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Biomedical subjects

O Smithies

Publications and source records attributed to O Smithies.

At least 55 records · Page 3Linked to original sources
PubMed1998

Reduced growth, abnormal kidney structure, and type 2 (AT2) angiotensin receptor-mediated blood pressure regulation in mice lacking both AT1A and AT1B receptors for angiotensin II.

The classically recognized functions of the renin-angiotensin system are mediated by type 1 (AT1) angiotensin receptors. Whereas man possesses a single AT1 receptor, there are two AT1 receptor isoforms in rodents (AT1A and AT1B) that are products of separate genes (Agtr1a and Agtr1b). We have generated mice lacking AT1B (Agtr1b -/-) and both AT1A and AT1B receptors (Agtr1a -/-Agtr1b -/-). Agtr1b -/- mice are healthy, without an abnormal phenotype. In contrast, Agtr1a -/-Agtr1b -/- mice have diminished growth, vascular thickening within the kidney, and atrophy of the inner renal medulla. This phenotype is virtually identical to that seen in angiotensinogen-deficient (Agt-/-) and angiotensin-converting enzyme-deficient (Ace -/-) mice that are unable to synthesize angiotensin II. Agtr1a -/-Agtr1b -/- mice have no systemic pressor response to infusions of angiotensin II, but they respond normally to another vasoconstrictor, epinephrine. Blood pressure is reduced substantially in the Agtr1a -/- Agtr1b -/- mice and following administration of an angiotensin converting enzyme inhibitor, their blood pressure increases paradoxically. We suggest that this is a result of interruption of AT2-receptor signaling. In summary, our studies suggest that both AT1 receptors promote somatic growth and maintenance of normal kidney structure. The absence of either of the AT1 receptor isoforms alone can be compensated in varying degrees by the other isoform. These studies reaffirm and extend the importance of AT1 receptors to mediate physiological functions of the renin-angiotensin system.

Adrenal Glands↗
PubMed1998

Generation and reproductive phenotypes of mice lacking estrogen receptor beta.

Estrogens influence the differentiation and maintenance of reproductive tissues and affect lipid metabolism and bone remodeling. Two estrogen receptors (ERs) have been identified to date, ERalpha and ERbeta. We previously generated and studied knockout mice lacking estrogen receptor alpha and reported severe reproductive and behavioral phenotypes including complete infertility of both male and female mice and absence of breast tissue development. Here we describe the generation of mice lacking estrogen receptor beta (ERbeta -/-) by insertion of a neomycin resistance gene into exon 3 of the coding gene by using homologous recombination in embryonic stem cells. Mice lacking this receptor develop normally and are indistinguishable grossly and histologically as young adults from their littermates. RNA analysis and immunocytochemistry show that tissues from ERbeta -/- mice lack normal ERbeta RNA and protein. Breeding experiments with young, sexually mature females show that they are fertile and exhibit normal sexual behavior, but have fewer and smaller litters than wild-type mice. Superovulation experiments indicate that this reduction in fertility is the result of reduced ovarian efficiency. The mutant females have normal breast development and lactate normally. Young, sexually mature male mice show no overt abnormalities and reproduce normally. Older mutant males display signs of prostate and bladder hyperplasia. Our results indicate that ERbeta is essential for normal ovulation efficiency but is not essential for female or male sexual differentiation, fertility, or lactation. Future experiments are required to determine the role of ERbeta in bone and cardiovascular homeostasis.

Animals↗
PubMed1998

Coagulation defects and altered hemodynamic responses in mice lacking receptors for thromboxane A2.

Thromboxane A2 (TXA2) is a labile metabolite of arachidonic acid that has potent biological effects. Its actions are mediated by G protein-coupled thromboxane-prostanoid (TP) receptors. TP receptors have been implicated in the pathogenesis of cardiovascular diseases. To investigate the physiological functions of TP receptors, we generated TP receptor-deficient mice by gene targeting. Tp-/- animals reproduce and survive in expected numbers, and their major organ systems are normal. Thromboxane agonist binding cannot be detected in tissues from Tp-/- mice. Bleeding times are prolonged in Tp-/- mice and their platelets do not aggregate after exposure to TXA2 agonists. Aggregation responses after collagen stimulation are also delayed, although ADP-stimulated aggregation is normal. Infusion of the TP receptor agonist U-46619 causes transient increases in blood pressure followed by cardiovascular collapse in wild-type mice, but U-46619 caused no hemodynamic effect in Tp-/- mice. Tp-/- mice are also resistant to arachidonic acid-induced shock, although arachidonic acid signifi-cantly reduced blood pressure in Tp-/- mice. In summary, Tp-/- mice have a mild bleeding disorder and altered vascular responses to TXA2 and arachidonic acid. Our studies suggest that most of the recognized functions of TXA2 are mediated by the single known Tp gene locus.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1998

Role of mouse cryptochrome blue-light photoreceptor in circadian photoresponses.

