Variability of clinical chemical and hematological parameters, immunological parameters, and behavioral tests in data sets of the Mouse Phenome Database.

The use of mice as animal models in biomedical research allows the standardization of genetic background, housing conditions as well as experimental protocols, which all affect phenotypic variability. The phenotypic variability within the experimental unit determines the choice of the group size which is necessary for achieving valid and reproducible results. In this study, the variability of clinical chemical and hematological parameters which represent a comprehensive blood screen of laboratory mice, as well as of immunological parameters and behavioral tests was analyzed in data sets which have been submitted to the Mouse Phenome Database for mouse strains which are predominantly used in biomedical research. Most of the clinical chemical and hematological parameters-except of some parameters being known for their high variability-showed an average coefficient of variation (CV = standard deviation / mean) below 0.25. Most immunological parameters measured in blood samples had a CV between 0.2 and 0.4. The behavioral tests showed a CV between 0.4 and 0.6, or higher. In addition, a large range of the CV was found for most parameters/tests between and within the selected projects. This clearly demonstrates the appearance of unpredictable major interactions between genotype, environment and experiment regarding the variability of the parameters and tests analyzed.

New C3H KitN824K/WT cancer mouse model develops late-onset malignant mammary tumors with high penetrance.

Gastro-intestinal stromal tumors and acute myeloid leukemia induced by activating stem cell factor receptor tyrosine kinase (KIT) mutations are highly malignant. Less clear is the role of KIT mutations in the context of breast cancer. Treatment success of KIT-induced cancers is still unsatisfactory because of primary or secondary resistance to therapy. Mouse models offer essential platforms for studies on molecular disease mechanisms in basic cancer research. In the course of the Munich N-ethyl-N-nitrosourea (ENU) mutagenesis program a mouse line with inherited polycythemia was established. It carries a base-pair exchange in the Kit gene leading to an amino acid exchange at position 824 in the activation loop of KIT. This KIT variant corresponds to the N822K mutation found in human cancers, which is associated with imatinib-resistance. C3H KitN824K/WT mice develop hyperplasia of interstitial cells of Cajal and retention of ingesta in the cecum. In contrast to previous Kit-mutant models, we observe a benign course of gastrointestinal pathology associated with prolonged survival. Female mutants develop mammary carcinomas at late onset and subsequent lung metastasis. The disease model complements existing oncology research platforms. It allows for addressing the role of KIT mutations in breast cancer and identifying genetic and environmental modifiers of disease progression.

Distinct Morphological and Behavioural Alterations in ENU-Induced Heterozygous Trpc7K810Stop Mutant Mice.

Trpc7 (transient receptor potential cation channel, subfamily C, member 7; 862 amino acids) knockout mice are described showing no clear phenotypic alterations, therefore, the functional relevance of the gene remains unclear. A complementary approach for the functional analysis of a given gene is the examination of individuals harbouring a mutant allele of the gene. In the phenotype-driven Munich ENU mouse mutagenesis project, a high number of phenotypic parameters was used for establishing novel mouse models on the genetic background of C3H inbred mice. The phenotypically dominant mutant line SMA002 was established and further examined. Analysis of the causative mutation as well as the phenotypic characterization of the mutant line were carried out. The causative mutation was detected in the gene Trpc7 which leads to the production of a truncated protein due to the novel stop codon at amino acid position 810 thereby affecting the highly conserved cytoplasmic C terminus of the protein. Trpc7 heterozygous mutant mice of both sexes were viable and fertile, but showed distinct morphological and behavioural alterations which is in contrast to the published phenotype of Trpc7 knockout mice. Thus, the Trpc7K810Stop mutation leads to a dominant negative effect of the mutant protein.

Analysis of the sex-specific variability of blood parameters in data sets of the Mouse Phenome Database.

OBJECTIVE: The use of mice as animal models in biomedical research allows the standardization of genetic background and environmental conditions, which both affect phenotypic variability. As the use of both sexes in experiments is strongly recommended, sex-specific phenotypic variability is discussed with regard to putative consequences on the group size which is necessary for achieving valid and reproducible results. In this study, the sex-specific variability of 25 clinical chemical and hematological parameters which represent a comprehensive blood screen of laboratory mice, was analyzed in data sets which have been submitted to the Mouse Phenome Database. RESULTS: The overall analysis comprising all 25 clinical chemical and hematological parameters showed no evidence for substantial and robust general sex-specific variability. A large range of the ratio of the female and male coefficient of variation (CV) was found for every parameter among the respective strain data sets. This clearly demonstrated the appearance of unpredictable major interactions between genotype and environment regarding the sex-specific variability of the blood parameters analyzed.

