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PubMed1999

Increased immunogenicity of tumor vaccines complexed with anti-Gal: studies in knockout mice for alpha1,3galactosyltransferase.

A major prerequisite for the success of tumor vaccines is their effective uptake by antigen-presenting cells (APCs) and transport of these APCs to the draining lymph nodes, where the processed and presented tumor-associated antigens activate tumor-specific naive T cells. We previously suggested that the immunogenicity of autologus tumor vaccines in humans may be augmented by engineering vaccinating tumor cell membranes to express alpha-galactosyl (alpha-gal) epitopes (i.e., Galalpha1,3Galbeta1,4GlcNAc-R). Subsequent in situ binding of natural anti-Gal IgG molecules to these epitopes would result in the formation of immune complexes that target tumor vaccines for uptake by APCs, via the interaction of the Fc portion of anti-Gal with Fcgamma receptors on APCs. This hypothesis was tested in a unique experimental animal model of knockout mice for alpha1,3galactosyltransferase (alpha1,3GT) and the mouse melanoma B16-BL6 (referred to here as BL6). Like humans, these mice lack alpha-gal epitopes and produce anti-GaL BL6 melanoma cels are highly tumorigenic, and like human tumor cells, they lack alpha-gal epitopes. Expression of alpha-gal epitopes on these melanoma cells was achieved by stable transfection with alpha,3GT cDNA. The transfected melanoma cells (termed BL6alphaGT) express approximately 2 x 10(6) alpha-gal epitopes per cell and readily form immune complexes with anti-Gal. Vaccination of the mice with 2 x 10(6) irradiated melanoma cells that express alpha-gal epitopes, followed by challenge with 0.5 x 10(6) live parental melanoma cells, resulted in protection for at least 2 months (i.e, no tumor growth) in one-third of the mice, whereas all mice immunized with irradiated parental melanoma cells developed tumors 21-26 days post-challenge. The proportion of protected mice doubled when the mice were immunized twice with irradiated melanoma cells expressing alpha-gal epitopes and challenged with 0.2 x 10(6) live BL6 cells. Histological studies on the developing tumors in challenged mice that were immunized with melanoma cells expressing alpha-gal epitopes demonstrated extensive infiltration of T lymphocytes and macrophages, whereas no mononuclear cell infiltrates were observed in tumors of mice immunized with parental tumor cells. Overall, these studies imply that immunization of alpha1,3GT knockout mice with BL6 melanoma cells that express alpha-gal epitopes elicits, in a proportion of the population, protective immune response against the same tumor lacking such epitopes. These studies further suggest that similar immunization of cancer patients with autologous tumor vaccines that are engineered to express alpha-gal epitopes may increase the immune response to autologous tumor-associated antigens and, thus, may elicit immune-mediated destruction of metastatic cells expressing these antigens.

Animals↗

PubMed1999

[Serotonin receptor knockout mice].

The neurotransmitter serotonin is known to play a key role in determining emotional state in higher animals, especially in humans. Pharmacological studies have highlighted a subset of serotonin receptor subtypes (1A, 2B and 2C) worthy of knockout analysis. Several groups investigated knockout phenotype and determined the physiological function of these receptors, although the phenotype of the mouse seems somewhat paradoxical. However, the results strongly suggest that serotonin receptors are mainly involved in emotion (i.e., anxiety, aggressive behavior, obesity and epilepsy).

Animals↗

PubMed2000

mu-Opioid receptor knockout mice do not self-administer alcohol.