Cryptochromes are photoactive pigments in the eye that have been proposed to function as circadian photopigments. Mice lacking the cryptochrome 2 blue-light photoreceptor gene (mCry2) were tested for circadian clock-related functions. The mutant mice had a lower sensitivity to acute light induction of mPer1 in the suprachiasmatic nucleus (SCN) but exhibited normal circadian oscillations of mPer1 and mCry1 messenger RNA in the SCN. Behaviorally, the mutants had an intrinsic circadian period about 1 hour longer than normal and exhibited high-amplitude phase shifts in response to light pulses administered at circadian time 17. These data are consistent with the hypothesis that CRY2 protein modulates circadian responses in mice and suggest that cryptochromes have a role in circadian photoreception in mammals.

Animals↗
PubMed1998

A common human beta globin splicing mutation modeled in mice.

The betaIVS-2-654 C-->T mutation accounts for approximately 20% of beta thalassemia mutations in southern China; it causes aberrant RNA splicing and leads to beta0 thalassemia. To provide an animal model for testing therapies for correcting splicing defects, we have used the "plug and socket" method of gene targeting in murine embryonic stem cells to replace the two (cis) murine adult beta globin genes with a single copy of the human betaIVS-2-654 gene. No homozygous mice survive postnatally. Heterozygous mice carrying this mutant gene produce reduced amounts of the mouse beta globin chains and no human beta globin, and have a moderate form of beta thalassemia. The heterozygotes show the same aberrant splicing as their human counterparts and provide an animal model for testing therapies to correct splicing defects at either the RNA or DNA level.

Animals↗
PubMed1998

Natriuretic peptide receptor 1 expression influences blood pressures of mice in a dose-dependent manner.

Activation of the natriuretic peptide system lowers blood pressure and causes the excretion of salt. Atrial natriuretic peptide and B-type natriuretic peptide are the humoral mediators of this effect; they act primarily by binding to membrane-bound natriuretic peptide receptor A (NPRA) and stimulating its intrinsic guanylate cyclase activity. To study whether genetically determined differences in NPRA expression affect blood pressure we have generated mice with one, two, three, or four copies of the gene encoding NPRA (Npr1 in the mouse). Atrial natriuretic peptide-dependent guanylate cyclase activity ranged progressively from approximately one-half normal in one-copy animals to twice normal in four-copy animals (P < 0.001). On different diets (0.05%, 2%, and 8% NaCl), the blood pressures of F1 male mice having only one copy of Npr1 averaged 9.1 mmHg (1 mmHg = 133 Pa) above those of wild-type two-copy males (P < 0.001), whereas males with three copies of the gene had blood pressures averaging 5.2 mmHg below normal (P < 0.01). The blood pressures of the one-copy F1 animals were significantly higher (by 6.2 mmHg; P < 0.01) on the high-salt than on the low-salt diet. The blood pressures of four-copy F3 males were significantly lower (by 7 mmHg; P < 0.05) on the high-salt than on the low-salt diet. These results demonstrate that below normal Npr1 expression leads to a salt-sensitive increase in blood pressure, whereas above normal Npr1 expression lowers blood pressures and protects against high dietary salt.

Animals↗
PubMed1998

Angiotensin-converting enzyme and male fertility.

The angiotensin-converting enzyme (ACE; EC 3.4.15.1) gene (Ace) encodes both a somatic isozyme found in blood and several other tissues, including the epididymis, and a testis-specific isozyme (testis ACE) found only in developing spermatids and mature sperm. We recently used gene targeting to disrupt the gene coding for both ACE isozymes in mice and reported that male homozygous mutants mate normally but have reduced fertility; the mutant females are fertile. Here we explore the male fertility defect. We demonstrate that ACE is important for achieving in vivo fertilization and that sperm from mice lacking both ACE isozymes show defects in transport within the oviducts and in binding to zonae pellucidae. Males generated by gene targeting that lack somatic ACE but retain testis ACE are normally fertile, establishing that somatic ACE in males is not essential for their fertility. Furthermore, male and female mice lacking angiotensinogen have normal fertility, indicating that angiotensin I is not a necessary substrate for testis ACE. Males heterozygous for the mutation inactivating both ACE isozymes sire wild-type and heterozygous offspring at an indistinguishable frequency, indicating no selection against sperm carrying the mutation.