Mild maternal hyperglycemia in INSC93S transgenic pigs causes impaired glucose tolerance and metabolic alterations in neonatal offspring.

Alongside the obesity epidemic, the prevalence of maternal diabetes is rising worldwide, and adverse effects on fetal development and metabolic disturbances in the offspring's later life have been described. To clarify whether metabolic programming effects are due to mild maternal hyperglycemia without confounding obesity, we investigated wild-type offspring of INSC93S transgenic pigs, which are a novel genetically modified large-animal model expressing mutant insulin (INS) C93S in pancreatic ß-cells. This mutation results in impaired glucose tolerance, mild fasting hyperglycemia and insulin resistance during late pregnancy. Compared with offspring from wild-type sows, piglets from hyperglycemic mothers showed impaired glucose tolerance and insulin resistance (homeostatic model assessment of insulin resistance: +3-fold in males; +4.4-fold in females) prior to colostrum uptake. Targeted metabolomics in the fasting and insulin-stimulated state revealed distinct alterations in the plasma metabolic profile of piglets from hyperglycemic mothers. They showed increased levels of acylcarnitines, gluconeogenic precursors such as alanine, phospholipids (in particular lyso-phosphatidylcholines) and α-aminoadipic acid, a potential biomarker for type 2 diabetes. These observations indicate that mild gestational hyperglycemia can cause impaired glucose tolerance, insulin resistance and associated metabolic alterations in neonatal offspring of a large-animal model born at a developmental maturation status comparable to human babies.

Standardized, systemic phenotypic analysis reveals kidney dysfunction as main alteration of Kctd1 I27N mutant mice.

BACKGROUND: Increased levels of blood plasma urea were used as phenotypic parameter for establishing novel mouse models for kidney diseases on the genetic background of C3H inbred mice in the phenotype-driven Munich ENU mouse mutagenesis project. The phenotypically dominant mutant line HST014 was established and further analyzed. METHODS: Analysis of the causative mutation as well as the standardized, systemic phenotypic analysis of the mutant line was carried out. RESULTS: The causative mutation was detected in the potassium channel tetramerization domain containing 1 (Kctd1) gene which leads to the amino acid exchange Kctd1 I27N thereby affecting the functional BTB domain of the protein. This line is the first mouse model harboring a Kctd1 mutation. Kctd1 I27N homozygous mutant mice die perinatally. Standardized, systemic phenotypic analysis of Kctd1 I27N heterozygous mutants was carried out in the German Mouse Clinic (GMC). Systematic morphological investigation of the external physical appearance did not detect the specific alterations that are described in KCTD1 mutant human patients affected by the scalp-ear-nipple (SEN) syndrome. The main pathological phenotype of the Kctd1 I27N heterozygous mutant mice consists of kidney dysfunction and secondary effects thereof, without gross additional primary alterations in the other phenotypic parameters analyzed. Genome-wide transcriptome profiling analysis at the age of 4 months revealed about 100 differentially expressed genes (DEGs) in kidneys of Kctd1 I27N heterozygous mutants as compared to wild-type controls. CONCLUSIONS: In summary, the main alteration of the Kctd1 I27N heterozygous mutants consists in kidney dysfunction. Additional analyses in 9-21 week-old heterozygous mutants revealed only few minor effects.

Missense Mutation of POU Domain Class 3 Transcription Factor 3 in Pou3f3L423P Mice Causes Reduced Nephron Number and Impaired Development of the Thick Ascending Limb of the Loop of Henle.

