Targeted disruption of soluble epoxide hydrolase reveals a role in blood pressure regulation

Renal microsomal cytochrome P-450 monooxygenase-dependent metabolism of arachidonic acid generates a series of regioisomeric epoxyeicosatrienoic acids that can be further metabolized by soluble epoxide hydrolase to the corresponding dihydroxyeicosatrienoic acids. Evidence exists that these metabolites affect renal function and, in particular, blood pressure regulation. To examine this possibility, blood pressure and renal arachidonic acid metabolism were examined in mice with a targeted disruption of the soluble epoxide hydrolase gene. Systolic blood pressure of male soluble epoxide hydrolase-null mice was lower compared with wild-type mice in both the absence and presence of dietary salt loading. Both female soluble epoxide hydrolase-null and wild-type female mice also had significantly lower systolic blood pressure than male wild-type mice. Renal formation of epoxyeicosatrienoic and dihydroxyeicosatrienoic acids was markedly lower for soluble epoxide hydrolase-null versus wild-type mice of both sexes. Although disruption of soluble epoxide hydrolase in female mice had minimal effects on blood pressure, deletion of this gene feminized male mice by lowering systolic blood pressure and altering arachidonic acid metabolism. These data provide the first direct evidence for a role for soluble epoxide hydrolase in blood pressure regulation and identify this enzyme as a novel and attractive target for therapeutic intervention in hypertension.     

Targeted disruption of the mouse gene encoding the V-ATPase accessory subunit Ac45

Acidification of organelles of the eukaryotic vacuolar system is important for multiple intracellular processes including receptor-mediated endocytosis, proteolytic activity in lysosomes, and prohormone sorting and processing in secretory granules. Responsible for the generation of a proton gradient across a membrane is vacuolar H(+)-ATPase (V-ATPase). How the activity of this multisubunit enzyme is regulated remains to be established. Accessory subunits of the V-ATPase may be involved in the organelle-specific regulation, one candidate being the chromaffin granular V-ATPase-associated protein Ac45. To assess the function of Ac45, we disrupted its gene by gene targeting in male mouse embryonic stem cells. We have successfully generated Ac45 null mutant (-IY) embryonic stem cells and injected them into C57BL/6 recipient blastocysts. The blastocysts were replaced into pseudopregnant foster mothers, giving rise to 16 littermates. One of these appeared to be a low-chimeric female mouse that died 6 weeks after birth. No signs of late abortion were detected in the foster mothers. The results suggest that the injected Ac45 null mutant embryonic stem cells affect the normal development of the blastocyst and are in line with knockout studies on other V-ATPase subunits that point to an essential role for the V-ATPase in early embryonic development.     

Phenotypic characterization of the murine Nkx2.6 homeobox gene by gene targeting

The NK-2 homeobox genes have been shown to play critical roles in the development of specific organs and tissues. Nkx2.6 is a member of the NK-2 homeobox gene family and is most closely related to the Drosophila tinman gene. Nkx2.6 is expressed in the caudal pharyngeal pouches, the caudal heart progenitors, the sinus venosus, and the outflow tract of the heart and in a short segment of the gut at early stages of embryogenesis. To investigate the function of Nkx2.6 in vivo, we generated mice with null mutations of Nkx2.6 by the gene targeting technique. Homozygous Nkx2.6 mutant mice were viable and fertile. There were no obvious abnormalities in the caudal pharyngeal pouch derivatives (the thymus, parathyroid glands, and thyroid gland), heart, and gut. Expression of Nkx2.6 overlaps that of Nkx2.5 in the pharynx and heart and that of Nkx2.3 in the pharynx. Interestingly, in mutant embryos homozygous for Nkx2.6, Nkx2.5 expression extended to the lateral side of the pharynx, suggesting a compensatory function of Nkx2.5 in the mutant pharyngeal pouches.     

