Vet Pathol 38:20-30 (2001)
© 2001 American College of Veterinary Pathologists
ANIMAL MODELS
Special Considerations in the Evaluation of the Hematology and Hemostasis of Mutant Mice
B. D. Car and
V. M. Eng
DuPont Pharmaceuticals Company, Stine–Haskell Research Center, Haskell-1, P.O. Box 30, Newark, DE (BDC); and University of Pennsylvania, University Laboratory Animal Resources, Philadelphia, PA (VME)
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Abstract
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The study of mutant mice with altered or deficient hematopoietic or hemostatic gene products provides a challenge to the researcher, particularly when genetic alterations lead to lethal phenotypes. The following review provides a framework for understanding murine hematopoiesis, based on work with mutant mice, and details experimental approaches used to evaluate these animals. Mice with deficiencies in hemostatic and fibrinolytic system proteins are discussed, and the investigation of their phenotypes is reviewed.
Key words: Coagulation; fibrinolysis; hematology; hematopoiesis; hemostasis; knockout; mice.
Embryonic stem (ES) cell technology, facilitating targeted murine gene deletion, replacement, or insertion, has permitted the generation of many mutant mice in the study of hematopoiesis, leukemia, the immune system, and hemostasis. Dissection of the phenotypes of such mutant mice requires the application of all instrumentation currently employed in the clinical pathology laboratory, including hematology, chemistry, and coagulation analyzers, and the histology laboratory (see Brayton and Montgomery,11 this issue). This review details approaches to the study of murine hematology and coagulation in addition to those typically employed in the veterinary clinical laboratory setting. Virtually all studies relating to the phenotyping of mice appear outside the veterinary literature and are undertaken in a wide variety of laboratories. To better serve the scientific community as veterinarians, it is important to acquire expertise with these technologies and suggest appropriate approaches based on a thorough understanding of the scientific questions to be addressed by analysis of mutant murine phenotypes. The ability to reproduce diseases by recapitulating human genetic alterations in mice has resulted in the creation of numerous animal models of human disease. Previously, the association of the Philadelphia chromosome with human chronic myelogenous leukemia (CML) was of academic interest in veterinary pathology. The reproduction of this1,43,45,50,116 and other diseases in mice has placed an onus on comparative pathologists to be cognizant of the pathogenesis of important human conditions.
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Murine Hematopoiesis
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Collectively, numerous studies of gene-deleted mice have provided a model for the ontogeny of hematopoiesis. Primitive hematopoiesis is initiated in the yolk sac around gestation day 7 (E7), with aggregates of mesodermal cells forming the extraembryonic blood islands. Such islands are comprised of internal erythropoietic elements and surrounded by cells that differentiate into endothelial cells.2 Coexpression of proteins (Flk-1) by endothelial cells and hematopoetic pluripotential stem cells led to the hypothesis that a pluripotent "hemangioblast" exists with potential to differentiate into hematopoietic and endothelial cells.21,101 In the Flk-1-/- mouse, primitive hematopoiesis and blood vasculature fail to develop, supporting this premise.101,127 During organogenesis, definitive hematopoietic cells arise and proliferate in the anterior part of the aorta–gonad–mesonephros (AGM) region around E9 until E11/12, gradually replacing yolk sac–derived cells.76 Lymphopoiesis arises in the closely associated intraembrionic paraortic splanchnopleura (PAS).26,33 Extensive interchange of hematopoietic stem cells occurs through the blood vasculature, with secondary population of yolk sac and liver. The fetal liver takes over as the principal site of hematopoiesis around E12–14 until term, when the bone marrow and spleen become the quantitatively most important sites of hematopoiesis and the thymus and bone marrow the most important sites of lymphopoiesis.26,33 A partial list of the phenotypes of gene-deleted mice employed in the study of hematopoiesis is provided (Table 1). Since most nonredundant genetic deletions with pronounced hematologic phenotype are either lethal or have reduced viability, novel approaches have been developed to understand the mechanisms of these phenotypes. Subtle phenotypic alterations may also be detected by sensitive methodologies in mice with apparently normal peripheral blood and marrow cytology.
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Approaches to Understanding Lethal Phenotypes
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Once inbred to homozygosity at the target gene, murine hematopoietic gene mutations frequently prove to be lethal phenotypes. Creative alternatives have been developed to understand the nature of the defect leading to embryonic or fetal death. Genetic approaches involving deletion or reinsertion of whole or mutated genes into mice under tissue-specific promoters (Cre-loxP system) are used to study normal and aberrant hematopoiesis.8,54,86 Approaches to the knocking in and out of genes and transgenes involved in murine hematopoiesis has recently been reviewed in detail.87
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Examination of Fetal Blood and Liver
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Blood smears prepared with 2 µl of heart blood and Wright-Giemsa–stained liver impressions made from E12+ fetuses are used to evaluate fetal blood and bone marrow morphologies, respectively. Mutant mice with defects resulting in impaired definitive hematopoiesis contain far more nucleated red cells in peripheral blood after E12 than wild-type mice. Embryonic (yolk sac–derived) hematopoiesis does not yield mature erythrocytes. By E12, enucleated discocytes appear in peripheral blood. By E14.5, approximately 80% of erythrocytes in peripheral blood are enucleated. In mutant mice with defects affecting primitive hematopoiesis, only 20% of the circulating erythron may be enucleated at E14.5.59 Primitive erythropoiesis is further distinguished in mice by the specific expression of an embryonic type of globin in nucleated erythrocytes.
