Translational Mouse Models of Autism: Advancing Toward Pharmacological Therapeutics

PubMed Central

Kazdoba, Tatiana M.; Leach, Prescott T.; Yang, Mu; Silverman, Jill L.; Solomon, Marjorie

2016-01-01

Animal models provide preclinical tools to investigate the causal role of genetic mutations and environmental factors in the etiology of autism spectrum disorder (ASD). Knockout and humanized knock-in mice, and more recently knockout rats, have been generated for many of the de novo single gene mutations and copy number variants (CNVs) detected in ASD and comorbid neurodevelopmental disorders. Mouse models incorporating genetic and environmental manipulations have been employed for preclinical testing of hypothesis-driven pharmacological targets, to begin to develop treatments for the diagnostic and associated symptoms of autism. In this review, we summarize rodent behavioral assays relevant to the core features of autism, preclinical and clinical evaluations of pharmacological interventions, and strategies to improve the translational value of rodent models of autism. PMID:27305922

Ventromedial Hypothalamic Nitric Oxide Production Is Necessary for Hypoglycemia Detection and Counterregulation

PubMed Central

Fioramonti, Xavier; Marsollier, Nicolas; Song, Zhentao; Fakira, Kurt A.; Patel, Reema M.; Brown, Stacey; Duparc, Thibaut; Pica-Mendez, Arnaldo; Sanders, Nicole M.; Knauf, Claude; Valet, Philippe; McCrimmon, Rory J.; Beuve, Annie; Magnan, Christophe; Routh, Vanessa H.

2010-01-01

OBJECTIVE The response of ventromedial hypothalamic (VMH) glucose-inhibited neurons to decreased glucose is impaired under conditions where the counterregulatory response (CRR) to hypoglycemia is impaired (e.g., recurrent hypoglycemia). This suggests a role for glucose-inhibited neurons in the CRR. We recently showed that decreased glucose increases nitric oxide (NO) production in cultured VMH glucose-inhibited neurons. These in vitro data led us to hypothesize that NO release from VMH glucose-inhibited neurons is critical for the CRR. RESEARCH DESIGN AND METHODS The CRR was evaluated in rats and mice in response to acute insulin-induced hypoglycemia and hypoglycemic clamps after modulation of brain NO signaling. The glucose sensitivity of ventromedial nucleus glucose-inhibited neurons was also assessed. RESULTS Hypoglycemia increased hypothalamic constitutive NO synthase (NOS) activity and neuronal NOS (nNOS) but not endothelial NOS (eNOS) phosphorylation in rats. Intracerebroventricular and VMH injection of the nonselective NOS inhibitor NG-monomethyl-l-arginine (l-NMMA) slowed the recovery to euglycemia after hypoglycemia. VMH l-NMMA injection also increased the glucose infusion rate (GIR) and decreased epinephrine secretion during hyperinsulinemic/hypoglycemic clamp in rats. The GIR required to maintain the hypoglycemic plateau was higher in nNOS knockout than wild-type or eNOS knockout mice. Finally, VMH glucose-inhibited neurons were virtually absent in nNOS knockout mice. CONCLUSIONS We conclude that VMH NO production is necessary for glucose sensing in glucose-inhibited neurons and full generation of the CRR to hypoglycemia. These data suggest that potentiating NO signaling may improve the defective CRR resulting from recurrent hypoglycemia in patients using intensive insulin therapy. PMID:19934009

Functional conservation of Gsdma cluster genes specifically duplicated in the mouse genome.

PubMed

Tanaka, Shigekazu; Mizushina, Youichi; Kato, Yoriko; Tamura, Masaru; Shiroishi, Toshihiko

2013-10-03

Mouse Gasdermin A3 (Gsdma3) is the causative gene for dominant skin mutations exhibiting alopecia. Mouse has two other Gsdma3-related genes, Gsdma and Gsdma2, whereas human and rat have only one related gene. To date, no skin mutation has been reported for human GSDMA and rat Gsdma as well as mouse Gsdma and Gsdma2. Therefore, it is possible that only Gsdma3 has gain-of-function type mutations to cause dominant skin phenotype. To elucidate functional divergence among the Gsdma-related genes in mice, and to infer the function of the human and rat orthologs, we examined in vivo function of mouse Gsdma by generating Gsdma knockout mice and transgenic mice that overexpress wild-type Gsdma or Gsdma harboring a point mutation (Alanine339Threonine). The Gsdma knockout mice shows no visible phenotype, indicating that Gsdma is not essential for differentiation of epidermal cells and maintenance of the hair cycle, and that Gsdma is expressed specifically both in the inner root sheath of hair follicles and in suprabasal cell layers, whereas Gsdma3 is expressed only in suprabasal layers. By contrast, both types of the transgenic mice exhibited epidermal hyperplasia resembling the Gsdma3 mutations, although the phenotype depended on the genetic background. These results indicate that the mouse Gsdma and Gsdma3 genes share common function to regulate epithelial maintenance and/or homeostasis, and suggest that the function of human GSDMA and rat Gsdma, which are orthologs of mouse Gsdma, is conserved as well.

CRISPR/Cas9 mediated generation of stable chondrocyte cell lines with targeted gene knockouts; analysis of an aggrecan knockout cell line.

PubMed

Yang, Maozhou; Zhang, Liang; Stevens, Jeff; Gibson, Gary

2014-12-01

The Swarm rat chondrosarcoma (RCS) cell lines derived from a spontaneous neoplasm in a rat spine several decades ago have provided excellent models of chondrosarcoma tumor development. In addition the robust chondrocyte phenotype (expression of a large panel of genes identical to that seen in normal rat cartilage) and the ability to generate cell clones have facilitated their extensive use in the identification of chondrocyte proteins and genes. The clustered regularly interspersed short palindromic repeat (CRISPR) technology employing the RNA-guided nuclease Cas9 has rapidly dominated the genome engineering field as a unique and powerful gene editing tool. We have generated a stable RCS cell line (RCS Cas9) expressing the nuclease Cas9 that enables the editing of any target gene or non-coding RNA by simple transfection with a guide RNA. As proof of principle, stable cell lines with targeted ablation of aggrecan expression (Acan KO) were generated and characterized. The studies show that stable chondrocyte cell lines with targeted genome editing can be quickly generated from RCS Cas9 cells using this system. The Acan KO cell lines also provided a tool for characterizing the response of chondrocytes to aggrecan loss and the role of aggrecan in chondrosarcoma development. Loss of aggrecan expression while not affecting the chondrocyte phenotype resulted in a much firmer attachment of cells to their substrate in culture. Large changes in the expression of several genes were observed in response to the absence of the proteoglycan matrix, including those for several small leucine rich proteoglycans (SLRPs), transcription factors and membrane transporters. Acan KO cells failed to form a substantial chondrosarcoma when injected subcutaneously in nude mice consistent with previous suggestions that the glycosaminoglycan-rich matrix surrounding the chondrosarcoma protects it from destruction by the host immune system. The studies provide new understanding of aggrecan function and the RCS Cas9 cell line is expected to provide a very valuable tool for the study gene function in chondrocytes. Copyright © 2014 Elsevier Inc. All rights reserved.

Functional Conservation of Gsdma Cluster Genes Specifically Duplicated in the Mouse Genome

PubMed Central

Tanaka, Shigekazu; Mizushina, Youichi; Kato, Yoriko; Tamura, Masaru; Shiroishi, Toshihiko

2013-01-01

Mouse Gasdermin A3 (Gsdma3) is the causative gene for dominant skin mutations exhibiting alopecia. Mouse has two other Gsdma3-related genes, Gsdma and Gsdma2, whereas human and rat have only one related gene. To date, no skin mutation has been reported for human GSDMA and rat Gsdma as well as mouse Gsdma and Gsdma2. Therefore, it is possible that only Gsdma3 has gain-of-function type mutations to cause dominant skin phenotype. To elucidate functional divergence among the Gsdma-related genes in mice, and to infer the function of the human and rat orthologs, we examined in vivo function of mouse Gsdma by generating Gsdma knockout mice and transgenic mice that overexpress wild-type Gsdma or Gsdma harboring a point mutation (Alanine339Threonine). The Gsdma knockout mice shows no visible phenotype, indicating that Gsdma is not essential for differentiation of epidermal cells and maintenance of the hair cycle, and that Gsdma is expressed specifically both in the inner root sheath of hair follicles and in suprabasal cell layers, whereas Gsdma3 is expressed only in suprabasal layers. By contrast, both types of the transgenic mice exhibited epidermal hyperplasia resembling the Gsdma3 mutations, although the phenotype depended on the genetic background. These results indicate that the mouse Gsdma and Gsdma3 genes share common function to regulate epithelial maintenance and/or homeostasis, and suggest that the function of human GSDMA and rat Gsdma, which are orthologs of mouse Gsdma, is conserved as well. PMID:23979942

Serotonin Transporter Knockout Rats Show Improved Strategy Set-Shifting and Reduced Latent Inhibition

ERIC Educational Resources Information Center

Nonkes, Lourens J. P.; van de Vondervoort, Ilse I. G. M.; de Leeuw, Mark J. C.; Wijlaars, Linda P.; Maes, Joseph H. R.; Homberg, Judith R.

2012-01-01

Behavioral flexibility is a cognitive process depending on prefrontal areas allowing adaptive responses to environmental changes. Serotonin transporter knockout (5-HTT[superscript -/-]) rodents show improved reversal learning in addition to orbitofrontal cortex changes. Another form of behavioral flexibility, extradimensional strategy set-shifting…

Effects of PDE4 Pathway Inhibition in Rat Experimental Stroke

PubMed Central

Yang, Fan; Sumbria, Rachita K.; Xue, Dong; Yu, Chuanhui; He, Dan; Liu, Shuo; Paganini-Hill, Annlia; Fisher, Mark J.

2015-01-01

PURPOSE The first genomewide association study indicated that variations in the phosphodiesterase 4D (PDE4D) gene confer risk for ischemic stroke. However, inconsistencies among the studies designed to replicate the findings indicated the need for further investigation to elucidate the role of the PDE4 pathway in stroke pathogenesis. Hence, we studied the effect of global inhibition of the PDE4 pathway in two rat experimental stroke models, using the PDE4 inhibitor rolipram. Further, the specific role of the PDE4D isoform in ischemic stroke pathogenesis was studied using PDE4D knockout rats in experimental stroke. METHODS Rats were subjected to either the ligation or embolic stroke model and treated with rolipram (3mg/kg; i.p.) prior to the ischemic insult. Similarly, the PDE4D knockout rats were subjected to experimental stroke using the embolic model. RESULTS Global inhibition of the PDE4 pathway using rolipram produced infarcts that were 225% (p<0.01) and 138% (p<0.05) of control in the ligation and embolic models, respectively. PDE4D knockout rats subjected to embolic stroke showed no change in infarct size compared to wild-type control. CONCLUSIONS Despite increase in infarct size after global inhibition of the PDE4 pathway with rolipram, specific inhibition of the PDE4D isoform had no effect on experimental stroke. These findings support a role for the PDE4 pathway, independent of the PDE4D isoform, in ischemic stroke pathogenesis. PMID:25224348

Estrogen receptors in skeletal metabolism: lessons from genetically modified models of receptor function.

PubMed

McCauley, Laurie K; Tözüm, Tolga F; Rosol, Thomas J

2002-01-01

Estrogens have long been known to be important for skeletal homeostasis, but their precise mechanisms of action in bone are still unclear. Mice with targeted deletions of the estrogen receptors alpha (ERalpha) and beta (ERbeta) have been generated by two research groups and several studies performed characterizing the phenotype of ERalpha knockout (ERKOalpha), ERbeta knockout (ERKObeta), or double deletion of ERalpha and ERbeta (DERKO) mice. Initial studies reported a reduction in bone mineral density in male ERKOalpha mice. More extensive analyses have been puzzling, likely because of compensatory mechanisms in ERKO mice. Furthermore, the existence of a third ER continues to be a potential explanation for some actions of estrogen in bone. Other rodent models, including the testicular feminized mouse and rat, the aromatase knockout mouse, and a rat with a dominant negative ER mutation, have added information regarding estrogen's actions in bone. This review summarizes many reports characterizing available rodent models with genetic alterations relevant to estrogen action. The sum of these reports suggests that the ERbeta is not highly protective in bone because loss of its function results in minimal alterations in the skeleton. Furthermore, loss of both the ERalpha and the ERbeta does not account for loss of estrogen action in bone, because the impact of DERKO is seemingly not as great as the impact of gonadectomy on the skeleton. Finally, through studies of ERKO mice and other rodent models of altered sex steroid action, it appears that estrogen may be more protective in the skeleton than androgens.

Comparison of the hepatic and thyroid gland effects of sodium phenobarbital in wild type and constitutive androstane receptor (CAR) knockout rats and pregnenolone-16α-carbonitrile in wild type and pregnane X receptor (PXR) knockout rats.

PubMed

Haines, Corinne; Chatham, Lynsey R; Vardy, Audrey; Elcombe, Clifford R; Foster, John R; Lake, Brian G

2018-05-01

A number of chemicals produce liver and thyroid gland tumours in rodents by nongenotoxic modes of action (MOAs). In this study the hepatic and thyroid gland effects of the constitutive androstane receptor (CAR) activator sodium phenobarbital (NaPB) were examined in male Sprague-Dawley wild type (WT) rats and in CAR knockout (CAR KO) rats and the effects of the pregnane X receptor (PXR) activator pregnenolone-16α-carbonitrile (PCN) were examined in WT and PXR knockout (PXR KO) rats. Rats were either fed diets containing 0 (control) or 500 ppm NaPB or were dosed with 0 (control) or 100 mg/kg/day PCN orally for 7 days. The treatment of WT rats with NaPB and PCN for 7 days resulted in increased relative liver weight, increased hepatocyte replicative DNA synthesis (RDS) and the induction of cytochrome P450 CYP2B and CYP3A subfamily enzyme, mRNA and protein levels. In marked contrast, the treatment of CAR KO rats with NaPB and PXR KO rats with PCN did not result in any increases in liver weight and induction of CYP2B and CYP3A enzymes. The treatment of CAR KO rats with NaPB had no effect on hepatocyte RDS, while PCN produced only a small increase in hepatocyte RDS in PXR KO rats. Treatment with NaPB had no effect on thyroid gland weight in WT and CAR KO rats, whereas treatment with PCN resulted in an increase in relative thyroid gland weight in WT, but not in PXR KO, rats. Thyroid gland follicular cell RDS was increased by the treatment of WT rats with NaPB and PCN, with NaPB also producing a small increase in thyroid gland follicular cell RDS in CAR KO rats. Overall, the present study with CAR KO rats demonstrates that a functional CAR is required for NaPB-mediated increases in liver weight, stimulation of hepatocyte RDS and induction of hepatic CYP enzymes. The studies with PXR KO rats demonstrate that a functional PXR is required for PCN-mediated increases in liver weight and induction of hepatic CYP enzymes; with induction of hepatocyte RDS also being largely mediated through PXR. The hepatic effects of NaPB in CAR KO rats and of PCN in PXR KO rats are in agreement with those observed in other recent literature studies. These results suggest that CAR KO and PXR KO rats are useful experimental models for liver MOA studies with rodent CAR and PXR activators and may also be useful for thyroid gland MOA studies. Copyright © 2018 Elsevier B.V. All rights reserved.

The serotonin transporter plays an important role in male sexual behavior: a study in serotonin transporter knockout rats.

PubMed

Chan, Johnny S W; Snoeren, Eelke M S; Cuppen, Edwin; Waldinger, Marcel D; Olivier, Berend; Oosting, Ronald S

2011-01-01

Serotonin (5-HT) is an important neurotransmitter for sexual behaviors. Heterozygous (+/-) serotonin transporter (SERT) rats and SERT knockout rats (-/-) have serotonergic disturbances with significant elevations of basal extracellular 5-HT levels. To investigate the putative role of the SERT in male sexual behavior. After extensive sexual training, the effects of the 5-HT(1A/7) receptor agonist ± 8-OH-DPAT, the 5-HT(1A) receptor antagonist WAY100 635 and a combination of both on sexual behaviors of SERT(-/-) and SERT(+/-) knockout and wildtype (SERT(+/+) ) male Wistar rats were examined. Male rat sexual behaviors of mounts, intromissions, and ejaculations. SERT(-/-) had lower basal ejaculation frequencies than SERT(+/-) and SERT(+/+) animals. ± 8-OH-DPAT enhanced sexual performance in all three genotypes to the same extent. WAY100635 dose-dependently inhibited sexual behavior in all three genotypes with significant dose to genotype interactions. WAY100635 exerted the strongest effects in SERT(-/-) animals. The combination of a dose range of ± 8-OH-DPAT and a selected dose of WAY100635 revealed only partial antagonism by ± 8-OH-DPAT of the sexual inhibitory effects of WAY100635. Absence of the serotonin transporter reduces basal ejaculatory performance in male rats. Pharmacological experiments suggest that separate pools of 5-HT(1A) receptors regulate different aspects of sexual performance in male rats. 5-HT(7) receptors may play a minor role in the partial recovery of sexual behavior after combination of ± 8-OH-DPAT and WAY100635. The SERT(-/-) rat may be a model for chronic SSRI treatment, delayed ejaculation, anorgasmia, and/or low libido. © 2010 International Society for Sexual Medicine.

Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases.

PubMed

Remy, Séverine; Tesson, Laurent; Menoret, Séverine; Usal, Claire; De Cian, Anne; Thepenier, Virginie; Thinard, Reynald; Baron, Daniel; Charpentier, Marine; Renaud, Jean-Baptiste; Buelow, Roland; Cost, Gregory J; Giovannangeli, Carine; Fraichard, Alexandre; Concordet, Jean-Paul; Anegon, Ignacio

2014-08-01

The generation of genetically modified animals is important for both research and commercial purposes. The rat is an important model organism that until recently lacked efficient genetic engineering tools. Sequence-specific nucleases, such as ZFNs, TALE nucleases, and CRISPR/Cas9 have allowed the creation of rat knockout models. Genetic engineering by homology-directed repair (HDR) is utilized to create animals expressing transgenes in a controlled way and to introduce precise genetic modifications. We applied TALE nucleases and donor DNA microinjection into zygotes to generate HDR-modified rats with large new sequences introduced into three different loci with high efficiency (0.62%-5.13% of microinjected zygotes). Two of these loci (Rosa26 and Hprt1) are known to allow robust and reproducible transgene expression and were targeted for integration of a GFP expression cassette driven by the CAG promoter. GFP-expressing embryos and four Rosa26 GFP rat lines analyzed showed strong and widespread GFP expression in most cells of all analyzed tissues. The third targeted locus was Ighm, where we performed successful exon exchange of rat exon 2 for the human one. At all three loci we observed HDR only when using linear and not circular donor DNA. Mild hypothermic (30°C) culture of zygotes after microinjection increased HDR efficiency for some loci. Our study demonstrates that TALE nuclease and donor DNA microinjection into rat zygotes results in efficient and reproducible targeted donor integration by HDR. This allowed creation of genetically modified rats in a work-, cost-, and time-effective manner. © 2014 Remy et al.; Published by Cold Spring Harbor Laboratory Press.

Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases

PubMed Central

Remy, Séverine; Tesson, Laurent; Menoret, Séverine; Usal, Claire; De Cian, Anne; Thepenier, Virginie; Thinard, Reynald; Baron, Daniel; Charpentier, Marine; Renaud, Jean-Baptiste; Buelow, Roland; Cost, Gregory J.; Giovannangeli, Carine; Fraichard, Alexandre; Concordet, Jean-Paul; Anegon, Ignacio

2014-01-01

The generation of genetically modified animals is important for both research and commercial purposes. The rat is an important model organism that until recently lacked efficient genetic engineering tools. Sequence-specific nucleases, such as ZFNs, TALE nucleases, and CRISPR/Cas9 have allowed the creation of rat knockout models. Genetic engineering by homology-directed repair (HDR) is utilized to create animals expressing transgenes in a controlled way and to introduce precise genetic modifications. We applied TALE nucleases and donor DNA microinjection into zygotes to generate HDR-modified rats with large new sequences introduced into three different loci with high efficiency (0.62%–5.13% of microinjected zygotes). Two of these loci (Rosa26 and Hprt1) are known to allow robust and reproducible transgene expression and were targeted for integration of a GFP expression cassette driven by the CAG promoter. GFP-expressing embryos and four Rosa26 GFP rat lines analyzed showed strong and widespread GFP expression in most cells of all analyzed tissues. The third targeted locus was Ighm, where we performed successful exon exchange of rat exon 2 for the human one. At all three loci we observed HDR only when using linear and not circular donor DNA. Mild hypothermic (30°C) culture of zygotes after microinjection increased HDR efficiency for some loci. Our study demonstrates that TALE nuclease and donor DNA microinjection into rat zygotes results in efficient and reproducible targeted donor integration by HDR. This allowed creation of genetically modified rats in a work-, cost-, and time-effective manner. PMID:24989021

Polycystin-1 Is a Cardiomyocyte Mechanosensor That Governs L-Type Ca2+ Channel Protein Stability.

PubMed

Pedrozo, Zully; Criollo, Alfredo; Battiprolu, Pavan K; Morales, Cyndi R; Contreras-Ferrat, Ariel; Fernández, Carolina; Jiang, Nan; Luo, Xiang; Caplan, Michael J; Somlo, Stefan; Rothermel, Beverly A; Gillette, Thomas G; Lavandero, Sergio; Hill, Joseph A

2015-06-16

L-type calcium channel activity is critical to afterload-induced hypertrophic growth of the heart. However, the mechanisms governing mechanical stress-induced activation of L-type calcium channel activity are obscure. Polycystin-1 (PC-1) is a G protein-coupled receptor-like protein that functions as a mechanosensor in a variety of cell types and is present in cardiomyocytes. We subjected neonatal rat ventricular myocytes to mechanical stretch by exposing them to hypo-osmotic medium or cyclic mechanical stretch, triggering cell growth in a manner dependent on L-type calcium channel activity. RNAi-dependent knockdown of PC-1 blocked this hypertrophy. Overexpression of a C-terminal fragment of PC-1 was sufficient to trigger neonatal rat ventricular myocyte hypertrophy. Exposing neonatal rat ventricular myocytes to hypo-osmotic medium resulted in an increase in α1C protein levels, a response that was prevented by PC-1 knockdown. MG132, a proteasomal inhibitor, rescued PC-1 knockdown-dependent declines in α1C protein. To test this in vivo, we engineered mice harboring conditional silencing of PC-1 selectively in cardiomyocytes (PC-1 knockout) and subjected them to mechanical stress in vivo (transverse aortic constriction). At baseline, PC-1 knockout mice manifested decreased cardiac function relative to littermate controls, and α1C L-type calcium channel protein levels were significantly lower in PC-1 knockout hearts. Whereas control mice manifested robust transverse aortic constriction-induced increases in cardiac mass, PC-1 knockout mice showed no significant growth. Likewise, transverse aortic constriction-elicited increases in hypertrophic markers and interstitial fibrosis were blunted in the knockout animals PC-1 is a cardiomyocyte mechanosensor that is required for cardiac hypertrophy through a mechanism that involves stabilization of α1C protein. © 2015 American Heart Association, Inc.

Concussion: comparison of humans and rats.