Animals↗
PubMed1998

Direct evidence for the importance of endothelium-derived nitric oxide in vascular remodeling.

The vascular endothelium mediates the ability of blood vessels to alter their architecture in response to hemodynamic changes; however, the specific endothelial-derived factors that are responsible for vascular remodeling are poorly understood. Here we show that endothelial-derived nitric oxide (NO) is a major endothelial-derived mediator controlling vascular remodeling. In response to external carotid artery ligation, mice with targeted disruption of the endothelial nitric oxide synthase gene (eNOS) did not remodel their ipsilateral common carotid arteries whereas wild-type mice did. Rather, the eNOS mutant mice displayed a paradoxical increase in wall thickness accompanied by a hyperplastic response of the arterial wall. These findings demonstrate a critical role for endogenous NO as a negative regulator of vascular smooth muscle proliferation in response to a remodeling stimulus. Furthermore, our data suggests that a primary defect in the NOS/NO pathway can promote abnormal remodeling and may facilitate pathological changes in vessel wall morphology associated with complex diseases such as hypertension and atherosclerosis.

Adaptation, Physiological↗
PubMed1998

Rapid genotyping of mice with hemoglobinopathies and globin transgenes.

The hematology of the laboratory mouse has been well characterized. Normal genetic differences at the alpha- and beta-globin gene loci serve as useful markers for a wide variety of types of experimental studies. There are a number of naturally occurring or induced mutations that disrupt globin expression and produce thalassemic phenotypes. In addition, much has been learned of the workings of the globin locus control region from studies of transgenic mice, including those with mutations induced by targeted site-specific modifications. After a new mutation or transgene has been created, it must be maintained in living mice, and the genotypes of the offspring must be ascertained. While it is possible to determine genotypes by DNA analyses, such assays are time consuming and relatively expensive. An osmotic challenge test--originally developed for the genotyping of large-deletion alpha-thalassemia mutations in mice--has proven useful in detecting both severe and milder alpha- and beta-thalassemias, as well as some transgenic genotypes in mice carrying human globin genes. Reliable genotyping can, in some cases, be completed within a few minutes with minimal expense. Quantification of red cell fragility for a variety of thalassemic and transgenic mice is described here, along with a simplified test suitable for rapid, routine genotyping. The osmotic challenge test is perfectly reliable for distinguishing genotypes that cause significantly decreased release of hemoglobin from the red cells, but it is also useful for some of the conditions in which overall erythrocyte osmotic fragility is essentially normal.

Animals↗
PubMed1998

Absence of macrophage inflammatory protein-1alpha prevents the development of blinding herpes stromal keratitis.

Prior studies in our laboratory have suggested that the CC chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) may be an important mediator in the blinding ocular inflammation which develops following herpes simplex virus type 1 (HSV-1) infection of the murine cornea. To directly test this hypothesis, MIP-1alpha-deficient (-/-) mice and their wild-type (+/+) counterparts were infected topically on the scarified cornea with 2.5 x 10(5) PFU of HSV-1 strain RE and subsequently graded for corneal opacity. Four weeks postinfection (p.i.), the mean corneal opacity score of -/- mice was 1.1 +/- 0.3 while that of the +/+ mice was 3.7 +/- 0.5. No detectable infiltrating CD4+ T cells were seen histologically at 14 or 21 days p.i. in -/- animals, whereas the mean CD4+ T-cell count per field (36 fields counted) in +/+ hosts was 26 +/- 2 (P < 0.001). In addition, neutrophil counts in the -/- mouse corneas were reduced by >80% in comparison to the wild-type controls. At 2 weeks p.i., no interleukin-2 or gamma interferon could be detected in six of seven -/- mice, whereas both T-cell cytokines were readily demonstrable in +/+ mouse corneas. Also, MIP-2 and monocyte chemoattractant protein-1 protein levels were significantly lower in MIP-1alpha -/- mouse corneas than in +/+ host corneas, suggesting that MIP-1alpha directly, or more likely indirectly, influences the expression of other chemokines. Interestingly, despite the paucity of infiltrating cells, HSV-1 clearance from the eyes of -/- mice was not significantly different from that observed in +/+ hosts. We conclude that MIP-1alpha is not needed to control virus growth in the cornea but is essential for the development of severe stromal keratitis.