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Bone Marrow Transplantation
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To evaluate the long-term repopulating hematopoietic stem cells (LTR-HSC) and multipotent hematopoietic progenitors (colony-forming units—spleen, CFU-S) of lethal mutants, cell suspensions of yolk sac, AGM region, or liver are cultured for 2 to 3 days followed by dispersion. These cells are injected intravenously into lethally irradiated mice exposed to 1,000 rads, or approximately 9 Gy of a 60C source. Generally, male cells are injected into female recipients. Following a period of recovery in isolators during which antibiotics are provided in the drinking water, mice are evaluated at 11 to 12 days postinjection for CFU-S and after several months for HSC. Hematopoietic clones form nodules readily enumerated on the surface of Bouin's fixed spleens. Splenic weight correlates highly with CFU-S. Long-term survival of lethally irradiated, transfused mice requires adequate HSC to be present. Periodic blood collections provide leukocyte DNA for PCR quantification of male determinants (YMT2/B) and construct inserts (typically the neomycin gene) or cytoplasmic protein for glucose 6-phosphate isomerase (GPI) isozyme patterning.76 These markers allow the determination of the relative contribution of engrafted HSC to marrow lineages. Routine hematology and bone marrow cytology are also evaluated. In addition to stem cell analyses, bone marrow transplantation allows defects in marrow stroma to be differentiated from effects on hematopoietic elements, such as the stromal cell defect (defective c-kit or stem cell factor receptor) in Sl/Sld (Steel anemia) and the complementary defect in W/Wv (white spotted) mice (kit ligand or stem cell factor [SCF] deficiency).32,51 The sites of definitive lymphohematopoiesis and, therefore, of LTR-HSC generation (AGM and PAS regions) were identified by transplantation into lethally irradiated mice.26,33,63
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Bone Marrow Cell Culture
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Three main bone marrow cell culture methodologies are used to evaluate hematopoiesis in vitro.
- ES cells may be differentiated into hematopoietic lineages when cultured in the absence of fibroblast feeder cells or leukemia inhibitory factor (LIF) with inclusion of hematopoietic cytokines. Embryoid bodies develop in such cultures after 9 to 14 days.9,36,57 These are dispersed by collagenase and subsequently cultured in semisolid methylcellulose-based media as described below.85,86
- Numbers of committed progenitor cells (those cells not differentiated by conventional cytology) in the hematopoietic tissues of yolk sac, AGM region, liver, or dispersed embryoid bodies from lethal mutants are best assayed in semisolid, methylcellulose-based tissue culture media.126 Cells are plated with hematopoietic cytokines (erythropoetin, G-CSF, GM-CSF, IL-1, IL-3, IL-11, SCF, etc.) for 7 to 9 days. Committed progenitor cells named for their morphology in tissue culture are visually enumerated based on appearance in phase contrast or direct illumination microscopy. These colonies carry abbreviated designations such as CFU-E (erythroid), BFU (blast-forming unit)-E, CFU-GM (granulocyte macrophage), CFU-Emega (erythroid megakaryocytic), CFU-GEMM (multilineage), etc. The colony designation is periodically checked by selecting and smearing cell clusters, then evaluating their morphology after staining with Wright-Giemsa.
- Long-term bone marrow cultures more closely recapitulate the hematopoietic microenvironment and may be used to assay for the presence of stem cells with potential for self-renewal.32,52,93 The Dexter culture system is commonly employed. This system utilizes an irradiated layer of fibroblasts, upon which bone marrow is seeded and cultured together with a complete media containing hematopoietic cytokines.93
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First Generation Chimeras
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Murine offspring generated by blastocyst injection of homozygous negative ES cells that have the brindled coat color combination of both parent (C57BL/6, black) and ES cell (SV129, agouti) strains are usually viable chimeras. The contribution of mutant cells to different tissues is a random process. The complete absence of mutant cells from a tissue of several chimeric mice suggests that the targeted gene is required for normal differentiation of that tissue. The isoenzyme differences between C57BL/6 (GPI-1B) and SV129 (GPI-1A) mice for the GPI gene are readily exploited to establish mutant versus parent cell contribution in hematopoietic and lymphoid tissues. After electrophoretic separation of tissue homogenate on cellulose acetate, membranes are incubated with substrate and color reagent to reveal lighter (1A) and heavier (1B) bands of GPI.5,85,92,101
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Complementation Chimeras
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Mice with severe combined immunodeficiency (SCID) are deficient in functional T and B cells and are therefore unable to reject xenogeneic organ grafts. Acute lymphoblastic (ALL), myeloblastic (AML), CML, or chronic lymphocytic leukemic (CLL) cells induce overt disease when injected into SCID or NOD (nonobese diabetic)-SCID mice, reproducing patterns reminiscent of the human disease.1,45,116 The absence of lymphocytes in SCID and Rag (recombinase activating gene)-deficient mice may be exploited in complementation assays. ES cells with mutations that produce embryonic or fetal lethality may be inserted into SCID or Rag-/- blastocysts. Lymphocyte populations in resultant chimeras will derive solely from injected ES cells. Complementation chimeras may also be created by injecting the blastocyst of a lethal mutant with modified ES cells used to generate the same mutant.85 Gene insertions in these ES cells are aimed at restoring normal hematopoietic function. The insertion of complementation genes together with the gene for bacterial ß-galactosidase (lacZ) enables ready localization of lacZ-containing cells based on the blue color reaction obtained when lacZ is incubated with substrate.101 This approach was used to analyze the angiogenic and hematopoietic functions of Flk-1.101
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In Vivo Stimulation of Hematologic Alterations
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Commonly used approaches to study accelerated hematopoiesis in mice include treatment with 5-fluorouracil (5FU, 150 mg/kg/day IP) or phenylhydazine (60 mg/kg IP).27,71 The former treatment effaces all dividing marrow elements, and cytopenias result from acute marrow ablation. Phenylhydrazine causes an acute Heinz body hemolytic anemia; recovery occurs with intact bone marrow.76,110 Alternatively, a fixed volume of blood (to 3% of body weight) may be removed from a rodent and replaced with intraperitoneal saline, stimulating erythropoiesis. Many mice with deficiencies of leukocyte adhesion molecules or chemotaxis receptors are leukocytotic. Due to extensive redundancy among adhesion molecules, the expected leukocytotic reactions may not manifest (Table 2). To assess the mobilization of granulocytes and accelerated granulopoiesis, Bacto-tryptone (a potent chemotactic casein digest), thioglycollate, or specific chemotactic agents may be administered interperitoneally39,75 with subsequent enumeration of peritoneal lavage cells. By intravenous dosing of G-CSF at a dose of 5 µg/kg,5 recruitment of neutrophils from the marrow may be assessed. To determine if leukocytosis relates to increased production or decreased margination, unanesthetized mutant and wildtype mice are injected intravenously with epinephrine (0.25 mg/kg) to demarginate cells. Blood is collected 35 minutes postinjection from the tail vein, and leukocytes are compared to baseline values.56,60
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Murine Erythrokinetics
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When interpreting changes in the murine erythron, consideration must be given to the short lifespan of the mouse erythrocyte (approximately 20 to 30 days), relative to larger domestic species and humans.44 Reduction in progenitor or precursor pools or increased turnover of mature red cells are, therefore, more rapidly reflected in anemia in mice.