PubMed

Parkinson, D; West, M; Pathiraja, T

1978-01-01

Concussion is the most primitive and probably the most simple central nervous system response to an outside agent. Although millions of neurons undergo a very rapid reversal with loss and restoration of function, many physicians argue that every knockout blow leaves some residual damage, i.e., some or all of these neurons stop short of recovery. Clinical experience shows a wide discrepancy in the appearance or persistence of certain signs and symptoms after all types of head injury, but particularly after concussing blows, when third party or grievance cases are involved. Using slow motion filmstrips of boxing ring knockouts, we established a grading system for concussion and duplicated these grades in nonanesthetized rats. When we compared the performance, learning, and memory of concussed rats with their preconcussion performance and with the performance of nonconcussed controls, we were unable to find any differences.

Chronic central serotonin depletion attenuates ventilation and body temperature in young but not adult Tph2 knockout rats.

PubMed

Kaplan, Kara; Echert, Ashley E; Massat, Ben; Puissant, Madeleine M; Palygin, Oleg; Geurts, Aron M; Hodges, Matthew R

2016-05-01

Genetic deletion of brain serotonin (5-HT) neurons in mice leads to ventilatory deficits and increased neonatal mortality during development. However, it is unclear if the loss of the 5-HT neurons or the loss of the neurochemical 5-HT led to the observed physiologic deficits. Herein, we generated a mutant rat model with constitutive central nervous system (CNS) 5-HT depletion by mutation of the tryptophan hydroxylase 2 (Tph2) gene in dark agouti (DA(Tph2-/-)) rats. DA(Tph2-/-) rats lacked TPH immunoreactivity and brain 5-HT but retain dopa decarboxylase-expressing raphe neurons. Mutant rats were also smaller, had relatively high mortality (∼50%), and compared with controls had reduced room air ventilation and body temperatures at specific postnatal ages. In adult rats, breathing at rest and hypoxic and hypercapnic chemoreflexes were unaltered in adult male and female DA(Tph2-/-) rats. Body temperature was also maintained in adult DA(Tph2-/-) rats exposed to 4°C, indicating unaltered ventilatory and/or thermoregulatory control mechanisms. Finally, DA(Tph2-/-) rats treated with the 5-HT precursor 5-hydroxytryptophan (5-HTP) partially restored CNS 5-HT and showed increased ventilation (P < 0.05) at a developmental age when it was otherwise attenuated in the mutants. We conclude that constitutive CNS production of 5-HT is critically important to fundamental homeostatic control systems for breathing and temperature during postnatal development in the rat. Copyright © 2016 the American Physiological Society.

Chronic central serotonin depletion attenuates ventilation and body temperature in young but not adult Tph2 knockout rats

PubMed Central

Kaplan, Kara; Echert, Ashley E.; Massat, Ben; Puissant, Madeleine M.; Palygin, Oleg; Geurts, Aron M.

2016-01-01

Genetic deletion of brain serotonin (5-HT) neurons in mice leads to ventilatory deficits and increased neonatal mortality during development. However, it is unclear if the loss of the 5-HT neurons or the loss of the neurochemical 5-HT led to the observed physiologic deficits. Herein, we generated a mutant rat model with constitutive central nervous system (CNS) 5-HT depletion by mutation of the tryptophan hydroxylase 2 (Tph2) gene in dark agouti (DATph2−/−) rats. DATph2−/− rats lacked TPH immunoreactivity and brain 5-HT but retain dopa decarboxylase-expressing raphe neurons. Mutant rats were also smaller, had relatively high mortality (∼50%), and compared with controls had reduced room air ventilation and body temperatures at specific postnatal ages. In adult rats, breathing at rest and hypoxic and hypercapnic chemoreflexes were unaltered in adult male and female DATph2−/− rats. Body temperature was also maintained in adult DATph2−/− rats exposed to 4°C, indicating unaltered ventilatory and/or thermoregulatory control mechanisms. Finally, DATph2−/− rats treated with the 5-HT precursor 5-hydroxytryptophan (5-HTP) partially restored CNS 5-HT and showed increased ventilation (P < 0.05) at a developmental age when it was otherwise attenuated in the mutants. We conclude that constitutive CNS production of 5-HT is critically important to fundamental homeostatic control systems for breathing and temperature during postnatal development in the rat. PMID:26869713

Developing a Mouse Model of Sensory and Cognitive Deficits for Multiple Sclerosis

DTIC Science & Technology

2012-07-01

ABRs and otoacoustic emissions. More sophisticates measures, such as neural processing of binaural responses are typically performed in rats, guinea...ears we are able to calculate the binaural component of the EEGs for comparison of wild type and Claudin 11 knockout responses. We are awaiting the...knockout of the Claudin 11 gene. 2. Development of a novel anesthesia protocol to measure binaural auditory signals in the superior olivary complex of

THE EFFECT OF A TARGETED KNOCKOUT MUTATION ON THE TRANSCRIPTIONAL PROFILE OF THE KIDNEY IN TSC2 MUTANT (EKER) RATS.

EPA Science Inventory

Renal cell carcinoma (RCC) is the most common tumor of the adult kidney, accounting for up to 80% of malignant renal neoplasms. Hereditary RCC in the Eker rat, which bear a number of cellular, molecular and phenotypic similarities to human RCC, results from an inherited insertion...

Essential role of Kir5.1 channels in renal salt handling and blood pressure control

PubMed Central

Levchenko, Vladislav; Ilatovskaya, Daria V.; Pavlov, Tengis S.; Pochynyuk, Oleh M.; Jacob, Howard J.; Geurts, Aron M.; Hodges, Matthew R.

2017-01-01

Supplementing diets with high potassium helps reduce hypertension in humans. Inwardly rectifying K+ channels Kir4.1 (Kcnj10) and Kir5.1 (Kcnj16) are highly expressed in the basolateral membrane of distal renal tubules and contribute to Na+ reabsorption and K+ secretion through the direct control of transepithelial voltage. To define the importance of Kir5.1 in blood pressure control under conditions of salt-induced hypertension, we generated a Kcnj16 knockout in Dahl salt-sensitive (SS) rats (SSKcnj16–/–). SSKcnj16–/– rats exhibited hypokalemia and reduced blood pressure, and when fed a high-salt diet (4% NaCl), experienced 100% mortality within a few days triggered by salt wasting and severe hypokalemia. Electrophysiological recordings of basolateral K+ channels in the collecting ducts isolated from SSKcnj16–/– rats revealed activity of only homomeric Kir4.1 channels. Kir4.1 expression was upregulated in SSKcnj16–/– rats, but the protein was predominantly localized in the cytosol in SSKcnj16–/– rats. Benzamil, but not hydrochlorothiazide or furosemide, rescued this phenotype from mortality on a high-salt diet. Supplementation of high-salt diet with increased potassium (2% KCl) prevented mortality in SSKcnj16–/– rats and prevented or mitigated hypertension in SSKcnj16–/– or control SS rats, respectively. Our results demonstrate that Kir5.1 channels are key regulators of renal salt handling in SS hypertension. PMID:28931751

PTTG expression in different experimental and human prolactinomas in relation to dopaminergic control of lactotropes

PubMed Central

Cristina, Carolina; Díaz-Torga, Graciela S; Goya, Rodolfo G; Kakar, Sham S; Perez-Millán, María I; Passos, Vanessa Q; Giannella-Neto, Daniel; Bronstein, Marcello D; Becu-Villalobos, Damasia

2007-01-01

Background Pituitary tumor transforming gene (pttg) is a novel oncogene that is expressed at higher level in most of the tumors analyzed to date compared to normal tissues. Nevertheless, its expression in prolactinomas and its relation with the pituitary dopamine receptor 2 (D2R) are not well defined. We sought to determine the pituitary level of pttg in three different experimental models of prolactinomas with altered dopaminergic control of the pituitary: the dopaminergic D2R knockout female mouse, the estrogen-treated rat, and the senescent female rat. These three models shared the characteristics of increased pituitary weight, hyperprolactinemia, lactotrope hyperplasia and reduced or absent dopaminergic action at the pituitary level. We also studied samples from human macroprolactinomas, which were characterized as responsive or resistant to dopamine agonist therapy. Results When compared to female wild-type mice, pituitaries from female D2R knockout mice had decreased PTTG concentration, while no difference in pttg mRNA level was found. In senescent rats no difference in pituitary PTTG protein expression was found when compared to young rats. But, in young female rats treated with a synthetic estrogen (Diethylstylbestrol, 20 mg) PTTG protein expression was enhanced (P = 0.029). Therefore, in the three experimental models of prolactinomas, pituitary size was increased and there was hyperprolactinemia, but PTTG levels followed different patterns. Patients with macroprolactinomas were divided in those in which dopaminergic therapy normalized or failed to normalize prolactin levels (responsive and resistant, respectively). When pituitary pttg mRNA level was analyzed in these macroprolactinomas, no differences were found. We next analyzed estrogen action at the pituitary by measuring pituitary estrogen receptor α levels. The D2R knockout female mice have low estrogen levels and in accordance, pituitary estrogen receptors were increased (P = 0.047). On the other hand, in senescent rats estrogen levels were slightly though not significantly higher, and estrogen receptors were similar between groups. The estrogen-treated rats had high pharmacological levels of the synthetic estrogen, and estrogen receptors were markedly lower than in controls (P < 0.0001). Finally, in patients with dopamine resistant or responsive prolactinomas no significant differences in estrogen receptor α levels were found. Therefore, pituitary PTTG was increased only if estrogen action was increased, which correlated with a decrease in pituitary estrogen receptor level. Conclusion We conclude that PTTG does not correlate with prolactin levels or tumor size in animal models of prolactinoma, and its pituitary content is not related to a decrease in dopaminergic control of the lactotrope, but may be influenced by estrogen action at the pituitary level. Therefore it is increased only in prolactinomas generated by estrogen treatment, and not in prolactinomas arising from deficient dopamine control, or in dopamine resistant compared with dopamine responsive human prolactinomas. These results are important in the search for reliable prognostic indicators for patients with pituitary adenomas which will make tumor-specific therapy possible, and help to elucidate the poorly understood phenomenon of pituitary tumorigenesis. PMID:17222350

Sarcocystis cymruensis: Discovery in Western hemisphere in the Brown rat (Rattus norvegicus) from Grenada, West Indies: Redescription, molecular characterization, transmission to IFN-gamma gene knockout mice via sporocysts

USDA-ARS?s Scientific Manuscript database

Rodents are intermediate hosts for many species of Sarcocystis. Little is known of Sarcocystis cymruensis that uses the Brown rat (Rattus norvegicus) as intermediate hosts and the domestic cat (Felis catus) as experimental definitive host. Here, we identified and described Sarcocystis cymruensis in ...

Acute Depletion of D2 Receptors from the Rat Substantia Nigra Alters Dopamine Kinetics in the Dorsal Striatum and Drug Responsivity

PubMed Central

Budygin, Evgeny A.; Oleson, Erik B.; Lee, Yun Beom; Blume, Lawrence C.; Bruno, Michael J.; Howlett, Allyn C.; Thompson, Alexis C.; Bass, Caroline E.

2017-01-01

Recent studies have used conditional knockout mice to selectively delete the D2 autoreceptor; however, these approaches result in global deletion of D2 autoreceptors early in development. The present study takes a different approach using RNA interference (RNAi) to knockdown the expression of the D2 receptors (D2R) in the substantia nigra (SN), including dopaminergic neurons, which project primarily to the dorsal striatum (dStr) in adult rats. This approach restricts the knockdown primarily to nigrostriatal pathways, leaving mesolimbic D2 autoreceptors intact. Analyses of dopamine (DA) kinetics in the dStr reveal a decrease in DA transporter (DAT) function in the knockdown rats, an effect not observed in D2 autoreceptor knockout mouse models. SN D2 knockdown rats exhibit a behavioral phenotype characterized by persistent enhancement of locomotor activity in a familiar open field, reduced locomotor responsiveness to high doses of cocaine and the ability to overcome haloperidol-induced immobility on the bar test. Together these results demonstrate that presynaptic D2R can be depleted from specific neuronal populations and implicates nigrostriatal D2R in different behavioral responses to psychotropic drugs. PMID:28154530

Enhanced Mitochondrial Transient Receptor Potential Channel, Canonical Type 3-Mediated Calcium Handling in the Vasculature From Hypertensive Rats.

PubMed

Wang, Bin; Xiong, Shiqiang; Lin, Shaoyang; Xia, Weijie; Li, Qiang; Zhao, Zhigang; Wei, Xing; Lu, Zongshi; Wei, Xiao; Gao, Peng; Liu, Daoyan; Zhu, Zhiming

2017-07-15

Mitochondrial Ca 2+ homeostasis is fundamental to the regulation of mitochondrial reactive oxygen species (ROS) generation and adenosine triphosphate production. Recently, transient receptor potential channel, canonical type 3 (TRPC3), has been shown to localize to the mitochondria and to play a role in maintaining mitochondrial calcium homeostasis. Inhibition of TRPC3 attenuates vascular calcium influx in spontaneously hypertensive rats (SHRs). However, it remains elusive whether mitochondrial TRPC3 participates in hypertension by increasing mitochondrial calcium handling and ROS production. In this study we demonstrated increased TRPC3 expression in purified mitochondria in the vasculature from SHRs, which facilitates enhanced mitochondrial calcium uptake and ROS generation compared with Wistar-Kyoto rats. Furthermore, inhibition of TRPC3 by its specific inhibitor, Pyr3, significantly decreased the vascular mitochondrial ROS production and H 2 O 2 synthesis and increased adenosine triphosphate content. Administration of telmisartan can improve these abnormalities. This beneficial effect was associated with improvement of the mitochondrial respiratory function through recovering the activity of pyruvate dehydrogenase in the vasculature of SHRs. In vivo, chronic administration of telmisartan suppressed TRPC3-mediated excessive mitochondrial ROS generation and vasoconstriction in the vasculature of SHRs. More importantly, TRPC3 knockout mice exhibited significantly ameliorated hypertension through reduction of angiotensin II-induced mitochondrial ROS generation. Together, we give experimental evidence for a potential mechanism by which enhanced TRPC3 activity at the cytoplasmic and mitochondrial levels contributes to redox signaling and calcium dysregulation in the vasculature from SHRs. Angiotensin II or telmisartan can regulate [Ca 2+ ] mito , ROS production, and mitochondrial energy metabolism through targeting TRPC3. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

The mGluR2/3 Agonist LY379268 Induced Anti-Reinstatement Effects in Rats Exhibiting Addiction-like Behavior

PubMed Central

Cannella, Nazzareno; Halbout, Briac; Uhrig, Stefanie; Evrard, Lionel; Corsi, Mauro; Corti, Corrado; Deroche-Gamonet, Veronique; Hansson, Anita C; Spanagel, Rainer

2013-01-01

Medication development for cocaine-addicted patients is difficult, and many promising preclinical candidates have failed in clinical trials. One reason for the difficulty in translating preclinical findings to the human condition is that drug testing is typically conducted in behavioral procedures in which animals do not show addiction-like traits. Recently, a DSM-IV-based animal model has been developed that allows studying the transition to an addiction-like behavior. Changes in synaptic plasticity are involved in the transition to cocaine addiction. In particular, it has been shown that metabotropic glutamate receptor 2/3 (mGluR2/3)-mediated long-term depression is suppressed in the prelimbic cortex in addict-like rats. We therefore hypothesized that cocaine-seeking in addict-like rats could be treated with an mGluR2/3 agonist. Indeed, addict-like rats that were treated systemically with the mGluR2/3 agonist LY379268 (0, 0.3, and 3 mg/kg) showed a pronounced reduction in cue-induced reinstatement of cocaine-seeking. In an attempt to dissect the role played by mGluR2 and mGluR3 in cue-induced reinstatement, we analyzed the mRNA expression patterns in several relevant brain areas but did not find any significant differences between cocaine addict-like and non-addict-like rats, suggesting that the behavioral differences observed are due to translational rather than transcriptional regulation. Another possibility to study the contributions of mGluR2 and mGluR3 in mediating addictive-like behavior is the use of knockout models. Because mGluR2 knockouts cannot be used in operant procedures due to motoric impairment, we only tested mGluR3 knockouts. These mice did not differ from controls in reinstatement, suggesting that mGluR2 receptors are critical in mediating addictive-like behavior. PMID:23624743

Antithrombin III/SerpinC1 insufficiency exacerbates renal ischemia/reperfusion injury

PubMed Central

Wang, Feng; Zhang, Guangyuan; Lu, Zeyuan; Geurts, Aron M; Usa, Kristie; Jacob, Howard J; Cowley, Allen W; Wang, Niansong; Liang, Mingyu

2015-01-01

Antithrombin III, encoded by SerpinC1, is a major anti-coagulation molecule in vivo and has anti-inflammatory effects. We found that patients with low antithrombin III activities presented a higher risk of developing acute kidney injury after cardiac surgery. To study this further, we generated SerpinC1 heterozygous knockout rats and followed the development of acute kidney injury in a model of modest renal ischemia/reperfusion injury. Renal injury, assessed by serum creatinine and renal tubular injury scores after 24 h of reperfusion, was significantly exacerbated in SerpinC1+/− rats compared to wild-type littermates. Concomitantly, renal oxidative stress, tubular apoptosis, and macrophage infiltration following this injury were significantly aggravated in SerpinC1+/− rats. However, significant thrombosis was not found in the kidneys of any group of rats. Antithrombin III is reported to stimulate the production of prostaglandin I2, a known regulator of renal cortical blood flow, in addition to having anti-inflammatory effects and to protect against renal failure. Prostaglandin F1α, an assayable metabolite of prostaglandin I2, was increased in the kidneys of the wild-type rats at 3 h after reperfusion. The increase of prostaglandin F1α was significantly blunted in SerpinC1+/− rats, which preceded increased tubular injury and oxidative stress. Thus, our study found a novel role of SerpinC1 insufficiency in increasing the severity of renal ischemia/reperfusion injury. PMID:26108065

Acute food deprivation reverses morphine-induced locomotion deficits in M5 muscarinic receptor knockout mice.

PubMed

Steidl, Stephan; Lee, Esther; Wasserman, David; Yeomans, John S

2013-09-01

Lesions of the pedunculopontine tegmental nucleus (PPT), one of two sources of cholinergic input to the ventral tegmental area (VTA), block conditioned place preference (CPP) for morphine in drug-naïve rats. M5 muscarinic cholinergic receptors, expressed by midbrain dopamine neurons, are critical for the ability of morphine to increase nucleus accumbens dopamine levels and locomotion, and for morphine CPP. This suggests that M5-mediated PPT cholinergic inputs to VTA dopamine neurons critically contribute to morphine-induced dopamine activation, reward and locomotion. In the current study we tested whether food deprivation, which reduces PPT contribution to morphine CPP in rats, could also reduce M5 contributions to morphine-induced locomotion in mice. Acute 18-h food deprivation reversed the phenotypic differences usually seen between non-deprived wild-type and M5 knockout mice. That is, food deprivation increased morphine-induced locomotion in M5 knockout mice but reduced morphine-induced locomotion in wild-type mice. Food deprivation increased saline-induced locomotion equally in wild-type and M5 knockout mice. Based on these findings, we suggest that food deprivation reduces the contribution of M5-mediated PPT cholinergic inputs to the VTA in morphine-induced locomotion and increases the contribution of a PPT-independent pathway. The contributions of cholinergic, dopaminergic and GABAergic neurons to the effects of acute food deprivation are discussed. Copyright © 2013 Elsevier B.V. All rights reserved.

Animal models of pituitary neoplasia

PubMed Central

Lines, K.E.; Stevenson, M.; Thakker, R.V.

2016-01-01

Pituitary neoplasias can occur as part of a complex inherited disorder, or more commonly as sporadic (non-familial) disease. Studies of the molecular and genetic mechanisms causing such pituitary tumours have identified dysregulation of >35 genes, with many revealed by studies in mice, rats and zebrafish. Strategies used to generate these animal models have included gene knockout, gene knockin and transgenic over-expression, as well as chemical mutagenesis and drug induction. These animal models provide an important resource for investigation of tissue-specific tumourigenic mechanisms, and evaluations of novel therapies, illustrated by studies into multiple endocrine neoplasia type 1 (MEN1), a hereditary syndrome in which ∼30% of patients develop pituitary adenomas. This review describes animal models of pituitary neoplasia that have been generated, together with some recent advances in gene editing technologies, and an illustration of the use of the Men1 mouse as a pre clinical model for evaluating novel therapies. PMID:26320859

Stem cell potency and the ability to contribute to chimeric organisms.

PubMed

Polejaeva, Irina; Mitalipov, Shoukhrat

2013-03-01

Mouse embryonic chimeras are a well-established tool for studying cell lineage commitment and pluripotency. Experimental chimeras were successfully produced by combining two or more preimplantation embryos or by introducing into host embryo cultured pluripotent embryonic stem cells (ESCs). Chimera production using genetically modified ESCs became the method of choice for the generation of knockout or knockin mice. Although the derivation of ESCs or ESC-like cells has been reported for other species, only mouse and rat pluripotent stem cells have been shown to contribute to germline-competent chimeras, which is the defining feature of ESCs. Herein, we describe different approaches employed for the generation of embryonic chimeras, define chimera-competent cell types, and describe cases of spontaneous chimerism in humans. We also review the current state of derivation of pluripotent stem cells in several species and discuss outcomes of various chimera studies when such cells are used.

Novel Therapeutic Targets to Inhibit Tumor Microenvironment-Induced Castration Resistant Prostate Cancer

DTIC Science & Technology

2016-10-01

MAPK4. We are also in the process of generating MAPK4-knockout LNCaP cells using the CRISPR /Cas9 system. Altogether, we hope that we can generate the...engineered LNCaP cells that are stable for either total loss of MAPK4 ( CRISPR /Cas9 knockout) or with inducible knockdown of MAPK4 (Dox-inducible...knockdown or CRISPR /Cas9 mediated knockout of MAPK4 (we are working on them). Major Task 2: Determine whether inhibition of MAPK4 (in PCa) and TGF-β

Acute and constitutive increases in central serotonin levels reduce social play behaviour in peri-adolescent rats

PubMed Central

Schiepers, Olga J. G.; Schoffelmeer, Anton N. M.; Cuppen, Edwin; Vanderschuren, Louk J. M. J.

2007-01-01

Rationale Serotonin is an important modulator of social behaviour. Individual differences in serotonergic signalling are considered to be a marker of personality that is stable throughout lifetime. While a large body of evidence indicates that central serotonin levels are inversely related to aggression and sexual behaviour in adult rats, the relationship between serotonin and social behaviour during peri-adolescence has hardly been explored. Objective To study the effect of acute and constitutive increases in serotonin neurotransmission on social behaviour in peri-adolescent rats. Materials and methods Social behaviour in peri-adolesent rats (28–35 days old) was studied after genetic ablation of the serotonin transporter, causing constitutively increased extra-neuronal serotonin levels, and after acute treatment with the serotonin reuptake inhibitor fluoxetine or the serotonin releasing agent 3,4-methylenedioxymethamphetamine (MDMA). A distinction was made between social play behaviour that mainly occurs during peri-adolescence, and non-playful social interactions that are abundant during the entire lifespan of rats. Results In serotonin transporter knockout rats, social play behaviour was markedly reduced, while non-playful aspects of social interaction were unaffected. Acute treatment with fluoxetine or MDMA dose-dependently inhibited social play behaviour. MDMA also suppressed non-playful social interaction but at higher doses than those required to reduce social play. Fluoxetine did not affect non-playful social interaction. Conclusions These data show that both acute and constitutive increases in serotonergic neurotransmission reduce social play behaviour in peri-adolescent rats. Together with our previous findings of reduced aggressive and sexual behaviour in adult serotonin transporter knockout rats, these data support the notion that serotonin modulates social behaviour in a trait-like manner. PMID:17661017

Bile Salt Homeostasis in Normal and Bsep Gene Knockout Rats with Single and Repeated Doses of Troglitazone.

