SEARCH · PubMed Health

Results for “knockout”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,117 records · Page 62Linked to original sources

PubMed2002

Appearance of atypical 3 alpha,6 beta,7 beta,12 alpha-tetrahydroxy-5 beta-cholan-24-oic acid in spgp knockout mice.

Bile formation and its canalicular secretion are essential functions of the mammalian liver. The sister-of-p-glycoprotein (spgp) gene was shown to encode the canalicular bile salt export protein, and mutations in spgp gene were identified as the cause of progressive familial intrahepatic cholestasis type 2. However, target inactivation of spgp gene in mice results in nonprogressive but persistent cholestasis and causes the secretion of unexpectedly large amounts of unknown tetrahydroxylated bile acid in the bile. The present study confirms the identity of this tetrahydroxylated bile acid as 3 alpha,6 beta,7 beta,12 alpha-tetrahydroxy-5 beta-cholan-24-oic acid. The data further show that in serum, liver, and urine of the spgp knockout mice, there is a significant increase in the concentration of total bile salts containing a large amount of tetrahydroxy-5 beta-cholan-24-oic acid. The increase in total bile acids was associated with up-regulation of the mRNA of cholesterol 7 alpha-hydroxylase in male mice only. It is suggested that the lower severity of the cholestasis in the spgp knockout mice may be due to the synthesis of 3 alpha,6 beta,7 beta,12 alpha-tetrahydroxy-5 beta-cholan-24-oic acid, which neutralizes in part the toxic effect of bile acids accumulated in the liver.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2004

Catecholamine synthesizing enzymes and their modulation by immobilization stress in knockout mice.

The c-fos knockout mice (c-fos KO) and corticotropin-releasing hormone knockout mice (CRH KO) can serve as interesting models for studying mechanisms involved in response of the organism to stress, focused mainly on the hypothalamic-pituitary-adrenal (HPA) axis and sympathoadrenal system (SAS). The present study focused on the investigation of changes in gene expression of catecholamine biosynthesizing enzymes tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT) in adrenal medulla of c-fos KO and CRH KO mice stressed by immobilization. Levels of TH, DBH, and PNMT mRNA were determined by reverse transcription-polymerase chain reaction (RT-PCR). Single immobilization for 2 h significantly increased adrenomedullary TH, DBH, and PNMT mRNA levels in both c-fos KO and wild-type (WT) mice compared to unstressed controls. In CRH KO mice, PNMT gene expression was not increased to the same extent after single, but especially after repeated immobilization as in WT mice, in contrast to TH and DBH mRNA levels. Thus, our data indicate that CRH deficiency can influence the PNMT mRNA level in adrenal medulla during stress, confirming the idea that the HPA axis plays the crucial role in PNMT gene regulation in mice. On the other hand, c-Fos protein probably does not play a crucial role in TH, DBH, and PNMT gene expression in adrenal medulla under stress conditions.

Adrenal Medulla↗

PubMed2004

Establishment of native insulin-negative NOD mice and the methodology to distinguish specific insulin knockout genotypes and a B:16 alanine preproinsulin transgene.

We hypothesize that NOD mice without native insulin, but with an altered insulin B:9-23 sequence, will be completely protected from diabetes/insulitis if insulin B:9-23 is an essential T cell epitope. To investigate this hypothesis, we have established initial insulin 1- and 2-negative NOD mice with a transgene directing production of preproinsulin with alanine at position B:16 rather than the native tyrosine of both insulin 1 and insulin 2. Sets of primers for PCR-based assays have been created and validated. They are able to distinguish the presence or absence of the insulin gene knockouts and of both native insulin 1 and insulin 2 (and thus distinguish heterozygous versus homozygous knockouts), as well as the presence of the altered insulin transgene, B:16 alanine preproinsulin. Four B:16 alanine transgenic founders were produced directly in NOD mice and, by intercrossing, initial live native insulin-negative B:16 alanine transgenic mice have been generated.

