Mouse Mutagenesis Research Articles

Recombinant Retroviral Production and Infection of B Cells

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control of a heterologous expression system to facilitate the analysis of the resulting phenotype. This approach can be used to express a gene that is not normally found in the organism, to express a mutant form of a gene product, or to over-express a dominant-negative form of the gene product. It is particularly useful in the study of the hematopoietic system, where transcriptional regulation is a major control mechanism in the development and differentiation of B cells, reviewed. Mouse genetics is a powerful tool for the study of human genes and diseases. A comparative analysis of the mouse and human genome reveals conservation of synteny in over 90% of the genome. Also, much of the technology used in mouse models is applicable to the study of human genes, for example, gene disruptions and allelic replacement. However, the creation of a transgenic mouse requires a great deal of resources of both a financial and technical nature. Several projects have begun to compile libraries of knock out mouse strains (KOMP, EUCOMM, NorCOMM) or mutagenesis induced strains (RIKEN), which require large-scale efforts and collaboration. Therefore, it is desirable to first study the phenotype of a desired gene in a cell culture model of primary cells before progressing to a mouse model. Retroviral DNA integrates into the host DNA, preferably within or near transcription units or CpG islands, resulting in stable and heritable expression of the packaged gene of interest while avoiding transcriptional silencing. The genes are then transcribed under the control of a high efficiency retroviral promoter, resulting in a high efficiency of transcription and protein production. Therefore, retroviral expression can be used with cells that are difficult to transfect, provided the cells are in an active state during mitosis. Because the structural genes of the virus are contained within the packaging cell line, the expression vectors used to clone the gene of interest contain no structural genes of the virus, which both eliminates the possibility of viral revertants and increases the safety of working with viral supernatants as no infectious virions are produced. Here we present a protocol for recombinant retroviral production and subsequent infection of splenic B cells. After isolation, the cultured splenic cells are stimulated with Th derived lymphokines and anti-CD40, which induces a burst of B cell proliferation and differentiation. This protocol is ideal for the study of events occurring late in B cell development and differentiation, as B cells are isolated from the spleen following initial hematopoietic events but prior to antigenic stimulation to induce plasmacytic differentiation.

The possibility of determining of anomalies and pathologies in the offspring of liquidators of Chernobyl accident by non-radiation factors

Hereditary effects of radiation on people are not revealed and estimations for humans have been carried out by extrapolation from mutagenesis of irradiated mice (UNSCEAR). Nevertheless, in Russia, Ukraine and Belarus disturbance and pathologies are found in the children of liquidators of the accident at the Chernobyl atomic power station. Authors connect effects only with irradiation (basically in low doses) though there were also other factors. So, the psychogenic stress leads to genomic and chromosomal damages and strengthens the mutagens. For the offspring of parents with psychoemotional stress, depression, etc. the set of alterations and diseases is registered. For liquidators probably there is also the influence of other non–radiation factors. The conclusion is made that effects on the children of liquidators have mainly not a radiation cause. Families of liquidators were in a stress (together with effects of other agents on fathers) and could give birth to less healthy children.

Dissemination of Advanced Mouse Resources and Technologies at RIKEN BioResource Center

RIKEN BioResource Center (BRC) has collected, preserved, conducted quality control of, and distributed mouse resources since 2002 as the core facility of the National BioResource Project by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. Our mouse resources include over 5,000 strains such as humanized disease models, fluorescent reporters, and knockout mice. We have developed novel mouse strains such as tissue-specific Cre-drivers and optogenetic strains that are in high demand by the research community. We have removed all our specified pathogens from the deposited mice and used our quality control tests to examine their genetic modifications and backgrounds. RIKEN BRC is a founding member of the Federation of International Mouse Resources and the Asian Mouse Mutagenesis and Resource Association, and provides mouse resources to the one-stop International Mouse Strain Resource database. RIKEN BRC also participates in the International Gene Trap Consortium, having registered 713 gene-trap clones and their sequences in a public library, and is an advisory member of the CREATE (Coordination of resources for conditional expression of mutated mouse alleles) consortium which represents major European and international mouse database holders for the integration and dissemination of Cre-driver strains. RIKEN BRC provides training courses in the use of advanced technologies for the quality control and cryopreservation of mouse strains to promote the effective use of mouse resources worldwide.