PubMed

Cheng, Yaofeng; Chen, Shenjue; Freeden, Chris; Chen, Weiqi; Zhang, Yueping; Abraham, Pamela; Nelson, David M; Humphreys, W Griffith; Gan, Jinping; Lai, Yurong

2017-09-01

The interference of bile acid secretion through bile salt export pump (BSEP) inhibition is one of the mechanisms for troglitazone (TGZ)-induced hepatotoxicity. Here, we investigated the impact of single or repeated oral doses of TGZ (200 mg/kg/day, 7 days) on bile acid homoeostasis in wild-type (WT) and Bsep knockout (KO) rats. Following oral doses, plasma exposures of TGZ were not different between WT and KO rats, and were similar on day 1 and day 7. However, plasma exposures of the major metabolite, troglitazone sulfate (TS), in KO rats were 7.6- and 9.3-fold lower than in WT on day 1 and day 7, respectively, due to increased TS biliary excretion. With Bsep KO, the mRNA levels of multidrug resistance-associated protein 2 (Mrp2), Mrp3, Mrp4, Mdr1, breast cancer resistance protein (Bcrp), sodium taurocholate cotransporting polypeptide, small heterodimer partner, and Sult2A1 were significantly altered in KO rats. Following seven daily TGZ treatments, Cyp7A1 was significantly increased in both WT and KO rats. In the vehicle groups, plasma exposures of individual bile acids demonstrated variable changes in KO rats as compared with WT. WT rats dosed with TGZ showed an increase of many bile acid species in plasma on day 1, suggesting the inhibition of Bsep. Conversely, these changes returned to base levels on day 7. In KO rats, alterations of most bile acids were observed after seven doses of TGZ. Collectively, bile acid homeostasis in rats was regulated through bile acid synthesis and transport in response to Bsep deficiency and TGZ inhibition. Additionally, our study is the first to demonstrate that repeated TGZ doses can upregulate Cyp7A1 in rats. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Effect of Dietary Treatment with Dimethylarsinous Acid (DMAIII) on the Urinary Bladder Epithelium of Arsenic (+3 Oxidation State) Methyltransferase (As3mt) Knockout and C57BL/6 Wild Type Female Mice

EPA Science Inventory

Abstract Chronic exposure to inorganic arsenic (iAs) is carcinogenic to the human urinary bladder. It produces urothelial cytotoxicity and proliferation in rats and mice. DMAv, a major methylated urinary metabolite of iAs, is a rat bladder carcinogen, but without effects on the...

Electroacupuncture preconditioning-induced neuroprotection may be mediated by glutamate transporter type 2

PubMed Central

Zhu, Xiaoling; Yin, Jinbo; Li, Liaoliao; Ma, Lei; Tan, Hongying; Deng, Jiao; Chen, Shaoyang; Zuo, Zhiyi

2013-01-01

Electroacupuncture has been shown to induce a preconditioning effect in the brain. The mechanisms for this protection are not fully elucidated. We hypothesize that this protection is mediated by excitatory amino acid transporters (EAATs) that have been shown to be neuroprotective. To test this hypothesis, two-month old male Sprague-Dawley rats and EAAT type 3 (EAAT3) knockout mice received or did not receive 30-min electroacupuncture once a day for 5 consecutive days. They were subjected to a 120-min middle cerebral arterial occlusion (MCAO) at 24 h after the last electroacupuncture. Neurological outcome was assessed 2 days after the MCAO. Brain tissues were harvested at 24 h after the last electroacupuncture for Western blotting. Rats subjected to electroacupuncture at the Baihui acupoint had smaller brain infarct volumes and better neurological deficit scores than control rats. Electroacupuncture increased EAAT type 2 (EAAT2) in the cerebral cortex, tended to increase EAAT3 in the hippocampus, and had no effect on EAAT type 1 expression. Dihydrokainate, an EAAT2 inhibitor, worsened the neurological outcome of rats with electroacupuncture pretreatment. Electroacupuncture pretreatment at the Baihui acupoint increased EAAT2 in the cerebral cortex and improved the neurological outcome of EAAT3 knockout mice. Together, our results suggest that EAAT2 may mediate the electroacupuncture preconditioning-induced neuroprotection. PMID:23831620

Cytoprotective role of autophagy against BH3 mimetic gossypol in ATG5 knockout cells generated by CRISPR-Cas9 endonuclease.

PubMed

Kim, Na-Yeon; Han, Byeal-I; Lee, Michael

2016-01-01

Previously, we demonstrated the association between autophagy and gossypol-induced growth inhibition of mutant BRAF melanoma cells. Here, we investigate the role of autophagy in ATG5 knockout cell lines generated by the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas-mediated genome editing. The MTT assay revealed that the inhibitory effect of gossypol was weaker on ATG5 knockout cells than that on the wild type (WT) cells. The conversion of non-autophagic LC3-I to autophagic LC3-II and RT-PCR confirmed the functional gene knockout. However, Cyto-ID autophagy assay revealed that gossypol induced ATG5- and LC3-independent autophagy in ATG5 knockout cells. Moreover, gossypol acts as an autophagy inducer in ATG5 knockout cells while blocking the later stages of the autophagy process in WT cells, which was determined by measuring autophagic flux after co-treatment of gossypol with chloroquine (late-stage autophagy inhibitor). On the other hand, inhibition of autophagy with 3-MA or Beclin-1 siRNA caused a partial increase in the sensitivity to gossypol in ATG5 knockout cells, but not in the WT cells. Together, our findings suggest that the resistance to gossypol in ATG5 knockout cells is associated with increased cytoprotective autophagy, independent of ATG5. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

One-step generation of complete gene knockout mice and monkeys by CRISPR/Cas9-mediated gene editing with multiple sgRNAs.

PubMed

Zuo, Erwei; Cai, Yi-Jun; Li, Kui; Wei, Yu; Wang, Bang-An; Sun, Yidi; Liu, Zhen; Liu, Jiwei; Hu, Xinde; Wei, Wei; Huo, Xiaona; Shi, Linyu; Tang, Cheng; Liang, Dan; Wang, Yan; Nie, Yan-Hong; Zhang, Chen-Chen; Yao, Xuan; Wang, Xing; Zhou, Changyang; Ying, Wenqin; Wang, Qifang; Chen, Ren-Chao; Shen, Qi; Xu, Guo-Liang; Li, Jinsong; Sun, Qiang; Xiong, Zhi-Qi; Yang, Hui

2017-07-01

The CRISPR/Cas9 system is an efficient gene-editing method, but the majority of gene-edited animals showed mosaicism, with editing occurring only in a portion of cells. Here we show that single gene or multiple genes can be completely knocked out in mouse and monkey embryos by zygotic injection of Cas9 mRNA and multiple adjacent single-guide RNAs (spaced 10-200 bp apart) that target only a single key exon of each gene. Phenotypic analysis of F0 mice following targeted deletion of eight genes on the Y chromosome individually demonstrated the robustness of this approach in generating knockout mice. Importantly, this approach delivers complete gene knockout at high efficiencies (100% on Arntl and 91% on Prrt2) in monkey embryos. Finally, we could generate a complete Prrt2 knockout monkey in a single step, demonstrating the usefulness of this approach in rapidly establishing gene-edited monkey models.

The importance of immunohistochemical analyses in evaluating the phenotype of Kv channel knockout mice.

PubMed

Menegola, Milena; Clark, Eliana; Trimmer, James S

2012-06-01

To gain insights into the phenotype of voltage-gated potassium (Kv)1.1 and Kv4.2 knockout mice, we used immunohistochemistry to analyze the expression of component principal or α subunits and auxiliary subunits of neuronal Kv channels in knockout mouse brains. Genetic ablation of the Kv1.1 α subunit did not result in compensatory changes in the expression levels or subcellular distribution of related ion channel subunits in hippocampal medial perforant path and mossy fiber nerve terminals, where high levels of Kv1.1 are normally expressed. Genetic ablation of the Kv4.2 α subunit did not result in altered neuronal cytoarchitecture of the hippocampus. Although Kv4.2 knockout mice did not exhibit compensatory changes in the expression levels or subcellular distribution of the related Kv4.3 α subunit, we found dramatic decreases in the cellular and subcellular expression of specific Kv channel interacting proteins (KChIPs) that reflected their degree of association and colocalization with Kv4.2 in wild-type mouse and rat brains. These studies highlight the insights that can be gained by performing detailed immunohistochemical analyses of Kv channel knockout mouse brains. Wiley Periodicals, Inc. © 2012 International League Against Epilepsy.

Sleep/Wake Physiology and Quantitative Electroencephalogram Analysis of the Neuroligin-3 Knockout Rat Model of Autism Spectrum Disorder.

PubMed

Thomas, Alexia M; Schwartz, Michael D; Saxe, Michael D; Kilduff, Thomas S

2017-10-01

Neuroligin-3 (NLGN3) is one of the many genes associated with autism spectrum disorder (ASD). Sleep dysfunction is highly prevalent in ASD, but has not been rigorously examined in ASD models. Here, we evaluated sleep/wake physiology and behavioral phenotypes of rats with genetic ablation of Nlgn3. Male Nlgn3 knockout (KO) and wild-type (WT) rats were assessed using a test battery for ASD-related behaviors and also implanted with telemeters to record the electroencephalogram (EEG), electromyogram, body temperature, and locomotor activity. 24-h EEG recordings were analyzed for sleep/wake states and spectral composition. Nlgn3 KO rats were hyperactive, exhibited excessive chewing behavior, and had impaired prepulse inhibition to an auditory startle stimulus. KO rats also spent less time in non-rapid eye movement (NREM) sleep, more time in rapid eye movement (REM) sleep, exhibited elevated theta power (4-9 Hz) during wakefulness and REM, and elevated delta power (0.5-4 Hz) during NREM. Beta (12-30 Hz) power and gamma (30-50 Hz) power were suppressed across all vigilance states. The sleep disruptions in Nlgn3 KO rats are consistent with observations of sleep disturbances in ASD patients. The EEG provides objective measures of brain function to complement rodent behavioral analyses and therefore may be a useful tool to study ASD. © Sleep Research Society 2017. Published by Oxford University Press on behalf of the Sleep Research Society. All rights reserved. For permissions, please e-mail journals.permissions@oup.com.

Evidence of Neurobiological Changes in the Presymptomatic PINK1 Knockout Rat.

PubMed

Ferris, Craig F; Morrison, Thomas R; Iriah, Sade; Malmberg, Samantha; Kulkarni, Praveen; Hartner, Jochen C; Trivedi, Malav

2018-01-01

Genetic models of Parkinson's disease (PD) coupled with advanced imaging techniques can elucidate neurobiological disease progression, and can help identify early biomarkers before clinical signs emerge. PTEN-induced putative kinase 1 (PINK1) helps protect neurons from mitochondrial dysfunction, and a mutation in the associated gene is a risk factor for recessive familial PD. The PINK1 knockout (KO) rat is a novel model for familial PD that has not been neuroradiologically characterized for alterations in brain structure/function, alongside behavior, prior to 4 months of age. To identify biomarkers of presymptomatic PD in the PINK1 -/- rat at 3 months using magnetic resonance imaging techniques. At postnatal weeks 12-13; one month earlier than previously reported signs of motor and cognitive dysfunction, this study combined imaging modalities, including assessment of quantitative anisotropy across 171 individual brain areas using an annotated MRI rat brain atlas to identify sites of gray matter alteration between wild-type and PINK1 -/- rats. The olfactory system, hypothalamus, thalamus, nucleus accumbens, and cerebellum showed differences in anisotropy between experimental groups. Molecular analyses revealed reduced levels of glutathione, ATP, and elevated oxidative stress in the substantia nigra, striatum and deep cerebellar nuclei. Mitochondrial genes encoding proteins in Complex IV, along with mRNA levels associated with mitochondrial function and genes involved in glutathione synthesis were reduced. Differences in brain structure did not align with any cognitive or motor impairment. These data reveal early markers, and highlight novel brain regions involved in the pathology of PD in the PINK1 -/- rat before behavioral dysfunction occurs.

Renin knockout rat: control of adrenal aldosterone and corticosterone synthesis in vitro and adrenal gene expression

PubMed Central

Gehrand, Ashley; Bruder, Eric D.; Hoffman, Matthew J.; Engeland, William C.; Moreno, Carol

2014-01-01

The classic renin-angiotensin system is partly responsible for controlling aldosterone secretion from the adrenal cortex via the peptide angiotensin II (ANG II). In addition, there is a local adrenocortical renin-angiotensin system that may be involved in the control of aldosterone synthesis in the zona glomerulosa (ZG). To characterize the long-term control of adrenal steroidogenesis, we utilized adrenal glands from renin knockout (KO) rats and compared steroidogenesis in vitro and steroidogenic enzyme expression to wild-type (WT) controls (Dahl S rat). Adrenal capsules (ZG; aldosterone production) and subcapsules [zona reticularis/fasciculata (ZFR); corticosterone production] were separately dispersed and studied in vitro. Plasma renin activity and ANG II concentrations were extremely low in the KO rats. Basal and cAMP-stimulated aldosterone production was significantly reduced in renin KO ZG cells, whereas corticosterone production was not different between WT and KO ZFR cells. As expected, adrenal renin mRNA expression was lower in the renin KO compared with the WT rat. Real-time PCR and immunohistochemical analysis showed a significant decrease in P450aldo (Cyp11b2) mRNA and protein expression in the ZG from the renin KO rat. The reduction in aldosterone synthesis in the ZG of the renin KO adrenal seems to be accounted for by a specific decrease in P450aldo and may be due to the absence of chronic stimulation of the ZG by circulating ANG II or to a reduction in locally released ANG II within the adrenal gland. PMID:25394830

Identifying the integrated neural networks involved in capsaicin-induced pain using fMRI in awake TRPV1 knockout and wild-type rats

PubMed Central

Yee, Jason R.; Kenkel, William; Caccaviello, John C.; Gamber, Kevin; Simmons, Phil; Nedelman, Mark; Kulkarni, Praveen; Ferris, Craig F.

2015-01-01

In the present study, we used functional MRI in awake rats to investigate the pain response that accompanies intradermal injection of capsaicin into the hindpaw. To this end, we used BOLD imaging together with a 3D segmented, annotated rat atlas and computational analysis to identify the integrated neural circuits involved in capsaicin-induced pain. The specificity of the pain response to capsaicin was tested in a transgenic model that contains a biallelic deletion of the gene encoding for the transient receptor potential cation channel subfamily V member 1 (TRPV1). Capsaicin is an exogenous ligand for the TRPV1 receptor, and in wild-type rats, activated the putative pain neural circuit. In addition, capsaicin-treated wild-type rats exhibited activation in brain regions comprising the Papez circuit and habenular system, systems that play important roles in the integration of emotional information, and learning and memory of aversive information, respectively. As expected, capsaicin administration to TRPV1-KO rats failed to elicit the robust BOLD activation pattern observed in wild-type controls. However, the intradermal injection of formalin elicited a significant activation of the putative pain pathway as represented by such areas as the anterior cingulate, somatosensory cortex, parabrachial nucleus, and periaqueductal gray. Notably, comparison of neural responses to capsaicin in wild-type vs. knock-out rats uncovered evidence that capsaicin may function in an antinociceptive capacity independent of TRPV1 signaling. Our data suggest that neuroimaging of pain in awake, conscious animals has the potential to inform the neurobiological basis of full and integrated perceptions of pain. PMID:25745388

Cntnap2 Knockout Rats and Mice Exhibit Epileptiform Activity and Abnormal Sleep-Wake Physiology.

PubMed

Thomas, Alexia M; Schwartz, Michael D; Saxe, Michael D; Kilduff, Thomas S

2017-01-01

Although recent innovations have enabled modification of the rat genome, it is unclear whether enhanced utility of rodents as human disease models will result. We compared electroencephalogram (EEG) and behavioral phenotypes of rats and mice with homozygous deletion of Cntnap2, a gene associated with cortical dysplasia-focal epilepsy (CDFE) and autism spectrum disorders (ASD). Male contactin-associated protein-like 2 (Cntnap2) knockout (KO) and wild-type (WT) rats and male Cntnap2 KO and WT mice were implanted with telemeters to record EEG, electromyogram, body temperature, and locomotor activity. Animals were subjected to a test battery for ASD-related behaviors, followed by 24-hr EEG recordings that were analyzed for sleep-wake parameters and subjected to spectral analysis. Cntnap2 KO rats exhibited severe motor seizures, hyperactivity, and increased consolidation of wakefulness and REM sleep. By contrast, Cntnap2 KO mice demonstrated absence seizure-like events, hypoactivity, and wake fragmentation. Although seizures observed in Cntnap2 KO rats were more similar to those in CDFE patients than in KO mice, neither model fully recapitulated the full spectrum of disease symptoms. However, KOs in both species had reduced spectral power in the alpha (9-12 Hz) range during wake, suggesting a conserved EEG biomarker. Deletion of Cntnap2 impacts similar behaviors and EEG measures in rats and mice, but with profound differences in nature and phenotypic severity. These observations highlight the importance of cross-species comparisons to understand conserved gene functions and the limitations of single- species models to provide translational insights relevant to human diseases. © Sleep Research Society 2016. Published by Oxford University Press on behalf of the Sleep Research Society. All rights reserved. For permissions, please e-mail journals.permissions@oup.com.

The Trace Amine 1 receptor knockout mouse: an animal model with relevance to schizophrenia.

PubMed

Wolinsky, T D; Swanson, C J; Smith, K E; Zhong, H; Borowsky, B; Seeman, P; Branchek, T; Gerald, C P

2007-10-01

Trace amines have been implicated in a number of neuropsychiatric disorders including depression and schizophrenia. Although long known to modulate neurotransmission indirectly through the release of catecholamines, the identification of the Trace Amine 1 receptor (TA1) offers a mechanism by which trace amines can influence synaptic activity directly. TA1 binds and is activated by trace amines such as beta-phenylethylamine and tyramine. Our pharmacological characterization of mouse TA1 showed that, as in rat and primate, amphetamine is an agonist at this receptor but with surprisingly high potency. Without selective ligands for TA1 that do not also possess catecholamine-releasing properties, however, it has not been possible to study its physiological role in the central nervous system. To that end, a line of mice lacking the TA1 receptor was generated to characterize its contribution to the regulation of behavior. Compared with wild-type littermates, TA1 knockout (KO) mice displayed a deficit in prepulse inhibition. Knockout animals, in which the TA1-agonist influence of amphetamine was absent, showed enhanced sensitivity to the psychomotor-stimulating effect of this drug, which was temporally correlated with significantly larger increases in the release of both dopamine and norepinephrine in the dorsal striatum and associated with a 262% increase in the proportion of striatal high-affinity D2 receptors. TA1 therefore appears to play a modulatory role in catecholaminergic function and represents a potentially novel mechanism for the treatment of neuropsychiatric disorders. Furthermore, the TA1 KO mouse may provide a useful model for the development of treatments for some positive symptoms of schizophrenia.

Motor coordination and balance in rodents.

PubMed

Carter, R J; Morton, J; Dunnett, S B

2001-08-01

Measurement of motor coordination and balance can be used not only to assess the effect of drugs or other experimental manipulations on mice and rats, but also to characterize the motor phenotype of transgenic or knock-out animals. Three well established and widely used protocols for measuring motor coordination and balance in mice and rats (rotarod, beam walking and footprint analysis) are described in this unit. The tests can be used equally well for rats and mice, and have been used both for the phenotypic characterization of transgenic mice and for evaluating the effects of lesions and aging in rats. The protocols are described in the primary context of testing mice, but modifications of the test apparatus or variations in the test parameters for assessment of rats are noted.

The Expression of TALEN before Fertilization Provides a Rapid Knock-Out Phenotype in Xenopus laevis Founder Embryos.

PubMed

Miyamoto, Kei; Suzuki, Ken-Ichi T; Suzuki, Miyuki; Sakane, Yuto; Sakuma, Tetsushi; Herberg, Sarah; Simeone, Angela; Simpson, David; Jullien, Jerome; Yamamoto, Takashi; Gurdon, J B

2015-01-01

Recent advances in genome editing using programmable nucleases have revolutionized gene targeting in various organisms. Successful gene knock-out has been shown in Xenopus, a widely used model organism, although a system enabling less mosaic knock-out in founder embryos (F0) needs to be explored in order to judge phenotypes in the F0 generation. Here, we injected modified highly active transcription activator-like effector nuclease (TALEN) mRNA to oocytes at the germinal vesicle (GV) stage, followed by in vitro maturation and intracytoplasmic sperm injection, to achieve a full knock-out in F0 embryos. Unlike conventional injection methods to fertilized embryos, the injection of TALEN mRNA into GV oocytes allows expression of nucleases before fertilization, enabling them to work from an earlier stage. Using this procedure, most of developed embryos showed full knock-out phenotypes of the pigmentation gene tyrosinase and/or embryonic lethal gene pax6 in the founder generation. In addition, our method permitted a large 1 kb deletion. Thus, we describe nearly complete gene knock-out phenotypes in Xenopus laevis F0 embryos. The presented method will help to accelerate the production of knock-out frogs since we can bypass an extra generation of about 1 year in Xenopus laevis. Meantime, our method provides a unique opportunity to rapidly test the developmental effects of disrupting those genes that do not permit growth to an adult able to reproduce. In addition, the protocol shown here is considerably less invasive than the previously used host transfer since our protocol does not require surgery. The experimental scheme presented is potentially applicable to other organisms such as mammals and fish to resolve common issues of mosaicism in founders.

Electroacupuncture preconditioning-induced neuroprotection may be mediated by glutamate transporter type 2.

PubMed

Zhu, Xiaoling; Yin, Jinbo; Li, Liaoliao; Ma, Lei; Tan, Hongying; Deng, Jiao; Chen, Shaoyang; Zuo, Zhiyi

2013-10-01

Electroacupuncture has been shown to induce a preconditioning effect in the brain. The mechanisms for this protection are not fully elucidated. We hypothesize that this protection is mediated by excitatory amino acid transporters (EAATs) that have been shown to be neuroprotective. To test this hypothesis, two-month old male Sprague-Dawley rats and EAAT type 3 (EAAT3) knockout mice received or did not receive 30-min electroacupuncture once a day for five consecutive days. They were subjected to a 120-min middle cerebral arterial occlusion (MCAO) at 24h after the last electroacupuncture. Neurological outcome was assessed 2days after the MCAO. Brain tissues were harvested at 24h after the last electroacupuncture for Western blotting. Rats subjected to electroacupuncture at the Baihui acupoint had smaller brain infarct volumes and better neurological deficit scores than control rats. Electroacupuncture increased EAAT type 2 (EAAT2) in the cerebral cortex, tended to increase EAAT3 in the hippocampus, and had no effect on EAAT type 1 expression. Dihydrokainate, an EAAT2 inhibitor, worsened the neurological outcome of rats with electroacupuncture pretreatment. Electroacupuncture pretreatment at the Baihui acupoint increased EAAT2 in the cerebral cortex and improved the neurological outcome of EAAT3 knockout mice. Together, our results suggest that EAAT2 may mediate the electroacupuncture preconditioning-induced neuroprotection. Copyright © 2013 Elsevier Ltd. All rights reserved.