Uptake of iron into hemoglobin is determined from the half-life of intravenously injected 59Fe.10 Plasma samples are taken at regular intervals up to 50 minutes after injection and counted in a scintillation spectrometer. Radioactivity is also measured on 16-µm-thick cryosections using a ß-particle imaging instrument. To determine erythrocyte life span, approximately 1 x 109 cells are fluorescently labeled with 5-chloromethylfluorescein diacetate (CMFDA) and injected into the tail veins of recipient mice. Blood samples are collected at various times up to 30 days postinjection and analyzed by flow cytometry for the percentage of labeled erythrocytes. Red blood cell (RBC) survival curves are constructed by plotting the circulating labeled cells as the percentage of the number of circulating erythrocytes at 2 hours postinjection of CMFDA.66
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Flow Cytometry
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The flow cytometric evaluation of murine bone marrow and fetal liver has become standardized (Table 3).98,126 From the differential count obtained by flow cytometry or cytologic examination and femoral bone marrow cellularity (approximately 15 to 30 million cells per normal mouse femur), the absolute numbers of identifiable femoral precursor cells may be readily quantified. Since the cells of a single femur contain approximately 6% of the total murine marrow elements, an estimate of absolute numbers of precursors per mouse may also be calculated (approximately 250 to 500 million hematopoietic cells per mouse).120 Except for rare reports,54 basophils are generally considered to be absent from the peripheral blood of mice and are best evaluated by bone marrow cytology or flow cytometry.7 To reduce background fluorescence related to receptor binding of the Fc portion of immunoglobulins, bone marrow cells are commonly blocked with antibodies to the two Fc
receptors, CD16 and CD32.
Pluripotent hematopoietic stem cells are frequently studied in mutant mice. The identification of markers for these cells will ultimately provide tools for transplantation of these cells with minimal contamination by nonpluripotent cells. HSCs are currently defined by their ability to repopulate the bone marrow of lethally irradiated mice (see above) and by a series of antigens or properties of cells revealed by flow cytometric evaluation (Table 4).
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Murine Hemostasis
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Megakaryocytes and platelets
Megakaryocytopoiesis, thrombopoiesis, and platelet function have been studied extensively in mutant mice. Alterations in megakaryocyte and/or platelet function commonly manifest as thrombocytosis or thrombocytopenia.
Thrombocytopenia in mutant mice results from increased consumption or decreased production of platelets. The assessment of decreased production is best ascertained by cytologic or histologic examination of marrow or other hematopoietic tissue or, alternatively, culture of hematopoietic tissue for megakaryocytic progenitors. Increased consumption is generally linked to increased production, for which mean platelet volume (MPV) is a sensitive indicator in mice. Following experimental acute induction of thrombocytopenia in mice or dosing with thrombopoietin, the MPV increases from 4.7 to 8.2 fl, peaking at 36 hours and returning to normal by approximately 3 days.81,117 A variety of methods are utilized to establish the cause of thrombocytopenia. Splenectomy allows the assessment of the effects of hypersplenism, altered monocyte–macrophage phagocytic activity, or immune-mediated thrombocytopenia on platelet turnover. Platelet life span is readily assessed in mice. Platelets are fluorescently labeled with CMFDA as described for red blood cells4,66 and injected into the tail veins of recipient mice. Blood samples are collected periodically for 4 days and analyzed by flow cytometry to determine the proportion of labeled platelets present at each time point. Alternatively, platelets may be biotinylated by in vivo injection of sulfo-NHS-biotin (35 mg/kg), with platelet disappearance studied periodically as for CMFDA.7,24 The life span of platelets in mice relative to other species is short (
5 days).3 Induction of thrombocytopenia is used to study accelerated megakaryocytopoiesis and thrombopoiesis in mice. To this end, antiplatelet antiserum or 5FU is administered intraperitoneally, and recovery of platelet numbers to pretreatment values is evaluated.106
To assess thrombopoiesis cytologically, blood is collected by cardiac puncture, and the morphology of platelets (discoid, elongated, beaded) in platelet-rich plasma19 is evaluated by phase contrast microscopy at 400x magnification.65 The total percent of nondiscoid platelets is considered the proplatelet fraction.
Megakaryocyte ploidy is commonly assessed in targeted mutants with altered megakaryocytopoiesis or mice treated with thrombopoietic cytokines. The ploidy distribution of cells is determined by flow cytometry following prolonged incubation (24 hours) with propidium iodide.4,109 Consistent with most species examined, the mean ploidy of murine megakaryocytes is 16N. This increases with acute thrombocytopenia and is higher in certain mouse strains such as the C3H.53,73,74 Given the large capacity for increasing production, increased megakaryocyte ploidy53 or mean platelet volume23 may be the only indicators belying compensated, increased platelet turnover in mice.