Alanine↗

PubMed2006

Attenuation of experimental aortic aneurysm formation in P-selectin knockout mice.

The aim of this study was to determine the role of P-selectin, an adhesion molecule found on the surface of activated platelets and endothelial cells during experimental aortic aneurysm formation. Infrarenal abdominal aortas of C57 black wild-type (WT) mice and P-selectin knockout (PKO) mice were measured in situ and then perfused with porcine pancreatic elastase (0.332 U/mL). Whole blood was drawn from the tail artery on day 2 pre-perfusion to determine total and differential white blood cell (WBC) counts. On day 14 postperfusion, aortic diameters (AD) of WT mice (N = 19) and PKO mice (N = 9) were measured. An aortic aneurysm was defined as a 100% or greater increase in AD from pre-perfusion measurement. Immunohistochemistry, including H&E, trichrome and von Gieson staining, was performed on harvested aortic tissue. Statistical analysis was performed by t-test and Fisher's exact test. There were no significant differences in peripheral leukocyte counts at baseline between the two groups. WT mice had significantly larger AD compared to PKO mice at day 14 postperfusion (116 % vs. 38 %, P < 0.001). Aortic aneurysm penetrance was 52% in WT mice, while 0% (P = 0.01) of PKO mice formed aneurysms. On histologic examination, WT mouse aortas were associated with a significant inflammatory response and degradation of elastin and collagen fibers, while PKO mouse aortas lacked signs of inflammation or vessel wall injury. P-selectin deficiency attenuates aneurysm formation in the elastase aortic perfusion model. This was associated with a blunting of the inflammatory response and preserved vessel wall intergrity following elastase perfusion in the P-selectin knockout mice. Further investigation to elucidate the independent contributions of endothelial cell and platelet P-selectin in experimental aortic aneurysm formation is required.

Animals↗

PubMed2004

Ventilatory responses to hypercapnia and hypoxia in conscious cystic fibrosis knockout mice Cftr-/-.

This study was designed to examine the ventilatory performance and the lung histopathology of cystic fibrosis knockout mice (Cftr-/-) compared with heterozygous (Cftr+/-) or wild-type (Cftr+/+) littermates. Ventilation was recorded in conscious animals using whole-body plethysmography. Tidal volume (VT), respiratory frequency (f), and minute ventilation (VE) were measured during air breathing and in response to various levels of hypercapnia (2, 4, 6, or 8% CO2) or hypoxia (14, 12, 10, or 8% O2). The results for Cftr+/- and Cftr+/+ were pooled into one control group because they did not differ. In air and in response to hypercapnia, VE, VT, and f were similar in Cftr-/- mice and in controls. During graded hypoxia, VE was decreased in Cftr-/- mice at 10 and 8% O2 because of a lower f. Histology showed neither inflammation nor obstruction of airways in Cftr-/- mice. Morphometric analysis showed alveolar dilation as a result of either distension or impaired development. In conclusion, cystic fibrosis knockout mice have normal baseline breathing and ventilatory response to hypercapnia but a decreased ventilatory response to severe hypoxia. This latter result associated with the morphometric analysis suggests that Cftr-/- mice may exhibit immaturity of the respiratory system.

Animals↗

PubMed2003

Prostate development and carcinogenesis in prolactin receptor knockout mice.

Hyperprolactinemia results in prostatic hypertrophy and hyperplasia, but it is not known whether prolactin plays an essential role in these processes in the prostate. To address this question, we investigated prostate development, gene expression, and simian virus 40 (SV40)T-induced prostate carcinogenesis in prolactin receptor knockout mice. These animals showed a small increase in dorsolateral and ventral prostate weight but no change in the weight of the anterior prostate. The dorsal but not ventral or lateral lobes showed a 12% loss of epithelial cells; all other morphological parameters were normal. The area of SV40T-induced prostate intraepithelial neoplasia was reduced by 28% in the ventral lobe but not the dorsal lobe, and no tumors were seen in 20 prolactin receptor knockout animals, compared with 1 of 11 detected in wild-type and 4 of 21 found in heterozygous animals. Oligonucleotide microarrays were used to identify essential transcriptional roles of prolactin and revealed a small set of genes with decreased expression involved in sperm/oocyte interaction and copulatory plug formation. Infertility or reduced fertility was apparent in these animals. These findings establish essential though subtle roles for prolactin in the regulation of prostate morphology, gene expression, SV40T-induced neoplasia, and reproductive function.