Opioid peptides long have been hypothesized to play a role in ethanol reinforcement. Neuropharmacological studies have shown that opioid receptor antagonists decrease ethanol self-administration in rodents and prevent relapse in humans. However, the exact mechanism for such powerful effects has remained elusive. The availability of mu-opioid receptor knockout mice has made possible the direct examination of the role of the mu-opioid receptor in mediating ethanol self-administration. In the present experiments, both nosepoke and lever operant ethanol self-administration and several tests of two bottle-choice ethanol drinking were studied in these genetically engineered mice. In no case did knockout mice show evidence of ethanol self-administration, and, in fact, these mice showed evidence of an aversion to ethanol under several experimental conditions. These data provide new evidence for a critical role for mu-opioid receptors in ethanol self-administration assessed with a variety of behavioral paradigms and new insights into the neuropharmacological basis for ethanol reinforcement.

Alcohol Drinking↗

PubMed2000

Laminar specificity of local circuits in barrel cortex of ephrin-A5 knockout mice.

Cortical circuits are characterized by layer-specific axonal arbors. Molecular laminar cues are believed to direct the development of this specificity. We have tested the hypothesis that ephrin-A5 is responsible for preventing layer 2/3 pyramidal cell axons from branching within layer 4 (Castellani et al., 1998) by assessing the laminar specificity of axonal arbors in ephrin-A5 knockout mice. We find that in barrel cortex of knockout mice, layer 2/3 pyramidal neurons form axonal arbors specifically in layers 2/3 and 5, avoiding layer 4. This pattern of arborization is indistinguishable from that of wild-type littermates. Furthermore, we find that in wild-type mice, laminar patterns of ephrin-A5 expression differ between cortical areas despite the similarity of layer-specific local cortical circuits across areas. Most notably, ephrin-A5 is not expressed preferentially in layer 4 of wild-type mouse barrel cortex. We conclude that ephrin-A5 is not responsible for preventing the development of layer 2/3 pyramidal cell axonal arbors in layer 4 of mouse barrel cortex. These observations also suggest that if ephrin-A5 plays a role in the emergence of layer-specific circuits, that role must differ between cortical areas.

Animals↗

PubMed2000

Ultrastructural analysis of megakaryocytes in GPV knockout mice.

Lesions in the genes for GPIb alpha, GPIb beta or GPIX result in a bleeding diathesis, the Bernard-Soulier syndrome (BSS), which associates a platelet adhesion defect with thrombocytopenia, giant platelets and abnormal megakaryocytes (MK). The role of GPV, also absent in BSS, was recently addressed by gene targeting in mice. While a negative modulator function for GPV on thrombin-induced platelet responses was found in one model, the absence of GP V had no effect on GPIb-IX expression or platelet adhesion. Our study extends previous results and reports that electron microscopy of bone marrow from the GPV knockout mice revealed a normal MK ultrastructure and development of the demarcation membrane system (DMS). There was a usual presence of MK fragments in the bone marrow vascular sinus. Immunogold labelling of MK from the knockout mice showed a normal distribution of GPIb-IX in the DMS and on the cell surface. The distribution of fibrinogen, vWF and P-selectin was unchanged with, interestingly, P-selectin also localised within the DMS in both situations. Thus GPV is not crucial to MK development and platelet production, consistent with the fact that no mutation in the GPV gene has as yet been described in BSS.

Animals↗

PubMed2001

Increased TGF-beta1 in the lungs of asbestos-exposed rats and mice: reduced expression in TNF-alpha receptor knockout mice.

Inhalation of numerous fibrogenic agents causes interstitial pulmonary fibrosis (IPF) in humans and in a number of animal models. Several of these models provide evidence that certain peptide growth factors (GF) are playing a role in the disease process. Transforming growth factor beta 1 (TGF-beta1) is a potent inducer of extracellular matrix production by mesenchymal cells, and we have shown that this peptide is produced in the lung after asbestos exposure. We used in situ hybridization to demonstrate that the mRNA for TGF-beta1 is rapidly expressed post-exposure at sites of initial asbestos-induced lung injury in both rats and mice. The TGF-beta1 is expressed by bronchiolar-alveolar epithelial cells as well as by mesenchymal cells and lung macrophages in exposed animals. Normal rats and mice express little TGF-beta1, as we have demonstrated previously for PDGF-A and -B, TGF-alpha, and TNF-alpha. TGF-beta1 expression is accompanied by collagen and fibronectin production in asbestos-exposed animals. Most interesting, TGF-beta1 expression is largely absent in the lungs of TNF-alpha receptor knockout mice that fail to develop asbestos-induced IPE We have shown previously that the mRNAs and cognate peptides of PDGF-A and -B and TGF-alpha, but not TNF-alpha, are reduced in the fibrosis-resistant knockout mice. In this article, we show that TGF-beta1 is included in this group of cytokines, supporting the postulate that TNF-alpha is necessary for the expression of other, more downstream growth factors, and the consequent development of idiopathic pulmonary fibrosis (IPF).