Intravascular activation of platelets may cause increased platelet clearance and result in thrombocytopenia. The activation state of platelets may be assessed by flow cytometric evaluation of the expression of surface P-selectin and fibrinogen binding by GpIIbIIIa.78 Flow cytometric approaches for evaluation of platelets utilize low volumes (µl) of whole blood and are ideal for use with mice. This approach also will readily detect changes in MPV or reveal subpopulations of large platelets observed during states of accelerated thrombopoiesis. Urinary assays for metabolites of thromboxane-A2 (TXA2), such as TXB2, and plasma assays for ß-thromboglobulin provide an indirect assessment of in vivo platelet activation.
Platelet aggregation may be evaluated in platelet-rich plasma or whole blood from mice. Adenosine diphosphate (ADP), collagen, arachidonate, and thrombin are potent agonists of platelet aggregation in the mouse and are potentiated by heparinized versus citrated plasma. In contrast to their effects in other species, epinephrine, serotonin and platelet activating factor (PAF) do not cause aggregation of mouse platelets.77,97,115
Bleeding time assays, as for higher species, are also used to assess platelet function in mice. However, unlike higher species, bleeding times in mice are also markedly prolonged by factor deficiencies that decrease thrombin generation (Table 4).115
Murine blood coagulation and fibrinolysis
In general, mechanisms of blood coagulation and fibrinolysis are common to domestic animal species and laboratory rodents, with limited differences. Mice deficient in most proteins known to be directly involved in human coagulation and fibrinolysis have been generated (Table 5). The human and domestic animal phenotypes of deficiencies of proteins involved in blood coagulation and fibrinolysis are among the best understood of inborn errors, lending the comparison to the phenotypes of orthologous murine mutants of particular interest. Murine hemostasis has recently been extensively reviewed.6,115
Most important in the evaluation of mutants with suspected or known hemostatic abnormalities is the careful assessment of fetuses or mice for macroscopic and microscopic evidence of hemorrhage or tissue infarction. In addition to tests of platelet function described above, standard tests for coagulation factors (prothrombin time, activated partial thromboplastin time, factor assays) may be readily applied to murine plasma samples. Murine fibrin degradation products and D-dimer are not detected by kits for humans. Careful blood collection to minimize partial clotting is imperative. The murine fibrinolytic system may be assessed by studying the effects of intravenous thrombin administration and resistance to microthrombotic challenge.17,18 Damage to or ligation of the middle cerebral artery with planimetric measurement of the infarcted area is commonly employed to evaluate thrombosis and fibrinolysis in mutant mice and mice given therapeutic agents.22,82 The disappearance of thrombin-coagulated, 125I-labeled normal murine plasma injected into the jugular vein in the presence or absence of intraperitoneally dosed endotoxin is also used to evaluate fibrinolysis in vivo. Clot lysis is evaluated by the measurement of the residual radioactivity in the heart and lungs ex vivo at 2 and 4 hours after injection.17,18,68
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General Technical Considerations
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The application of typical laboratory instrumentation to the assessment of mutant mouse hematology and hemostasis is valuable but must be undertaken cautiously. Where specific software settings are available for mice, they should be used. Frequently, mutations causing altered cell size or differentiation (e.g., murine GpIb deficiency118) result in incorrect or overlapping gating of cell populations by analyzers—blood smears should always be evaluated during initial phenotyping to confirm instrument interpretations. The volume of blood or other samples collected from mice will often fall below the dead volume of laboratory instrumentation and require dilution. Potential artifacts of linearity with dilutions of small quantities can be overcome by comparing results from treated or genetically altered mice with similarly diluted volumes from control or wild-type mice, permitting valid conclusions.
The technology that permits the generation of mice deficient in single genes has provided the tools necessary to probe hematopoiesis and hemostasis in great detail. Given the small size of mice and the frequency with which embryonic lethal or unexpected phenotypes manifest, experimental pathologists must be knowledgeable and creative in exploring the pathophysiology of observed effects.
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Acknowledgments
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E. Calvert is gratefully acknowledged for preparation of citations.
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References
