mouse mutant resource, the RIKEN ENU-based gene-driven mutagenesis system in the mouse has been available to the research community since 2002. By using random base-substitution mutagenesis with ENU, a new reverse genetics infrastructure has been developed as a next-generation gene-targeting system. The construction of a large-scale mutant mouse library and high-throughput mutation discovery systems were the keys making it practically feasible. The RIKEN mutant mouse library consists of ~ 10,000 G1 mice, within which 100-150 mutant strains have been established based on users' requests every year. Use of the system is very simple: users 1) download an application form from our web site and send to us, and 2) design the PCR primers for the target gene. Then, we screen the RIKEN mutant mouse library and report all the detected mutations to the user. From among the allelic series of discovered mutations, users decide which mutant strain(s) to analyze and request the live mutant strain for functional studies of the target gene. Users have been reporting various functional mutations in the RIKEN mutant mouse library: e.g., missense, knockout-type and even functional non-coding mutations. In the near future, next-generation re-sequencing systems should drastically enhance the utility of the ENU-based gene-driven mutagenesis not only for the mouse but also for other species.

Comparing effects of mTR and mTERT deletion on gene expression and DNA damage response: a critical examination of telomere length maintenance-independent roles of telomerase

Telomerase, the essential enzyme that maintains telomere length, contains two core components, TERT and TR. Early studies in yeast and mouse showed that loss of telomerase leads to phenotypes only after several generations, due to telomere shortening. However, recent studies have suggested additional roles for telomerase components in transcription and the response to DNA damage. To examine these potential telomere length maintenance-independent roles of telomerase components, we examined first generation mTR−/− and mTERT−/− mice with long telomeres. We used gene expression profiling and found no genes that were differentially expressed in mTR−/− G1 mice and mTERT−/− G1 mice compared with wild-type mice. We also compared the response to DNA damage in mTR−/−G1 and mTERT−/− G1 mouse embryonic fibroblasts, and found no increase in the response to DNA damage in the absence of either telomerase component compared to wild-type. We conclude that, under physiologic conditions, neither mTR nor mTERT acts as a transcription factor or plays a role in the DNA damage response.

Genotypic Features of Lentivirus Transgenic Mice

Lentivector-mediated transgenesis is increasingly used, whether for basic studies as an alternative to pronuclear injection of naked DNA or to test candidate gene therapy vectors. In an effort to characterize the genetic features of this approach, we first measured the frequency of germ line transmission of individual proviruses established by infection of fertilized mouse oocytes. Seventy integrants from 11 founder (G0) mice were passed to 111 first generation (G1) pups, for a total of 255 events corresponding to an average rate of transmission of 44%. This implies that integration had most often occurred at the one- or two-cell stage and that the degree of genotypic mosaicism in G0 mice obtained through this approach is generally minimal. Transmission analysis of eight individual proviruses in 13 G2 mice obtained by a G0-G1 cross revealed only 8% of proviral homozygosity, significantly below the 25% expected from purely Mendelian transmission, suggesting counter-selection due to interference with the functions of targeted loci. Mapping of 239 proviral integration sites in 49 founder animals revealed that about 60% resided within annotated genes, with a marked tendency for clustering in the middle of the transcribed region, and that integration was not influenced by the transcriptional orientation. Transcript levels of a set of arbitrarily chosen target genes were significantly higher in two-cell embryos than in embryonic stem cells or adult somatic cells, suggesting that, as previously noted in other settings, lentiviral vectors integrate preferentially into regions of the genome that are transcriptionally active or poised for activation.

Studies on N-Ethyl-N-nitrosourea Mutagenesis in BALB/c Mice.

N-ethyl-N-nitrosoures (ENU) is effective in inducing hypermorphic mutation as well as hypomorphic and antimorphic mutations. Therefore, this mutagen is used to the production of mutant in the mice. In order to perform an effective ENU mutagenesis using BALB/cAnN mice, determination of optimal dosage and dosage regimen of ENU is necessary. And this study tried to develop a suitable screening method and searched for novel and various mutants as model animals in phenotypedriven ENU mutagenesis. We have carried out dosage regimen for mutagenizing dose of 200 mg/kg ENU in the BALB/c mice. Total screened mice were 30,133. As the results of Esaki and Cho’s Phenotype Screening, we got 2,516 phenotypic and behavior abnormalities in G1, G2 and G3 mice. One hundred thirty five G1 phenodeviants were tested for inheritance and 16 dominant mutants were discovered. Forty two recessive mutants were also found in tested 201 micropedigrees. Early-onset mutant mice included the dysmorphology of face, eye, tail, limb, skin, and foot and abnormal behavior like circling, swimming, head tossing, stiff-walking, high cholesterol level, and tremor etc. In this study we could effectively screen G3 recessive mutants. The frequent and concise early-onset screening before weaning will be available for ENU mutagenesis.