Animals↗
PubMed1998

Renal growth and development in mice lacking AT1A receptors for angiotensin II.

To examine the role of the type 1A (AT1A) angiotensin receptor in renal growth and development, we analyzed F2 progeny from a series of crosses between F1 mice that were heterozygous for a targeted disruption of the AT1A receptor gene [Agtr1A-(+/-)]. Among 21-day-old weanling F2 mice, we found that 194 (32%) were homozygous for the wild-type allele Agtr1A-(+/+), 299 (49%) were Agtr1A-(+/-), and 119 (19%) were Agtr1A-(-/-). This differed significantly from the proportions predicted by Mendelian genetics (P = 0.01), suggesting that the complete absence of AT1A receptors is associated with a mild survival disadvantage. Agtr1A-(-/-) mice grew normally, and we found no significant differences in body weight or heart and kidney weights in Agtr1A-(+/+) and Agtr1A-(-/-) mice examined at 21, 60, and 100 days. Protein and DNA content of kidneys and hearts were also similar in weanling or adult Agtr1A-(+/+) and Agtr1A-(-/-) mice. By light microscopy with immunohistochemistry, kidneys from Agtr1A-(-/-) were essentially normal, with two exceptions: 1) there was marked hypertrophy of the juxtaglomerular apparatus (JGA) and proximal expansion of renin-producing cells along the afferent arterioles, and 2) some glomeruli showed evidence of mesangial expansion. We did not find the severe renal vascular lesions or papillary atrophy that have been observed in angiotensinogen- or angiotensin converting enzyme-deficient animals. We conclude that the AT1A receptor is not essential for the normal organogenesis of the kidney; however, its absence is associated with mild mesangial expansion and JGA hypertrophy.

Angiotensin II↗
PubMed1997

Hypertension, cardiac hypertrophy, and sudden death in mice lacking natriuretic peptide receptor A.

Natriuretic peptides, produced in the heart, bind to the natriuretic peptide receptor A (NPRA) and cause vasodilation and natriuresis important in the regulation of blood pressure. We here report that mice lacking a functional Npr1 gene coding for NPRA have elevated blood pressures and hearts exhibiting marked hypertrophy with interstitial fibrosis resembling that seen in human hypertensive heart disease. Echocardiographic evaluation of the mice demonstrated a compensated state of systemic hypertension in which cardiac hypertrophy and dilatation are evident but with no reduction in ventricular performance. Nevertheless, sudden death, with morphologic evidence indicative in some animals of congestive heart failure and in others of aortic dissection, occurred in all 15 male mice lacking Npr1 before 6 months of age, and in one of 16 females in our study. Thus complete absence of NPRA causes hypertension in mice and leads to cardiac hypertrophy and, particularly in males, lethal vascular events similar to those seen in untreated human hypertensive patients.

Animals↗
PubMed1997

A coagulation factor IX-deficient mouse model for human hemophilia B.

Coagulation factor IX deficiency causes hemophilia B in humans. We have used gene targeting to develop a coagulation factor IX-deficient (factor IX-knockout) mouse strain. Mouse embryonic stem (ES) cells were targeted by a socket-containing vector that replaces the promoter through exon 3 of the factor IX gene by neoDeltaHPRT, which is a functional neo gene plus a partially deleted hypoxanthine phosphoribosyl transferase minigene. Chimeric mice generated using these socket-containing ES cells transmitted the targeted factor IX gene to their female offspring. Male offspring from these females were characterized and shown to exhibit a phenotype similar to hemophilia B. This factor IX-deficient mouse strain will be useful for studying gene therapy methods and structure-function relationships of recombinant factor IX proteins in vivo.

Animals↗
PubMed1997

Severe reduction in leukocyte adhesion and monocyte extravasation in mice deficient in CC chemokine receptor 2.