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- Ailles LE, Gerhard B, Kawagoe H, Hogge DE: Growth characteristics of acute myelogenous leukemia progenitors that initiate malignant hematopoiesis in nonobese diabetic/severe combined immunodeficient mice. Blood 94:1761-1772, 1999[Abstract/Free Full Text]
- Auerbach R, Gilligan B, Lu LS, Wang SJ: Cell interactions in the mouse yolk sac: vasculogenesis and hematopoiesis. J Cell Physiol 173:202-205, 1997[CrossRef][Medline]
- Ault KA, Knowles C: In vivo biotinylation demonstrates that reticulated platelets are the youngest platelets in circulation. Exp Hematol 23:996-1001, 1995[Medline]
- Baker GR, Levin J: Transient thrombocytopenia produced by administration of macrophage colony-stimulating factor: investigations of the mechanism. Blood 91:89-99, 1998[Abstract/Free Full Text]
- Barker JE, Compton ST: Hematopoietic repopulation of adult mice with beta-thalassemia. Blood 83:828-832, 1994[Abstract/Free Full Text]
- Benestad HB, Strom-Gundersen I, Ole Iversen P, Haug E, Nja A: No neuronal regulation of murine bone marrow function. Blood 91:1280-1287, 1998[Abstract/Free Full Text]
- Berger G, Hartwell DW, Wagner DD: P-Selectin and platelet clearance. Blood 92:4446-4452, 1998[Abstract/Free Full Text]
- Bi L, Lawler AM, Antonarakis SE, High KA, Gearhart JD, Kazazian HH, Jr: Targeted disruption of the mouse factor VIII gene produces a model of haemophilia A. Nat Genet 10:119-121, 1995[CrossRef][Medline]
- Bielinska M, Narita N, Heikinheimo M, Porter SB, Wilson DB: Erythropoiesis and vasculogenesis in embryoid bodies lacking visceral yolk sac endoderm. Blood 88:3720-3730, 1996[Abstract/Free Full Text]
- Bohl D, Bosch A, Cardona A, Salvetti A, Heard JM: Improvement of erythropoiesis in ß-thalassemic mice by continuous erythropoietin delivery from muscle. Blood 95:2793-2798, 2000[Abstract/Free Full Text]
- Brayton C, Justice M, Montgomery CA: Evaluating mutant mice: anatomic pathology. Vet Pathol 37:1-19, 2000[Abstract/Free Full Text]
- Bugge TH, Flick MJ, Daugherty CC, Degen JL: Plasminogen deficiency causes severe thrombosis but is compatible with development and reproduction. Genes Dev 9:794-807, 1995[Abstract/Free Full Text]
- Bugge TH, Kombrinck HW, Flick MJ, Daugherty CC, Danaton MJS, Degen JL: Loss of fibrinogen rescues mice from the pleiotropic effects of plasminogen deficiency. Cell 87:709-719, 1996[CrossRef][Medline]
- Bugge TH, Suh TT, Flick MJ, Daugherty CC, Romer J, Solberg H, Ellis V, Dano K, Degen JL: The receptor for urokinase-type plasminogen activator is not essential for mouse development or fertility. J Biol Chem 270:16886-16894, 1995[Abstract/Free Full Text]
- Bugge TH, Yiao Q, Kombrinck KW, Flick MJ, Holmback K, Danton MJ, Colbert MC, Witte DP, Fujikawa K, Davie EW, Degen JL: Fatal embryonic bleeding events in mice lacking tissue factor, the cell-associated initiator of blood coagulation. Proc Natl Acad Sci USA 93:6258-6563, 1996[Abstract/Free Full Text]
- Cacakano G, Lee J, Kikly K, Ryan AM, Pitts-Meek S, Hultgren B, Wood WI, Moore MW: Neutrophil and B cell expansion in mice that lack the murine IL-8 receptor homolog. Science 265:682-684, 1994[Abstract/Free Full Text]
- Carmeliet P, Collen D: Gene targeting and gene transfer studies of the biological role of the plasminogen/plasmin system. Thromb Haemost 74:429-436, 1995[Medline]
- Carmeliet P, Stassen JM, Schoonjans L, Ream B, Van de Oord JJ, De Mol M, Mulligan RC, Collen D: Plasminogen activator inhibitor-1 gene deficient mice. II. Effects on hemostasis, thrombosis and thrombolysis. J Clin Invest 92:2756-2760, 1993
- Catalfamo JL, Dodds WJ: Isolation of platelets from laboratory animals. Methods Enzymol 169:27-34, 1989[Medline]
- Cheng J, Baumhueter S, Caclano G, Carver-Moore K, Thibodeaux H, Thomas R, Broxmeyer HE, Cooper S, Hague N, Moore M, Lasky LA: Hematopoietic defects in mice lacking the sialomucin CD34. Blood 87:479-490, 1996[Abstract/Free Full Text]
- Choi K: Hemangioblast development and regulation. Biochem Cell Biol 76:947-956, 1998[CrossRef][Medline]
- Choudhri TF, Hoh BL, Prestigiacomo CJ, Huang J, Kim LJ, Schmidt AM, Kisiel W, Connolly ES, Jr Oinsky DJ: Targeted inhibition of intrinsic coagulation limits cerebral injury in stroke without increasing intracerebral hemorrhage. J Exp Med 190:91-99, 1999[Abstract/Free Full Text]
- Corash L, Mok Y, Levin J, Baker G: Regulation of platelet heterogeneity effects of thrombocytopenia on platelet volume and density. Exp Hematol 18:205-212, 1990[Medline]
- Coxon A, Rieu P, Barkalow FJ, Askari S, Sharpe AH, von Andrian UH, Arnaout MA, Mayadas TN: A novel role for the beta 2 integrin CD11b/CD18 in neutrophil apoptosis: a homeostatic mechanism in inflammation. Immunity 5:653-66, 1996[CrossRef][Medline]
- Cui J, O'Shea KS, Purkayastha A, Saunders TL, Ginsburg D: Fatal haemorrhage and incomplete block to embryogenesis in mice lacking coagulation factor V. Nature 384:66-68, 1996[CrossRef][Medline]
- Cumano A, Dieterlen-Lievre F, Godin I: Lymphoid potential, probed before circulation in mouse, is restricted to caudal intraembryonic splanchnopleura. Cell 86:907-916, 1996[CrossRef][Medline]
- De Haan G, Donte B, Engel C, Loeffler M, Nijhof W: Prophylactic pretreatment of mice with hematopoietic growth factors induces expansion of primitive cell compartments and results in protection against 5-fluorouracil-induced toxicity. Blood 87:4581-4588, 1996[Abstract/Free Full Text]