Normal radial migration and lamination are maintained in dyslexia-susceptibility candidate gene homolog Kiaa0319 knockout mice.

PubMed

Martinez-Garay, Isabel; Guidi, Luiz G; Holloway, Zoe G; Bailey, Melissa A G; Lyngholm, Daniel; Schneider, Tomasz; Donnison, Timothy; Butt, Simon J B; Monaco, Anthony P; Molnár, Zoltán; Velayos-Baeza, Antonio

2017-04-01

Developmental dyslexia is a common disorder with a strong genetic component, but the underlying molecular mechanisms are still unknown. Several candidate dyslexia-susceptibility genes, including KIAA0319, DYX1C1, and DCDC2, have been identified in humans. RNA interference experiments targeting these genes in rat embryos have shown impairments in neuronal migration, suggesting that defects in radial cortical migration could be involved in the disease mechanism of dyslexia. Here we present the first characterisation of a Kiaa0319 knockout mouse line. Animals lacking KIAA0319 protein do not show anatomical abnormalities in any of the layered structures of the brain. Neurogenesis and radial migration of cortical projection neurons are not altered, and the intrinsic electrophysiological properties of Kiaa0319-deficient neurons do not differ from those of wild-type neurons. Kiaa0319 overexpression in cortex delays radial migration, but does not affect final neuronal position. However, knockout animals show subtle differences suggesting possible alterations in anxiety-related behaviour and in sensorimotor gating. Our results do not reveal a migration disorder in the mouse model, adding to the body of evidence available for Dcdc2 and Dyx1c1 that, unlike in the rat in utero knockdown models, the dyslexia-susceptibility candidate mouse homolog genes do not play an evident role in neuronal migration. However, KIAA0319 protein expression seems to be restricted to the brain, not only in early developmental stages but also in adult mice, indicative of a role of this protein in brain function. The constitutive and conditional knockout lines reported here will be useful tools for further functional analyses of Kiaa0319.

Cdh13 and AdipoQ gene knockout alter instrumental and Pavlovian drug conditioning.

PubMed

King, C P; Militello, L; Hart, A; St Pierre, C L; Leung, E; Versaggi, C L; Roberson, N; Catlin, J; Palmer, A A; Richards, J B; Meyer, P J

2017-09-01

Genome-wide association studies in humans have suggested that variants of the cadherin-13 (CDH13) gene are associated with substance use disorder, subjective response to amphetamine, and attention deficit hyperactivity disorder. To examine the role of the Cdh13 and its peptide ligand adiponectin (AdipoQ) in addiction-related behaviors, we assessed Cdh13 knockout (KO) rats and AdipoQ KO mice using intravenous cocaine self-administration and conditioned place preference (CPP) paradigms. During intravenous cocaine self-administration, male Cdh13 heterozygous (+/-) and KO (-/-) rats showed increased cue-induced reinstatement compared with wild-type (WT) rats when presented with a cocaine-paired stimulus, whereas female Cdh13 rats showed no differences across genotype. Cdh13 -/- rats showed higher responding for a saccharin reinforcer and learned the choice reaction time (RT) task more slowly than WTs. However, we found no differences between Cdh13 -/- and +/+ rats in responding for sensory reinforcement, number of premature responses in the RT task, tendency to approach a Pavlovian food cue, CPP and locomotor activation to cocaine (10 or 20 mg/kg). In AdipoQ -/- mice, there was a significant increase in CPP to methamphetamine (1 mg/kg) but not to a range of d-amphetamine doses (0.5, 1, 2 and 4 mg/kg). Taken together, these data suggest that Cdh13 and AdipoQ regulate sensitivity to psychomotor stimulants and palatable rewards without producing major changes in other behaviors. In humans, these two genes may regulate sensitivity to natural and drug rewards, thus influencing susceptibility to the conditioned drug effects and relapse. © 2017 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

Development and initial characterization of a novel ghrelin receptor CRISPR/Cas9 knockout wistar rat model.

PubMed

Zallar, L J; Tunstall, B J; Richie, C T; Zhang, Y J; You, Z B; Gardner, E L; Heilig, M; Pickel, J; Koob, G F; Vendruscolo, L F; Harvey, B K; Leggio, L

2018-01-30

Ghrelin, a stomach-derived hormone implicated in numerous behaviors including feeding, reward, stress, and addictive behaviors, acts by binding to the growth hormone secretagogue receptor (GHSR). Here, we present the development, verification, and initial characterization of a novel GHSR knockout (KO) Wistar rat model created with CRISPR genome editing. Using CRISPR/Cas9, we developed a GHSR KO in a Wistar background. Loss of GHSR mRNA expression was histologically verified using RNAscope in wild-type (WT; n = 2) and KO (n = 2) rats. We tested the effects of intraperitoneal acyl-ghrelin administration on food consumption and plasma growth hormone (GH) concentrations in WT (n = 8) and KO (n = 8) rats. We also analyzed locomotion, food consumption, and body fat composition in these animals. Body weight was monitored from early development to adulthood. The RNAscope analysis revealed an abundance of GHSR mRNA expression in the hypothalamus, midbrain, and hippocampus in WTs, and no observed probe binding in KOs. Ghrelin administration increased plasma GH levels (p = 0.0067) and food consumption (p = 0.0448) in WT rats but not KOs. KO rats consumed less food overall at basal conditions and weighed significantly less compared with WTs throughout development (p = 0.0001). Compared with WTs, KOs presented higher concentrations of brown adipose tissue (BAT; p = 0.0322). We have verified GHSR deletion in our KO model using histological, physiological, neuroendocrinological, and behavioral measures. Our findings indicate that GHSR deletion in rats is not only associated with a lack of response to ghrelin, but also associated with decreases in daily food consumption and body growth, and increases in BAT. This GHSR KO Wistar rat model provides a novel tool for studying the role of the ghrelin system in obesity and in a wide range of medical and neuropsychiatric disorders.

Of Men and Mice: Modeling the Fragile X Syndrome

PubMed Central

Dahlhaus, Regina

2018-01-01

The Fragile X Syndrome (FXS) is one of the most common forms of inherited intellectual disability in all human societies. Caused by the transcriptional silencing of a single gene, the fragile x mental retardation gene FMR1, FXS is characterized by a variety of symptoms, which range from mental disabilities to autism and epilepsy. More than 20 years ago, a first animal model was described, the Fmr1 knock-out mouse. Several other models have been developed since then, including conditional knock-out mice, knock-out rats, a zebrafish and a drosophila model. Using these model systems, various targets for potential pharmaceutical treatments have been identified and many treatments have been shown to be efficient in preclinical studies. However, all attempts to turn these findings into a therapy for patients have failed thus far. In this review, I will discuss underlying difficulties and address potential alternatives for our future research. PMID:29599705

Validation of microinjection methods for generating knockout mice by CRISPR/Cas-mediated genome engineering.

PubMed

Horii, Takuro; Arai, Yuji; Yamazaki, Miho; Morita, Sumiyo; Kimura, Mika; Itoh, Masahiro; Abe, Yumiko; Hatada, Izuho

2014-03-28

The CRISPR/Cas system, in which the Cas9 endonuclease and a guide RNA complementary to the target are sufficient for RNA-guided cleavage of the target DNA, is a powerful new approach recently developed for targeted gene disruption in various animal models. However, there is little verification of microinjection methods for generating knockout mice using this approach. Here, we report the verification of microinjection methods of the CRISPR/Cas system. We compared three methods for injection: (1) injection of DNA into the pronucleus, (2) injection of RNA into the pronucleus, and (3) injection of RNA into the cytoplasm. We found that injection of RNA into the cytoplasm was the most efficient method in terms of the numbers of viable blastocyst stage embryos and full-term pups generated. This method also showed the best overall knockout efficiency.

A lentivirus-free inducible CRISPR-Cas9 system for efficient targeting of human genes.

PubMed

Bisht, Kamlesh; Grill, Sherilyn; Graniel, Jacqueline; Nandakumar, Jayakrishnan

2017-08-01

CRISPR-Cas9 is a cutting-edge tool for modifying genomes. The efficacy with which Cas9 recognizes its target has revolutionized the engineering of knockouts. However this efficacy complicates the knocking out of important genes in cultured cells. Unedited cells holding a survival advantage within an edited population can confound the knockout phenotype. Here we develop a HeLa-based system that overcomes this limitation, incorporating several attractive features. First, we use Flp-recombinase to generate clones stably integrated for Cas9 and guide RNAs, eliminating the possibility of unedited cells. Second, Cas9 can be induced uniformly in the clonal cultures using doxycycline to measure the knockout phenotype. Third, two genes can be simultaneously knocked out using this approach. Finally, by not involving lentiviruses, our method is appealing to a broad research audience. Using this methodology we generated an inducible AGO2-knockout cell line showing normal RNA interference in the absence of doxycycline. Upon induction of Cas9, the AGO2 locus was cleaved, the AGO2 protein was depleted, and RNA interference was compromised. In addition to generating inducible knockouts, our technology can be adapted to improve other applications of Cas9, including transcriptional/epigenetic modulation and visualization of cellular DNA loci. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Vulnerability to mild predator stress in serotonin transporter knockout mice.

PubMed

Adamec, Robert; Burton, Paul; Blundell, Jacqueline; Murphy, Dennis L; Holmes, Andrew

2006-06-03

Effect of predator stress on rat and mouse anxiety-like behavior may model aspects of post traumatic stress disorder (PTSD). A single cat exposure of wild type (C57, CFW) mice can produce lasting anxiety-like effects in the elevated plus maze, light/dark box tests and startle. In addition, female but not male C57 mice are made more anxious in the plus maze by exposure to predator odors alone, suggesting differential vulnerability to predator stressors of differing intensity. There is a link between genetic variation in the serotonin (5-HT) transporter (SERT) and anxiety in humans. This prompted the generation of SERT knockout mice [see Holmes A, Murphy DL, Crawley, JN. Biol Psychiatry 2003;54(10):953-9]. Present work used these mice to determine if there was a link between vulnerability to the anxiogenic effects of predator odors and abnormalities of 5-HT transmission induced by a life long reduction in 5-HT reuptake. Wild type (WT, C57 background), heterozygous (SERT +/-, HET) mice and homozygous knockout (SERT -/-, KO) were assigned to handled control groups or groups exposed for 10 min to a large testing room rich in cat odor. One week after handling or room exposure, anxiety testing took place in the dark phase of the light/dark cycle, in red light. Predator odor exposure was selectively anxiogenic in the plus maze and light/dark box tests in SERT -/- mice. Exposure to predator odor did not potentiate startle. Findings suggest a role for abnormalities in 5-HT transmission in vulnerability to some of the lasting anxiogenic effects of species relevant stressors and possibly in vulnerability to PTSD.

Myostatin knockout using zinc-finger nucleases promotes proliferation of ovine primary satellite cells in vitro.

PubMed

Salabi, Fatemeh; Nazari, Mahmood; Chen, Qing; Nimal, Jonathan; Tong, Jianming; Cao, Wen G

2014-12-20

Myostatin (MSTN) has previously been shown to negatively regulate the proliferation and differentiation of skeletal muscle cells. Satellite cells are quiescent muscle stem cells that promote muscle growth and repair. Because the mechanism of MSTN in the biology of satellite cells is not well understood, this study was conducted to generate MSTN mono-allelic knockout satellite cells using the zinc-finger nuclease mRNA (MSTN-KO ZFN mRNA) and also to investigate the effect of this disruption on the proliferation and differentiation of sheep primary satellite cells (PSCs). Nineteen biallelic and four mono-allelic knockout cell clones were obtained after sequence analysis. The homologous mono-allelic knockout cells with 5-bp deletion were used to further evaluations. The results demonstrated that mono-allelic knockout of MSTN gene leads to translation inhibition. Real-time quantitative PCR results indicated that knockout of MSTN contributed to an increase in CDK2 and follistatin and a decrease in p21 at the transcript level in proliferation conditions. Moreover, MSTN knockout significantly increased the proliferation of mutant clones (P < 0.01). Consistent with the observed increase in CDK2 and decrease in p21 in cells lacking MSTN, cell cycle analysis showed that MSTN negatively regulated the G1 to S progression. In addition, knockout of myostatin resulted in a remarkable increase in MyoD and MyoG expression under differentiating conditions but had no effect on Myf5 expression. These results expanded our understanding of the regulation mechanism of MSTN. Furthermore, the MSTN-KO ZFN mRNA system in PSCs could be used to generate transgenic sheep in the future.

Degraded speech sound processing in a rat model of fragile X syndrome

PubMed Central

Engineer, Crystal T.; Centanni, Tracy M.; Im, Kwok W.; Rahebi, Kimiya C.; Buell, Elizabeth P.; Kilgard, Michael P.

2014-01-01

Fragile X syndrome is the most common inherited form of intellectual disability and the leading genetic cause of autism. Impaired phonological processing in fragile X syndrome interferes with the development of language skills. Although auditory cortex responses are known to be abnormal in fragile X syndrome, it is not clear how these differences impact speech sound processing. This study provides the first evidence that the cortical representation of speech sounds is impaired in Fmr1 knockout rats, despite normal speech discrimination behavior. Evoked potentials and spiking activity in response to speech sounds, noise burst trains, and tones were significantly degraded in primary auditory cortex, anterior auditory field and the ventral auditory field. Neurometric analysis of speech evoked activity using a pattern classifier confirmed that activity in these fields contains significantly less information about speech sound identity in Fmr1 knockout rats compared to control rats. Responses were normal in the posterior auditory field, which is associated with sound localization. The greatest impairment was observed in the ventral auditory field, which is related to emotional regulation. Dysfunction in the ventral auditory field may contribute to poor emotional regulation in fragile X syndrome and may help explain the observation that later auditory evoked responses are more disturbed in fragile X syndrome compared to earlier responses. Rodent models of fragile X syndrome are likely to prove useful for understanding the biological basis of fragile X syndrome and for testing candidate therapies. PMID:24713347

Knockout of Eva1a leads to rapid development of heart failure by impairing autophagy

PubMed Central

Zhang, Shu; Lin, Xin; Li, Ge; Shen, Xue; Niu, Di; Lu, Guang; Fu, Xin; Chen, Yingyu; Cui, Ming; Bai, Yun

2017-01-01

EVA1A (Eva-1 homologue A) is a novel lysosome and endoplasmic reticulum-associated protein that can regulate cell autophagy and apoptosis. Eva1a is expressed in the myocardium, but its function in myocytes has not yet been investigated. Therefore, we generated inducible, cardiomyocyte-specific Eva1a knockout mice with an aim to determine the role of Eva1a in cardiac remodelling in the adult heart. Data from experiments showed that loss of Eva1a in the adult heart increased cardiac fibrosis, promoted cardiac hypertrophy, and led to cardiomyopathy and death. Further investigation suggested that this effect was associated with impaired autophagy and increased apoptosis in Eva1a knockout hearts. Moreover, knockout of Eva1a activated Mtor signalling and the subsequent inhibition of autophagy. In addition, Eva1a knockout hearts showed disorganized sarcomere structure and mitochondrial misalignment and aggregation, leading to the lack of ATP generation. Collectively, these data demonstrated that Eva1a improves cardiac function and inhibits cardiac hypertrophy and fibrosis by increasing autophagy. In conclusion, our results demonstrated that Eva1a may have an important role in maintaining cardiac homeostasis. PMID:28151473

Caveolin-1 is enriched in the peroxisomal membrane of rat hepatocytes.

PubMed

Woudenberg, Jannes; Rembacz, Krzysztof P; van den Heuvel, Fiona A J; Woudenberg-Vrenken, Titia E; Buist-Homan, Manon; Geuken, Mariska; Hoekstra, Mark; Deelman, Leo E; Enrich, Carlos; Henning, Rob H; Moshage, Han; Faber, Klaas Nico

2010-05-01

Caveolae are a subtype of cholesterol-enriched lipid microdomains/rafts that are routinely detected as vesicles pinching off from the plasma membrane. Caveolin-1 is an essential component of caveolae. Hepatic caveolin-1 plays an important role in liver regeneration and lipid metabolism. Expression of caveolin-1 in hepatocytes is relatively low, and it has been suggested to also reside at other subcellular locations than the plasma membrane. Recently, we found that the peroxisomal membrane contains lipid microdomains. Like caveolin-1, hepatic peroxisomes are involved in lipid metabolism. Here, we analyzed the subcellular location of caveolin-1 in rat hepatocytes. The subcellular location of rat hepatocyte caveolin-1 was analyzed by cell fractionation procedures, immunofluorescence, and immuno-electron microscopy. Green fluorescent protein (GFP)-tagged caveolin-1 was expressed in rat hepatocytes. Lipid rafts were characterized after Triton X-100 or Lubrol WX extraction of purified peroxisomes. Fenofibric acid-dependent regulation of caveolin-1 was analyzed. Peroxisome biogenesis was studied in rat hepatocytes after RNA interference-mediated silencing of caveolin-1 and caveolin-1 knockout mice. Cell fractionation and microscopic analyses reveal that caveolin-1 colocalizes with peroxisomal marker proteins (catalase, the 70 kDa peroxisomal membrane protein PMP70, the adrenoleukodystrophy protein ALDP, Pex14p, and the bile acid-coenzyme A:amino acid N-acyltransferase BAAT) in rat hepatocytes. Artificially expressed GFP-caveolin-1 accumulated in catalase-positive organelles. Peroxisomal caveolin-1 is associated with detergent-resistant microdomains. Caveolin-1 expression is strongly repressed by the peroxisome proliferator-activated receptor-alpha agonist fenofibric acid. Targeting of peroxisomal matrix proteins and peroxisome number and shape were not altered in rat hepatocytes with 70%-80% reduced caveolin-1 levels and in livers of caveolin-1 knockout mice. Caveolin-1 is enriched in peroxisomes of hepatocytes. Caveolin-1 is not required for peroxisome biogenesis, but this unique subcellular location may determine its important role in hepatocyte proliferation and lipid metabolism.

Neurolysin Knockout Mice Generation and Initial Phenotype Characterization*

PubMed Central

Cavalcanti, Diogo M. L. P.; Castro, Leandro M.; Rosa Neto, José C.; Seelaender, Marilia; Neves, Rodrigo X.; Oliveira, Vitor; Forti, Fábio L.; Iwai, Leo K.; Gozzo, Fabio C.; Todiras, Mihail; Schadock, Ines; Barros, Carlos C.; Bader, Michael; Ferro, Emer S.

2014-01-01

The oligopeptidase neurolysin (EC 3.4.24.16; Nln) was first identified in rat brain synaptic membranes and shown to ubiquitously participate in the catabolism of bioactive peptides such as neurotensin and bradykinin. Recently, it was suggested that Nln reduction could improve insulin sensitivity. Here, we have shown that Nln KO mice have increased glucose tolerance, insulin sensitivity, and gluconeogenesis. KO mice have increased liver mRNA for several genes related to gluconeogenesis. Isotopic label semiquantitative peptidomic analysis suggests an increase in specific intracellular peptides in gastrocnemius and epididymal adipose tissue, which likely is involved with the increased glucose tolerance and insulin sensitivity in the KO mice. These results suggest the exciting new possibility that Nln is a key enzyme for energy metabolism and could be a novel therapeutic target to improve glucose uptake and insulin sensitivity. PMID:24719317

Mechanistic Links between PARP, NAD, and Brain Inflammation after TBI

DTIC Science & Technology

2014-10-01

metabolite which we have in prior studies shown to also suppress poly(ADP-ribose) polymerase activity and inflammatory responses) and ketogenic diet . CtBP1/2...knockout mice will be generated to test a specific mechanisms by which ketogenic diet can have anti-inflammatory effects. For all studies, outcome...inflammatory responses. (3) Ketogenic diet , begun 12 hours after TBI. CtBP1/2 knockout mice will be generated to test a specific mechanisms by which

Electroacupuncture at LI11 promotes jejunal motility via the parasympathetic pathway.

PubMed

Hu, Xuanming; Yuan, Mengqian; Yin, Yin; Wang, Yidan; Li, Yuqin; Zhang, Na; Sun, Xueyi; Yu, Zhi; Xu, Bin

2017-06-21

Gastrointestinal motility disorder has been demonstrated to be regulated by acupuncture treatment. The mechanisms underlying the effects of acupuncture stimulation of abdominal and lower limb acupoints on gastrointestinal motility have been thoroughly studied; however, the physiology underlying the effects of acupuncture on the forelimbs to mediate gastrointestinal motility requires further exploration. The aim of this study was to determine whether electroacupuncture (EA) at LI11 promotes jejunal motility, whether the parasympathetic pathway participates in this effect, and if so, which somatic afferent nerve fibres are involved. A manometric balloon was used to observe jejunal motility. The effects and mechanisms of EA at LI11 were explored in male Sprague-Dawley rats with or without drug administration (propranolol, clenbuterol, acetylcholine, and atropine) and with or without vagotomy. Three types of male mice (β 1 β 2 receptor-knockout [β 1 β 2 -/- ] mice, M 2 M 3 receptor-knockout [M 2 M 3 -/- ] mice and wild-type [WT] mice) were also studied by using different EA intensities (1, 2, 4, 6, and 8 mA). A total of 72 rats and 56 mice were included in the study. EA at LI11 increased the contractile amplitude of jejunal motility in the majority of both rats and mice. However, EA at LI11 did not enhance jejunal motility in rats administered atropine, rats that underwent vagotomy, and M 2 M 3 -‍‍/- mice (at all intensities). In WT mice, EA at LI11 significantly increased jejunal motility at all intensities except 1 mA, and a plateau was reached at intensities greater than 4 mA. Our results suggest that EA at LI11 promotes jejunal motility primarily by exciting the parasympathetic pathway, and that Aδ-fibres and C-fibres may play important roles in the process.

Reversal of mineral ion homeostasis and soft-tissue calcification of klotho knockout mice by deletion of vitamin D 1α-hydroxylase

PubMed Central

Ohnishi, Mutsuko; Nakatani, Teruyo; Lanske, Beate; Razzaque, M. Shawkat

2011-01-01

Changes in the expression of klotho, a β-glucuronidase, contribute to the development of features that resemble those of premature aging, as well as chronic renal failure. Klotho knockout mice have increased expression of the sodium/phosphate cotransporter (NaPi2a) and 1α-hydroxylase in their kidneys, along with increased serum levels of phosphate and 1,25-dihydroxyvitamin D. These changes are associated with widespread soft-tissue calcifications, generalized tissue atrophy, and a shorter lifespan in the knockout mice. To determine the role of the increased vitamin D activities in klotho knockout animals, we generated klotho and 1α-hydroxylase double-knockout mice. These double mutants regained body weight and developed hypophosphatemia with a complete elimination of the soft-tissue and vascular calcifications that were routinely found in klotho knockout mice. The markedly increased serum fibroblast growth factor 23 and the abnormally low serum parathyroid hormone levels, typical of klotho knockout mice, were significantly reversed in the double-knockout animals. These in vivo studies suggest that vitamin D has a pathologic role in regulating abnormal mineral ion metabolism and soft-tissue anomalies of klotho-deficient mice. PMID:19225558

Generation of knockout rabbits using transcription activator-like effector nucleases.