Administration, Inhalation↗

PubMed2001

[Pulmonary hypertension in endothelial NO synthase knockout mice].

Inhalation of nitric oxide (NO) effectively reduces pulmonary arterial pressure in patients with pulmonary hypertension. However, it is controversial as to the role of endothelium-derived NO in pulmonary hypertension. Disruption of endothelial NO synthase (eNOS) gene causes systemic hypertension and diminishes endothelium-dependent vasodilatation. Even at normoxia eNOS knockout mice showed slightly increased right ventricular systolic pressure, but normal right ventricular weight. Hypoxia increased right ventricular systolic pressure, hematocrit, right ventricular weight in a concentration-dependent manner. The hypoxic condition induced pulmonary vascular remodeling; increases in the ratio of fully muscular vessels. Thus, pulmonary hypertension in eNOS knockout mice may be due to exaggerated vasoconstriction in response to hypoxia. In contrast, eNOS gene transduction reduces pulmonary vascular resistance.

Animals↗

PubMed2001

Transgenic and knockout mouse models clarify pituitary development, function and disease.

Mouse models have been used to study various aspects of pituitary development, function and disease. Transgenic or knockout technology has been applied to examine the regulation of hormone gene expression and the pathophysiology of its alterations, to ascertain the factors that determine cell differentiation, and to manipulate oncogenesis. Transgenic mice have elucidated the necessary elements required for the tissue- and cell-specific expression of pituitary hormones. Transgenic and knockout technologies have derived mice with hormone overexpression or abrogation of hormone action, and have identified novel hormones. The role of precursor cells in cell differentiation has been confirmed by genetic ablation of cell lineages. Inactivation of transcription factors implicated in pituitary organogenesis and cytogenesis has proven their critical roles in pituitary development. Pituitary oncogenesis has been studied by promoter-directed oncogene expression or tumor suppressor gene ablation, by adenohypophysiotropic hormone overexpression, or by growth factor or receptor overexpression. The tumors have provided a number of cell lines for use in the continuing study of pituitary physiology and pathology. These models may also be used in the future to examine novel therapeutic strategies for the management of patients with pituitary disorders.

Animals↗

PubMed2001

Insights into molecular mechanisms of contact hypersensitivity gained from gene knockout studies.

Contact hypersensitivity (CHS), a dendritic-cell (DC)-dependent, T-cell-mediated skin immune response to reactive haptens, has been a subject of intense research for many years. The molecular mechanisms underlying CHS are complicated and are not fully understood. During the past few years, varieties of gene-targeted knockout mice have been used in the study of CHS. Such studies have contributed significantly to our understanding of the mechanisms responsible for the initiation of CHS. This review focuses on insights into molecular requirements for CHS gained from knockout studies.

Animals↗

PubMed2001

[The role of tetrodotoxin-resistant sodium channels in pain sensation studied on sns-knockout mice].