- de Sousa M, Reimao R, Lacerda R, Hugo P, Kaufmann SH, Porto G: Iron overload in beta 2-microglobulin-deficient mice. Immunol Lett 39:105-111, 1994[CrossRef][Medline]
- Degen SJ, Sun WY: The biology of prothrombin. Crit Rev Eukaryot Gene Expr 8:203-224, 1998[Medline]
- Denis C, Methia N, Frenette PS, Rayburn H, Ullmann-Cullere M, Hynes RO, Wagner DD: A mouse model of severe von Willebrand disease: defects in hemostasis and thrombosis. Proc Natl Acad Sci USA 95:9524-9529, 1998[Abstract/Free Full Text]
- Dewerchin M, Liang Z, Moons L, Carmeliet P, Castellino FJ, Collen D, Rosen ED: Blood coagulation factor X deficiency causes partial embryonic lethality and fatal neonatal bleeding in mice. Thromb Haemost 83:185-190, 2000[Medline]
- Dexter TM, Moore MAS: In vitro duplication and "cure" of haematopoietic defects in genetically anaemic mice. Nature 269:412-414, 1977[CrossRef][Medline]
- Dranoff G, Crawford AD, Sadelain M, Ream B, Rashid A, Bronson RT, Dickersin GR, Bachurski CJ, Mark EL, Whitsett JA, Mulligan RC: Involvement of granulocyte–macrophage colony-stimulating factor in pulmonary homeostasis. Science 264:713-716, 1994[Abstract/Free Full Text]
- Dzierzak E, Medvinsky A, de Bruijn M: Qualitative and quantitative aspects of haematopoietic cell development in the mammalian embryo. Immunol Today 19:228-236, 1998[CrossRef][Medline]
- Enjyoji K, Sevigny J, Lin Y, Frenette PS, Christie PD, Esch JS, 2nd Imai M, Edelberg JM, Rayburn H, Lech M, Beeler DL, Csizmadia E, Wagner DD, Robson SC, Rosenberg RD: Targeted disruption of cd39/ATP diphosphohydrolase results in disordered hemostasis and thromboregulation. Nat Med 5:1010-1017, 1999[CrossRef][Medline]
- Era T, Takagi T, Takahashi T, Bories JC, Nakano T: Characterization of hematopoietic lineage-specific gene expression by ES cell in vitro differentiation induction system. Blood 95:870-878, 2000[Abstract/Free Full Text]
- Ferra N, Carver-Moore K, Chen H, Dowd M, Lu L, O'Shea KS, Powel-Braxton L, Hillan KJ, Moore MW: Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 280:439-442, 1996[CrossRef]
- Freedman JE, Sauter R, Battimelli EM, Ault K, Knowles C, Huang PL, Loscalzo J: Deficient platelet-derived nitric oxide and enhanced hemostasis in mice lacking the NOSIII gene. Circ Res 84:1416-1421, 1999[Abstract/Free Full Text]
- Frenette PS, Mayadas TN, Rayburn H, Hynes RO, Wagner DD: Susceptibility to infection and altered hematopoiesis in mice deficient in both P- and E- selections. Cell 84:563-574, 1996[CrossRef][Medline]
- Georgopoulos K, Bigby M, Wang JH, Molnar A, Wu P, Winandy S, Sharpe A: The Ikaros gene is required for the development of all lymphoid lineages. Cell 79:143-156, 1994[CrossRef][Medline]
- Gerwin N, Gonzalo JA, Lloyd C, Coyle AJ, Reiss Y, Banu N, Wang B, Xu H, Avraham H, Engelhardt B, Springer TA, Gutierrez-Ramos JC: Prolonged eosinophil accumulation in allergic lung interstitium of ICAM-2 deficient mice results in extended hyperresponsiveness. Immunity 10:9-19, 1999[CrossRef][Medline]
- Gurney AL, Carver-Moore K, de Sauvage FJ, Moore MW: Thrombocytopenia in c-mpl-deficient mice. Science 265:1445-1447, 1994[Abstract/Free Full Text]
- Hao SX, Ren R: Expression of interferon consensus sequence binding protein (ICSBP) is downregulated in Bcr-Abl-induced murine chronic myelogenous leukemia-like disease, and forced coexpression of ICSBP inhibits Bcr-Abl-induced myeloproliferative disorder. Mol Cell Biol 20:1149-1161, 2000[Abstract/Free Full Text]
- Harvey JW: Erythrocyte Metabolism. In: Clinical Biochemistry of Domestic Animals, ed. Kaneko JJ, 4th ed., pp 196-197, Academic Press, San Diego, CA 1989
- Hendrickson EA: The SCID mouse: relevance as an animal model system for studying human disease. Am J Pathol 143:1511-1522, 1993[Abstract]
- Hermans MH, Ward AC, Antonissen C, Karis A, Lowenberg B, Touw IP: Perturbed granulopoiesis in mice with a targeted mutation in the granulocyte colony-stimulating factor receptor gene associated with severe chronic neutropenia. Blood 92:32-39, 1998[Abstract/Free Full Text]
- Hess JL, Yu BD, Li B, Hanson R, Korsmeyer SJ: Defects in yolk sac hematopoiesis in Mll-null embryos. Blood 90:1799-1806, 1997[Abstract/Free Full Text]
- Hirsch E, Iglesias A, Potocnik AJ, Hartmann U, Fassler R: Impaired migration but not differentiation of haematopoietic stem cells in the absence of beta1 integrins. Nature 380:171-175, 1996[CrossRef][Medline]
- Hodivala-Dilke KM, McHugh KP, Tsakiris DA, Rayburn H, Crowley D, Ullman-Cullere M, Ross FP, Coller BS, Teitelbaum S, Hynes RO: Beta3-integrin–deficient mice are a model for Glanzmann thrombasthenia showing placental defects and reduced survival. J Clin Invest 103:229-238, 1999[Medline]
- Holtschke T, Lohler J, Kanno Y, Fehr T, Giese N, Rosenbauer F, Lou J, Knobeloch KP, Gabriele L, Waring Jf, Bachmann MF, Zinkernagel RM, Morse HC, 3rd Ozato K, Horak I: Immunodeficiency and chronic myelogenous leukemia-like syndrome in mice with a targeted mutation of the ICSBP gene. Cell 87:307-317, 1996[CrossRef][Medline]