PubMed

Wang, Yu; Fan, Nana; Song, Jun; Zhong, Juan; Guo, Xiaogang; Tian, Weihua; Zhang, Quanjun; Cui, Fenggong; Li, Li; Newsome, Philip N; Frampton, Jon; Esteban, Miguel A; Lai, Liangxue

2014-01-01

Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platforms contributing to redefine the boundaries of modern biological research. They are composed of a non-specific cleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications by inducing efficient DNA double-strand breaks at desired loci. Among other remarkable uses, these nucleases have been employed to produce gene knockouts in mid-size and large animals, such as rabbits and pigs, respectively. This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies, biotechnology or agricultural purposes. Here we describe a protocol for the efficient generation of knockout rabbits using transcription activator-like effector nucleases, and a perspective of the field.

Generation of a Knockout Mouse Embryonic Stem Cell Line Using a Paired CRISPR/Cas9 Genome Engineering Tool.

PubMed

Wettstein, Rahel; Bodak, Maxime; Ciaudo, Constance

2016-01-01

CRISPR/Cas9, originally discovered as a bacterial immune system, has recently been engineered into the latest tool to successfully introduce site-specific mutations in a variety of different organisms. Composed only of the Cas9 protein as well as one engineered guide RNA for its functionality, this system is much less complex in its setup and easier to handle than other guided nucleases such as Zinc-finger nucleases or TALENs.Here, we describe the simultaneous transfection of two paired CRISPR sgRNAs-Cas9 plasmids, in mouse embryonic stem cells (mESCs), resulting in the knockout of the selected target gene. Together with a four primer-evaluation system, it poses an efficient way to generate new independent knockout mouse embryonic stem cell lines.

iNOS-Derived Nitric Oxide Stimulates Osteoclast Activity and Alveolar Bone Loss in Ligature-Induced Periodontitis in Rats

PubMed Central

Herrera, Bruno S.; Martins-Porto, Rodrigo; Maia-Dantas, Aline; Campi, Paula; Spolidorio, Luis C.; Costa, Soraia K.P.; Van Dyke, Thomas E.; Gyurko, Robert; Muscara, Marcelo N.

2012-01-01

Background Inflammatory stimuli activate inducible nitric oxide synthase (iNOS) in a variety of cell types, including osteoclasts (OC) and osteoblasts, resulting in sustained NO production. In this study, we evaluate the alveolar bone loss in rats with periodontitis under long-term iNOS inhibition, and the differentiation and activity of OC from iNOS-knockout (KO) mice in vitro. Methods Oral aminoguanidine (an iNOS inhibitor) or water treatment was started 2 weeks before induction of periodontitis. Rats were sacrificed 3, 7, or 14 days after ligature placement, and alveolar bone loss was evaluated. In vitro OC culture experiments were also performed to study the differentiation of freshly isolated bone marrow cells from both iNOS KO and wild-type C57BL/6 mice. OC were counted 6 days later after tartrate-resistant acid phosphatase staining (a marker of osteoclast identity), and bone resorption activity was assessed by counting the number of resorption pits on dentin disks. Results Rats with ligature showed progressive and significant alveolar bone loss compared to sham animals, and aminoguanidine treatment significantly inhibited ligature-induced bone loss at 7 and 14 days after the induction. In comparison to bone marrow cells from wild-type mice, cells from iNOS KO mice showed decreased OC growth and the resulting OC covered a smaller culture dish area and generated fewer resorption pit counts. Conclusion Our results demonstrate that iNOS inhibition prevents alveolar bone loss in a rat model of ligature-induced periodontitis, thus confirming that iNOS-derived NO plays a crucial role in the pathogenesis of periodontitis, probably by stimulating OC differentiation and activity. PMID:21417589

Single-step generation of gene knockout-rescue system in pluripotent stem cells by promoter insertion with CRISPR/Cas9.

PubMed

Matsunaga, Taichi; Yamashita, Jun K

2014-02-07

Specific gene knockout and rescue experiments are powerful tools in developmental and stem cell biology. Nevertheless, the experiments require multiple steps of molecular manipulation for gene knockout and subsequent rescue procedures. Here we report an efficient and single step strategy to generate gene knockout-rescue system in pluripotent stem cells by promoter insertion with CRISPR/Cas9 genome editing technology. We inserted a tetracycline-regulated inducible gene promoter (tet-OFF/TRE-CMV) upstream of the endogenous promoter region of vascular endothelial growth factor receptor 2 (VEGFR2/Flk1) gene, an essential gene for endothelial cell (EC) differentiation, in mouse embryonic stem cells (ESCs) with homologous recombination. Both homo- and hetero-inserted clones were efficiently obtained through a simple selection with a drug-resistant gene. The insertion of TRE-CMV promoter disrupted endogenous Flk1 expression, resulting in null mutation in homo-inserted clones. When the inserted TRE-CMV promoter was activated with doxycycline (Dox) depletion, Flk1 expression was sufficiently recovered from the downstream genomic Flk1 gene. Whereas EC differentiation was almost completely perturbed in homo-inserted clones, Flk1 rescue with TRE-CMV promoter activation restored EC appearance, indicating that phenotypic changes in EC differentiation can be successfully reproduced with this knockout-rescue system. Thus, this promoter insertion strategy with CRISPR/Cas9 would be a novel attractive method for knockout-rescue experiments. Copyright © 2014 Elsevier Inc. All rights reserved.

An FBXO40 knockout generated by CRISPR/Cas9 causes muscle hypertrophy in pigs without detectable pathological effects.

PubMed

Zou, Yunlong; Li, Zhiyuan; Zou, Yunjing; Hao, Haiyang; Li, Ning; Li, Qiuyan

2018-04-15

The regulatory function of Fbxo40 has been well characterized in mice. As a key component of the SCF-E3 ubiquitin ligase complex, Fbxo40 induces IRS1 ubiquitination, thus inactivating the IGF1/Akt pathway. The expression of Fbxo40 is restricted to muscle, and mice with an Fbxo40 null mutation exhibit muscle hypertrophy. However, the function of FBXO40 has not been elucidated in pigs, and it is not known whether FBXO40 mutations affect their health. We therefore generated FBXO40 knockout pigs using somatic cell nuclear transfer (SCNT) technology. CRISPR/Cas9 technology was combined with G418 selection, making it possible to generate donor cells at an efficiency of 75.86%. In muscle from FBXO40 knockout pigs, IRS1 levels were higher, and the IGF1/Akt pathway was stimulated. Mutant animals also had approximately 4% more muscle mass compared to WT controls. The knockout pigs developed normally and no pathological changes were found in major organs. These results demonstrate that FBXO40 is a promising candidate gene for improving production traits in agricultural livestock and for developing therapeutic interventions for muscle diseases. Copyright © 2018. Published by Elsevier Inc.

Acute multi-sgRNA knockdown of KEOPS complex genes reproduces the microcephaly phenotype of the stable knockout zebrafish model.

PubMed

Jobst-Schwan, Tilman; Schmidt, Johanna Magdalena; Schneider, Ronen; Hoogstraten, Charlotte A; Ullmann, Jeremy F P; Schapiro, David; Majmundar, Amar J; Kolb, Amy; Eddy, Kaitlyn; Shril, Shirlee; Braun, Daniela A; Poduri, Annapurna; Hildebrandt, Friedhelm

2018-01-01

Until recently, morpholino oligonucleotides have been widely employed in zebrafish as an acute and efficient loss-of-function assay. However, off-target effects and reproducibility issues when compared to stable knockout lines have compromised their further use. Here we employed an acute CRISPR/Cas approach using multiple single guide RNAs targeting simultaneously different positions in two exemplar genes (osgep or tprkb) to increase the likelihood of generating mutations on both alleles in the injected F0 generation and to achieve a similar effect as morpholinos but with the reproducibility of stable lines. This multi single guide RNA approach resulted in median likelihoods for at least one mutation on each allele of >99% and sgRNA specific insertion/deletion profiles as revealed by deep-sequencing. Immunoblot showed a significant reduction for Osgep and Tprkb proteins. For both genes, the acute multi-sgRNA knockout recapitulated the microcephaly phenotype and reduction in survival that we observed previously in stable knockout lines, though milder in the acute multi-sgRNA knockout. Finally, we quantify the degree of mutagenesis by deep sequencing, and provide a mathematical model to quantitate the chance for a biallelic loss-of-function mutation. Our findings can be generalized to acute and stable CRISPR/Cas targeting for any zebrafish gene of interest.

Reprogramming of Sheep Fibroblasts into Pluripotency under a Drug-Inducible Expression of Mouse-Derived Defined Factors

PubMed Central

Li, Yang; Cang, Ming; Lee, Andrew Stephen; Zhang, Kehua; Liu, Dongjun

2011-01-01

Animal embryonic stem cells (ESCs) provide powerful tool for studies of early embryonic development, gene targeting, cloning, and regenerative medicine. However, the majority of attempts to establish ESC lines from large animals, especially ungulate mammals have failed. Recently, another type of pluripotent stem cells, known as induced pluripotent stem cells (iPSCs), have been successfully generated from mouse, human, monkey, rat and pig. In this study we show sheep fibroblasts can be reprogrammed to pluripotency by defined factors using a drug-inducible system. Sheep iPSCs derived in this fashion have a normal karyotype, exhibit morphological features similar to those of human ESCs and express AP, Oct4, Sox2, Nanog and the cell surface marker SSEA-4. Pluripotency of these cells was further confirmed by embryoid body (EB) and teratoma formation assays which generated derivatives of all three germ layers. Our results also show that the substitution of knockout serum replacement (KSR) with fetal bovine serum in culture improves the reprogramming efficiency of sheep iPSCs. Generation of sheep iPSCs places sheep on the front lines of large animal preclinical trials and experiments involving modification of animal genomes. PMID:21253598

Extracellular vesicles from human-induced pluripotent stem cell-derived mesenchymal stromal cells (hiPSC-MSCs) protect against renal ischemia/reperfusion injury via delivering specificity protein (SP1) and transcriptional activating of sphingosine kinase 1 and inhibiting necroptosis.

PubMed

Yuan, Xiaodong; Li, Dawei; Chen, Xiaosong; Han, Conghui; Xu, Longmei; Huang, Tao; Dong, Zhen; Zhang, Ming

2017-12-11

Renal ischemia-reperfusion is a main cause of acute kidney injury (AKI), which is associated with high mortality. Here we show that extracellular vesicles (EVs) secreted from hiPSC-MSCs play a critical role in protection against renal I/R injury. hiPSC-MSCs-EVs can fuse with renal cells and deliver SP1 into target cells, subsequently active SK1 expression and increase S1P formation. Chromatin immunoprecipitation (ChIP) analyses and luciferase assay were used to confirm SP1 binds directly to the SK1 promoter region and promote promoter activity. Moreover, SP1 inhibition (MIT) or SK1 inhibition (SKI-II) completely abolished the renal protective effect of hiPSC-MSCs-EVs in rat I/R injury mode. However, pre-treatment of necroptosis inhibitor Nec-1 showed no difference with the administration of hiPSC-MSCs-EVs only. We then generated an SP1 knockout hiPSC-MSC cell line by CRISPR/Cas9 system and found that SP1 knockout failed to show the protective effect of hiPSC-MSCs-EVs unless restoring the level of SP1 by Ad-SP1 in vitro and in vivo. In conclusion, this study describes an anti-necroptosis effect of hiPSC-MSCs-EVs against renal I/R injury via delivering SP1 into target renal cells and intracellular activating the expression of SK1 and the generation of S1P. These findings suggest a novel mechanism for renal protection against I/R injury, and indicate a potential therapeutic approach for a variety of renal diseases and renal transplantation.

3-MCPD 1-Palmitate Induced Tubular Cell Apoptosis In Vivo via JNK/p53 Pathways

PubMed Central

Liu, Man; Huang, Guoren; Wang, Thomas T.Y.; Sun, Xiangjun; Yu, Liangli (Lucy)

2016-01-01

Fatty acid esters of 3-chloro-1, 2-propanediol (3-MCPD esters) are a group of processing induced food contaminants with nephrotoxicity but the molecular mechanism(s) remains unclear. This study investigated whether and how the JNK/p53 pathway may play a role in the nephrotoxic effect of 3-MCPD esters using 3-MCPD 1-palmitate (MPE) as a probe compound in Sprague Dawley rats. Microarray analysis of the kidney from the Sprague Dawley rats treated with MPE, using Gene Ontology categories and KEGG pathways, revealed that MPE altered mRNA expressions of the genes involved in the mitogen-activated protein kinase (JNK and ERK), p53, and apoptotic signal transduction pathways. The changes in the mRNA expressions were confirmed by qRT-PCR and Western blot analyses and were consistent with the induction of tubular cell apoptosis as determined by histopathological, TUNEL, and immunohistochemistry analyses in the kidneys of the Sprague Dawley rats. Additionally, p53 knockout attenuated the apoptosis, and the apoptosis-related protein bax expression and cleaved caspase-3 activation induced by MPE in the p53 knockout C57BL/6 mice, whereas JNK inhibitor SP600125 but not ERK inhibitor U0126 inhibited MPE-induced apoptosis, supporting the conclusion that JNK/p53 might play a critical role in the tubular cell apoptosis induced by MPE and other 3-MCPD fatty acid esters. PMID:27008853

CRISPR-Cas9 Mediated NOX4 Knockout Inhibits Cell Proliferation and Invasion in HeLa Cells.

PubMed

Jafari, Naser; Kim, Hyunju; Park, Rackhyun; Li, Liqing; Jang, Minsu; Morris, Andrew J; Park, Junsoo; Huang, Cai

2017-01-01

Increased expression of NOX4 protein is associated with cancer progression and metastasis but the role of NOX4 in cell proliferation and invasion is not fully understood. We generated NOX4 knockout HeLa cell lines using the CRISPR-Cas9 gene editing system to explore the cellular functions of NOX4. After transfection of CRISPR-Cas9 construct, we performed T7 endonuclease 1 assays and DNA sequencing to generate and identify insertion and deletion of the NOX4 locus. We confirmed the knockout of NOX4 by Western blotting. NOX4 knockout cell lines showed reduced cell proliferation with an increase of sub-G1 cell population and the decrease of S/G2/M population. Moreover, NOX4 deficiency resulted in a dramatic decrease in invadopodium formation and the invasive activity. In addition, NOX4 deficiency also caused a decrease in focal adhesions and cell migration in HeLa cells. These results suggest that NOX4 is required for both efficient proliferation and invasion of HeLa cells.

Nitric oxide negatively regulates mammalian adult neurogenesis

NASA Astrophysics Data System (ADS)

Packer, Michael A.; Stasiv, Yuri; Benraiss, Abdellatif; Chmielnicki, Eva; Grinberg, Alexander; Westphal, Heiner; Goldman, Steven A.; Enikolopov, Grigori

2003-08-01

Neural progenitor cells are widespread throughout the adult central nervous system but only give rise to neurons in specific loci. Negative regulators of neurogenesis have therefore been postulated, but none have yet been identified as subserving a significant role in the adult brain. Here we report that nitric oxide (NO) acts as an important negative regulator of cell proliferation in the adult mammalian brain. We used two independent approaches to examine the function of NO in adult neurogenesis. In a pharmacological approach, we suppressed NO production in the rat brain by intraventricular infusion of an NO synthase inhibitor. In a genetic approach, we generated a null mutant neuronal NO synthase knockout mouse line by targeting the exon encoding active center of the enzyme. In both models, the number of new cells generated in neurogenic areas of the adult brain, the olfactory subependyma and the dentate gyrus, was strongly augmented, which indicates that division of neural stem cells in the adult brain is controlled by NO and suggests a strategy for enhancing neurogenesis in the adult central nervous system.

Breast Cancer and Early Onset Childhood Obesity: Cell Specific Gene Expression in Mammary Epithelia and Adipocytes

DTIC Science & Technology

2007-07-01

resistance on neuroendocrine and reproductive functions in transgenic and knock-out mice. Proc Soc Exp Biol Med. 1999 Nov;222(2):113-23. 11. Thordarson ...libitum overfeeding of Sprague- Dawley rats and its modulation by moderate and marked dietary restriction. Toxicol Pathol 2005;33:650–74. 26. Thordarson

Vascular dysfunction and fibrosis in stroke-prone spontaneously hypertensive rats: The aldosterone-mineralocorticoid receptor-Nox1 axis.

PubMed

Harvey, Adam P; Montezano, Augusto C; Hood, Katie Y; Lopes, Rheure A; Rios, Francisco; Ceravolo, Graziela; Graham, Delyth; Touyz, Rhian M

2017-06-15

We questioned whether aldosterone and oxidative stress play a role in vascular damage in severe hypertension and investigated the role of Nox1 in this process. We studied mesenteric arteries, aortas and vascular smooth muscle cells (VSMC) from WKY and SHRSP rats. Vascular effects of eplerenone or canrenoic acid (CA) (mineralocorticoid receptor (MR) blockers), ML171 (Nox1 inhibitor) and EHT1864 (Rac1/2 inhibitor) were assessed. Nox1-knockout mice were also studied. Vessels and VSMCs were probed for Noxs, reactive oxygen species (ROS) and pro-fibrotic/inflammatory signaling. Blood pressure and plasma levels of aldosterone and galectin-3 were increased in SHRSP versus WKY. Acetylcholine-induced vasorelaxation was decreased (61% vs 115%) and phenylephrine-induced contraction increased in SHRSP versus WKY (E max 132.8% vs 96.9%, p<0.05). Eplerenone, ML171 and EHT1864 attenuated hypercontractility in SHRSP. Vascular expression of collagen, fibronectin, TGFβ, MCP-1, RANTES, MMP2, MMP9 and p66Shc was increased in SHRSP versus WKY. These changes were associated with increased ROS generation, 3-nitrotyrosine expression and Nox1 upregulation. Activation of vascular p66Shc and increased expression of Nox1 and collagen I were prevented by CA in SHRSP. Nox1 expression was increased in aldosterone-stimulated WKY VSMCs, an effect that was amplified in SHRSP VSMCs (5.2vs9.9 fold-increase). ML171 prevented aldosterone-induced VSMC Nox1-ROS production. Aldosterone increased vascular expression of fibronectin and PAI-1 in wild-type mice but not in Nox1-knockout mice. Our findings suggest that aldosterone, which is increased in SHRSP, induces vascular damage through MR-Nox1-p66Shc-mediated processes that modulate pro-fibrotic and pro-inflammatory signaling pathways. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Heterogeneity of transverse-axial tubule system in mouse atria: Remodeling in atrial-specific Na+-Ca2+ exchanger knockout mice.

PubMed

Yue, Xin; Zhang, Rui; Kim, Brian; Ma, Aiqun; Philipson, Kenneth D; Goldhaber, Joshua I

2017-07-01

Transverse-axial tubules (TATs) are commonly assumed to be sparse or absent in atrial myocytes from small animals. Atrial myocytes from rats, cats and rabbits lack TATs, which results in a characteristic "V"-shaped Ca release pattern in confocal line-scan recordings due to the delayed rise of Ca in the center of the cell. To examine TAT expression in isolated mouse atrial myocytes, we loaded them with the membrane dye Di-4-ANEPPS to label TATs. We found that >80% of atrial myocytes had identifiable TATs. Atria from male mice had a higher TAT density than female mice, and TAT density correlated with cell width. Using the fluorescent Ca indicator Fluo-4-AM and confocal imaging, we found that wild type (WT) mouse atrial myocytes generate near-synchronous Ca transients, in contrast to the "V"-shaped pattern typically reported in other small animals such as rat. In atrial-specific Na-Ca exchanger (NCX) knockout (KO) mice, which develop sinus node dysfunction and atrial hypertrophy with dilation, we found a substantial loss of atrial TATs in isolated atrial myocytes. There was a greater loss of transverse tubules compared to axial tubules, resulting in a dominance of axial tubules. Consistent with the overall loss of TATs, NCX KO atrial myocytes displayed a "V"-shaped Ca transient with slower and reduced central (CT) Ca release and uptake in comparison to subsarcolemmal (SS) Ca release. We compared chemically detubulated (DT) WT cells to KO, and found similar slowing of CT Ca release and uptake. However, SS Ca transients in the WT DT cells had faster uptake kinetics than KO cells, consistent with the presence of NCX and normal sarcolemmal Ca efflux in the WT DT cells. We conclude that the remodeling of NCX KO atrial myocytes is accompanied by a loss of TATs leading to abnormal Ca release and uptake that could impact atrial contractility and rhythm. Copyright © 2017 Elsevier Ltd. All rights reserved.

Generation of Stable Knockout Mammalian Cells by TALEN-Mediated Locus-Specific Gene Editing.

PubMed

Mahata, Barun; Biswas, Kaushik

2017-01-01

Precise and targeted genome editing using Transcription Activator-Like Effector Endonucleases (TALENs) has been widely used and proven to be an extremely effective and specific knockout strategy in both cultured cells and animal models. The current chapter describes a protocol for the construction and generation of TALENs using serial and hierarchical digestion and ligation steps, and using the synthesized TALEN pairs to achieve locus-specific targeted gene editing in mammalian cell lines using a modified clonal selection strategy in an easy and cost-efficient manner.

Dopamine transporter and vesicular monoamine transporter knockout mice : implications for Parkinson's disease.

PubMed

Miller, G W; Wang, Y M; Gainetdinov, R R; Caron, M G

2001-01-01

One of the most valuable methods for understanding the function of a particular protein is the generation of animals that have had the gene encoding for the protein of interest disrupted, commonly known as a "quo;knockout"quo; or null mutant. By incorporating a sequence of DNA (typically encoding antibiotic resistance to aid in the selection of the mutant gene) into embryonic stem cells by homologous recombination, the normal transcription of the gene is effectively blocked (Fig. 1). Since a particular protein is encoded by two copies of a gene, it is necessary to have the gene on both alleles "quo;knocked out."quo; This is performed by cross-breeding animals with one affected allele (heterozygote) to generate offspring that have inherited two mutant alleles (homozygote). This procedure has been used to generate animals lacking either the plasma membrane dopamine transporter (DAT; Fig. 2) or the vesicular monoamine transporter (VMAT2; Fig. 3). Both DAT and VMAT2 are essential for dopamine homeostasis and are thought to participate in the pathogenesis of Parkinson's disease (1-5). Fig. 1. Maps of the targeting vector and the mock construct. The mouse genomic fragment (clone 11) was isolated from a Stratagene 129 SvJ library by standard colony hybridization using a PCR probe from the 5' end of rat cDNA. The restriction site abbreviations are as follows: H, HindIII; N, NotI; Sc, SacI; Sn, SnaI; X, XbaI; and Xh, XhoI. The region between HindIII and SnaI on clone 11 containing the coding sequence from transmembrane domains 3 and 4 of VMAT2 was deleted and replaced with PGK-neo. The 3' fragment of clone 11 was reserved as an external probe for Southern analysis. To facilitate PCR screening of embryonic stem cell clones, a mock construct containing the SnaI/XbaI fragment and part of the Neo cassette was generated as a positive control. pPNT and pGEM4Z were used to construct knockout and mock vectors, respectively. (Reproduced with permission from ref. 1). Fig. 2. DAT and VMAT2 expression in wild-type and DAT knockout midbrain. DAT immunoreactivity in wild-type (A) and DAT knockout midbrain (B). VMAT2 immunoreactivity in wild-type (C) and DAT knockout midbrain (D). Robust immunoreactivity was observed in the ventral tegmental area and substantia nigra pars compacta and reticulata in the wild-type brain. Note absence of DAT immunoreactivity and modest reduction of VMAT2 immunoreactivity in the DAT knockout. Fig. 3. Characterization of VMAT2 gene disruption. (A) Southern blot analysis of mouse genomic DNA. The Southern blot was prepared with 15 μg of genomic DNA per lane and probed with a 1.4-kb 3' external genomic fragment. +/+, wild type littermates; +/-, heterozygote; -/-, homozygote. (B) RT-PCR analysis of mouse brain poly(A)+ RNA. For each reverse transcription assay, 0.5 μg of poly(A)+ RNA was used. Equal volumes of cDNA templates were used for each PCR assay. The PCR primers used flank the neomycin cassette for the purpose of detecting potential readthrough of the neomycin DNA. The heterozygote has a reduced amount of transcripts compared with the wild-type littermate; the homozygote is devoid of VMAT2 transcripts. G3PDH was used as internal control. (C) Western blot analysis of wholebrain synaptic vesicles. Samples (25 μg) of vesicles were solubilized and separated by SDS-PAGE, transferred to nitrocellulose, subjected to Western blot analysis with anti-VMAT2-Ct (top) or anti-a-tubulin (bottom) antibodies, and developed with chemiluminescence. Molecular mass markers (kDa) are shown to the left. To confirm equal loading and transfer of proteins, the blots were stripped and reprobed with an antibody to α-tubulin. (Reproduced with permission from ref. 1). The importance of DAT in neuronal function is highlighted in animals in which DAT has been genetically deleted (DAT KO) (3). In the homozygote DAT KO mice, released dopamine remains in the extracellular space up to 300 times longer than normal. As expected, these animals display behaviors consistent with persistent activation of dopamine receptors, such as hyperlocomotion. Genetic deletion of VMAT2 reveals the essential role of vesicular storage and release of monoamines. Homozygote VMAT2 knockout mice survive for only a few days, whereas heterozygotes appear normal. Studies performed in homozygote pups and heterozygote adults clearly show that the level of VMAT2 expression calibrates the level of vesicular filling (1,2,bi4). With only 50% of normal VMAT2, heterozygote animals have reduced vesicular filling and release. These alterations in presynaptic monoamine function in the heterozygotes are thought to be responsible for the observed sensitization to the psychostimulants cocaine and amphetamine and to ethanol (1). Knockout animals also appear to parallel the changes that occur in reserpinized animals, suggesting that the adverse actions of this drug are mediated by VMAT2.