During nephrogenesis, POU domain class 3 transcription factor 3 (POU3F3 aka BRN1) is critically involved in development of distinct nephron segments, including the thick ascending limb of the loop of Henle (TAL). Deficiency of POU3F3 in knock-out mice leads to underdevelopment of the TAL, lack of differentiation of TAL cells, and perinatal death due to renal failure. Pou3f3L423P mutant mice, which were established in the Munich ENU Mouse Mutagenesis Project, carry a recessive point mutation in the homeobox domain of POU3F3. Homozygous Pou3f3L423P mutants are viable and fertile. The present study used functional, as well as qualitative and quantitative morphological analyses to characterize the renal phenotype of juvenile (12 days) and aged (60 weeks) homo- and heterozygous Pou3f3L423P mutant mice and age-matched wild-type controls. In both age groups, homozygous mutants vs. control mice displayed significantly smaller kidney volumes, decreased nephron numbers and mean glomerular volumes, smaller TAL volumes, as well as lower volume densities of the TAL in the kidney. No histological or ultrastructural lesions of TAL cells or glomerular cells were observed in homozygous mutant mice. Aged homozygous mutants displayed increased serum urea concentrations and reduced specific urine gravity, but no evidence of glomerular dysfunction. These results confirm the role of POU3F3 in development and function of the TAL and provide new evidence for its involvement in regulation of the nephron number in the kidney. Therefore, Pou3f3L423P mutant mice represent a valuable research model for further analyses of POU3F3 functions, or for nephrological studies examining the role of congenital low nephron numbers.

Generation and Standardized, Systemic Phenotypic Analysis of Pou3f3L423P Mutant Mice.

Increased levels of blood plasma urea were used as phenotypic parameter for establishing novel mouse models for kidney diseases on the genetic background of C3H inbred mice in the phenotype-driven Munich ENU mouse mutagenesis project. The phenotypically recessive mutant line HST011 was established and further analyzed. The causative mutation was detected in the POU domain, class 3 transcription factor 3 (Pou3f3) gene, which leads to the amino acid exchange Pou3f3L423P thereby affecting the conserved homeobox domain of the protein. Pou3f3 homozygous knockout mice are published and show perinatal death. Line Pou3f3L423P is a viable mouse model harboring a homozygous Pou3f3 mutation. Standardized, systemic phenotypic analysis of homozygous mutants was carried out in the German Mouse Clinic. Main phenotypic changes were low body weight and a state of low energy stores, kidney dysfunction and secondary effects thereof including low bone mineralization, multiple behavioral and neurological defects including locomotor, vestibular, auditory and nociceptive impairments, as well as multiple subtle changes in immunological parameters. Genome-wide transcriptome profiling analysis of kidney and brain of Pou3f3L423P homozygous mutants identified significantly regulated genes as compared to wild-type controls.

Screen for alterations of iron related parameters in N-ethyl-N-nitrosourea-treated mice identified mutant lines with increased plasma ferritin levels.

Iron is essential for numerous cellular processes. For diagnostic purposes iron-related parameters in patients are assessed by clinical chemical blood analysis including the analysis of ferritin, transferrin and iron levels. Here, we retrospectively evaluated the use of these parameters in the phenotype-driven Munich N-ethyl-N-nitrosourea mouse mutagenesis project for the generation of novel animal models for human diseases. The clinical chemical blood analysis was carried out on more than 10,700 G1 and G3 offspring of chemically mutagenized inbred C3H mice to detect dominant and recessive mutations leading to deviations in the plasma levels of iron-related plasma parameters. We identified animals consistently exhibiting altered plasma ferritin or transferrin values. Transmission of the phenotypic deviations to the subsequent generations led to the successful establishment of three mutant lines with increased plasma ferritin levels. For two of these lines the causative mutations were identified in the Fth1gene and the Ireb2 gene, respectively. Thus, novel mouse models for the functional analysis of iron homeostasis were established by a phenotype-driven screen for mutant mice.

Uromodulin retention in thick ascending limb of Henle's loop affects SCD1 in neighboring proximal tubule: renal transcriptome studies in mouse models of uromodulin-associated kidney disease.

Uromodulin-associated kidney disease (UAKD) is a hereditary progressive renal disease which can lead to renal failure and requires renal replacement therapy. UAKD belongs to the endoplasmic reticulum storage diseases due to maturation defect of mutant uromodulin and its retention in the enlarged endoplasmic reticulum in the cells of the thick ascending limb of Henle's loop (TALH). Dysfunction of TALH represents the key pathogenic mechanism of UAKD causing the clinical symptoms of this disease. However, the molecular alterations underlying UAKD are not well understood. In this study, transcriptome profiling of whole kidneys of two mouse models of UAKD, UmodA227T and UmodC93F, was performed. Genes differentially abundant in UAKD affected kidneys of both Umod mutant lines at different disease stages were identified and verified by RT-qPCR. Additionally, differential protein abundances of SCD1 and ANGPTL7 were validated by immunohistochemistry and Western blot analysis. ANGPTL7 expression was down-regulated in TALH cells of Umod mutant mice which is the site of the mutant uromodulin maturation defect. SCD1 was expressed selectively in the S3 segment of proximal tubule cells, and SCD1 abundance was increased in UAKD affected kidneys. This finding demonstrates that a cross talk between two functionally distinct tubular segments of the kidney, the TALH segment and the S3 segment of proximal tubule, exists.