Androgens↗

PubMed2003

Molecular characterization of postnatal development of testicular steroidogenesis in luteinizing hormone receptor knockout mice.

We recently demonstrated that the sexual development of LH receptor (LHR) knockout mice is normal until birth, but is totally arrested thereafter. To study further the functional defects of LHR knockout mice, the expression of selected Leydig cell-specific genes was studied in (-/-) and control (+/+) mice between birth and adulthood. Testis weights were similar at birth in both types of mice, but after about 3 wk, the (-/-) testes remained significantly lighter, weighing only 18% of (+/+) testes on d 70. Testicular testosterone (T) content on d 1 was also similar in (-/-) and (+/+) testes, but it was 97% reduced by d 70 in the former. Likewise, testicular T production in vitro was similar in neonatal (-/-) and (+/+) testes, but became undetectable in adult (-/-) testes. The mRNA expression of cytochrome P450 side-chain cleavage, 17 alpha-hydroxylase cytochrome P450, 17 beta-hydroxysteroid dehydrogenase type III, 3 beta-hydroxysteroid dehydrogenase I (3 beta HSDI), steroidogenic acute regulatory protein, and relaxin-like factor were similar in newborn (-/-) and (+/+) testes, but became gradually very low/undetectable in (-/-) mice. The only exception was the persistently high expression of 3 beta HSDI in peritubular Leydig precursor and mesenchymal cells of the (-/-) testes at all ages. Immunohistochemistry and Western hybridization studies confirmed the above findings. In conclusion, LH action is not essential for the differentiation and function of mouse fetal Leydig cells, but, with the exception of 3 beta HSDI, the expression of the key genes of endocrine function of adult Leydig cells is dependent on LH signaling.

3-Hydroxysteroid Dehydrogenases↗

PubMed2004

De novo testosterone production in luteinizing hormone receptor knockout mice after transplantation of leydig stem cells.

Mesenchymal stem cells or Leydig cell progenitors are rare and difficult to isolate from adult testes. The property of differential efflux of Hoechst 33342 dye by the multi-drug-like transporter enriches murine hematopoietic stem cells from bone marrow. Our work on testicular cell transplantation suggests that the "Hoechst dim" side population (SP) also contains Leydig stem cells or progenitors that proliferate and differentiate into mature functional Leydig cells. We harvested testicular cells from cryptorchid ROSA26 mice, stained them with Hoechst dye, and isolated the cell population that excludes the dye using flow cytometry. Mice with targeted deletion of the LH receptor (LHR) gene were used as the recipients of the transplanted cells. These mice are hypogonadal and infertile. Both testicular SP and non-SP cells were transplanted into the interstitium of the LHR knockout recipients' testes. Serial serum testosterone assays revealed a significant increase in the circulating testosterone levels and restoration of spermatogenesis in the LHR-knockout recipients transplanted with the SP cells compared with that of those transplanted with non-SP. A SP cell concentration- and time-dependent increase in circulating testosterone was observed. This demonstrates the successful transplantation of functional putative Leydig stem cells into a hypogonadal recipient. The increase in testosterone concentration indicates the de novo synthesis of androgen by the transplanted SP cells. This method offers a novel technique to isolate Leydig stem cells and to study Leydig cell development.

Animals↗

PubMed2004

Effect of Agouti-related protein in regulation of the hypothalamic-pituitary-thyroid axis in the melanocortin 4 receptor knockout mouse.