Critical role of FcR gamma-chain, LAT, PLCgamma2 and thrombin in arteriolar thrombus formation upon mild, laser-induced endothelial injury in vivo.

PubMed

Kalia, Neena; Auger, Jocelyn M; Atkinson, Ben; Watson, Steve P

2008-05-01

The role of collagen receptor complex GPVI-FcR gamma-chain, PLCgamma2 and LAT in laser-induced thrombosis is unclear. Controversy surrounds whether collagen is exposed in this model or whether thrombosis is dependent on thrombin. This study hypothesized that collagen exposure plays a critical role in thrombus formation in this model, which was tested by investigating contributions of FcR gamma-chain, LAT, PLCgamma2 and thrombin. Thrombi were monitored using intravital microscopy in anesthetized wild-type and FcR gamma-chain, LAT and PLCgamma2 knockout mice. Hirudin (thrombin inhibitor) was administered to wild-type and FcR gamma-chain knockout mice. Significantly reduced thrombus formation was observed in FcR gamma-chain and PLCgamma2 knockouts with a greater decrease observed in LAT knockouts. Dramatic reduction was observed in wild-types treated with hirudin, with abolished thrombus formation only observed in FcR gamma-chain knockouts treated with hirudin. GPVI-FcR gamma-chain, LAT and PLCgamma2 are essential for thrombus generation and stability in this laser-induced model of injury. More importantly, a greater role for LAT was identified, which may reflect a role for it downstream of a second matrix protein receptor. However, inhibition of platelet activation by matrix proteins and thrombin generation are both required to maximally prevent thrombus formation.

Acute multi-sgRNA knockdown of KEOPS complex genes reproduces the microcephaly phenotype of the stable knockout zebrafish model

PubMed Central

Schneider, Ronen; Hoogstraten, Charlotte A.; Schapiro, David; Majmundar, Amar J.; Kolb, Amy; Eddy, Kaitlyn; Shril, Shirlee; Braun, Daniela A.; Poduri, Annapurna

2018-01-01

Until recently, morpholino oligonucleotides have been widely employed in zebrafish as an acute and efficient loss-of-function assay. However, off-target effects and reproducibility issues when compared to stable knockout lines have compromised their further use. Here we employed an acute CRISPR/Cas approach using multiple single guide RNAs targeting simultaneously different positions in two exemplar genes (osgep or tprkb) to increase the likelihood of generating mutations on both alleles in the injected F0 generation and to achieve a similar effect as morpholinos but with the reproducibility of stable lines. This multi single guide RNA approach resulted in median likelihoods for at least one mutation on each allele of >99% and sgRNA specific insertion/deletion profiles as revealed by deep-sequencing. Immunoblot showed a significant reduction for Osgep and Tprkb proteins. For both genes, the acute multi-sgRNA knockout recapitulated the microcephaly phenotype and reduction in survival that we observed previously in stable knockout lines, though milder in the acute multi-sgRNA knockout. Finally, we quantify the degree of mutagenesis by deep sequencing, and provide a mathematical model to quantitate the chance for a biallelic loss-of-function mutation. Our findings can be generalized to acute and stable CRISPR/Cas targeting for any zebrafish gene of interest. PMID:29346415

Production of α1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene double-deficient pigs by CRISPR/Cas9 and handmade cloning.

PubMed

Gao, Hanchao; Zhao, Chengjiang; Xiang, Xi; Li, Yong; Zhao, Yanli; Li, Zesong; Pan, Dengke; Dai, Yifan; Hara, Hidetaka; Cooper, David K C; Cai, Zhiming; Mou, Lisha

2017-02-16

Gene-knockout pigs hold great promise as a solution to the shortage of organs from donor animals for xenotransplantation. Several groups have generated gene-knockout pigs via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) and somatic cell nuclear transfer (SCNT). Herein, we adopted a simple and micromanipulator-free method, handmade cloning (HMC) instead of SCNT, to generate double gene-knockout pigs. First, we applied the CRISPR/Cas9 system to target α1,3-galactosyltransferase (GGTA1) and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) genes simultaneously in porcine fetal fibroblast cells (PFFs), which were derived from wild-type Chinese domestic miniature Wuzhishan pigs. Cell colonies were obtained by screening and were identified by Surveyor assay and sequencing. Next, we chose the GGTA1/CMAH double-knockout (DKO) cells for HMC to produce piglets. As a result, we obtained 11 live bi-allelic GGTA1/CMAH DKO piglets with the identical phenotype. Compared to cells from GGTA1-knockout pigs, human antibody binding and antibody-mediated complement-dependent cytotoxicity were significantly reduced in cells from GGTA1/CMAH DKO pigs, which demonstrated that our pigs would exhibit reduced humoral rejection in xenotransplantation. These data suggested that the combination of CRISPR/Cas9 and HMC technology provided an efficient and new strategy for producing pigs with multiple genetic modifications.

A modulatory role of the Rax homeobox gene in mature pineal gland function: Investigating the photoneuroendocrine circadian system of a Rax conditional knockout mouse.

PubMed

Rohde, Kristian; Bering, Tenna; Furukawa, Takahisa; Rath, Martin Fredensborg

2017-10-01

The retinal and anterior neural fold homeobox gene (Rax) controls development of the eye and the forebrain. Postnatal expression of Rax in the brain is restricted to the pineal gland, a forebrain structure devoted to melatonin synthesis. The role of Rax in pineal function is unknown. In order to investigate the role of Rax in pineal function while circumventing forebrain abnormalities of the global Rax knockout, we generated an eye and pineal-specific Rax conditional knockout mouse. Deletion of Rax in the pineal gland did not affect morphology of the gland, suggesting that Rax is not essential for pineal gland development. In contrast, deletion of Rax in the eye generated an anophthalmic phenotype. In addition to the loss of central visual pathways, the suprachiasmatic nucleus of the hypothalamus housing the circadian clock was absent, indicating that the retinohypothalamic tract is required for the nucleus to develop. Telemetric analyses confirmed the lack of a functional circadian clock. Arylalkylamine N-acetyltransferase (Aanat) transcripts, encoding the melatonin rhythm-generating enzyme, were undetectable in the pineal gland of the Rax conditional knockout under normal conditions, whereas the paired box 6 homeobox gene, known to regulate pineal development, was up-regulated. By injecting isoproterenol, which mimics a nocturnal situation in the pineal gland, we were able to induce pineal expression of Aanat in the Rax conditional knockout mouse, but Aanat transcript levels were significantly lower than those of Rax-proficient mice. Our data suggest that Rax controls pineal gene expression and via Aanat may modulate melatonin synthesis. © 2017 International Society for Neurochemistry.

Hesr1 and Hesr3 are essential to generate undifferentiated quiescent satellite cells and to maintain satellite cell numbers

PubMed Central

Fukada, So-ichiro; Yamaguchi, Masahiko; Kokubo, Hiroki; Ogawa, Ryo; Uezumi, Akiyoshi; Yoneda, Tomohiro; Matev, Miroslav M.; Motohashi, Norio; Ito, Takahito; Zolkiewska, Anna; Johnson, Randy L.; Saga, Yumiko; Miyagoe-Suzuki, Yuko; Tsujikawa, Kazutake; Takeda, Shin’ichi; Yamamoto, Hiroshi

2011-01-01

Satellite cells, which are skeletal muscle stem cells, divide to provide new myonuclei to growing muscle fibers during postnatal development, and then are maintained in an undifferentiated quiescent state in adult skeletal muscle. This state is considered to be essential for the maintenance of satellite cells, but their molecular regulation is unknown. We show that Hesr1 (Hey1) and Hesr3 (Heyl) (which are known Notch target genes) are expressed simultaneously in skeletal muscle only in satellite cells. In Hesr1 and Hesr3 single-knockout mice, no obvious abnormalities of satellite cells or muscle regenerative potentials are observed. However, the generation of undifferentiated quiescent satellite cells is impaired during postnatal development in Hesr1/3 double-knockout mice. As a result, myogenic (MyoD and myogenin) and proliferative (Ki67) proteins are expressed in adult satellite cells. Consistent with the in vivo results, Hesr1/3-null myoblasts generate very few Pax7+ MyoD– undifferentiated cells in vitro. Furthermore, the satellite cell number gradually decreases in Hesr1/3 double-knockout mice even after it has stabilized in control mice, and an age-dependent regeneration defect is observed. In vivo results suggest that premature differentiation, but not cell death, is the reason for the reduced number of satellite cells in Hesr1/3 double-knockout mice. These results indicate that Hesr1 and Hesr3 are essential for the generation of adult satellite cells and for the maintenance of skeletal muscle homeostasis. PMID:21989910

USE OF THE SPONTANEOUS TSC2 KNOCKOUT (EKER) RAT MODEL OF HEREDITARY RENAL CELL CARCINOMA FOR THE STUDY OF RENAL CARCINOGENS

EPA Science Inventory

The kidney is a frequent site for chemically induced cancers in rodents and among the ten most frequent sites for cancer in human patients. Renal cell carcinoma (RCC) is the most frequent upper urinary tract cancer in humans and accounts for 80-85% of malignant renal tumors. He...

Regeneration of the Exocrine Pancreas Is Delayed in Telomere-Dysfunctional Mice

PubMed Central

von Figura, Guido; Wagner, Martin; Nalapareddy, Kodandaramireddy; Hartmann, Daniel; Kleger, Alexander; Guachalla, Luis Miguel; Rolyan, Harshvardhan; Adler, Guido; Rudolph, Karl Lenhard

2011-01-01

Introduction Telomere shortening is a cell-intrinsic mechanism that limits cell proliferation by induction of DNA damage responses resulting either in apoptosis or cellular senescence. Shortening of telomeres has been shown to occur during human aging and in chronic diseases that accelerate cell turnover, such as chronic hepatitis. Telomere shortening can limit organ homeostasis and regeneration in response to injury. Whether the same holds true for pancreas regeneration in response to injury is not known. Methods In the present study, pancreatic regeneration after acute cerulein-induced pancreatitis was studied in late generation telomerase knockout mice with short telomeres compared to telomerase wild-type mice with long telomeres. Results Late generation telomerase knockout mice exhibited impaired exocrine pancreatic regeneration after acute pancreatitis as seen by persistence of metaplastic acinar cells and markedly reduced proliferation. The expression levels of p53 and p21 were not significantly increased in regenerating pancreas of late generation telomerase knockout mice compared to wild-type mice. Conclusion Our results indicate that pancreatic regeneration is limited in the context of telomere dysfunction without evidence for p53 checkpoint activation. PMID:21364961

Substance P mediates inflammatory oedema in acute pancreatitis via activation of the neurokinin-1 receptor in rats and mice

PubMed Central

Grady, Eileen F; Yoshimi, Shandra K; Maa, John; Valeroso, Dahlia; Vartanian, Robert K; Rahim, Shamila; Kim, Edward H; Gerard, Craig; Gerard, Norma; Bunnett, Nigel W; Kirkwood, Kimberly S

2000-01-01

Pancreatic oedema occurs early in the development of acute pancreatitis, and the overall extent of fluid loss correlates with disease severity. The tachykinin substance P (SP) is released from sensory nerves, binds to the neurokinin-1 receptor (NK1-R) on endothelial cells and induces plasma extravasation, oedema, and neutrophil infiltration, a process termed neurogenic inflammation. We sought to determine the importance of neurogenic mechanisms in acute pancreatitis.Pancreatic plasma extravasation was measured using the intravascular tracers Evans blue and Monastral blue after administration of specific NK1-R agonists/antagonists in rats and NK1-R(+/+)/(−/−) mice. The effects of NK1-R genetic deletion/antagonism on pancreatic plasma extravasation, amylase, myeloperoxidase (MPO), and histology in cerulein-induced pancreatitis were characterized.In rats, both SP and the NK1-R selective agonist [Sar9 Met(O2)11]SP stimulated pancreatic plasma extravasation, and this response was blocked by the NK1-R antagonist CP 96,345. Selective agonists of the NK-2 or NK-3 receptors had no effect.In rats, cerulein stimulated pancreatic plasma extravasation and serum amylase. These responses were blocked by the NK1-R antagonist CP 96,345.In wildtype mice, SP induced plasma extravasation while SP had no effect in NK1-R knockout mice.In NK1-R knockout mice, the effects of cerulein on pancreatic plasma extravasation and hyperamylasemia were reduced by 60%, and pancreatic MPO by 75%, as compared to wildtype animals.Neurogenic mechanisms of inflammation are important in the development of inflammatory oedema in acute interstitial pancreatitis. PMID:10821777

FOCuS: a metaheuristic algorithm for computing knockouts from genome-scale models for strain optimization.

PubMed

Mutturi, Sarma

2017-06-27

Although handful tools are available for constraint-based flux analysis to generate knockout strains, most of these are either based on bilevel-MIP or its modifications. However, metaheuristic approaches that are known for their flexibility and scalability have been less studied. Moreover, in the existing tools, sectioning of search space to find optimal knocks has not been considered. Herein, a novel computational procedure, termed as FOCuS (Flower-pOllination coupled Clonal Selection algorithm), was developed to find the optimal reaction knockouts from a metabolic network to maximize the production of specific metabolites. FOCuS derives its benefits from nature-inspired flower pollination algorithm and artificial immune system-inspired clonal selection algorithm to converge to an optimal solution. To evaluate the performance of FOCuS, reported results obtained from both MIP and other metaheuristic-based tools were compared in selected case studies. The results demonstrated the robustness of FOCuS irrespective of the size of metabolic network and number of knockouts. Moreover, sectioning of search space coupled with pooling of priority reactions based on their contribution to objective function for generating smaller search space significantly reduced the computational time.

Generation and characterisation of a parkin-Pacrg knockout mouse line and a Pacrg knockout mouse line.

PubMed

Stephenson, Sarah E M; Aumann, Timothy D; Taylor, Juliet M; Riseley, Jessica R; Li, Ruili; Mann, Jeffrey R; Tomas, Doris; Lockhart, Paul J

2018-05-14

Mutations in PARK2 (parkin) can result in Parkinson's disease (PD). Parkin shares a bidirectional promoter with parkin coregulated gene (PACRG) and the transcriptional start sites are separated by only ~200 bp. Bidirectionally regulated genes have been shown to function in common biological pathways. Mice lacking parkin have largely failed to recapitulate the dopaminergic neuronal loss and movement impairments seen in individuals with parkin-mediated PD. We aimed to investigate the function of PACRG and test the hypothesis that parkin and PACRG function in a common pathway by generating and characterizing two novel knockout mouse lines harbouring loss of both parkin and Pacrg or Pacrg alone. Successful modification of the targeted allele was confirmed at the genomic, transcriptional and steady state protein levels for both genes. At 18-20 months of age, there were no significant differences in the behaviour of parental and mutant lines when assessed by openfield, rotarod and balance beam. Subsequent neuropathological examination suggested there was no gross abnormality of the dopaminergic system in the substantia nigra and no significant difference in the number of dopaminergic neurons in either knockout model compared to wildtype mice.

Efficient generation of P53 biallelic knockout Diannan miniature pigs via TALENs and somatic cell nuclear transfer.

PubMed

Shen, Youfeng; Xu, Kaixiang; Yuan, Zaimei; Guo, Jianxiong; Zhao, Heng; Zhang, Xuezeng; Zhao, Lu; Qing, Yubo; Li, Honghui; Pan, Weirong; Jia, Baoyu; Zhao, Hong-Ye; Wei, Hong-Jiang

2017-11-03

Pigs have many features that make them attractive as biomedical models for various diseases, including cancer. P53 is an important tumor suppressor gene that exerts a central role in protecting cells from oncogenic transformation and is mutated in a large number of human cancers. P53 mutations occur in almost every type of tumor and in over 50% of all tumors. In a recent publication, pigs with a mutated P53 gene were generated that resulted in lymphoma and renal and osteogenic tumors. However, approximately 80% of human tumors have dysfunctional P53. A P53-deficient pig model is still required to elucidate. Transcription activator-like effector nucleases (TALENs) were designed to target porcine P53 exon 4. The targeting activity was evaluated using a luciferase SSA recombination assay. P53 biallelic knockout (KO) cell lines were established from single-cell colonies of fetal fibroblasts derived from Diannan miniature pigs followed by electroporation with TALENs plasmids. One cell line was selected as the donor cell line for somatic cell nuclear transfer (SCNT) for the generation of P53 KO pigs. P53 KO stillborn fetuses and living piglets were obtained. Gene typing of the collected cloned individuals was performed by T7EI assay and sequencing. Fibroblast cells from Diannan miniature piglets with a P53 biallelic knockout or wild type were analyzed for the P53 response to doxorubicin treatment by confocal microscopy and western blotting. The luciferase SSA recombination assay revealed that the targeting activities of the designed TALENs were 55.35-fold higher than those of the control. Eight cell lines (8/19) were mutated for P53, and five of them were biallelic knockouts. One of the biallelic knockout cell lines was selected as nuclear donor cells for SCNT. The cloned embryos were transferred into five recipient gilts, three of them becoming pregnant. Five live fetuses were obtained from one surrogate by caesarean section after 38 days of gestation for genotyping. Finally, six live piglets and one stillborn piglet were collected from two recipients by caesarean section. Sequencing analyses of the target site confirmed the P53 biallelic knockout in all fetuses and piglets, consistent with the genotype of the donor cells. The qPCR analysis showed that the expression of the P53 mRNA had significant reduction in various tissues of the knockout piglets. Furthermore, confocal microscopy and western blotting analyses demonstrated that the fibroblast cells of Diannan miniature piglets with a P53 biallelic knockout were defective in mediating DNA damage when incubated with doxorubicin. TALENs combined with SCNT was successfully used to generate P53 KO Diannan miniature pigs. Although these genetically engineered Diannan miniature pigs had no tumorigenic signs, the P53 gene was dysfunctional. We believe that these pigs will provide powerful new resources for preclinical oncology and basic cancer research.

NR2C in the thalamic reticular nucleus; effects of the NR2C knockout.

PubMed

Zhang, Yuchun; Buonanno, Andres; Vertes, Robert P; Hoover, Walter B; Lisman, John E

2012-01-01

NMDAR antagonists can evoke delta frequency bursting in the nucleus reticularis of the thalamus (nRT). The mechanism of this oscillation was determined; antagonist blocks an NR2C-like conductance that has low Mg block at resting potential and thus can contribute a resting inward current in response to ambient glutamate. Block of this current hyperpolarizes the cell, deinactivating T-type Ca channels and thus triggering delta frequency bursting. The basis for assuming a NR2C-like conductance was that (1) transcripts for NR2C are abundant in the thalamus and (2) the current-voltage curve of the synaptically evoked NMDAR current has the low rectification characteristic of NR2C. In the current study, we have sought to determine whether the channels that generate the NMDAR current are NR2C-like or are actually comprised of receptors containing NR2C. We studied the current-voltage curve of synaptically evoked NMDAR current in the nRT of NR2C knockout mice. In wild-type mice, the current was weakly voltage dependent, as previously observed in rats. This weak rectification was absent in NR2C KO mice. In contrast, NR2C KO had no effect on the strongly rectifying NMDAR current in pyramidal cells of the prefrontal cortex. These results demonstrate that the low rectification normally observed in the nRT is due to NR2C.

CRISPR-Mediated Triple Knockout of SLAMF1, SLAMF5 and SLAMF6 Supports Positive Signaling Roles in NKT Cell Development.

PubMed

Huang, Bonnie; Gomez-Rodriguez, Julio; Preite, Silvia; Garrett, Lisa J; Harper, Ursula L; Schwartzberg, Pamela L

2016-01-01

The SLAM family receptors contribute to diverse aspects of lymphocyte biology and signal via the small adaptor molecule SAP. Mutations affecting SAP lead to X-linked lymphoproliferative syndrome Type 1, a severe immunodysregulation characterized by fulminant mononucleosis, dysgammaglobulinemia, and lymphoproliferation/lymphomas. Patients and mice having mutations affecting SAP also lack germinal centers due to a defect in T:B cell interactions and are devoid of invariant NKT (iNKT) cells. However, which and how SLAM family members contribute to these phenotypes remains uncertain. Three SLAM family members: SLAMF1, SLAMF5 and SLAMF6, are highly expressed on T follicular helper cells and germinal center B cells. SLAMF1 and SLAMF6 are also implicated in iNKT development. Although individual receptor knockout mice have limited iNKT and germinal center phenotypes compared to SAP knockout mice, the generation of multi-receptor knockout mice has been challenging, due to the genomic linkage of the genes encoding SLAM family members. Here, we used Cas9/CRISPR-based mutagenesis to generate mutations simultaneously in Slamf1, Slamf5 and Slamf6. Genetic disruption of all three receptors in triple-knockout mice (TKO) did not grossly affect conventional T or B cell development and led to mild defects in germinal center formation post-immunization. However, the TKO worsened defects in iNKT cells development seen in SLAMF6 single gene-targeted mice, supporting data on positive signaling and potential redundancy between these receptors.

Characterizing a Rat Brca2 Knockout Model

DTIC Science & Technology

2007-05-01

synechiae (adhesion of iris to lens), and anterior segment dysgenesis (adhesion of lens to cornea). The cataractous lens cells extend circumferentially...around the lens capsule to the posterior (arrowheads). ac, anterior chamber; c, cornea; i, iris ; l, lens; lc, lens capsule; r, retina; m, mineralization...to DSB forma - tion, chromosomal damage, and cell death resulting from ionizing radiation (reviewed by Pellegrini and Venkitaraman, 2004). Evidence from

Pyviko: an automated Python tool to design gene knockouts in complex viruses with overlapping genes.