Standardized, systemic phenotypic analysis of Slc12a1I299F mutant mice.

BACKGROUND: Type I Bartter syndrome is a recessive human nephropathy caused by loss-of-function mutations in the SLC12A1 gene coding for the Na+-K+-2Cl- cotransporter NKCC2. We recently established the mutant mouse line Slc12a1I299F exhibiting kidney defects highly similar to the late-onset manifestation of this hereditary human disease. Besides the kidney defects, low blood pressure and osteopenia were revealed in the homozygous mutant mice which were also described in humans. Beside its strong expression in the kidney, NKCC2 has been also shown to be expressed in other tissues in rodents i.e. the gastrointestinal tract, pancreatic beta cells, and specific compartments of the ear, nasal tissue and eye. RESULTS: To examine if, besides kidney defects, further organ systems and/or metabolic pathways are affected by the Slc12a1I299F mutation as primary or secondary effects, we describe a standardized, systemic phenotypic analysis of the mutant mouse line Slc12a1I299F in the German Mouse Clinic. Slc12a1I299F homozygous mutant mice and Slc12a1I299F heterozygous mutant littermates as controls were tested at the age of 4-6 months. Beside the already published changes in blood pressure and bone metabolism, a significantly lower body weight and fat content were found as new phenotypes for Slc12a1I299F homozygous mutant mice. Small additional effects included a mild erythropenic anemia in homozygous mutant males as well as a slight hyperalgesia in homozygous mutant females. For other functions, such as immunology, lung function and neurology, no distinct alterations were observed. CONCLUSIONS: In this systemic analysis no clear primary effects of the Slc12a1I299F mutation appeared for the organs other than the kidneys where Slc12a1 expression has been described. On the other hand, long-term effects additional and/or secondary to the kidney lesions might also appear in humans harboring SLC12A1 mutations.

No amelioration of uromodulin maturation and trafficking defect by sodium 4-phenylbutyrate in vivo: studies in mouse models of uromodulin-associated kidney disease.

Uromodulin (UMOD)-associated kidney disease (UAKD) belongs to the hereditary progressive ER storage diseases caused by maturation defects of mutant UMOD protein. Current treatments of UAKD patients are symptomatic and cannot prevent disease progression. Two in vitro studies reported a positive effect of the chemical chaperone sodium 4-phenylbutyrate (4-PBA) on mutant UMOD maturation. Thus, 4-PBA was suggested as a potential treatment for UAKD. This study evaluated the effects of 4-PBA in two mouse models of UAKD. In contrast to previous in vitro studies, treatment with 4-PBA did not increase HSP70 expression or improve maturation and trafficking of mutant UMOD in vivo. Kidney function of UAKD mice was actually deteriorated by 4-PBA treatment. In transfected tubular epithelial cells, 4-PBA did not improve maturation but increased the expression level of both mutant and wild-type UMOD protein. Activation of NF-κB pathway in thick ascending limb of Henle's loop cells of UAKD mice was detected by increased abundance of RelB and phospho-IκB kinase α/ß, an indirect activator of NF-κB. Furthermore, the abundance of NF-κB1 p105/p50, NF-κB2 p100/p52, and TRAF2 was increased in UAKD. NF-κB activation was identified as a novel disease mechanism of UAKD and might be a target for therapeutic intervention.

Standardized, systemic phenotypic analysis of Umod(C93F) and Umod(A227T) mutant mice.