Targeted disruption of NBS1 reveals its roles in mouse development and DNA repair

Nijmegen breakage syndrome (NBS) is an autosomal recessive hereditary disease that shares some common defects with ataxia-telangiectasia. The gene product mutated in NBS, named NBS1, is a component of the Mre11 complex that is involved in DNA strand-break repair. To elucidate the physiological roles of NBS1, we disrupted the N-terminal exons of the NBS1 gene in mice. NBS1(m/m) mice are viable, growth retarded and hypersensitive to ionizing radiation (IR). NBS1(m/m) mice exhibit multiple lymphoid developmental defects, and rapidly develop thymic lymphoma. In addition, female NBS1(m/m) mice are sterile due to oogenesis failure. NBS1(m/m) cells are impaired in cellular responses to IR and defective in cellular proliferation. Most systematic and cellular defects identified in NBS1(m/m) mice recapitulate those in NBS patients, and are essentially identical to those observed in Atm(-/-) mice. In contrast to Atm(-/-) mice, spermatogenesis is normal in NBS1(m/m) mice, indicating that distinct roles of ATM have differential requirement for NBS1 activity. Thus, NBS1 and ATM have overlapping and distinct functions in animal development and DNA repair.     

Targeted gene disruption reveals the role of cysteinyl leukotriene 1 receptor in the enhanced vascular permeability of mice undergoing acute inflammatory responses

The cysteinyl leukotrienes (cysLTs), leukotriene (LT) C(4), LTD(4), and LTE(4), are proinflammatory lipid mediators generated in the mouse by hematopoietic cells such as macrophages and mast cells. There are two mouse receptors for the cysLTs, CysLT(1) receptor (CysLT(1)R) and CysLT(2)R, which are 38% homologous and are located on mouse chromosomes X and 14, respectively. To clarify the different roles of the CysLT(1)R and CysLT(2)R in inflammatory responses in vivo, we generated CysLT(1)R-deficient mice by targeted gene disruption. These mice developed normally and were fertile. In an intracellular calcium mobilization assay with fura-2 acetoxymethyl ester, peritoneal macrophages from wild-type littermates, which express both CysLT(1)R and CysLT(2)R, responded substantially to 1 x 10(-6) m LTD(4) and slightly to 1 x 10(-6) m LTC(4), whereas the macrophages from CysLT(1)R-deficient mice did not respond to either LTD(4) or LTC(4). Plasma protein extravasation, but not neutrophil infiltration, was significantly reduced in CysLT(1)R-deficient mice subjected to zymosan A-induced peritoneal inflammation. Plasma protein extravasation was also significantly diminished in CysLT(1)R-deficient mice undergoing IgE-mediated passive cutaneous anaphylaxis as compared with the wild-type mice. Thus, the cysLTs generated in vivo by either monocytes/macrophages or mast cells utilize CysLT(1)R for the response of the microvasculature in acute inflammation.     

Targeted gene disruption reveals the role of the cysteinyl leukotriene 2 receptor in increased vascular permeability and in bleomycin-induced pulmonary fibrosis in mice

The cysteinyl leukotrienes (cys-LTs) mediate both acute and chronic inflammatory responses in mice, as demonstrated by the attenuation of the IgE/antigen-mediated increase in microvascular permeability and of bleomycin-induced pulmonary fibrosis, respectively, in a strain with targeted disruption of leukotriene C(4) synthase to prevent cys-LT synthesis. Our earlier finding that the acute, but not the chronic, injury was attenuated in a strain with targeted disruption of the cysteinyl leukotriene 1 (CysLT(1)) receptor suggested that the chronic injury might be mediated through the CysLT(2) receptor. Thus, we generated CysLT(2) receptor-deficient mice by targeted gene disruption. These mice developed normally and were fertile. The increased vascular permeability associated with IgE-dependent passive cutaneous anaphylaxis was significantly reduced in CysLT(2) receptor-null mice as compared with wild-type mice, whereas plasma protein extravasation in response to zymosan A-induced peritoneal inflammation was not altered. Alveolar septal thickening after intratracheal injection of bleomycin, characterized by interstitial infiltration with macrophages and fibroblasts and the accumulation of collagen fibers, was significantly reduced in CysLT(2) receptor-null mice as compared with the wild-type mice. The amounts of cys-LTs in bronchoalveolar lavage fluid after bleomycin injection were similar in the CysLT(2) receptor-null mice and the wild-type mice. Thus, in response to a particular pathobiologic event the CysLT(2) receptor can mediate an increase in vascular permeability in some tissues or promote chronic pulmonary inflammation with fibrosis.     