Nociceptive dorsal root ganglion neurons express sensory neuron-specific tetrodotoxin (TTX)-resistant voltage-gated sodium channel(SNS). The role of SNS in nociception has been studied by constructing sns-knockout mice. The sns-knockout mice expressed only TTX-sensitive sodium currents on step depolarizations from normal resting potentials, demonstrating that the slow TTX-resistant currents are mediated by the sns gene. The mutant mice were viable, fertile and apparently normal, although lowered thresholds of electrical activation of C-fibers and increased current densities of TTX-sensitive sodium channels demonstrated compensatory up-regulation of TTX-sensitive currents in DRG neurons. Behavioral studies demonstrated a pronounced analgesia to noxious mechanical stimuli, small deficits in noxious thermoreception and delayed development of inflammatory hyperalgesia. These data show that SNS is involved in pain sensation.

Animals↗

PubMed2001

Prenatal lens development in connexin43 and connexin50 double knockout mice.

PURPOSE: To determine the roles of intercellular communication in embryonic eye growth and development, mice with a targeted deletion of the Cx43 gene were examined, and mice without both Cx43 and Cx50 were generated and analyzed. METHODS: Embryonic eyes and lenses from wild-type mice, or mice deficient in Cx43, Cx50, or both Cx43 and Cx50 were collected and analyzed structurally by light and electron microscopy, immunohistochemically using connexin-specific antibodies, biochemically by Western blot analysis, and physiologically by measuring patterns of junctional communication revealed by iontophoretic injection of junction-permeable reporter molecules. RESULTS: Cx50 expression was limited to the ocular lens and was not detected in either the cornea or the retina. Cx43(-/-) embryos showed development of structurally normal lenses and eyes when examined by light and electron microscopy through embryonic day (E)18.5. In addition, Cx43(-/-) lenses synthesized four different markers of lens differentiation: MIP26, alphaA-crystallin, alphaB-crystallin, and gamma-crystallin. Double-knockout lenses were also histologically normal through E18.5 and synthesized the four lens differentiation markers. When assayed by intracellular injection with Lucifer yellow (Molecular Probes, Eugene, OR) and neurobiotin at E15.5, Cx43(-/-)/Cx50(-/-) lenses retained gap junction-mediated dye transfer between fiber cells. In contrast, dye transfer in double-knockout lenses was dramatically reduced between epithelial cells and was eliminated between epithelial cells and fibers. CONCLUSIONS: These data indicate that the unique functional properties of both Cx43 and Cx50 are not required for prenatal lens development and that connexin diversity is required for regulation of postnatal growth and homeostasis.

Animals↗

PubMed2001

Effect of Rpe65 knockout on accumulation of lipofuscin fluorophores in the retinal pigment epithelium.

PURPOSE: In all mammalian species examined to date the retinal pigment epithelium (RPE) has been found to accumulate autofluorescent lysosomal storage bodies (lipofuscin) during senescence. Substantial evidence indicates that retinoids in the RPE-retina complex play a major role in RPE lipofuscin formation. Indeed, at least one RPE lipofuscin fluorophore is derived in part from vitamin A aldehyde. However, the precise mechanisms by which retinoids modulate RPE lipofuscin accumulation have not been elucidated. In mice without a functional Rpe65 gene, isomerization of all-trans- to 11-cis-retinol is blocked. Experiments were performed to determine whether this impairment of retinoid metabolism alters RPE lipofuscin accumulation. METHODS: RPE lipofuscin fluorophore content was compared in 12- to 13-month-old Rpe65(+/+), Rpe65(+/-), and Rpe65(-/-) mice. Lipofuscin fluorophore content was determined using quantitative fluorometric measurements. RPE lipofuscin content was also estimated with quantitative ultrastructural techniques. RESULTS: In the Rpe65(-/-) mice, RPE lipofuscin fluorophore accumulation was almost abolished. In addition, a significantly reduced accumulation of lipofuscin fluorophores was also observed in the Rpe65(+/-) animals. The inability of the RPE of)Rpe65(-/-) mice to supply 11-cis-retinal from the RPE to the retinal photoreceptors was accompanied by a massive accumulation of lipid droplets in the RPE that appeared to contain substantial amounts of retinoids. CONCLUSIONS: These findings indicate that formation of RPE lipofuscin fluorophores is almost completely dependent on a normal visual cycle. The absence of retinal (both all-trans and 11-cis) in Rpe65 knockout mice drastically reduced formation of lipofuscin fluorophores in these animals. Even an excessive accumulation of retinyl fatty acid esters in the RPE of Rpe65 knockout mice did not contribute to lipofuscin accumulation.