A recessive screen for genes regulating hematopoietic stem cells

AbstractIdentification of genes that regulate the development, self-renewal, and differentiation of stem cells is of vital importance for understanding normal organogenesis and cancer; such knowledge also underpins regenerative medicine. Here we demonstrate that chemical mutagenesis of mice combined with advances in hematopoietic stem cell reagents and genome resources can efficiently recover recessive mutations and identify genes essential for generation and proliferation of definitive hematopoietic stem cells and/or their progeny. We used high-throughput fluorescence-activated cell sorter to analyze 9 subsets of blood stem cells, progenitor cells, circulating red cells, and platelets in more than 1300 mouse embryos at embryonic day (E) 14.5. From 45 pedigrees, we recovered 6 strains with defects in definitive hematopoiesis. We demonstrate rapid identification of a novel mutation in the c-Myb transcription factor that results in thrombocythemia and myelofibrosis as proof of principal of the utility of our fluorescence-activated cell sorter–based screen. Such phenotype-driven approaches will provide new knowledge of the genes, protein interactions, and regulatory networks that underpin stem cell biology.

Mitochondrial Fe-S cluster biogenesis, frataxin and the modulation of susceptibility to drug-induced cardiomyopathy

The role of energy deficits in the development and progression of heart failure is well-established and cardiac high-energy phosphate levels correlate directly with survival in cardiomyopathy patients [1]. In light of these clinical observations it has been posited that the modulation of mitochondrial function may be a feasible strategy to ameliorate heart failure. Aspects of mitochondrial function that have been manipulated to investigate this include: modulation of energy substrate utilization; activation of mitochondrial biogenesis; induction of mitochondrial antioxidant defenses and suppression of mitochondrial death programs including apoptosis and mitochondrial permeability transition. As briefly discussed below, targeting distinct aspects of these programs result in variable levels of success and suggest that our understanding of the role of the mitochondria in the pathophysiology remains incomplete. Pharmacologic approaches to modulate mitochondrial function have, to date, been limited and have recently been reviewed [2,3]. To identify potential novel therapeutic targets, genetic approaches have been used to directly modulate mitochondrial programming with the subsequent assessment of cardiac effects. These include the genetic augmentation of PGC-1α, the master regulator of mitochondrial biogenesis. Here, somewhat counterintuitively, acute and chronic upregulation of PGC-1α results in deleterious cardiac phenotype [4,5]. In contrast, the overexpression of mitochondrial transcription factor A ameliorates the development of ischemia-induced heart failure [6]. The genetic modulation of substrate utilization similarly exhibit divergent effects. The upregulation of atranscription factor promoting fatty acid oxidation, a known major cardiac source of energy, is detrimental [7], whereas the promotion of glucose uptake enhances cardiac tolerance to ischemia [8]. Other approaches include augmentation of anti-oxidant enzymes, e.g., by the upregulation of cardiac manganese superoxide. These transgenic mice are protected against doxorubicin-induced [9] and diabetic cardiomyopathy [10]. Augmentation of cardiac anti-apoptotic programming by the overexpression of Bcl-2 similarly restores mitochondrial function and reduces cardiomyopathy in desmin null mice [11]. In light of incomplete characterization of proteins involved in the mitochondrial permeability transition (MPT) the genetic modulation of this program to prevent cardiomyopathy has to my knowledge not being explored. However, pharmacologic inhibition of MPT by cyclosporine A attenuates cardiomyopathy and mitochondrial genomic mutagenesis in mice harboring a cardiac specific defect in mitochondrial DNA proofreading [12]. Taken together, these pharmacologic and genetic approaches are beginning to discern distinct levels at which mitochondrial reprogramming may have beneficial effects in preventing the development and progression of heart failure. An intriguing new finding, by Shultz et al is published in AGING regarding the induction of frataxin. Mutations in frataxin result in the development of Friedreich's Ataxia, an inherited neurodegenerative disease associated with the development of severe cardiomyopathy. Frataxin, itself is involved in mitochondrial iron-sulphur cluster biogenesis which functions, in part, to incorporate appropriate amounts of iron into mitochondrial proteins including aconitase and succinate dehydrogenase [13]. Whether frataxin functions as an iron-chaperone protein or plays a regulatory role in controlling iron and sulphur flux within mitochondria is not yet completely characterized. Nevertheless, the study by Shultz and colleagues [14] shows that increased cardiac frataxin enhances tricarboxylic acid cycle function resulting in increased cardiac ATP, NADH, NADPH and reduced glutathione levels. This array of features is consistent with an enhanced bioenergetic capacity and increased antioxidant defenses. The authors go on to demonstrate that overexpression of frataxin is cardioprotective against doxorubicin-induced cardiomyopathy. This intriguing study shows that the modulation of the mitochondria at the fundamental level of integrating cofactors required for protein functional integrity have beneficial effects in disease processes that are exacerbated by mitochondrial dysfunction. This study further highlights the complexity of mitochondrial function and adds a new level of regulation operational in the pathophysiology of heart failure that may be amenable to therapeutic modulation.