Agouti-related protein (AGRP) is thought to be one of the neuropeptides mediating the effects of leptin on appetite and satiety. The central administration of AGRP not only stimulates food intake, but also inhibits the hypothalamic-pituitary-thyroid axis (HPT) axis, closely replicating the central hypothyroid state induced by fasting. AGRP binds as an endogenous antagonist or inverse agonist of the central melanocortin receptors but has also been hypothesized to have melanocortin receptor-independent effects. Thus, we determined whether the central effects of AGRP on the HPT axis are altered in mice with selective deletion of the melanocortin 4 receptor (MC4-R). AGRP or artificial cerebrospinal fluid was administered daily into the lateral ventricle of adult, male MC4-R knockout and wild-type (WT) mice for 3 d. AGRP significantly increased the cumulative food intake and weight of white and brown adipose tissue, suppressed circulating levels of T(4) [control vs. AGRP in WT (microg/dl): 4.54 +/- 0.16 vs. 3.87 +/- 21], and inhibited proTRH mRNA content in the hypothalamic paraventricular nucleus of WT mice (control vs. AGRP in WT (density units +/- sem): 4.65 +/- 0.50 vs. 2.47 +/- 0.17). In contrast, no significant effects of AGRP were observed in any of these parameters in the MC4-R knockout mice. These data suggest that AGRP signaling to TRH hypophysiotropic neurons in the paraventricular nucleus is primarily mediated by the MC4-R and therefore, binding to the MC3-R or other putative AGRP receptors may have only a minor role.

Adipose Tissue↗

PubMed2006

S14: insights from knockout mice.

Spot 14 (S14) is a protein whose mRNA is rapidly up-regulated by lipogenic stimuli including thyroid hormone and a high-carbohydrate diet. Previous investigation into the role of S14 suggested that it is involved in de novo lipogenesis. Knockout of the gene in mice has given further support to this hypothesis. The lack of S14 in different tissues resulted in varying phenotypic effects. In the lactating mammary gland, levels of lipogenesis, specifically the production of medium chain fatty acids, were decreased, whereas hepatic lipogenesis was not decreased. In fact, hepatic lipogenesis was increased, and the increase may be due to compensation by a paralog of S14 called S14-R. S14-R is expressed in the liver but not the mammary gland. Importantly, S14 knockout mice did not have reduced levels of lipogenic enzymes, implying that it does not affect the transcriptional rate of those enzymes. Instead, S14 may act in the cytoplasm to affect lipogenesis.

Animals↗

PubMed2002

Immune deviation away from Th1 in interferon-gamma knockout mice does not enhance TSH receptor antibody production after naked DNA vaccination.

TSH receptor (TSHR) DNA vaccination induces high TSHR antibody levels in BALB/c mice housed in a conventional facility. However, under pathogen-free conditions, we observed a Th1 cellular response to TSHR antigen characterized by interferon-gamma (IFN gamma) production. In the present study we investigated the effect on TSHR DNA vaccination of diverting the cytokine milieu away from Th1 using 1) IFN gamma knockout BALB/c mice, and 2) wild-type mice covaccinated with DNA for the TSHR and for IFN gamma/receptor-Fc protein that prevents IFN gamma from binding to its receptor. Neither approach enhanced TSHR antibody levels, although splenocyte IFN gamma production in response to TSHR antigen was absent (IFN gamma knockouts) or reduced (IFN gamma receptor-Fc). Moreover, production of IL-2, another Th1 cytokine, but not Th2 cytokines, indicated that neither strategy overcame the Th1 bias of im DNA vaccination. Importantly, splenocyte production of IFN gamma and IL-2 provides a sensitive detection system for TSHR-specific T cells. Unexpectedly, higher TSHR antibody levels developed in rare mice. High titer animals had TSHR-specific responses of both Th2 and Th1 types, whereas low titer animals had Th1-restricted TSHR responses. The heterogeneity of responses induced by TSHR DNA vaccination in mice may provide insight into the titers and IgG subclasses of spontaneous autoantibodies in humans.