CC chemokine receptor 2 (CCR2) is a prominent receptor for the monocyte chemoattractant protein (MCP) group of CC chemokines. Mice generated by gene targeting to lack CCR2 exhibit normal leukocyte rolling but have a pronounced defect in MCP-1-induced leukocyte firm adhesion to microvascular endothelium and reduced leukocyte extravasation. Constitutive macrophage trafficking into the peritoneal cavity was not significantly different between CCR2-deficient and wild-type mice. However, after intraperitoneal thioglycollate injection, the number of peritoneal macrophages in CCR2-deficient mice did not rise above basal levels, whereas in wild-type mice the number of macrophages at 36 h was approximately 3.5 times the basal level. The CCR2-deficient mice showed enhanced early accumulation and delayed clearance of neutrophils and eosinophils. However, by 5 days neutrophils and eosinophils in both CCR2-deficient and wild-type mice had returned to near basal levels, indicating that resolution of this inflammatory response can occur in the absence of macrophage influx and CCR2-mediated activation of the resident peritoneal macrophages. After intravenous injection with yeast beta-glucan, wild-type mice formed numerous large, well-defined granulomas throughout the liver parenchyma, whereas CCR2-deficient mice had much fewer and smaller granulomas. These results demonstrate that CCR2 is a major regulator of induced macrophage trafficking in vivo.

Animals↗
PubMed1997

Inactivation of the mouse HPRT locus by a 203-bp retroposon insertion and a 55-kb gene-targeted deletion: establishment of new HPRT-deficient mouse embryonic stem cell lines.

To obtain useful hypoxanthine phosphoribosyl-transferase (HPRT)-deficient mouse ES cell lines, two different methods were employed: (i) selection of spontaneous 6-TG-resistant mutants and (ii) gene targeting of the HPRT locus. The first approach resulted in the establishment of E14.1TG3B1, a spontaneous HPRT-deficient cell line with an insertional mutation of 203 bp in the third exon of the HPRT gene. The insert is highly homologous to the B2 mouse repetitive element and has all the expected retroposon characteristics, thus providing an example of gene inactivation by retroposon insertion. This clone exhibited stable 6-TG resistance and high germ-line transmission frequency. Thus E14.1TG3B1 is a useful ES cell line for modifying the mouse genome using the HPRT gene as a selection marker and for transmission at a high frequency into the mouse germ line. The second approach resulted in a 55-kb deletion of the mouse HPRT locus, demonstrating the feasibility of replacement-targeting vectors to generate large genomic DNA deletions.

Animals↗
PubMed1997

Complementation of reduced survival, hypotension, and renal abnormalities in angiotensinogen-deficient mice by the human renin and human angiotensinogen genes.

The aim of this study was to determine whether elements of the human renin-angiotensin system (RAS) could functionally replace elements of the mouse RAS by complementing the reduced survival and renal abnormalities observed in mice carrying a gene-targeted deletion of the mouse angiotensinogen gene (mAgt). Double transgenic mice containing the human renin (HREN) and human angiotensinogen (HAGT) genes were bred to mice heterozygous for the mAgt deletion and the compound heterozygotes were identified and intercrossed. The resulting progeny (n = 139) were genotyped at each locus and the population was stratified into two groups: the first containing both human transgenes (RA+) and the second containing zero or one, but not both human transgenes (RA-). Despite appropriate Mendelian ratios of RA- mice that were wildtype (+/+), heterozygous (+/-), and homozygous (-/-) for the deletion of mAgt at birth, there was reduced survival of RA- mAgt-/- mice to adulthood (P < 0.001 by chi2). In contrast, we observed appropriate Mendelian ratios of RA+ mAgt+/+, RA+ mAgt+/-, and RA+ mAgt-/- mice at birth and in adults (P > 0.05 by chi2). These results demonstrate that the presence of both human transgenes rescues the postnatal lethality in mAgt-/- mice. The renal histopathology exhibited by RA- mAgt-/- mice, including thickened arterial walls, severe fibrosis, lymphocytic infiltration, and atrophied parenchyma, was also rescued in the RA+ mAgt-/- mice. Direct arterial blood pressure recordings in conscious freely moving mice revealed that BP (in mmHg) varied proportionally to mAgt gene copy number in RA+ mice (approximately 20 mmHg per mAgt gene copy, P < 0.001). BP in RA+ mAgt-/- mice (132+/-3, n = 14) was intermediate between wild-type (RA- mAgt+/+, 105+/-2, n = 9) and RA+ mAgt+/+ (174+/-3, n = 10) mice. These studies establish that the human renin and angiotensinogen genes can functionally replace the mouse angiotensinogen gene, and provides proof in principle that we can examine the regulation of elements of the human RAS and test the significance of human RAS gene variants by a combined transgenic and gene targeting approach.

Angiotensinogen↗