Animals↗

PubMed2002

Suppression of UV carcinogenesis by difluoromethylornithine in nucleotide excision repair-deficient Xpa knockout mice.

Xeroderma pigmentosum (XP) patients are deficient in nucleotide excision repair (NER) because of mutations in one of the genes coding for NER enzymes. This results predominantly in high frequency of UV-induced skin tumors at an early age; the most severe phenotype is found in patients of complementation group A (XPA). However, in a subset of these XPA patients no skin tumors appear, even at advanced age. Fibroblasts of this subset of patients are not capable of raising UV-induced enhanced reactivation (ER) of viruses and up-regulation of ornithine decarboxylase (ODC). We hypothesized that prevention of ODC induction would protect NER-deficient patients from cancer. To simulate the situation in XPA patients, we used a hairless Xpa knockout mouse model and down-regulated the ODC activity by difluoromethylornithine (DFMO) administered in the drinking water. The DFMO treatment significantly suppressed UV-induced carcinogenesis. In a crossover study, we additionally found that discontinuation of the DFMO treatment resulted in a rapid appearance of skin tumors, up to levels found in mice not treated with DFMO. Late-stage DFMO treatment significantly reduced the number of carcinomas by a factor of 2-3, and it appeared to select for carcinomas with high ODC activity. These results indicate that DFMO suppresses the outgrowth but not the initiation of UV-induced tumors. The DFMO treatment reduced the tumor load but did not offer the Xpa knockout mice full protection against UV carcinogenesis.

Animals↗

PubMed2002

Effects of major histocompatibility complex class II knockout on mouse bone mechanical properties during development.

We investigated the effect of major histocompatibility complex class II (MHC II) knockout on the development of the mouse peripheral skeleton. These C2D mice had less skeletal development at 8, 12 and 16 weeks of age compared to wild-type C57BL/6J (B6) male mice. The C2D mice had decreased femur mechanical, geometric and compositional measurements compared to wild type mice at each of these ages. C2D femur stiffness (S), peak force in 3-pt bending (Pm), and mineral mass (Min-M) were 74%, 64% and 66%, respectively, of corresponding B6 values at 8 weeks of age. Similar differences were measured at 12 weeks (for which C2D femoral S, Pm and Min-M were 71%, 72% and 73%, respectively, of corresponding B6 values) and at 16 weeks (for which C2D femoral S, Pm and Min-M were 80%, 66% and 61%, respectively, of corresponding B6 values). MHC II knockout delays the development of adult bone properties and is accompanied by lower body mass compared to wild-type controls.

Animals↗

PubMed2002

Hepatocellular carcinoma caused by loss of heterozygosity in Lkb1 gene knockout mice.

Germline mutations of the LKB1 gene are associated with Peutz-Jeghers syndrome, which is characterized by mucocutaneous pigmentation and gastrointestinal hamartoma with an increased risk of cancer development. To investigate the role of LKB1 in vivo, we have recently constructed Lkb1 gene knockout mice. Because of Lkb1 gene haploinsufficiency, the heterozygous Lkb1 mice develop gastrointestinal polyps of which the histological characteristics resemble those of the Peutz-Jeghers syndrome hamartomas. Here we demonstrate that the Lkb1 (+/-) mice develop hepatocellular carcinomas (HCCs). In Lkb1 (+/-) mice >50 weeks of age, >70% of the male mice developed HCCs, whereas only 20% of the females had HCCs, showing a sex difference in the susceptibility. Histological examinations revealed various types of HCCs, such as "trabecular," "clear cell," "pseudoglandular," and "sarcomatous" types, which were strikingly similar to those found in human HCCs. Western blotting and PCR analyses showed loss of Lkb1 heterozygosity in all of the HCC tissues examined, indicating a tumor suppressor role of LKB1 in the mouse liver. These results suggest that lack of LKB1 is a novel mechanism for HCC development. Thus, the Lkb1 (+/-) knockout mutant should be an important and useful model for human HCC.