Random mutagenesis of the mouse genome: a strategy for discovering gene function and the molecular basis of disease

Mutagenesis of mice with N-ethyl-N-nitrosourea (ENU) is a phenotype-driven approach to unravel gene function and discover new biological pathways. Phenotype-driven approaches have the advantage of making no assumptions about the function of genes and their products and have been successfully applied to the discovery of novel gene-phenotype relationships in many physiological systems. ENU mutagenesis of mice is used in many large-scale and more focused projects to generate and identify novel mouse models for the study of gene functions and human disease. This review examines the strategies and tools used in ENU mutagenesis screens to efficiently generate and identify functional mutations.

Screening of Ethylnitrosourea Mice With Fatty Acid Oxidation Disorders by a Candidate Gene Approach After Proteome Analysis

Background/Purpose Ethylnitrosourea (ENU) is an alkylating agent and primarily induces point mutations such as AT to TA transversions and AT to GC transitions. Due to its high mutagenicity, ENU mouse mutagenesis enables the generation and identification of mouse mutants with aberrance in various phenotypes and to identify novel genes relevant for the expression of the phenotype. The purpose of this study was to investigate the candidate genes involved in fatty acid oxidation disorders by the proteomic approach. Methods We screened ENU mice from 39 families from previously published data and identified two mutant mice that had a striking elevation in blood C4-OH short chain fatty acids compared with ENU controls. Total mitochondrial proteins were extracted from the gastrocnemius for two-dimensional electrophoresis, and two downregulated proteins, adenylate kinase isoenzyme 1 (AK1) and adenosine-5′-triphosphate (ATP) synthase D chain (ATP5H), were identified in the mutant mice through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Results After genomic polymerase chain reaction and direct sequencing of Ak1 and Atp5h , no variation was found in both gene sequence analyses. Conclusion Proteomic profiling can be a useful approach for detecting dynamic protein expression in ENU-induced mice. It is important to further clarify mechanisms of the mutant C4-OH disorder responsible for this expression.

Mouse Sperm Cryopreservation and Effective Embryo Production Using Cryopreserved C57BL/6 Mouse Sperm

Mouse sperm cryopreservation has greatly developed and spread throughout the world since it was the first successfully performed in 1990. The technology of sperm cryopreservation is widely used for storage of genetically engineered mice produced by global large-scale knockout mice or mouse mutagenesis projects in mouse banks. There are several benefits to using sperm cryopreservation. These include the ability to archive a great number of genetically engineered mice at a low cost in a small space; the ease with which freezing and thawing procedures can be carried out; and the convenience of performing mouse reproduction via in vitro fertilization. Sperm cryopreservation provides the research community with permanent access to genetically engineered mice and as such is an essential part of research activities. Many researchers have actively been investigating the techniques surrounding mouse sperm cryopreservation and embryo production using cryopreserved sperm. In particular, the topic of improving the low fertility rate of cryopreserved sperm in the C57BL/6 strain of mouse, the most common inbred strain used as a base for genetically engineered mice, has been garnering much attention. This review focuses on recent work regarding various attractive technologies in the fields of sperm cryopreservation and efficient embryo production using cryopreserved C57BL/6 mouse sperm.