PubMed

Taylor, Louis J; Strebel, Klaus

2017-01-07

Gene knockouts are a common tool used to study gene function in various organisms. However, designing gene knockouts is complicated in viruses, which frequently contain sequences that code for multiple overlapping genes. Designing mutants that can be traced by the creation of new or elimination of existing restriction sites further compounds the difficulty in experimental design of knockouts of overlapping genes. While software is available to rapidly identify restriction sites in a given nucleotide sequence, no existing software addresses experimental design of mutations involving multiple overlapping amino acid sequences in generating gene knockouts. Pyviko performed well on a test set of over 240,000 gene pairs collected from viral genomes deposited in the National Center for Biotechnology Information Nucleotide database, identifying a point mutation which added a premature stop codon within the first 20 codons of the target gene in 93.2% of all tested gene-overprinted gene pairs. This shows that Pyviko can be used successfully in a wide variety of contexts to facilitate the molecular cloning and study of viral overprinted genes. Pyviko is an extensible and intuitive Python tool for designing knockouts of overlapping genes. Freely available as both a Python package and a web-based interface ( http://louiejtaylor.github.io/pyViKO/ ), Pyviko simplifies the experimental design of gene knockouts in complex viruses with overlapping genes.

Erythropoiesis and Blood Pressure Are Regulated via AT1 Receptor by Distinctive Pathways.

PubMed

Kato, Hideki; Ishida, Junji; Matsusaka, Taiji; Ishimaru, Tomohiro; Tanimoto, Keiji; Sugiyama, Fumihiro; Yagami, Ken-Ichi; Nangaku, Masaomi; Fukamizu, Akiyoshi

2015-01-01

The renin-angiotensin system (RAS) plays a central role in blood pressure regulation. Although clinical and experimental studies have suggested that inhibition of RAS is associated with progression of anemia, little evidence is available to support this claim. Here we report that knockout mice that lack angiotensin II, including angiotensinogen and renin knockout mice, exhibit anemia. The anemia of angiotensinogen knockout mice was rescued by angiotensin II infusion, and rescue was completely blocked by simultaneous administration of AT1 receptor blocker. To genetically determine the responsible receptor subtype, we examined AT1a, AT1b, and AT2 knockout mice, but did not observe anemia in any of them. To investigate whether pharmacological AT1 receptor inhibition recapitulates the anemic phenotype, we administered AT1 receptor antagonist in hypotensive AT1a receptor knockout mice to inhibit the remaining AT1b receptor. In these animals, hematocrit levels barely decreased, but blood pressure further decreased to the level observed in angiotensinogen knockout mice. We then generated AT1a and AT1b double-knockout mice to completely ablate the AT1 receptors; the mice finally exhibited the anemic phenotype. These results provide clear evidence that although erythropoiesis and blood pressure are negatively controlled through the AT1 receptor inhibition in vivo, the pathways involved are complex and distinct, because erythropoiesis is more resistant to AT1 receptor inhibition than blood pressure control.

Thyroid Hormone Receptor α- and β-Knockout Xenopus tropicalis Tadpoles Reveal Subtype-Specific Roles During Development.

PubMed

Nakajima, Keisuke; Tazawa, Ichiro; Yaoita, Yoshio

2018-02-01

Thyroid hormone (TH) binds TH receptor α (TRα) and β (TRβ) to induce amphibian metamorphosis. Whereas TH signaling has been well studied, functional differences between TRα and TRβ during this process have not been characterized. To understand how each TR contributes to metamorphosis, we generated TRα- and TRβ-knockout tadpoles of Xenopus tropicalis and examined developmental abnormalities, histology of the tail and intestine, and messenger RNA expression of genes encoding extracellular matrix-degrading enzymes. In TRβ-knockout tadpoles, tail regression was delayed significantly and a healthy notochord was observed even 5 days after the initiation of tail shortening (stage 62), whereas in the tails of wild-type and TRα-knockout tadpoles, the notochord disappeared after ∼1 day. The messenger RNA expression levels of genes encoding extracellular matrix-degrading enzymes (MMP2, MMP9TH, MMP13, MMP14, and FAPα) were obviously reduced in the tail tip of TRβ-knockout tadpoles, with the shortening tail. The reduction in olfactory nerve length and head narrowing by gill absorption were also affected. Hind limb growth and intestinal shortening were not compromised in TRβ-knockout tadpoles, whereas tail regression and olfactory nerve shortening appeared to proceed normally in TRα-knockout tadpoles, except for the precocious development of hind limbs. Our results demonstrated the distinct roles of TRα and TRβ in hind limb growth and tail regression, respectively. Copyright © 2018 Endocrine Society.

Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells.

PubMed

Seki, Akiko; Rutz, Sascha

2018-03-05

CRISPR (clustered, regularly interspaced, short palindromic repeats)/Cas9 (CRISPR-associated protein 9) has become the tool of choice for generating gene knockouts across a variety of species. The ability for efficient gene editing in primary T cells not only represents a valuable research tool to study gene function but also holds great promise for T cell-based immunotherapies, such as next-generation chimeric antigen receptor (CAR) T cells. Previous attempts to apply CRIPSR/Cas9 for gene editing in primary T cells have resulted in highly variable knockout efficiency and required T cell receptor (TCR) stimulation, thus largely precluding the study of genes involved in T cell activation or differentiation. Here, we describe an optimized approach for Cas9/RNP transfection of primary mouse and human T cells without TCR stimulation that results in near complete loss of target gene expression at the population level, mitigating the need for selection. We believe that this method will greatly extend the feasibly of target gene discovery and validation in primary T cells and simplify the gene editing process for next-generation immunotherapies. © 2018 Genentech.

Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors.

PubMed

Miura, Hiromi; Quadros, Rolen M; Gurumurthy, Channabasavaiah B; Ohtsuka, Masato

2018-01-01

CRISPR/Cas9-based genome editing can easily generate knockout mouse models by disrupting the gene sequence, but its efficiency for creating models that require either insertion of exogenous DNA (knock-in) or replacement of genomic segments is very poor. The majority of mouse models used in research involve knock-in (reporters or recombinases) or gene replacement (e.g., conditional knockout alleles containing exons flanked by LoxP sites). A few methods for creating such models have been reported that use double-stranded DNA as donors, but their efficiency is typically 1-10% and therefore not suitable for routine use. We recently demonstrated that long single-stranded DNAs (ssDNAs) serve as very efficient donors, both for insertion and for gene replacement. We call this method efficient additions with ssDNA inserts-CRISPR (Easi-CRISPR) because it is a highly efficient technology (efficiency is typically 30-60% and reaches as high as 100% in some cases). The protocol takes ∼2 months to generate the founder mice.

Vaccination with recombinant adenoviruses expressing Ebola virus glycoprotein elicits protection in the interferon alpha/beta receptor knock-out mouse.

PubMed

O'Brien, Lyn M; Stokes, Margaret G; Lonsdale, Stephen G; Maslowski, David R; Smither, Sophie J; Lever, Mark S; Laws, Thomas R; Perkins, Stuart D

2014-03-01

The resistance of adult immunocompetent mice to infection with ebolaviruses has led to the development of alternative small animal models that utilise immunodeficient mice, for example the interferon α/β receptor knock-out mouse (IFNR(-/-)). IFNR(-/-) mice have been shown to be susceptible to infection with ebolaviruses by multiple routes but it is not known if this murine model is suitable for testing therapeutics that rely on the generation of an immune response for efficacy. We have tested recombinant adenovirus vectors for their ability to protect IFNR(-/-) mice from challenge with Ebola virus and have analysed the humoral response generated after immunisation. The recombinant vaccines elicited good levels of protection in the knock-out mouse and the antibody response in IFNR(-/-) mice was similar to that observed in vaccinated wild-type mice. These results indicate that the IFNR(-/-) mouse is a relevant small animal model for studying ebolavirus-specific therapeutics. Copyright © 2014. Published by Elsevier Inc.

True-breeding targeted gene knock-out in barley using designer TALE-nuclease in haploid cells.

PubMed

Gurushidze, Maia; Hensel, Goetz; Hiekel, Stefan; Schedel, Sindy; Valkov, Vladimir; Kumlehn, Jochen

2014-01-01

Transcription activator-like effector nucleases (TALENs) are customizable fusion proteins able to cleave virtually any genomic DNA sequence of choice, and thereby to generate site-directed genetic modifications in a wide range of cells and organisms. In the present study, we expressed TALENs in pollen-derived, regenerable cells to establish the generation of instantly true-breeding mutant plants. A gfp-specific TALEN pair was expressed via Agrobacterium-mediated transformation in embryogenic pollen of transgenic barley harboring a functional copy of gfp. Thanks to the haploid nature of the target cells, knock-out mutations were readily detected, and homozygous primary mutant plants obtained following genome duplication. In all, 22% of the TALEN transgenics proved knocked out with respect to gfp, and the loss of function could be ascribed to the deletions of between four and 36 nucleotides in length. The altered gfp alleles were transmitted normally through meiosis, and the knock-out phenotype was consistently shown by the offspring of two independent mutants. Thus, here we describe the efficient production of TALEN-mediated gene knock-outs in barley that are instantaneously homozygous and non-chimeric in regard to the site-directed mutations induced. This TALEN approach has broad applicability for both elucidating gene function and tailoring the phenotype of barley and other crop species.

Deficiency in the manganese efflux transporter SLC30A10 induces severe hypothyroidism in mice.

PubMed

Hutchens, Steven; Liu, Chunyi; Jursa, Thomas; Shawlot, William; Chaffee, Beth K; Yin, Weiling; Gore, Andrea C; Aschner, Michael; Smith, Donald R; Mukhopadhyay, Somshuvra

2017-06-09

Manganese is an essential metal that becomes toxic at elevated levels. Loss-of-function mutations in SLC30A10, a cell-surface-localized manganese efflux transporter, cause a heritable manganese metabolism disorder resulting in elevated manganese levels and parkinsonian-like movement deficits. The underlying disease mechanisms are unclear; therefore, treatment is challenging. To understand the consequences of loss of SLC30A10 function at the organism level, we generated Slc30a10 knock-out mice. During early development, knock-outs were indistinguishable from controls. Surprisingly, however, after weaning and compared with controls, knock-out mice failed to gain weight, were smaller, and died prematurely (by ∼6-8 weeks of age). At 6 weeks, manganese levels in the brain, blood, and liver of the knock-outs were ∼20-60-fold higher than controls. Unexpectedly, histological analyses revealed that the brain and liver of the knock-outs were largely unaffected, but their thyroid exhibited extensive alterations. Because hypothyroidism leads to growth defects and premature death in mice, we assayed for changes in thyroid and pituitary hormones. At 6 weeks and compared with controls, the knock-outs had markedly reduced thyroxine levels (∼50-80%) and profoundly increased thyroid-stimulating hormone levels (∼800-1000-fold), indicating that Slc30a10 knock-out mice develop hypothyroidism. Importantly, a low-manganese diet produced lower tissue manganese levels in the knock-outs and rescued the phenotype, suggesting that manganese toxicity was the underlying cause. Our unanticipated discovery highlights the importance of determining the role of thyroid dysfunction in the onset and progression of manganese-induced disease and identifies Slc30a10 knock-out mice as a new model for studying thyroid biology. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Role of thin descending limb urea transport in renal urea handling and the urine concentrating mechanism

PubMed Central

Lei, Tianluo; Zhou, Lei; Layton, Anita T.; Zhou, Hong; Zhao, Xuejian; Bankir, Lise

2011-01-01

Urea transporters UT-A2 and UT-B are expressed in epithelia of thin descending limb of Henle's loop and in descending vasa recta, respectively. To study their role and possible interaction in the context of the urine concentration mechanism, a UT-A2 and UT-B double knockout (UT-A2/B knockout) mouse model was generated by targeted deletion of the UT-A2 promoter in embryonic stem cells with UT-B gene knockout. The UT-A2/B knockout mice lacked detectable UT-A2 and UT-B transcripts and proteins and showed normal survival and growth. Daily urine output was significantly higher in UT-A2/B knockout mice than that in wild-type mice and lower than that in UT-B knockout mice. Urine osmolality in UT-A2/B knockout mice was intermediate between that in UT-B knockout and wild-type mice. The changes in urine osmolality and flow rate, plasma and urine urea concentration, as well as non-urea solute concentration after an acute urea load or chronic changes in protein intake suggested that UT-A2 plays a role in the progressive accumulation of urea in the inner medulla. These results suggest that in wild-type mice UT-A2 facilitates urea absorption by urea efflux from the thin descending limb of short loops of Henle. Moreover, UT-A2 deletion in UT-B knockout mice partially remedies the urine concentrating defect caused by UT-B deletion, by reducing urea loss from the descending limbs to the peripheral circulation; instead, urea is returned to the inner medulla through the loops of Henle and the collecting ducts. PMID:21849488

Role of thin descending limb urea transport in renal urea handling and the urine concentrating mechanism.

PubMed

Lei, Tianluo; Zhou, Lei; Layton, Anita T; Zhou, Hong; Zhao, Xuejian; Bankir, Lise; Yang, Baoxue

2011-12-01

Urea transporters UT-A2 and UT-B are expressed in epithelia of thin descending limb of Henle's loop and in descending vasa recta, respectively. To study their role and possible interaction in the context of the urine concentration mechanism, a UT-A2 and UT-B double knockout (UT-A2/B knockout) mouse model was generated by targeted deletion of the UT-A2 promoter in embryonic stem cells with UT-B gene knockout. The UT-A2/B knockout mice lacked detectable UT-A2 and UT-B transcripts and proteins and showed normal survival and growth. Daily urine output was significantly higher in UT-A2/B knockout mice than that in wild-type mice and lower than that in UT-B knockout mice. Urine osmolality in UT-A2/B knockout mice was intermediate between that in UT-B knockout and wild-type mice. The changes in urine osmolality and flow rate, plasma and urine urea concentration, as well as non-urea solute concentration after an acute urea load or chronic changes in protein intake suggested that UT-A2 plays a role in the progressive accumulation of urea in the inner medulla. These results suggest that in wild-type mice UT-A2 facilitates urea absorption by urea efflux from the thin descending limb of short loops of Henle. Moreover, UT-A2 deletion in UT-B knockout mice partially remedies the urine concentrating defect caused by UT-B deletion, by reducing urea loss from the descending limbs to the peripheral circulation; instead, urea is returned to the inner medulla through the loops of Henle and the collecting ducts.

Renal Dysfunction Induced by Kidney-Specific Gene Deletion of Hsd11b2 as a Primary Cause of Salt-Dependent Hypertension.

PubMed

Ueda, Kohei; Nishimoto, Mitsuhiro; Hirohama, Daigoro; Ayuzawa, Nobuhiro; Kawarazaki, Wakako; Watanabe, Atsushi; Shimosawa, Tatsuo; Loffing, Johannes; Zhang, Ming-Zhi; Marumo, Takeshi; Fujita, Toshiro

2017-07-01

Genome-wide analysis of renal sodium-transporting system has identified specific variations of Mendelian hypertensive disorders, including HSD11B2 gene variants in apparent mineralocorticoid excess. However, these genetic variations in extrarenal tissue can be involved in developing hypertension, as demonstrated in former studies using global and brain-specific Hsd11b2 knockout rodents. To re-examine the importance of renal dysfunction on developing hypertension, we generated kidney-specific Hsd11b2 knockout mice. The knockout mice exhibited systemic hypertension, which was abolished by reducing salt intake, suggesting its salt-dependency. In addition, we detected an increase in renal membrane expressions of cleaved epithelial sodium channel-α and T53-phosphorylated Na + -Cl - cotransporter in the knockout mice. Acute intraperitoneal administration of amiloride-induced natriuresis and increased urinary sodium/potassium ratio more in the knockout mice compared with those in the wild-type control mice. Chronic administration of amiloride and high-KCl diet significantly decreased mean blood pressure in the knockout mice, which was accompanied with the correction of hypokalemia and the resultant decrease in Na + -Cl - cotransporter phosphorylation. Accordingly, a Na + -Cl - cotransporter blocker hydrochlorothiazide significantly decreased mean blood pressure in the knockout mice. Chronic administration of mineralocorticoid receptor antagonist spironolactone significantly decreased mean blood pressure of the knockout mice along with downregulation of cleaved epithelial sodium channel-α and phosphorylated Na + -Cl - cotransporter expression in the knockout kidney. Our data suggest that kidney-specific deficiency of 11β-HSD2 leads to salt-dependent hypertension, which is attributed to mineralocorticoid receptor-epithelial sodium channel-Na + -Cl - cotransporter activation in the kidney, and provides evidence that renal dysfunction is essential for developing the phenotype of apparent mineralocorticoid excess. © 2017 American Heart Association, Inc.

Slc25a12 disruption alters myelination and neurofilaments: a model for a hypomyelination syndrome and childhood neurodevelopmental disorders.

PubMed

Sakurai, Takeshi; Ramoz, Nicolas; Barreto, Marta; Gazdoiu, Mihaela; Takahashi, Nagahide; Gertner, Michael; Dorr, Nathan; Gama Sosa, Miguel A; De Gasperi, Rita; Perez, Gissel; Schmeidler, James; Mitropoulou, Vivian; Le, H Carl; Lupu, Mihaela; Hof, Patrick R; Elder, Gregory A; Buxbaum, Joseph D

2010-05-01

SLC25A12, a susceptibility gene for autism spectrum disorders that is mutated in a neurodevelopmental syndrome, encodes a mitochondrial aspartate-glutamate carrier (aspartate-glutamate carrier isoform 1 [AGC1]). AGC1 is an important component of the malate/aspartate shuttle, a crucial system supporting oxidative phosphorylation and adenosine triphosphate production. We characterized mice with a disruption of the Slc25a12 gene, followed by confirmatory in vitro studies. Slc25a12-knockout mice, which showed no AGC1 by immunoblotting, were born normally but displayed delayed development and died around 3 weeks after birth. In postnatal day 13 to 14 knockout brains, the brains were smaller with no obvious alteration in gross structure. However, we found a reduction in myelin basic protein (MBP)-positive fibers, consistent with a previous report. Furthermore, the neocortex of knockout mice contained abnormal neurofilamentous accumulations in neurons, suggesting defective axonal transport and/or neurodegeneration. Slice cultures prepared from knockout mice also showed a myelination defect, and reduction of Slc25a12 in rat primary oligodendrocytes led to a cell-autonomous reduction in MBP expression. Myelin deficits in slice cultures from knockout mice could be reversed by administration of pyruvate, indicating that reduction in AGC1 activity leads to reduced production of aspartate/N-acetylaspartate and/or alterations in the dihydronicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide(+) ratio, resulting in myelin defects. Our data implicate AGC1 activity in myelination and in neuronal structure and indicate that while loss of AGC1 leads to hypomyelination and neuronal changes, subtle alterations in AGC1 expression could affect brain development, contributing to increased autism susceptibility. Copyright 2010 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Maternal loading with very low-density lipoproteins stimulates fetal surfactant synthesis.

PubMed

Ryan, Alan J; Medh, Jheem D; McCoy, Diann M; Salome, Ronald G; Mallampalli, Rama K

2002-08-01

We examined whether administration of very low-density lipoproteins (VLDL) to pregnant rats increases surfactant phosphatidylcholine (PtdCho) content in fetal pre-type II alveolar epithelial cells. VLDL-triglycerides are hydrolyzed to fatty acids by lipoprotein lipase (LPL), an enzyme activated by heparin. Fatty acids released by LPL can incorporate into the PtdCho molecule or activate the key biosynthetic enzyme cytidylyltransferase (CCT). Dams were given BSA, heparin, VLDL, or VLDL with heparin intravenously. Radiolabeled VLDL given to the pregnant rat crossed the placenta and was distributed systemically in the fetus and incorporated into disaturated PtdCho (DSPtdCho) in pre-type II cells. Maternal administration of VLDL with heparin increased DSPtdCho content in cells by 45% compared with control (P < 0.05). VLDL produced a dose-dependent, saturable, and selective increase in CCT activity. VLDL did not significantly alter immunoreactive CCT content but increased palmitic, stearic, and oleic acids in pre-type II cells. Furthermore, hypertriglyceridemic apolipoprotein E knockout mice contained significantly greater levels of DSPtdCho content in alveolar lavage and CCT activity compared with either LDL receptor knockout mice or wild-type controls that have normal serum triglycerides. Thus the nutritional or genetic modulation of serum VLDL-triglycerides provides specific fatty acids that stimulate PtdCho synthesis and CCT activity thereby increasing surfactant content.

Glutamine synthetase gene knockout-human embryonic kidney 293E cells for stable production of monoclonal antibodies.

PubMed

Yu, Da Young; Lee, Sang Yoon; Lee, Gyun Min

2018-05-01

Previously, it was inferred that a high glutamine synthetase (GS) activity in human embryonic kidney (HEK) 293E cells results in elevated resistance to methionine sulfoximine (MSX) and consequently hampers GS-mediated gene amplification and selection by MSX. To overcome this MSX resistance in HEK293E cells, a GS-knockout HEK293E cell line was generated using the CRISPR/Cas9 system to target the endogenous human GS gene. The GS-knockout in the HEK293E cell line (RK8) was confirmed by Western blot analysis of GS and by observation of glutamine-dependent growth. Unlike the wild type HEK293E cells, the RK8 cells were successfully used as host cells to generate a recombinant HEK293E cell line (rHEK293E) producing a monoclonal antibody (mAb). When the RK8 cells were transfected with the GS expression vector containing the mAb gene, rHEK293E cells producing the mAb could be selected in the absence as well as in the presence of MSX. The gene copies and mRNA expression levels of the mAb in rHEK293E cells were also quantified using qRT-PCR. Taken together, the GS-knockout HEK293E cell line can be used as host cells to generate stable rHEK293E cells producing a mAb through GS-mediated gene selection in the absence as well as in the presence of MSX. © 2018 Wiley Periodicals, Inc.

Fabp4-Cre-mediated Sirt6 deletion impairs adipose tissue function and metabolic homeostasis in mice.

PubMed

Xiong, Xiwen; Zhang, Cuicui; Zhang, Yang; Fan, Rui; Qian, Xinlai; Dong, X Charlie

2017-06-01

SIRT6 is a member of sirtuin family of deacetylases involved in diverse processes including genome stability, metabolic homeostasis and anti-inflammation. However, its function in the adipose tissue is not well understood. To examine the metabolic function of SIRT6 in the adipose tissue, we generated two mouse models that are deficient in Sirt6 using the Cre-lox approach. Two commonly used Cre lines that are driven by either the mouse Fabp4 or Adipoq gene promoter were chosen for this study. The Sirt6- knockout mice generated by the Fabp4-Cre line ( Sirt6 f/f : Fabp4-Cre) had a significant increase in both body weight and fat mass and exhibited glucose intolerance and insulin resistance as compared with the control wild-type mice. At the molecular levels, the Sirt6 f/f :Fabp4-Cre-knockout mice had increased expression of inflammatory genes including F4/80, TNFα, IL-6 and MCP-1 in both white and brown adipose tissues. Moreover, the knockout mice showed decreased expression of the adiponectin gene in the white adipose tissue and UCP1 in the brown adipose tissue, respectively. In contrast, the Sirt6 knockout mice generated by the Adipoq-Cre line ( Sirt6 f/f :Adipoq-Cre) only had modest insulin resistance. In conclusion, our data suggest that the function of SIRT6 in the Fabp4-Cre-expressing cells in addition to mature adipocytes plays a critical role in body weight maintenance and metabolic homeostasis. © 2017 Society for Endocrinology.