AMP-Activated Protein Kinases↗

PubMed2002

[Susceptibility of wild and knockout p53 FVB/N line mice to benz(a)pyrene-induced subcutaneous sarcoma].

Susceptibility to benz(a)pyrene (BP) has been compared between wild and knockout p53 homo- and heterozygous FVB/N, C57BL/6 and NMRI line mice. Each group included 34-80 animals. Each animal was injected 2 mg BP, s.c., in sunflower oil solution. The animals were followed up for 24 weeks. Fibrosarcomas arose at the site of injection in: wild FVB/N line mice--93%; FVB/N heterozygous--100%; p53 homozygous knockout--95%; C57B1/6--84%, and NMRI mice--83%. Mean latent period was 68, 66, 69, 112 and 109 days, respectively. Hence, FVB/N line mice developed tumors much earlier than C57B1/6 or NMRI did, suggesting their higher susceptibility to carcinogenic BP injection. No significant difference in susceptibility to carcinogen versus p53 expression was reported.

Animals↗

PubMed2003

Retarded outer segment development in TrkB knockout mouse retina organ culture.

PURPOSE: To determine the effects of trkB deficiency in the mouse retina on photoreceptor development and retinal organization, in the absence of confounding systemic effects. METHODS: Newborn mice that carried two null trkB alleles (trkB-/-) and their wild type (WT) littermates were used for retinal organ cultures. On Day 21, rod development was assessed histologically in plastic sections (outer segment length) and retinal organization was analyzed using retinal cell-type specific antibodies. Anatomical data obtained from the organ cultures were compared to previously published histological results from in vivo data. RESULTS: (1) Rod outer segment length was significantly shorter in retinas from trkB-/- mice in the presence of normal numbers of rods. (2) No dopaminergic amacrine cells were observed in the knockout retina. (3) Unlike in the in vivo condition, recoverin-positive OFF-cone bipolar cells were present in trkB-/- retinas grown in culture. CONCLUSIONS: (1) These results demonstrate that rod outer segment development is compromised in the absence of trkB in the retina. (2) This study further supports our previous conclusion that the elimination of trkB expression alters rod development, because the presence of trkB receptors within the retina is essential for normal rod maturation and not because of confounding systemic effects. (3) More generally, this study stresses the importance of investigating complex phenotypes in gene knockout mice under conditions that isolate the organ under investigation from unrelated systemic variations.

Animals↗

PubMed2003

[Study on the gene knockout model mice of Duchenne muscular dystrophy].

OBJECTIVE: With a view to further experimental studies on Duchenne muscular dystrophy (DMD), we investigated the motor function, serum creatine kinase (CK) level and pathological characteristics of muscular tissue in C57, mdx, dystrophin/utrophin gene double knockout (dko)mice. METHODS: Gene identification was performed for the filial generation of heterozygote of utrophin gene knockout of mdx mice for acquiring mdx and dko mice. The motor function, serum CK level, and the pathology of muscular tissue of C57, mdx and dko mice were compared. RESULTS: There were significant differences in all detecting aspects between C57 mouse and dko mouse. In the aspects of serum CK level and pathology of muscular tissue, there were differences between C57 mouse and mdx mouse; however, no significant difference in motor function was observed between them. CONCLUSION: Compared with mdx mouse, dko mouse is in a more serious illness state which is nearer to the natural state of DMD. Therefore, dko mouse is a more ideal model for studying the praxiology of DMD.

Animals↗