Uromodulin-associated kidney disease (UAKD) summarizes different clinical features of an autosomal dominant heritable disease syndrome in humans with a proven uromodulin (UMOD) mutation involved. It is often characterized by hyperuricemia, gout, alteration of urine concentrating ability, as well as a variable rate of disease progression inconstantly leading to renal failure and histological alterations of the kidneys. We recently established the two Umod mutant mouse lines Umod(C93F) and Umod(A227T) on the C3H inbred genetic background both showing kidney defects analogous to those found in human UAKD patients. In addition, disease symptoms were revealed that were not yet described in other published mouse models of UAKD. To examine if further organ systems and/or metabolic pathways are affected by Umod mutations as primary or secondary effects, we describe a standardized, systemic phenotypic analysis of the two mutant mouse lines Umod(A227T) and Umod(C93F) in the German Mouse Clinic. Different genotypes as well as different ages were tested. Beside the already published changes in body weight, body composition and bone metabolism, the influence of the Umod mutation on energy metabolism was confirmed. Hematological analysis revealed a moderate microcytic and erythropenic anemia in older Umod mutant mice. Data of the other analyses in 7-10 month-old mutant mice showed single small additional effects.

Assessment of the genomic variation in a cattle population by re-sequencing of key animals at low to medium coverage.

BACKGROUND: Genome- and population-wide re-sequencing would allow for most efficient detection of causal trait variants. However, despite a strong decrease of costs for next-generation sequencing in the last few years, re-sequencing of large numbers of individuals is not yet affordable. We therefore resorted to re-sequencing of a limited number of bovine animals selected to explain a major proportion of the population's genomic variation, so called key animals, in order to provide a catalogue of functional variants and a substrate for population- and genome-wide imputation of variable sites. RESULTS: Forty-three animals accounting for about 69 percent of the genetic diversity of the Fleckvieh population, a cattle breed of Southern Germany and Austria, were sequenced with coverages ranging from 4.17 to 24.98 and averaging 7.46. After alignment to the reference genome (UMD3.1) and multi-sample variant calling, more than 17 million variant positions were identified, about 90 percent biallelic single nucleotide variants (SNVs) and 10 percent short insertions and deletions (InDels). The comparison with high-density chip data revealed a sensitivity of at least 92 percent and a specificity of 81 percent for sequencing based genotyping, and 97 percent and 93 percent when a imputation step was included. There are 91,733 variants in coding regions of 18,444 genes, 46 percent being non-synonymous exchanges, of which 575 variants are predicted to cause premature stop codons. Three variants are listed in the OMIA database as causal for specific phenotypes. CONCLUSIONS: Low- to medium-coverage re-sequencing of individuals explaining a major fraction of a population's genomic variation allows for the efficient and reliable detection of most variants. Imputation strongly improves genotype quality of lowly covered samples and thus enables maximum density genotyping by sequencing. The functional annotation of variants provides the basis for exhaustive genotype imputation in the population, e.g., for highest-resolution genome-wide association studies.

Type of uromodulin mutation and allelic status influence onset and severity of uromodulin-associated kidney disease in mice.

Uromodulin-associated kidney disease (UAKD) is a dominant heritable renal disease in humans which is caused by mutations in the uromodulin (UMOD) gene and characterized by heterogeneous clinical appearance. To get insights into possible causes of this heterogeneity of UAKD, we describe the new mutant mouse line Umod(C93F), leading to disruption of a putative disulfide bond which is also absent in a known human UMOD mutation, and compare the phenotype of this new mouse line with the recently published mouse line Umod(A227T). In both mutant mouse lines, which were both bred on the C3H background, the Umod mutations cause a gain-of-toxic function due to a maturation defect of the mutant uromodulin leading to a dysfunction of thick ascending limb of Henle's loop (TALH) cells of the kidney. Umod mutant mice exhibit increased plasma urea and Cystatin levels, impaired urinary concentration ability, reduced fractional excretion of uric acid and nephropathological alterations including uromodulin retention in TALH cells, interstitial fibrosis and inflammatory cell infiltrations, tubular atrophy and occasional glomerulo- und tubulocystic changes, a phenotype highly similar to UAKD in humans. The maturation defect of mutant uromodulin leads to the accumulation of immature uromodulin in the endoplasmic reticulum (ER) and to ER hyperplasia. Further, this study was able to demonstrate for the first time in vivo that the severity of the uromodulin maturation defect as well as onset and speed of progression of renal dysfunction and morphological alterations are strongly dependent on the particular Umod mutation itself and the zygosity status.

Imputation of high-density genotypes in the Fleckvieh cattle population.