Animals↗

PubMed2005

Keratinocytes display normal proliferation, survival and differentiation in conditional beta4-integrin knockout mice.

The alpha6beta4 integrin is located at the basal surface of keratinocytes, in hemidesmosomal structures that mediate stable adhesion of epidermal cells to the underlying basement membrane component laminin-5. The absence of alpha6beta4 integrin causes junctional epidermolysis bullosa, a severe blistering disease of the skin leading to perinatal death, confirming its essential role in mediating strong keratinocyte adhesion. Several studies have suggested that alpha6beta4 integrin can also regulate signaling cascades that control cell proliferation, survival and migration through a mechanism independent of its adhesive function. We have generated a conditional knockout mouse strain, in which the gene encoding the beta4 integrin subunit (Itgb4) was inactivated only in small stretches of the skin. These mice were viable and permitted an accurate analysis of the consequences of the loss of beta4 on various biological processes by comparing beta4-positive and -negative parts of the skin in the same animal. Despite the complete loss of hemidesmosomes in regions lacking alpha6beta4 integrin, the distribution of a range of adhesion receptors and basement membrane proteins was unaltered. Moreover, loss of alpha6beta4 did not affect squamous differentiation, proliferation or survival, except for areas in which keratinocytes had detached from the basement membrane. These in vivo observations were confirmed in vitro by using immortalized keratinocytes - derived from beta4-subunit conditional knockout mice - from which the gene encoding beta4 had been deleted by Cre-mediated recombination. Consistent with the established role of alpha6beta4 in adhesion strengthening, its loss from cells was found to increase their motility. Our findings clearly demonstrate that, after birth, epidermal differentiation, proliferation and survival all proceed normally in the absence of alpha6beta4, provided that cell adhesion is not compromised.

Alleles↗

PubMed2000

Mammalian skeletogenesis and extracellular matrix: what can we learn from knockout mice?

Formation of the vertebrate skeleton and the proper functions of bony and cartilaginous elements are determined by extracellular, cell surface and intracellular molecules. Genetic and biochemical analyses of human heritable skeletal disorders as well as the generation of knockout mice provide useful tools to identify the key players of mammalian skeletogenesis. This review summarises our recent work with transgenic animals carrying ablated genes for cartilage extracellular matrix proteins. Some of these mice exhibit a lethal phenotype associated with severe skeletal defects (type II collagen-null, perlecan-null), whereas others show mild (type IX collagen-null) or no skeletal abnormalities (matrilin-1-null, fibromodulin-null, tenascin-C-null). The appropriate human genetic disorders are discussed and contrasted with the knockout mice phenotypes.

Animals↗

PubMed2001

[Functional analysis of phospholipase A2 receptor by gene knockout studies].

Phospholipase A2 receptor (PLA2R) is a type I transmembrane glycoprotein related to the C-type animal lectin family and mediates a variety of biological responses elicited by group IB secretory phospholipase A2 (sPLA2-IB). In the present study, we have shown the evidence that a novel type of sPLA2, sPLA2-X, also acts as one of the high-affinity ligands for mouse PLA2R. We then generated PLA2R-deficient mice and found that the knockout mice exhibited the resistance to an endotoxic shock with reduced plasma levels of proinflammatory cytokines, such as TNF-alpha and IL-1 beta. In situ hybridization analysis revealed that the expression of PLA2R transcript was markedly enhanced in type II alveolar epithelial cells and a subset of splenic lymphocytes in accordance with the elevated expression of sPLA2-IB and TNF-alpha mRNAs during endotoxic shock. In addition, the elevated expression level of TNF-alpha transcript was significantly reduced by the deficiency of PLA2R, suggesting that PLA2R plays a role in the regulation of TNF-alpha expression in these cell types. We then synthesized a specific sPLA2 inhibitor, indoxam, which blocked the binding of sPLA2-IB and X to PLA2R. Indoxam was found to suppress the elevation of the plasma level of TNF-alpha and prolonged the survival of endotoxin-challenged mice. The inhibitory effects of indoxam were abolished by the deficiency of PLA2R, demonstrating the involvement of PLA2R in the progression of endotoxic shock. We also detected and characterized a soluble form of PLA2R protein in the plasma of mouse with anti-PLA2R antibody, and showed its potential role as an endogenous sPLA2 inhibitor. Taken together, a series of studies with PLA2R-knockout mice have elucidated a critical role of PLA2R in the regulation of the development of endotoxic shock.