PanR1, a Dominant Negative Missense Allele of the Gene Encoding TNF-α (Tnf), Does Not Impair Lymphoid Development

A dominant hypomorphic allele of Tnf, PanR1, was identified in a population of G(1) mice born to N-ethyl-N-nitrosourea-mutagenized sires. Macrophages from homozygotes produced no detectable TNF bioactivity, although normal quantities of immunoreactive TNF were secreted. The phenotype was confined to a critical region on mouse chromosome 17, and then ascribed to a C-->A transversion at position 3480 of the Tnf gene, corresponding to the amino acid substitution P138T. As a result of subunit exchange, the protein exerts a dominant-negative effect on normal TNF trimers, interfering with the trimer/receptor interaction. Homozygotes are highly susceptible to infection by Listeria monocytogenes, confirming the essential role of TNF in innate immune defense. However, PanR1 mutant mice show normal architecture of the spleen and Peyer's patches, suggesting that TNF is not essential for the formation of these lymphoid structures.

Effects of Sulfamethoxazole on Murine Ocular Toxoplasmosis in Interferon-γ Knockout Mice

To evaluate the effects of sulfamethoxazole (SMX) on experimental ocular toxoplasmosis by quantitative competitive polymerase chain reaction (QC-PCR) assay. Wild-type (WT) C57BL/6 and WT BALB/c mice and interferon-gamma knockout (GKO) mice were infected orally with Toxoplasma gondii of the Fukaya strain. Mice were classified into groups. The first group (G1) remained untreated, the second group (G2) had a short SMX treatment period, and the third group (G3) received treatment continuously. WT and GKO mice were divided into G1 and G3, and G1, G2, and G3, respectively. T. gondii burdens were evaluated by QC-PCR assay. The effect on stage distribution was analyzed by reverse transcription-PCR. SMX significantly decreased mortality among the infected WT C57BL/6 and GKO mice. In WT G1 mice, T. gondii DNA was detected in all organs and tissues, although in G3 mice it was detected only in the brain. In GKO C57BL/6 G1 mice, the protozoan proliferated much more actively than in the WT mice. In the GKO C57BL/6 G2 mice, the number of T. gondii was less than in G1 during the treatment, although the protozoan reappeared after cessation of treatment. In GKO C57BL/6 G3 mice, T. gondii DNA was detected in the brain, optic nerve, and retina, but not in the iris, choroid, sclera, and blood. In GKO BALB/c mice, the patterns of the kinetics of protozoan abundance in various organs were similar or were milder than those in GKO C57BL/6 mice. In SMX-treated GKO mice, the percentage of bradyzoites increased and that of tachyzoites decreased in the organs and tissues. SMX decreased the parasitic load in both WT and GKO mice. SMX decreased the tachyzoite load but did not completely eliminate bradyzoites in GKO mice. The present mouse model was used successfully to assess treatment effects in a quantitative fashion.

Short Telomeres, even in the Presence of Telomerase, Limit Tissue Renewal Capacity

Autosomal-dominant dyskeratosis congenita is associated with heterozygous mutations in telomerase. To examine the dosage effect of telomerase, we generated a line of mTR+/- mice on the CAST/EiJ background, which has short telomeres. Interbreeding of heterozygotes resulted in progressive telomere shortening, indicating that limiting telomerase compromises telomere maintenance. In later-generation heterozygotes, we observed a decrease in tissue renewal capacity in the bone marrow, intestines, and testes that resembled defects seen in dyskeratosis congenita patients. The progressive worsening of disease with decreasing telomere length suggests that short telomeres, not telomerase level, cause stem cell failure. Further, wild-type mice derived from the late-generation heterozygous parents, termed wt*, also had short telomeres and displayed a germ cell defect, indicating that telomere length determines these phenotypes. We propose that short telomeres in mice that have normal telomerase levels can cause an occult form of genetic disease.

Identification of Novel Erythropoietic Genes and Alleles Using a Large-Scale Chemical Mutagenesis Screen in Mice.