- Huang E, Nocka K, Beier DR, Chu T-Y, Buck J, Lahm H-W, Wellner D, Leder P, Besmer P: The hematopoietic growth factor KL is encoded by the SL locus and is the ligand of the c-kit receptor, the gene product of the W locus. Cell 63:225-233, 1990[CrossRef][Medline]
- Huang PL, Huang Z, Mashimo H, Block KD, Moskowitz MA, Bevan JA, Fishman MC: Hypertension in mice lacking the gene for endothelial nitric oxide synthase. Nature 377:239-242, 1995[CrossRef][Medline]
- Jackson CW, Steward SA, Chenaille PJ, Ashmun RA, McDonald TP: Analysis of megakaryocytopoiesis in the C3H mouse: an animal model whose megakaryocytes have 32N as the modal DNA class. Blood 76:690-696, 1990[Abstract/Free Full Text]
- Jacoby W, Cammarata PV, Findlay S, Pincus SH: Anaphylaxis in mast cell–deficient mice. J Invest Dermatol 83:302-304, 1984[CrossRef][Medline]
- Jalbert LR, Rosen ED, Careliet P, Moons L, Chan JC, Collen D, Castellino FJ: Inactivation of the gene for anticoagulant protein C causes lethal perinatal consumptive coagulopathy in mice. J Clin Invest 192:1481-1488, 1998
- Johnson RC, Mayadas TN, Frenette PS, Mebius RE, Subramaniam M, Lacasce A, Hynes RO, Wagner DD: Blood cell dynamics in P-selection–deficient mice. Blood 86:1106-1114, 1995[Abstract/Free Full Text]
- Keller G, Kennedy M, Papayannopoulou T, Wiles MV: Hematopoietic commitment during embryonic stem cell differentiation in culture. Mol Cell Biol 13:473-486, 1993[Abstract/Free Full Text]
- Kim M, Cooper DD, Hayes SF, Spangrude GJ: Rhodamine-123 staining in hematopoietic stem cells of young mice indicates mitochondrial activation rather than dye efflux. Blood 91:4106-4117, 1998[Abstract/Free Full Text]
- Kitajima K, Kojima M, Nakajima K, Kondo S, Hara T, Miyajima A, Takeuchi T: Definitive but not primitive hematopoiesis is impaired in jumonji mutant mice. Blood 93:87-95, 1999[Abstract/Free Full Text]
- Labow MA, Norton CR, Rumberger JM, Lombard-Gillooly KM, Shuster DJ, Hubbard J, Bertko R, Knaack PA, Terry RW, Harbison ML, Kontgen F, Stewart CL, McIntyre KW, Will PC, Burns DK, Wolitsky BA: Characterization of E-selectin–deficient mice: demonstration of overlapping function of the endothelial selectins. Immunity 1:709-720, 1994[CrossRef][Medline]
- Lantz CS, Boesigner J, Song CH, Mach N, Kobayashi T, Mulligan RC, Nawa Y, Dranoff G, Galli SJ: Role for interleukin-3 in mast cells and basophil development and in immunity to parasities. Nature 392:90-93, 1998[CrossRef][Medline]
- Lawler J, Sunday M, Thibert V, Duquette M, George EL, Rayburn H, Hynes RO: Thrombospondin-1 is required for normal murine pulmonary homeostasis and its absence causes pneumonia. J Clin Invest 101:982-992, 1998[Medline]
- Lawrence HJ, Helgason CD, Sauvageau G, Fong S, Izon DJ, Humphries RK, Largman C: Mice bearing a targeted interruption of the homeobox gene HOXA9 have defects in myeloid, erythroid, and lymphoid hematopoiesis. Blood 89:1922-1930, 1997[Abstract/Free Full Text]
- Lecine P, Villeval JL, Vyas P, Swencki B, Xu Y, Shivdasani RA: Mice lacking transcription factor NF-E2 provide in vivo validation of the proplatelet model of thrombocytopoiesis and show a platelet production defect that is intrinsic to megakaryocytes. Blood 92:1608-1616, 1998[Abstract/Free Full Text]
- Leveen P, Pekny M, Gerbre-Medhin S, Swolin B, Larsson E, Betsholtz C: Mice deficient for PDGF-ß show renal, cardiovascular, and hematological abnormalities. Genes Dev 8:1875-1887, 1994[Abstract/Free Full Text]
- Levin J, Peng JP, Baker GR, Villeval JL, Lecine P, Burstein SA, Shivdasani RA: Pathophysiology of thrombocytopenia and anemia in mice lacking transcription factor NF-E2. Blood 94:3037-3047, 1999[Abstract/Free Full Text]
- Lieschke GJ, Grail D, Hodgson G, Metcalf D, Stanley E, Cheers C, Fowler KJ, Basu S, Zhan YF, Dunn AR: Mice lacking granulocyte colony-stimulating factor have chronic neutropenia, granulocyte and macrophage progenitor cell deficiency, and impaired neutrophil mobilization. Blood 84:1737-1746, 1994[Abstract/Free Full Text]
- Lijnen HR, Okada K, Matsuo O, Collen D, Dewerchin M: Alpha2-antiplasmin gene deficiency in mice associated with enhanced fibrinolytic potential without overt bleeding. Blood 93:2274-2281, 1999[Abstract/Free Full Text]
- Lim SK, Kim H, Lim SK, bin Ali A, Lim YK, Wang Y, Chong SM, Costantini F, Baumman H: Increased susceptibility in Hp knockout mice during acute hemolysis. Blood 92:1870-1877, 1998[Abstract/Free Full Text]
- Lin HF, Maeda N, Smithies O, Straight DL, Stafford DW: A coagulation factor IX-deficient mouse model for human hemophilia B. Blood 90:3962-3966, 1997[Abstract/Free Full Text]
- Lorenz M, Slaughter HS, Wescott DM, Carter SI, Schnyder B, Dinchuk JE, Car BD: Cyclooxygenase-2 is essential for normal recovery from 5-fluorouracil-induced myelotoxicity in mice. Exp Hematol 27:1494-1502, 1999[CrossRef][Medline]
- Lu H, Smith CW, Perrard J, Bullard D, Tang L, Shappell SB, Entman ML, Beaudet AL, Ballantyne CM: LFA-1 is sufficient in mediating neutrophil emigration in Mac-1–deficient mice. J Clin Invest 99:1340-1350, 1997[Medline]
- Manning KL, McDonald TP: C3H mice have larger spleens, lower platelet counts, and shorter platelet lifespans than C57BL mice: an animal model for the study of hypersplenism. Exp Hematol 25:1019-1024, 1997[Medline]