Animals↗

PubMed2005

Histochemical characteristics of soleus muscle in angiotensin-converting enzyme gene knockout mice.

We examined the histochemical characteristics of soleus muscle in the angiotensin-converting enzyme (ACE) gene (Ace in mice, ACE in humans) knockout mice. Serial sections of soleus muscle of wild-type (Ace+/+, n=20) and heterozygous mutant (Ace+/-, n=24) mice were stained for myosin adenosine triphosphatase activity to identify different muscle fiber types. Capillaries were visualized by amylase-periodic acid-Schiff staining. ACE activity in the serum and gastrocnemius muscle was higher in male mice than in female mice. Female and male Ace+/- mice had markedly lower ACE activity in the serum and the gastrocnemius muscle than did female and male Ace+/+ mice, respectively. In both male and female mice, the composition of fiber types (type I and IIa) did not differ significantly between Ace+/+ and Ace+/- mice. There was no significant gender difference in capillary density. Ace+/- mice had significantly more capillaries around type IIa fibers (5.44 +/- 0.18 vs. 5.01 +/- 0.13, p<0.05) than Ace+/+ mice. The differences in the number of capillaries around type I fibers and in the number of capillaries around per fiber (capillary:fiber ratio) between Ace+/- and Ace+/+ mice were not significant (p<0.1). There was no significant difference in the mean cross-sectional area occupied by one capillary and the number of capillaries per fiber area between Ace+/+ and Ace+/- mice. In conclusion, knockout of the Ace gene in mice increased capillary density, as expressed by the mean number of capillaries around type IIa fibers. This finding suggests a possible mechanism for the cardioprotective effects of ACE inhibitors.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed2006

Relative impact of androgen and estrogen receptor activation in the effects of androgens on trabecular and cortical bone in growing male mice: a study in the androgen receptor knockout mouse model.

UNLABELLED: The relative importance of AR and ER activation has been studied in pubertal male AR knockout and WT mice after orchidectomy and androgen replacement therapy, either with or without an aromatase inhibitor. AR activation dominates normal trabecular bone development and cortical bone modeling in male mice. Moreover, optimal periosteal bone expansion is only observed in the presence of both AR and ER activation. INTRODUCTION: Androgen receptor (AR)-mediated androgen action has traditionally been considered a key determinant of male skeletal growth. Increasing evidence, however, suggests that estrogens are also essential for normal male bone growth. Therefore, the relative importance of AR-mediated and estrogen receptor (ER)-mediated androgen action after aromatization remains to be clarified. MATERIALS AND METHODS: Trabecular and cortical bone was studied in intact or orchidectomized pubertal AR knockout (ARKO) and male wildtype (WT) mice, with or without replacement therapy (3-8 weeks of age). Nonaromatizable (dihydrotestosterone [DHT]) and aromatizable (testosterone [T]) androgens and T plus an aromatase inhibitor (anastrazole) were administered to orchidectomized ARKO and WT mice. Trabecular and cortical bone modeling were evaluated by static and dynamic histomorphometry, respectively. RESULTS: AR inactivation or orchidectomy induced a similar degree of trabecular bone loss (-68% and -71%, respectively). Both DHT and T prevented orchidectomy-induced bone loss in WT mice but not in ARKO mice. Administration of an aromatase inhibitor did not affect T action on trabecular bone. AR inactivation and orchidectomy had similar negative effects on cortical thickness (-13% and -8%, respectively) and periosteal bone formation (-50% and -26%, respectively). In orchidectomized WT mice, both DHT and T were found to stimulate periosteal bone formation and, as a result, to increase cortical thickness. In contrast, the periosteum of ARKO mice remained unresponsive to either DHT or T. Interestingly, administration of an aromatase inhibitor partly reduced T action on periosteal bone formation in orchidectomized WT mice (-34% versus orchidectomized WT mice on T), but not in ARKO mice. This effect was associated with a significant decrease in serum IGF-I (-21% versus orchidectomized WT mice on T). CONCLUSIONS: These findings suggest a major role for AR activation in normal development of trabecular bone and periosteal bone growth in male mice. Moreover, optimal stimulation of periosteal growth is only obtained in the presence of both AR and ER activation.