Chick homeobox gene cbx and its role in retinal development

Homeobox genes play important roles in animal development. We isolated a chick homeobox gene, cbx, and studied its function during embryonic development. The deduced Cbx protein contained 376 amino acid residues. Its homeodomain was related (with 65-71% sequence identity) to that of human Crx, human Cart-1, and chick Alx-4. On searching the human genome sequence, a human homologue was found, which had 78% overall sequence identity and a 100% identical homeodomain. In the developing chick retina, cbx was expressed in a small fraction of post-mitotic cells residing at anatomical locations typical of bipolar cells. These cells were Goalpha(+) and protein kinase C(-), suggesting that they were probably cone bipolar cells. cbx mRNA was also detected outside the retina, particularly in the tectum and Rathke's pouch. Replication-competent retrovirus was used to drive misexpression of cbx and of an Engrailed repression construct. Engrailed-mediated repression of Cbx was embryonic lethal, while misexpression of cbx itself was tolerated. In the retina, misexpression of cbx resulted in fewer PKC(+) bipolar cells. Our data suggest that cbx is essential for embryonic survival and may participate in the development of bipolar, probably cone bipolar, cells in the retina.     

Targeted disruption of the methionine synthase gene in mice

Alterations in homocysteine, methionine, folate, and/or B12 homeostasis have been associated with neural tube defects, cardiovascular disease, and cancer. Methionine synthase, one of only two mammalian enzymes known to require vitamin B12 as a cofactor, lies at the intersection of these metabolic pathways. This enzyme catalyzes the transfer of a methyl group from 5-methyl-tetrahydrofolate to homocysteine, generating tetrahydrofolate and methionine. Human patients with methionine synthase deficiency exhibit homocysteinemia, homocysteinuria, and hypomethioninemia. They suffer from megaloblastic anemia with or without some degree of neural dysfunction and mental retardation. To better study the pathophysiology of methionine synthase deficiency, we utilized gene-targeting technology to inactivate the methionine synthase gene in mice. On average, heterozygous knockout mice from an outbred background have slightly elevated plasma homocysteine and methionine compared to wild-type mice but seem to be otherwise indistinguishable. Homozygous knockout embryos survive through implantation but die soon thereafter. Nutritional supplementation during pregnancy was unable to rescue embryos that were completely deficient in methionine synthase. Whether any human patients with methionine synthase deficiency have a complete absence of enzyme activity is unclear. These results demonstrate the importance of this enzyme for early development in mice and suggest either that methionine synthase-deficient patients have residual methionine synthase activity or that humans have a compensatory mechanism that is absent in mice.     

Targeted gene disruption in the development of mouse models to elucidate the role of receptor guanylyl cyclase signaling pathways in physiological function

The physiological role of receptor guanylyl cyclases (GCs), which transduce a signal via the generation of intracellular cyclic GMP, has been somewhat speculative since there are few specific inhibitors that discriminate among various receptor isoforms. Although the natriuretic peptide receptors have been thought to regulate cardiovascular and renal function, the exact contribution of the receptor subtypes has not been clarified. The normal role of the heat-stable enterotoxin receptor guanylyl cyclase remains undefined, and several orphan members of the family await the identification of ligands as well as function. Targeted gene disruption, familiarly known as gene knockout, has emerged during the past decade as a powerful technique for probing the function of gene products, and has been used to develop animal models of inherited human diseases. We are just beginning to apply gene targeting technology to the guanylyl cyclase receptor family. Reviewed here is the information gained to date from the targeted disruption of several members of the guanylyl cyclase receptor family, their ligands, or effector molecules.