Now and future of mouse mutagenesis for human disease models

One of the major objectives of the Human Genome Project is to understand the biological function of the gene and genome as well as to develop clinical applications for human diseases. For this purpose, the experimental validations and preclinical trails by using animal models are indispensable. The mouse (Mus musculus) is one of the best animal models because genetics is well established in the mouse and embryonic manipulation technologies are also well developed. Large-scale mouse mutagenesis projects have been conducted to develop various mouse models since 1997. Originally, the phenotype-driven mutagenesis with N-ethyl-N-nitrosourea (ENU) has been the major efforts internationally then knockout/conditional mouse projects and gene-driven mutagenesis have been following. At the beginning, simple monogenic traits in the experimental condition have been elucidated. Then, more complex traits with variety of environmental interactions and gene-to-gene interactions (epistasis) have been challenged with mutant mice. In addition, chromosomal substitution strains and collaborative cross strains are also available to elucidate the complex traits in the mouse. Altogether, mouse models with mutagenesis and various laboratory strains will accelerate the studies of functional genomics in the mouse as well as in human.

ROSA26Flpo deleter mice promote efficient inversion of conditional gene traps in vivo

Gene trap mutagenesis in embryonic stem cells is an important tool to help elucidate gene function in current mouse mutagenesis efforts. Vector systems based on inversion of the gene trap module have recently been devised to allow for conditional mutagenesis. However, additional efforts are needed to improve this technology including improving the efficiency of site-specific recombinases required to manipulate these conditional vectors in vivo. Here we describe a mouse line carrying the codon-optimized FLP recombinase Flpo at the ROSA26 locus that functions at higher efficiency than a similar Flpe line in mediating the DNA inversion of a conditional gene trap cassette in vivo.

008 Spontaneous cardiac hypertrophy and identification of novel hypertrophic pathways in a genetic mouse model that emulates human diabetes

Animal models are key in the exploration of the pathophysiological mechanisms and complications of diabetes mellitus (DM). Most current models have considerable limitations in that they do not faithfully replicate human forms of DM. Recently, a novel human relevant mouse model of diabetes (GENA 348) was identified through the Medical Research Council, Harwell, UK mouse mutagenesis programme in which a point mutation in the glucokinase gene results in severely impaired glucokinase function and significant hyperglycaemia. Similar mutations in the glucokinase gene are known to underlie Maturity Onset Diabetes of the Young Type 2 (MODY 2) in humans. We studied the GENA 348 mouse to determine whether it expresses a cardiac phenotype to provide insight into the pathophysiological mechanisms underlying the development of diabetic cardiomyopathy. Fifteen wild type (WT) controls and eight homozygote mutant (HO) mice had serial echocardiography performed at 3 and 6 months. At 3 months no evidence of cardiac hypertrophy or contractile dysfunction was demonstrated in HO compared to WT mice. By 6 months of age, echo demonstrated development of cardiac hypertrophy in GENA 348 HO mice characterised by a 16% increase in left ventricular mass/body weight (wt 4.28±0.15 vs HO 4.95±0.26, p<0.05), a 25% increase in dPW/dD (WT 23.3±1.0 vs HO 29.1±1.1, p<0.01) and an 18% increase in left ventricular relative wall thickness (WT 0.45±0.01 vs HO 0.53±0.02, p<0.05). No differences in systolic function were observed although significant diastolic dysfunction was also evident with a 31% reduction in the E:A ratio (WT 2.65±0.2 vs HO 1.84±0.19, p<0.05) and a 34% increase in the IVRT (WT 15.2±1.1 ms vs HO 20.5±0.37 ms, p<0.01). Histological staining illustrated cellular hypertrophy with a 60% increase in cardiomyocyte size (WT 238±14 μm² vs HO 383±26 μm², p<0.001). Hypertrophic pathways were examined through western blot analysis, a 140% increase in Akt phosphorylation, a key mediator of cardiac hypertrophy and a 65% increase in GSK3β phosphorylation one of its key regulators was observed. We also demonstrate that advanced glycation end products (AGE) were elevated by 86% in the serum of mutant GENA 348 mice. Collectively, these data indicate GENA 348 mutant mice develop a cardiac phenotype including hypertrophy and diastolic dysfunction similar to the clinical manifestations of diabetic cardiomyopathy. Furthermore, a novel hypertrophic pathway has been identified in which AGE stimulation through GSK3β leads to activation of the intracellular Akt pathway. GENA 348 may thus provide a valuable, human-relevant tool for studying the molecular determinants of DM related cardiovascular complications.