Genome-wide chemical mutagenesis screens have been successfully performed in zebrafish to identify genes involved in erythropoiesis. We have now executed a large-scale screen for genes involved in red cell production in mice by using N-ethyl-N-nitrosurea mutagenesis. Five thousand G1 mice were screened by automated haematological analysis at 7 weeks of age for abnormalities of their red cell indices that lay more than 3 standard deviations outside the normal population average. Mice with co-incident abnormalities in white cell or platelet counts were excluded. Six mutant lines were identified with microcytosis (RBC1-6) that was heritable with subsequent backcrossing. Three of these lines (RBC 1, 4, 5) were found by gene mapping and candidate gene sequence analysis to harbour different mutations in the transferrin receptor gene (TfR) that led to embryonic lethality in their homozygous state. These mutations were clustered in the sequence encoding the domain involved in receptor homodimerisation, providing, for the first time, in vivo functional correlations of the previous structural analyses. The RBC3 line was also mapped to the interval containing the TfR gene, although no mutations were present in the TfR coding region or splice donor/acceptor sites in this line, raising the possibility of a regulatory sequence mutation, which we are investigating. The RBC2 line was mapped to chromosome 8, and found to contain a splice acceptor mutation in the ankyrin gene. This mutation results in a 22 bp deletion with a resultant frame shift in the regulatory domain. Mice homozygous for this mutation are born jaundiced with massive splenomegaly and a blood film consistent with severe spherocytosis, and die within 6 weeks of progressive anemia. As such, this line represents a new mouse model of severe hereditary spherocytosis. The final line, RBC6 displays a profound hypochromic, microcytic anemia in the homozygous state at birth, with apparent normality of all other organ systems. The mutation has been mapped to a chromosomal interval containing only a few genes, none of which have been previously linked to red cell production or differentiation, and therefore represents a novel erythropoietic gene/allele.

Induction of Transient Cytopenia to Detect Hematopoietic Defects in the Mouse.

In an attempt to generate new mouse models of human hematopoietic diseases and increase our knowledge of the genetic networks that orchestrate hematopoiesis, we are developing a sensitized screen that will more effectively identify hematopoietic in heterozygous mutants. The chemical mutagen ethylnitrososurea (ENU) is a highly efficient mutagen in the mouse and other model organisms, generating primarily point mutations. As a part of the Centre for Modeling Human Disease (CMHD), we have used simple clinical assays such as blood morphology and automated blood cell counts to test saphenous vein collected peripheral blood from approximately 6500 offspring of ENU mutagenized mice to identify clinically relevant dominantly inherited hematopoietic disease models. Phenodeviant G1 animals are bred to wild type mice to generate G2 mice, and G2 strains demonstrating high penetrance undergo a genome scan. This strategy has yielded 14 heritable dominant mutants to date. However, many genes and pathways will not express phenotypes in a dominant manner due to the homeostatic balance maintained by complex genetic networks, possessing excess capacity or redundancy, a problem exacerbated by using a viable peripheral blood assay. For example, only one of the 14 heritable hematopoietic mutants demonstrates anemia. Because environmental sensitization can exacerbate latent phenotypes, such as the effect of a high salt diet in an individual with prehypertension, sensitized screens should identify subtle and additional phenotypes of dominant mutations. Thus, we are developing a sensitized screen to uncover subtle dominant phenotypes by inducing transient cytopenia and following the kinetics and magnitude of the recovery. We are using a variety of drugs (5-fluorouracil, phenylhydrazine, and hydroxyurea) to induce cytopenia and recruit hematopoietic stem cells and progenitors to repopulate the blood with differentiated cells. We hypothesize that animals with defects in blood development will demonstrate altered recovery kinetics. This strategy will allow us to determine if there is a defect in blood cell progenitors, while keeping the animals viable for further screening and breeding. We are testing these drugs on known dominant and recessive mutants to validate the assay, determine the best dosages, and compare the effects of each drug. However, we have already begun to use 5-fluoruracil on a cohort of G1 animals and have identified two G1s that display latent recovery in at least 1 blood lineage. The two G1s are confirmed heritable and are currently undergoing a genome scan to map position the mutation. We believe this strategy will further our understanding of the molecular mechanisms that regulate hematopoietic development, as well as generate new mouse models of human hematopoietic diseases and identify the underlying molecular defects of diseases whose etiological origins are currently unknown.