BACKGROUND: Currently, genome-wide evaluation of cattle populations is based on SNP-genotyping using ~ 54,000 SNP. Increasing the number of markers might improve genomic predictions and power of genome-wide association studies. Imputation of genotypes makes it possible to extrapolate genotypes from lower to higher density arrays based on a representative reference sample for which genotypes are obtained at higher density. METHODS: Genotypes using 639 214 SNP were available for 797 bulls of the Fleckvieh cattle breed. The data set was divided into a reference and a validation population. Genotypes for all SNP except those included in the BovineSNP50 Bead chip were masked and subsequently imputed for animals of the validation population. Imputation of genotypes was performed with Beagle, findhap.f90, MaCH and Minimac. The accuracy of the imputed genotypes was assessed for four different scenarios including 50, 100, 200 and 400 animals as reference population. The reference animals were selected to account for 78.03%, 89.21%, 97.47% and > 99% of the gene pool of the genotyped population, respectively. RESULTS: Imputation accuracy increased as the number of animals and relatives in the reference population increased. Population-based algorithms provided highly reliable imputation of genotypes, even for scenarios with 50 and 100 reference animals only. Using MaCH and Minimac, the correlation between true and imputed genotypes was > 0.975 with 100 reference animals only. Pre-phasing the genotypes of both the reference and validation populations not only provided highly accurate imputed genotypes but was also computationally efficient. Genome-wide analysis of imputation accuracy led to the identification of many misplaced SNP. CONCLUSIONS: Genotyping key animals at high density and subsequent population-based genotype imputation yield high imputation accuracy. Pre-phasing the genotypes of the reference and validation populations is computationally efficient and results in high imputation accuracy, even when the reference population is small.

Clinical Chemistry and Other Laboratory Tests on Mouse Plasma or Serum.

Besides hematological analyses, many other parameters, including clinical chemistry and endocrinological values, can be determined from mouse blood samples. For most of these tests, plasma or serum samples are used. Data obtained by these investigations provide indications of genotype effects on metabolism and organ functions. Here we describe in detail the considerations that have to be taken into account to get adequate samples for plasma or serum analyses and the recommended sample processing for different investigations. Furthermore, we describe established methods used in the German Mouse Clinic (GMC) to determine clinical chemical parameters; for more in-depth analysis of specific classes of biomarkers, we provide instructions for ELISAs (sandwich and competitive) as well as LC-MS/MS, focusing on markers associated with bone or steroid metabolism in the mouse as working examples. Curr. Protoc. Mouse Biol. 3:69-100 © 2013 by John Wiley & Sons, Inc.

Blood Collection from Mice and Hematological Analyses on Mouse Blood.

Basic phenotyping of inbred mouse strains and genetically modified mouse models usually includes the determination of blood-based parameters as a diagnostic screen for genotype effects on metabolism and organ function. A broad range of analytes, including hematological parameters, can be reliably determined in mouse blood, if appropriate samples are available. Here we describe recommended techniques for blood collection from mice and the considerations that have to be taken into account to get adequate samples for hematological investigations. Furthermore, we describe established methods used in the German Mouse Clinic (GMC) to determine hematological parameters in the mouse. Curr. Protoc. Mouse Biol. 3:101-119 © 2013 by John Wiley & Sons, Inc.

Generation of N-ethyl-N-nitrosourea-induced mouse mutants with deviations in hematological parameters.

Research on hematological disorders relies on suitable animal models. We retrospectively evaluated the use of the hematological parameters hematocrit (HCT), hemoglobin (HGB), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), red blood cell count (RBC), white blood cell count (WBC), and platelet count (PLT) in the phenotype-driven Munich N-ethyl-N-nitrosourea (ENU) mouse mutagenesis project as parameters for the generation of novel animal models for human diseases. The analysis was carried out on more than 16,000 G1 and G3 offspring of chemically mutagenized inbred C3H mice to detect dominant and recessive mutations leading to deviations in the levels of the chosen parameters. Identification of animals exhibiting altered values and transmission of the phenotypic deviations to the subsequent generations led to the successful establishment of mutant lines for the parameters MCV, RBC, and PLT. Analysis of the causative mutation was started in selected lines, thereby revealing a novel mutation in the transferrin receptor gene (Tfrc) in one line. Thus, novel phenotype-driven mouse models were established to analyze the genetic components of hematological disorders.