Abstract LB-153: PDE4D as a new prostate cancer drug target

Abstract We have been conducting a two-species screen for prostate cancer oncogenes that are suitable for use as new prostate cancer drug targets. The initial screen utilized transposon-based somatic mutagenesis in mice to induce prostate tumors and molecularly tag candidate oncogenes. This approach identified several candidate prostate cancer genes including known oncogenes such as Nras and Braf as well as new candidate oncogenes including Pde4d. A secondary screen used human prostate tissue microarrays (TMAs) to validate the relevance of new candidate oncogenes for understanding human prostate cancer. Automated quantitative analysis of PDE4D protein expression on the human TMAs confirmed its over-expression in human prostate cancer patients. The Pde4d mRNA is alternatively spliced to encode a family of related PDE4D enzymes. These enzymes share a common catalytic domain that gives all of the enzymes cAMP-specific 3′,5′-cyclic phosphodiesterase activity. Protein domains outside the catalytic domain allow for differential protein regulation and sub-cellular localization of different PDE4D isoforms. Several small molecules have been identified that are highly selective inhibitors of PDE4D activity including cilomilast and NVP-ABE171. In vitro testing of these small molecules has shown that they inhibit the growth of some but not all commonly used prostate cancer cell lines. Growth-inhibition was associated with down-regulation of the MEK-ERK and Shh signaling pathways. Current studies are investigating the molecular mechanisms associated with differential sensitivity/resistance to PDE4D inhibition as well as the potential use of PDE4D inhibitors as anti-prostate cancer drugs in vivo. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-153.

Three years in reviews

as we ring in 2010, I am delighted to highlight a few of our accomplishments, and I take the opportunity to congratulate our authors, reviewers, and Associate Editors for their continued excellence and collegiality. It is undeniable that AJP - Endocrinology and Metabolism has played a pivotal role

Sequential requirement of Sox4 and Sox11 during development of the sympathetic nervous system

The highly related transcription factors Sox4 and Sox11 are expressed in the developing sympathetic nervous system. In the mouse, Sox11 appears first, whereas Sox4 is prevalent later. Using mouse mutagenesis and overexpression strategies in chicken, we studied the role of both SoxC proteins in this tissue. Neither Sox4 nor Sox11 predominantly functioned by promoting pan-neuronal or noradrenergic differentiation of sympathetic neurons as might have been expected from studies in neuronal precursors of the central nervous system. The transcriptional network that regulates the differentiation of sympathetic neurons remained intact and expression of noradrenergic markers showed only minor alterations. Instead, Sox11 was required in early sympathetic ganglia for proliferation of tyrosine hydroxylase-expressing cells, whereas Sox4 ensured the survival of these cells at later stages. In the absence of both Sox4 and Sox11, sympathetic ganglia remained hypoplastic throughout embryogenesis because of consecutive proliferation and survival defects. As a consequence, sympathetic ganglia were rudimentary in the adult and sympathetic innervation of target tissues was impaired leading to severe dysautonomia.