Androgens↗

PubMed2004

Osteoblast-like cells from estrogen receptor alpha knockout mice have deficient responses to mechanical strain.

INTRODUCTION: In vivo, bones' osteogenic response to mechanical loading involves proliferation of surface osteoblasts. This response is replicated in vitro and involves ERK-mediated activation of the estrogen receptor (ER) alpha and upregulation of estrogen response element activity. This proliferative response can be blocked by selective estrogen receptor modulators and increased by transfection of additional ERalpha. MATERIALS AND METHODS: We have now investigated the mechanisms of ER involvement in osteoblast-like cells' early responses to strain by comparing the responses of primary cultures of these cells derived from homozygous ERalpha knockout (ERKO) mice (ERalpha-/-) with those from their wildtype (ERalpha+/+) and heterozygous (ERalpha+/-) littermates and from ER/beta knockout (BERKO) mice (ERbeta+/+, ERbeta+/-, and ERbeta-/-). RESULTS: Whereas ERalpha+/+, ERalpha+/-, ERbeta+/+, and ERbeta-/- cells proliferate in response to a single 10-minute period of cyclic strain, ERalpha-/- cells do not. Transfection of fully functional, but not mutant, ERalpha rescues the proliferative response to strain in these cells. The strain-related response of ERalpha-/- cells is also deficient in that they show no increased activity of an AP-I driven reporter vector and no strain-related increases in NO production. Their strain-related increase in prostacyclin production is retained. They proliferate in response to fibroblast growth factor-2 but not insulin-like growth factor (IGF)-I or IGF-II, showing the importance of ERalpha in the IGF axis and the ability of ERalpha-/- cells to proliferate normally in response to a mitogenic stimulus that does not require functional ERalpha. CONCLUSIONS: These data indicate ERalpha's obligatory involvement in a number of early responses to mechanical strain in osteoblast-like cells, including those that result in proliferation. They support the hypothesis that reduction in ERalpha expression or activity after estrogen withdrawal results in a less osteogenic response to loading. This could be important in the etiology of postmenopausal osteoporosis.

Animals↗

PubMed2006

Generation of genetically modified embryonic stem cells for the development of knockout mouse animal model systems.

The aim of our lab is to understand the contributions made by cell adhesion molecules in the processes of disease. Much of our recent work has focused on the role played by beta3-integrin in mediating pathological angiogenesis. It is fair to state that without the ability to manipulate the mouse genome, and specifically to create knockout mice, the advances we have made in this field would not be nearly as significant as they are. The ability to generate knockout mice depends on the two technological breakthroughs of the ability to isolate and culture mouse embryonic stem (ES) cells and the methods employed for achieving targeted gene replacement in these cells by homologous recombination. Here, we present the methods we have found to be successful, and that we routinely employ to grow and manipulate ES cells, as well as those to screen and identify homologous recombinants.

Animals↗