- Manning KL, Novinger S, Sullivan PS, McDonald TP: Successful determination of platelet lifespan in C3H mice by in vivo biotinylation. Lab Anim Sci 46:545-548, 1996[Medline]
- Mayadas TN, Johnson RC, Rayburn H, Hynes RO, Wagner DD: Leukocyte rolling and extravasation are severely compromised in P selectin–deficient mice. Cell 74:541-554, 1993[CrossRef][Medline]
- Medvinsky A, Dzierzak E: Definitive hematopoiesis is autonomously initiated by the AGM region. Cell 86:897-906, 1996[CrossRef][Medline]
- Meyers KM: Pathobiology of animal platelets. Adv Vet Sci Comp Med 30:131-165, 1985[Medline]
- Michelson AD: Flow cytometry: a clinical test of platelet function. Blood 87:4925-4936, 1996[Free Full Text]
- Mizgerd JP, Kubo H, Kutkoski GJ, Bhagwan SD, Scharffetter-Kochanek K, Beaudet AL, Doerschuk CM: Neutrophil emigration in the skin, lungs, and peritoneum: different requirements for CD11/CD18 revealed by CD18-deficient mice. J Exp Med 186:1357-1364, 1997[Abstract/Free Full Text]
- Mucenski ML, McLain K, Kier AB, Swerdlow SH, Schreiner CM, Miller TA, Pietryga DW, Scott WJ, Jr Potter SS: Functional c-myb gene is required for normal murine fetal hepatic hematopoiesis. Cell 65:677-69, 1991[CrossRef][Medline]
- Murone M, Carpenter DA, de Sauvage FJ: Hematopoietic deficiencies in c-mpl and TPO knockout mice. Stem Cells 16:1-6, 1998[Abstract/Free Full Text]
- Nagai N, De Mol M, Lijnen HR, Carmeliet P, Collen D: Role of plasminogen system components in focal cerebral ischemic infarction: a gene targeting and gene transfer study in mice. Circulation 99:2440-2444, 1999[Abstract/Free Full Text]
- Nishinakamura R, Miyajima A, Mee PJ, Tybulewicz VL, Murray R: Hematopoiesis in mice lacking the entire granulocyte–macrophage colony-simulating factor/interleukin-3/interleukin-5 functions. Blood 88:2458-2464, 1996[Abstract/Free Full Text]
- Oike Y, Takakura N, Hata A, Kaname T, Akizuki M, Yamaguchi Y, Yasue H, Araki K, Yamamura K, Suda T: Mice homozygous for a truncated form of CREB-binding protein exhibit defects in hematopoiesies and vasculo-angiogenesis. Blood 93:2771-2779, 1999[Abstract/Free Full Text]
- Okuda T, Takeda K, Fujita Y, Nishimura Yagyu S, Yoshida M, Akira S, Downing JR, Abe T: Biological characteristics of the leukemia-associated transcriptional factor AML1 disclosed by hematopoietic rescue of AML1-deficient embryonic stem cells by using a knock-in strategy. Mol Cell Biol 20:319-328, 2000[Abstract/Free Full Text]
- Okuda T, van Deursen J, Hiebert SW, Grosveld G, Downing JR: AML1, the target of multiple chromosomal translocations in human leukemia, is essential for normal fetal liver hematopoiesis. Cell 84:321-330, 1996[CrossRef][Medline]
- Pandolfi PP: Knocking in and out genes and trans genes: the use of the engineered mouse to study normal and aberrant hemopoiesis. Semin Hematol 35:136-148, 1998[Medline]
- Peschon JJ, Morrissey PJ, Grabstein KH, Ramsdell FJ, Maraskovsky E, Gliniak BC, Park LS, Ziegler SF, Williams DE, Ware CB, Meyer JD, Davidson BL: Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice. J Exp Med 180:1955-1960, 1994[Abstract/Free Full Text]
- Peters LL, Shivdasani RA, Liu SC, Hanspal M, John KM, Gonzalez JM, Brugnara C, Gwynn B, Mohandas N, Alper SL, Orkin SH, Lux SE: Anion exchanger 1 (band 3) is required to prevent erythrocyte membrane surface loss but not to form the membrane skeleton. Cell 86:917-927, 1996[CrossRef][Medline]
- Ploplis VA, French EL, Carmeliet P, Collen D, Plow EF: Plasminogen deficiency differentially affects recruitment of inflammatory cell populations in mice. Blood 91:2005-2009, 1998[Abstract/Free Full Text]
- Porcher C, Swat W, Rockwell K, Fujiwara Y, Alt FW, Orkin SH: The T cell leukemia oncoprotein SCL/tal-1 is essential for development of all hematopoietic lineages. Cell 86:47-57, 1996[CrossRef][Medline]
- Qu CK, Yu WM, Azzarelli B, Cooper S, Broxmeyer HE, Feng GS: Biased suppression of hematopoiesis and multiple developmental defects in chimeric mice containing Shp-2 mutant cells. Mol Cell Biol 18:6075-6082, 1998[Abstract/Free Full Text]
- Quesenberry PJ, Temeles D, McGrath H, Lowry P, Meyer D, Kittler E, Deacon D, Kister K, Crittenden R, Srikumar K: Stroma-dependent hematolymphopoietic stem cells. Curr Top Microbiol Immunol 177:151-166, 1992[Medline]
- Reya T, Contractor NV, Couzens MS, Wasik MA, Emerson SG, Carding SR: Abnormal myelocytic cell development in interleukin-2 (IL-2)-deficient mice: evidence for the involvement of IL-2 in myelopoiesis. Blood 91:2935-2947, 1998[Abstract/Free Full Text]
- Rosen ED, Chan JC, Idusogie E, Clotman F, Vlasuk G, Luther T, Jalbert LR, Albrecht S, Zhong L, Lissens A, Schoonjans L, Moons L, Collen D, Castellino FJ, Carmeliet P: Mice lacking factor VII develop normally but suffer fatal perinatal bleeding. Nature 390:290-294, 1997[CrossRef][Medline]
- Rosenberg RD: Thrombomodulin gene disruption and mutation in mice. Thromb Haemost 78:705-709, 1997[Medline]
- Rosenblum WI, Nelson GH, Cockrell CS, Ellis EF: Some properties of mouse platelets. Thromb Res 30:347-355, 1983[CrossRef][Medline]
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Abstract
Murine Hematopoiesis