Novel aspects of the roles of Rac1 GTPase in the cardiovascular system

Rac1 GTPase is an established master regulator of cell motility through cortical actin re-organization and of reactive oxygen species generation through regulation of NADPH oxidase activity. Numerous molecular and cellular studies have implicated Rac1 in various cardiovascular pathologies: vascular smooth muscle proliferation, cardiomyocyte hypertrophy, and endothelial cell shape change. The physiological relevance of these in vitro findings, however, is just beginning to be reassessed with the newly developed, conditional mouse mutagenesis technology. Conditional gene targeting has also revealed unexpected, cell type-specific roles of Rac1. The aim of this review is to summarize the recent advance made in Rac1 research in the cardiovascular system, with special focus on its novel roles in the regulation of endothelial function, angiogenesis, and endothelium-mediated neuroprotection.

Control of Cognition and Adaptive Behavior by the GLP/G9a Epigenetic Suppressor Complex

The genetic basis of cognition and behavioral adaptation to the environment remains poorly understood. Here we demonstrate that the histone methyltransferase complex GLP/G9a controls cognition and adaptive responses in a region-specific fashion in the adult brain. Using conditional mutagenesis in mice, we show that postnatal, neuron-specific deficiency of GLP/G9a leads to derepression of numerous nonneuronal and neuron progenitor genes in adult neurons. This transcriptional alteration is associated with complex behavioral abnormalities, including defects in learning, motivation, and environmental adaptation. The behavioral changes triggered by GLP/G9a deficiency are similar to key symptoms of the human 9q34 mental retardation syndrome that is associated with structural alterations of the GLP/EHMT1 gene. The likely causal role of GLP/G9a in mental retardation in mice and humans suggests a key role for the GLP/G9a-controlled histone H3K9 dimethylation in regulation of brain function through maintenance of the transcriptional homeostasis in adult neurons.

Homozygous lethality and heterozygous spotting due to a novel missense mutation in the mouse Kit gene

Abstract N-ethyl-N-nitrosourea (ENU) mutagenesis in mice can be used to study gene function in vivo and to establish genetic mouse models of human disease. In this study, a white spotted mouse (named KitW-1 Bao) was obtained by ENU-induced mutagenesis. Inheritance testing showed a single-gene dominant mutation and lethality in the KitW-1 Bao homozygous mice. The mutation was mapped to Chromosome 5 between markers D5 Mit356 and D5 Mit308. The region contains the Kit gene, whose mutations are known to lead to pigmentation defects in mice. Sequence analysis of the Kit cDNA from KitW-1 Bao heterozygotes revealed an A to T missense mutation resulting in an amino acid substitution of Asp (D) by Val (V) at amino acid position 849 within a highly conserved tyrosine kinase domain. The combined phenotype displayed by the KitW-1 Bao heterozygous and homozygous mutant mice demonstrates the critical function of the highly conserved aspartic acid residue at position 849 in the Kit gene product.

Role of skeletal muscle in the epigenetic shaping of motor neuron fate choices.

We study the role of muscle in the epigenetic (N.B., we use this term with the broader and more integrative meaning) shaping of developing motor neuron fate choices employing an approach based on mouse mutagenesis and pathology. The developmental role of skeletal muscle is studied in the whole mouse embryo by knocking out myogenic regulatory factors Myf5 and MyoD, to obtain an embryo without any skeletal musculature (Rudnicki et al., 1993). Our goal is to find muscle-provided trigger(s) of motor neuron death relevant to motor neuron diseases such as amyotrophic lateral sclerosis. The reason for this kind of thinking is the fact that a complete absence of lower and upper motor neurons, which is the pathological definition of amyotrophic lateral sclerosis, is only achieved in the complete absence of the muscle (Kablar and Rudnicki, 1999). Mutual embryonic inductive interactions between different tissue types and organs, between individual cell types belonging to the same or different lineages, and between various kinds of molecular players, are only some examples of the complex machinery that operates to connect genotype and phenotype. So far, our studies indicate that some aspects of this interplay can indeed be studied as proposed in this review article, suggesting the role of skeletal muscle in the epigenetic shaping of motor neuron fate choices. We will therefore continue this investigation as outlined to gain more insight into the nature of the epigenetic events that lead to the emergent properties of a phenotype.

