Subject(s)
Cell Culture Techniques/methods , Cell Separation/methods , Stem Cells/cytology , Animals , Embryo, Mammalian , MiceABSTRACT
Recently, the human ATP-binding cassette transporter-1 (ABC1) gene has been demonstrated to be mutated in patients with Tangier disease. To investigate the role of the ABC1 protein in an experimental in vivo model, we used gene targeting in DBA-1J embryonic stem cells to produce an ABC1-deficient mouse. Expression of the murine Abc1 gene was ablated by using a nonisogenic targeting construct that deletes six exons coding for the first nucleotide-binding fold. Lipid profiles from Abc1 knockout (-/-) mice revealed an approximately 70% reduction in cholesterol, markedly reduced plasma phospholipids, and an almost complete lack of high density lipoproteins (HDL) when compared with wild-type littermates (+/+). Fractionation of lipoproteins by FPLC demonstrated dramatic alterations in HDL cholesterol (HDL-C), including the near absence of apolipoprotein AI. Low density lipoprotein (LDL) cholesterol (LDL-C) and apolipoprotein B were also significantly reduced in +/- and -/- compared with their littermate controls. The inactivation of the Abc1 gene led to an increase in the absorption of cholesterol in mice fed a chow or a high-fat and -cholesterol diet. Histopathologic examination of Abc1-/- mice at ages 7, 12, and 18 mo demonstrated a striking accumulation of lipid-laden macrophages and type II pneumocytes in the lungs. Taken together, these findings demonstrate that Abc1-/- mice display pathophysiologic hallmarks similar to human Tangier disease and highlight the capacity of ABC1 transporters to participate in the regulation of dietary cholesterol absorption.
Subject(s)
ATP-Binding Cassette Transporters/genetics , Foam Cells/cytology , Glycoproteins/genetics , Lipoproteins, HDL/deficiency , Mutation , ATP Binding Cassette Transporter 1 , Animals , Base Sequence , Cholesterol/blood , DNA Primers , Humans , Lipoproteins, HDL/blood , Mice , Mice, KnockoutABSTRACT
Collagen-induced arthritis in the DBA/1 mouse is an experimental model of human rheumatoid arthritis. To examine the role of leukotrienes in the pathogenesis of this disease, we have developed embryonic stem (ES) cells from this mouse strain. Here, we report that DBA/1 mice made deficient in 5-lipoxygenase-activating protein (FLAP) by gene targeting in ES cells develop and grow normally. Zymosan-stimulated leukotriene production in the peritoneal cavity of these mice is undetectable, whereas they produce substantial amounts of prostaglandins. The inflammatory response to zymosan is reduced in FLAP-deficient mice. The severity of collagen-induced arthritis in the FLAP-deficient mice was substantially reduced when compared with wild-type or heterozygous animals. This was not due to an immunosuppressive effect, because anti-collagen antibody levels were similar in wild-type and FLAP-deficient mice. These data demonstrate that leukotrienes play an essential role in both the acute and chronic inflammatory response in mice.
Subject(s)
Arthritis, Experimental/physiopathology , Carrier Proteins/metabolism , Carrier Proteins/physiology , Collagen/immunology , Membrane Proteins/deficiency , Membrane Proteins/metabolism , Membrane Proteins/physiology , 5-Lipoxygenase-Activating Proteins , Animals , Antibody Formation , Arthritis, Experimental/pathology , Arthritis, Experimental/prevention & control , Blood Proteins/metabolism , Female , Heterozygote , Humans , Joints/immunology , Joints/pathology , Leukotrienes/biosynthesis , Leukotrienes/physiology , Male , Mice , Mice, Inbred DBA , Mice, Knockout , Peritoneal Cavity , Stem Cells , Zymosan/pharmacologyABSTRACT
The development of embryonic stem (ES) cells and their capacity to generate mice with mutations at specific loci has provided a powerful resource for functional analysis of genes in pathological processes. However, the ability to combine this technology with the large number of existing murine models of human genetic disease has been complicated by the inability to routinely generate ES cell lines from strains other than 129. Here, we report the production of a novel ES cell line derived from an inbred mouse, DBA/1lacJ. This new ES cell line undergoes homologous recombination and efficient colonization of the germline of male chimeric offspring with ES cell microinjection into C57B1/6 embryos. The DBA/1lacJ mouse is a murine model of human inflammation, therefore genetic modifications in the DBA ES cells will allow evaluation of the target gene's role in the inflammatory process.
Subject(s)
Disease Models, Animal , Gene Targeting/methods , Inflammation , Mice, Inbred DBA , Stem Cells/cytology , 5-Lipoxygenase-Activating Proteins , Animals , Blastocyst , Carrier Proteins/genetics , Cell Line , Chimera , Female , Genetic Vectors/genetics , Humans , Male , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Inbred DBA/embryology , Microinjections , Recombination, GeneticABSTRACT
Extensive DM contamination was found on Dacron bags that were incubated for prolonged periods of time in the rumen of steers fed alfalfa hay. The ash content of the contaminant was high, and most of it was acid-soluble X-ray analysis indicated the presence of hydroxylapatite and synthetic calcium magnesium phosphate or whitlockite. The contaminant appeared as a smooth coating on the Dacron fiber, suggesting that contamination was a gradual process rather than the result of entrapment of dislodged crystals from plant material. Contamination seemed to occur exponentially within the range of observations (0 to 42 d). Contamination also occurred in steers fed orchardgrass, although to a lesser extent than in steers fed alfalfa hay. The DM contamination was less than .04 g per bag (average bag weight was 1.2 g) during the first 10 d of incubation. However, correction for contamination might be required for studies involving longterm incubation or mineral digestion.