Phenotypic and pathomorphological characteristics of a novel mutant mouse model for maturity-onset diabetes of the young type 2 (MODY 2)

Several mutant mouse models for human diseases such as diabetes mellitus have been generated in the large-scale Munich ENU (N-ethyl-N-nitrosourea) mouse mutagenesis project. The aim of this study was to identify the causal mutation of one of these strains and to characterize the resulting diabetic phenotype. Mutants exhibit a T to G transversion mutation at nt 629 in the glucokinase (Gck) gene, leading to an amino acid exchange from methionine to arginine at position 210. Adult Munich Gck(M210R) mutant mice demonstrated a significant reduction of hepatic glucokinase enzyme activity but equal glucokinase mRNA and protein abundances. While homozygous mutant mice exhibited growth retardation and died soon after birth in consequence of severe hyperglycemia, heterozygous mutant mice displayed only slightly elevated blood glucose levels, present from birth, with development of disturbed glucose tolerance and glucose-induced insulin secretion. Additionally, insulin sensitivity and fasting serum insulin levels were slightly reduced in male mutant mice from an age of 90 days onward. While beta-cell mass was unaltered in neonate heterozygous and homozygous mutant mice, the total islet and beta-cell volumes and the total volume of isolated beta-cells were significantly decreased in 210-day-old male, but not female heterozygous mutant mice despite undetectable apoptosis. These findings indicate that reduced total islet and beta-cell volumes of male mutants might emerge from disturbed postnatal islet neogenesis. Considering the lack of knowledge about the pathomorphology of maturity-onset diabetes of the young type 2 (MODY 2), this glucokinase mutant model of reduced total islet and total beta-cell volume provides the opportunity to elucidate the impact of a defective glucokinase on development and maintenance of beta-cell mass and its relevance in MODY 2 patients.

A Recessive Embryonic Screen for Genes Regulating Hematopoietic Stem Cell and Blood Cell Generation and Function.

Abstract 2527Poster Board II-504Identifying the genes that regulate the development, self-renewal and differentiation of stem cells is of vital importance for understanding normal organogenesis, tailoring tissue engineering for regenerative medicine, cellular reprogramming and cancer. A forward genetic screen for aberrant long-term hematopoietic stem cells and progenitors provides an unbiased and tractable approach to finding genes responsible for stem cell homeostasis and differentiation. Here we demonstrate that chemical mutagenesis of mice combined with advances in hematopoietic stem cell reagents and genome/mapping resources can identify genes essential for mammalian stem cells and blood development. A pilot flow cytometry-based recessive screen comprehensively analyzed nine subsets of hematopoietic stem, progenitor, and red cells in over one thousand mouse embryos at embryonic day (E) 14.5 from 34 pedigrees and recovered five strains with defects in early hematopoiesis. One mutant strain (Booreana - an Australian Aboriginal name meaning white) which has excess long-term hematopoietic stem cells and platelets but reduced myelo-erythroid progenitors was outcrossed and the genetic mutation mapped and identified as a novel mis-sense mutation in the transcription factor c-Myb. The mutation in the trans-activation domain (TA) completely ablates transcriptional activation in a reporter assay which contrasts with other TA domain mutants which are partly dysfunctional[TJG1] . Moreover, the Booreana (Boo) mutation completely abrogates interaction with the transcriptional co-activator, CBP. Boo/Boo homozygous mutant mice survive into adulthood, albeit with severe anemia and massively increased platelet counts, whereas c-Myb−/− mice die by E15.5 of development, suggesting c-Myb has essential functions in vivo which are independent of transcriptional activation. This ENU-generated mutation provides another allele of c-Myb with a phenotype in between the complete loss-of-function allele and previously identified mutant alleles from other ENU screens. ENU-generated point mutants such as the Booreana mutation can provide novel informative insights into key functional domains of proteins, and protein interactions and networks, which are missed in gene knockout mice. Other phenodeviants generated in our screen are currently being mapped and will be presented.[TJG1]Not so in our hands, at least -– the Sandberg M303V is just as dead. Disclosures:No relevant conflicts of interest to declare.