Generation of ER{alpha}-floxed and knockout mice using the Cre/LoxP system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Antonson, P., E-mail: per.antonson@ki.se; Omoto, Y.; Humire, P.

2012-08-10

Highlights: Black-Right-Pointing-Pointer ER{alpha} floxed and knockout mice were generated. Black-Right-Pointing-Pointer Disruption of the ER{alpha} gene results in sterility in both male and female mice. Black-Right-Pointing-Pointer ER{alpha}{sup -/-} mice have ovaries with hemorrhagic follicles and hypoplastic uterus. Black-Right-Pointing-Pointer Female ER{alpha}{sup -/-} mice develop obesity. -- Abstract: Estrogen receptor alpha (ER{alpha}) is a nuclear receptor that regulates a range of physiological processes in response to estrogens. In order to study its biological role, we generated a floxed ER{alpha} mouse line that can be used to knock out ER{alpha} in selected tissues by using the Cre/LoxP system. In this study, we established amore » new ER{alpha} knockout mouse line by crossing the floxed ER{alpha} mice with Cre deleter mice. Here we show that genetic disruption of the ER{alpha} gene in all tissues results in sterility in both male and female mice. Histological examination of uterus and ovaries revealed a dramatically atrophic uterus and hemorrhagic cysts in the ovary. These results suggest that infertility in female mice is the result of functional defects of the reproductive tract. Moreover, female knockout mice are hyperglycemic, develop obesity and at the age of 4 months the body weight of these mice was more than 20% higher compared to wild type littermates and this difference increased over time. Our results demonstrate that ER{alpha} is necessary for reproductive tract development and has important functions as a regulator of metabolism in females.« less

Effect of KnockOut serum replacement on germ cell development of immature testis tissue culture.

PubMed

Liu, Feng; Cai, Chunhong; Wu, Xin; Cheng, Yanxia; Lin, Tao; Wei, Guanghui; He, Dawei

2016-01-15

To compare KnockOut serum replacement (KSR) and fetal bovine serum (FBS) for the development of germ cells. Testicular tissues from Sprague-Dawley rats were cultured for 4 weeks in culture media supplemented with FBS or KSR. Tissue area was measured at the beginning and end of the culturing period. Testicular histology, development of the germ cells, and the diameter of seminiferous tubules were analyzed by hematoxylin and eosin staining. After 4 weeks in culture, apoptosis and expression of the stage-specific spermatogenesis marker genes Kit, Sycp3, and Crisp1 were assayed. Tissues cultured in KSR-supplemented media were able to sustain growth and gradually increase seminiferous tubule diameter during the culture period. In addition, spermatogonia, primary spermatocytes, secondary spermatocytes, and round spermatids were observed after 4 weeks in culture, and reverse transcription-PCR confirmed expression of the marker genes. In comparison, tissues cultured in FBS-supplemented media showed dwindling testicular organization, necrotic seminiferous tubules, and expression of Kit, but inconsistent expression of Sycp3 and Crisp1 KnockOut serum replacement outperforms FBS as a growth media supplements for culturing immature spermatogonial tissue culture. Copyright © 2016 Elsevier Inc. All rights reserved.

Ultra-superovulation for the CRISPR-Cas9-mediated production of gene-knockout, single-amino-acid-substituted, and floxed mice.

PubMed

Nakagawa, Yoshiko; Sakuma, Tetsushi; Nishimichi, Norihisa; Yokosaki, Yasuyuki; Yanaka, Noriyuki; Takeo, Toru; Nakagata, Naomi; Yamamoto, Takashi

2016-08-15

Current advances in producing genetically modified mice using genome-editing technologies have indicated the need for improvement of limiting factors including zygote collection for microinjection and their cryopreservation. Recently, we developed a novel superovulation technique using inhibin antiserum and equine chorionic gonadotropin to promote follicle growth. This method enabled the increased production of fertilized oocytes via in vitro fertilization compared with the conventional superovulation method. Here, we verify that the ultra-superovulation technique can be used for the efficient generation of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated knockout mice by microinjection of plasmid vector or ribonucleoprotein into zygotes. We also investigated whether single-amino-acid-substituted mice and conditional knockout mice could be generated. Founder mice bearing base substitutions were generated more efficiently by co-microinjection of Cas9 protein, a guide RNA and single-stranded oligodeoxynucleotide (ssODN) than by plasmid microinjection with ssODN. The conditional allele was successfully introduced by the one-step insertion of an ssODN designed to carry an exon flanked by two loxP sequences and homology arms using a double-cut CRISPR-Cas9 strategy. Our study presents a useful method for the CRISPR-Cas9-based generation of genetically modified mice from the viewpoints of animal welfare and work efficiency. © 2016. Published by The Company of Biologists Ltd.

Connexin 43 reboots meiosis and reseals blood-testis barrier following toxicant-mediated aspermatogenesis and barrier disruption.

PubMed

Li, Nan; Mruk, Dolores D; Mok, Ka-Wai; Li, Michelle W M; Wong, Chris K C; Lee, Will M; Han, Daishu; Silvestrini, Bruno; Cheng, C Yan

2016-04-01

Earlier studies have shown that rats treated with an acute dose of 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide (adjudin, a male contraceptive under development) causes permanent infertility due to irreversible blood-testis barrier (BTB) disruption even though the population of undifferentiated spermatogonia remains similar to normal rat testes, because spermatogonia fail to differentiate into spermatocytes to enter meiosis. Since other studies have illustrated the significance of connexin 43 (Cx43)-based gap junction in maintaining the homeostasis of BTB in the rat testis and the phenotypes of Sertoli cell-conditional Cx43 knockout mice share many of the similarities of the adjudin-treated rats, we sought to examine if overexpression of Cx43 in these adjudin-treated rats would reseal the disrupted BTB and reinitiate spermatogenesis. A full-length Cx43 cloned into mammalian expression vector pCI-neo was used to transfect testes of adjudin-treated ratsversusempty vector. It was found that overexpression of Cx43 indeed resealed the Sertoli cell tight junction-permeability barrier based on a functionalin vivoassay in tubules displaying signs of meiosis as noted by the presence of round spermatids. Thus, these findings suggest that overexpression of Cx43 reinitiated spermatogenesis at least through the steps of meiosis to generate round spermatids in testes of rats treated with an acute dose of adjudin that led to aspermatogenesis. It was also noted that the round spermatids underwent eventual degeneration with the formation of multinucleated cells following Cx43 overexpression due to the failure of spermiogenesis because no elongating/elongated spermatids were detected in any of the tubules examined. The mechanism by which overexpression of Cx43 reboots meiosis and rescues BTB function was also examined. In summary, overexpression of Cx43 in the testis with aspermatogenesis reboots meiosis and reseals toxicant-induced BTB disruption, even though it fails to support round spermatids to enter spermiogenesis.-Li, N., Mruk, D. D., Mok, K.-W., Li, M. W. M., Wong, C. K. C., Lee, W. M., Han, D., Silvestrini, B., Cheng, C. Y. Connexin 43 reboots meiosis and reseals blood-testis barrier following toxicant-mediated aspermatogenesis and barrier disruption. © FASEB.

Degraded neural and behavioral processing of speech sounds in a rat model of Rett syndrome

PubMed Central

Engineer, Crystal T.; Rahebi, Kimiya C.; Borland, Michael S.; Buell, Elizabeth P.; Centanni, Tracy M.; Fink, Melyssa K.; Im, Kwok W.; Wilson, Linda G.; Kilgard, Michael P.

2015-01-01

Individuals with Rett syndrome have greatly impaired speech and language abilities. Auditory brainstem responses to sounds are normal, but cortical responses are highly abnormal. In this study, we used the novel rat Mecp2 knockout model of Rett syndrome to document the neural and behavioral processing of speech sounds. We hypothesized that both speech discrimination ability and the neural response to speech sounds would be impaired in Mecp2 rats. We expected that extensive speech training would improve speech discrimination ability and the cortical response to speech sounds. Our results reveal that speech responses across all four auditory cortex fields of Mecp2 rats were hyperexcitable, responded slower, and were less able to follow rapidly presented sounds. While Mecp2 rats could accurately perform consonant and vowel discrimination tasks in quiet, they were significantly impaired at speech sound discrimination in background noise. Extensive speech training improved discrimination ability. Training shifted cortical responses in both Mecp2 and control rats to favor the onset of speech sounds. While training increased the response to low frequency sounds in control rats, the opposite occurred in Mecp2 rats. Although neural coding and plasticity are abnormal in the rat model of Rett syndrome, extensive therapy appears to be effective. These findings may help to explain some aspects of communication deficits in Rett syndrome and suggest that extensive rehabilitation therapy might prove beneficial. PMID:26321676

Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells.

PubMed

Fan, Lianchun; Kadura, Ibrahim; Krebs, Lara E; Hatfield, Christopher C; Shaw, Margaret M; Frye, Christopher C

2012-04-01

Although Chinese hamster ovary (CHO) cells, with their unique characteristics, have become a major workhorse for the manufacture of therapeutic recombinant proteins, one of the major challenges in CHO cell line generation (CLG) is how to efficiently identify those rare, high-producing clones among a large population of low- and non-productive clones. It is not unusual that several hundred individual clones need to be screened for the identification of a commercial clonal cell line with acceptable productivity and growth profile making the cell line appropriate for commercial application. This inefficiency makes the process of CLG both time consuming and laborious. Currently, there are two main CHO expression systems, dihydrofolate reductase (DHFR)-based methotrexate (MTX) selection and glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection, that have been in wide industrial use. Since selection of recombinant cell lines in the GS-CHO system is based on the balance between the expression of the GS gene introduced by the expression plasmid and the addition of the GS inhibitor, L-MSX, the expression of GS from the endogenous GS gene in parental CHOK1SV cells will likely interfere with the selection process. To study endogenous GS expression's potential impact on selection efficiency, GS-knockout CHOK1SV cell lines were generated using the zinc finger nuclease (ZFN) technology designed to specifically target the endogenous CHO GS gene. The high efficiency (∼2%) of bi-allelic modification on the CHO GS gene supports the unique advantages of the ZFN technology, especially in CHO cells. GS enzyme function disruption was confirmed by the observation of glutamine-dependent growth of all GS-knockout cell lines. Full evaluation of the GS-knockout cell lines in a standard industrial cell culture process was performed. Bulk culture productivity improved two- to three-fold through the use of GS-knockout cells as parent cells. The selection stringency was significantly increased, as indicated by the large reduction of non-producing and low-producing cells after 25 µM L-MSX selection, and resulted in a six-fold efficiency improvement in identifying similar numbers of high-productive cell lines for a given recombinant monoclonal antibody. The potential impact of GS-knockout cells on recombinant protein quality is also discussed. Copyright © 2011 Wiley Periodicals, Inc.

Knockout of the BK β2 subunit abolishes inactivation of BK currents in mouse adrenal chromaffin cells and results in slow-wave burst activity

PubMed Central

Martinez-Espinosa, Pedro L.; Yang, Chengtao; Gonzalez-Perez, Vivian; Xia, Xiao-Ming

2014-01-01

Rat and mouse adrenal medullary chromaffin cells (CCs) express an inactivating BK current. This inactivation is thought to arise from the assembly of up to four β2 auxiliary subunits (encoded by the kcnmb2 gene) with a tetramer of pore-forming Slo1 α subunits. Although the physiological consequences of inactivation remain unclear, differences in depolarization-evoked firing among CCs have been proposed to arise from the ability of β2 subunits to shift the range of BK channel activation. To investigate the role of BK channels containing β2 subunits, we generated mice in which the gene encoding β2 was deleted (β2 knockout [KO]). Comparison of proteins from wild-type (WT) and β2 KO mice allowed unambiguous demonstration of the presence of β2 subunit in various tissues and its coassembly with the Slo1 α subunit. We compared current properties and cell firing properties of WT and β2 KO CCs in slices and found that β2 KO abolished inactivation, slowed action potential (AP) repolarization, and, during constant current injection, decreased AP firing. These results support the idea that the β2-mediated shift of the BK channel activation range affects repetitive firing and AP properties. Unexpectedly, CCs from β2 KO mice show an increased tendency toward spontaneous burst firing, suggesting that the particular properties of BK channels in the absence of β2 subunits may predispose to burst firing. PMID:25267913

Vision Integrating Strategies in Ophthalmology and Neurochemistry (VISION)

DTIC Science & Technology

2014-02-01

ganglion cells from pressure-induced damage in a rat model of glaucoma . Brn3b also induced optic nerve regeneration in this model (Stankowska et al. 2013...of glaucoma o Gene therapy with Neuritin1 structurally and functionally protected the retina in ONC model o CHOP knockout mice were structurally and...retinocollicular pathway of mice in a novel model of glaucoma . 2013 Annual Meeting of Association for Research in Vision and Ophthalmology, Abstract 421. Liu

Knockout of the Na,K-ATPase α2-isoform in cardiac myocytes delays pressure overload-induced cardiac dysfunction

PubMed Central

Rindler, Tara N.; Lasko, Valerie M.; Nieman, Michelle L.; Okada, Motoi; Lorenz, John N.

2013-01-01

The α2-isoform of the Na,K-ATPase (α2) is the minor isoform of the Na,K-ATPase expressed in the cardiovascular system and is thought to play a critical role in the regulation of cardiovascular hemodynamics. However, the organ system/cell type expressing α2 that is required for this regulation has not been fully defined. The present study uses a heart-specific knockout of α2 to further define the tissue-specific role of α2 in the regulation of cardiovascular hemodynamics. To accomplish this, we developed a mouse model using the Cre/loxP system to generate a tissue-specific knockout of α2 in the heart using β-myosin heavy chain Cre. We have achieved a 90% knockout of α2 expression in the heart of the knockout mice. Interestingly, the heart-specific knockout mice exhibit normal basal cardiac function and systolic blood pressure, and in addition, these mice develop ACTH-induced hypertension in response to ACTH treatment similar to control mice. Surprisingly, the heart-specific knockout mice display delayed onset of cardiac dysfunction compared with control mice in response to pressure overload induced by transverse aortic constriction; however, the heart-specific knockout mice deteriorated to control levels by 9 wk post-transverse aortic constriction. These results suggest that heart expression of α2 does not play a role in the regulation of basal cardiovascular function or blood pressure; however, heart expression of α2 plays a role in the hypertrophic response to pressure overload. This study further emphasizes that the tissue localization of α2 determines its unique roles in the regulation of cardiovascular function. PMID:23436327

Neuron-specific (pro)renin receptor knockout prevents the development of salt-sensitive hypertension

PubMed Central

Li, Wencheng; Peng, Hua; Mehaffey, Eamonn P.; Kimball, Christie D.; Grobe, Justin L.; van Gool, Jeanette M.G.; Sullivan, Michelle N.; Earley, Scott; Danser, A.H. Jan; Ichihara, Atsuhiro; Feng, Yumei

2013-01-01

The (pro)renin receptor, which binds both renin and prorenin, is a newly discovered component of the renin angiotensin system that is highly expressed in the central nervous system. The significance of brain PRRs in mediating local angiotensin II formation and regulating blood pressure remains unclear. The current study was performed to test the hypothesis that PRR-mediated, non-proteolytic activation of prorenin is the main source of angiotensin II in the brain. Thus, PRR knockout in the brain is expected to prevent angiotensin II formation and development of deoxycorticosterone acetate salt induced hypertension. A neuron-specific PRR (ATP6AP2) knockout mouse model was generated using the Cre-LoxP system. Physiological parameters were recorded by telemetry. (Pro)renin receptor expression, detected by immunostaining and RT-PCR, was significantly decreased in the brains of knockout compared with wide-type mice. Intracerebroventricular infusion of mouse prorenin increased blood pressure and angiotensin II formation in wild type mice. This hypertensive response was abolished in (pro)renin receptor knockout mice in association with a reduction in angiotensin II levels. Deoxycorticosterone acetate salt increased (pro)renin receptor expression and angiotensin II formation in the brains of wild-type mice, an effect that was attenuated in (pro)renin receptor knockout mice. (Pro)renin receptor knockout in neurons prevented the development of Deoxycorticosterone acetate salt-induced hypertension as well as activation of cardiac and vasomotor sympathetic tone. In conclusion, non-proteolytic activation of prorenin through binding to the PRR mediates angiotensin II formation in the brain. Neuron-specific PRR knockout prevents the development of deoxycorticosterone acetate salt-induced hypertension, possibly through diminished angiotensin II formation. PMID:24246383

A minor role of WNK3 in regulating phosphorylation of renal NKCC2 and NCC co-transporters in vivo.

PubMed

Oi, Katsuyuki; Sohara, Eisei; Rai, Tatemitsu; Misawa, Moko; Chiga, Motoko; Alessi, Dario R; Sasaki, Sei; Uchida, Shinichi

2012-02-15

Mutations in WNK1 and WNK4 kinase genes have been shown to cause a human hereditary hypertensive disease, pseudohypoaldosteronism type II (PHAII). We previously discovered that WNK kinases phosphorylate and activate OSR1/SPAK kinases that regulate renal SLC12A family transporters such as NKCC2 and NCC, and clarified that the constitutive activation of this cascade causes PHAII. WNK3, another member of the WNK kinase family, was reported to be a strong activator of NCC/NKCC2 when assayed in Xenopus oocytes, suggesting that WNK3 also plays a major role in regulating blood pressure and sodium reabsorption in the kidney. However, it remains to be determined whether WNK3 is in fact involved in the regulation of these transporters in vivo. To clarify this issue, we generated and analyzed WNK3 knockout mice. Surprisingly, phosphorylation and expression of OSR1, SPAK, NKCC2 and NCC did not decrease in knockout mouse kidney under normal and low-salt diets. Similarly, expression of epithelial Na channel and Na/H exchanger 3 were not affected in knockout mice. Na(+) and K(+) excretion in urine in WNK3 knockout mice was not affected under different salt diets. Blood pressure in WNK3 knockout mice was not lower under normal diet. However, lower blood pressure was observed in WNK3 knockout mice fed low-salt diet. WNK4 and WNK1 expression was slightly elevated in the knockout mice under low-salt diet, suggesting compensation for WNK3 knockout by these WNKs. Thus, WNK3 may have some role in the WNK-OSR1/SPAK-NCC/NKCC2 signal cascade in the kidney, but its contribution to total WNK kinase activity may be minimal.

Deconstructing mammalian reproduction: using knockouts to define fertility pathways.

PubMed

Roy, Angshumoy; Matzuk, Martin M

2006-02-01

Reproduction is the sine qua non for the propagation of species and continuation of life. It is a complex biological process that is regulated by multiple factors during the reproductive life of an organism. Over the past decade, the molecular mechanisms regulating reproduction in mammals have been rapidly unraveled by the study of a vast number of mouse gene knockouts with impaired fertility. The use of reverse genetics to generate null mutants in mice through targeted disruption of specific genes has enabled researchers to identify essential regulators of spermatogenesis and oogenesis in vivo and model human disorders affecting reproduction. This review focuses on the merits, utility, and the variations of the knockout technology in studies of reproduction in mammals.

Toxicological significance of renal Bcrp: Another potential transporter in the elimination of mercuric ions from proximal tubular cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bridges, Christy C., E-mail: bridges_cc@mercer.edu; Zalups, Rudolfs K.; Joshee, Lucy

Secretion of inorganic mercury (Hg{sup 2+}) from proximal tubular cells into the tubular lumen has been shown to involve the multidrug resistance-associated protein 2 (Mrp2). Considering similarities in localization and substrate specificity between Mrp2 and the breast cancer resistance protein (Bcrp), we hypothesize that Bcrp may also play a role in the proximal tubular secretion of mercuric species. In order to test this hypothesis, the uptake of Hg{sup 2+} was examined initially using inside-out membrane vesicles containing Bcrp. The results of these studies suggest that Bcrp may be capable of transporting certain conjugates of Hg{sup 2+}. To further characterize themore » role of Bcrp in the handling of mercuric ions and in the induction of Hg{sup 2+}-induced nephropathy, Sprague–Dawley and Bcrp knockout (bcrp{sup −/−}) rats were exposed intravenously to a non-nephrotoxic (0.5 μmol·kg{sup −1}), a moderately nephrotoxic (1.5 μmol·kg{sup −1}) or a significantly nephrotoxic (2.0 μmol·kg{sup −1}) dose of HgCl{sub 2}. In general, the accumulation of Hg{sup 2+} was greater in organs of bcrp{sup −/−} rats than in Sprague–Dawley rats, suggesting that Bcrp may play a role in the export of Hg{sup 2+} from target cells. Within the kidney, cellular injury and necrosis was more severe in bcrp{sup −/−} rats than in controls. The pattern of necrosis, which was localized in the inner cortex and the outer stripe of the outer medulla, was significantly different from that observed in Mrp2-deficient animals. These findings suggest that Bcrp may be involved in the cellular export of select mercuric species and that its role in this export may differ from that of Mrp2. - Highlights: • Bcrp may mediate transport of mercury out of proximal tubular cells. • Hg-induced nephropathy was more severe in Bcrp knockout rats. • Bcrp and Mrp2 may differ in their ability to transport Hg.« less

Highly Efficient Genome Editing via CRISPR/Cas9 to Create Clock Gene Knockout Cells.

PubMed

Korge, Sandra; Grudziecki, Astrid; Kramer, Achim

2015-10-01

Targeted genome editing using CRISPR/Cas9 is a relatively new, revolutionary technology allowing for efficient and directed alterations of the genome. It has been widely used for loss-of-function studies in animals and cell lines but has not yet been used to study circadian rhythms. Here, we describe the application of CRISPR/Cas9 genome editing for the generation of an F-box and leucine-rich repeat protein 3 (Fbxl3) knockout in a human cell line. Genomic alterations at the Fbxl3 locus occurred with very high efficiency (70%-100%) and specificity at both alleles, resulting in insertions and deletions that led to premature stop codons and hence FBXL3 knockout. Fbxl3 knockout cells displayed low amplitude and long period oscillations of Bmal1-luciferase reporter activity as well as increased CRY1 protein stability in line with previously published phenotypes for Fbxl3 knockout in mice. Thus, CRISPR/Cas9 genome editing should be highly valuable for studying circadian rhythms not only in human cells but also in classic model systems as well as nonmodel organisms. © 2015 The Author(s).

Evidence for the involvement of ASIC3 in sensory mechanotransduction in proprioceptors

PubMed Central

Lin, Shing-Hong; Cheng, Yuan-Ren; Banks, Robert W.; Min, Ming-Yuan; Bewick, Guy S.; Chen, Chih-Cheng

2016-01-01

Acid-sensing ion channel 3 (ASIC3) is involved in acid nociception, but its possible role in neurosensory mechanotransduction is disputed. We report here the generation of Asic3-knockout/eGFPf-knockin mice and subsequent characterization of heterogeneous expression of ASIC3 in the dorsal root ganglion (DRG). ASIC3 is expressed in parvalbumin (Pv+) proprioceptor axons innervating muscle spindles. We further generate a floxed allele of Asic3 (Asic3f/f) and probe the role of ASIC3 in mechanotransduction in neurite-bearing Pv+ DRG neurons through localized elastic matrix movements and electrophysiology. Targeted knockout of Asic3 disrupts spindle afferent sensitivity to dynamic stimuli and impairs mechanotransduction in Pv+ DRG neurons because of substrate deformation-induced neurite stretching, but not to direct neurite indentation. In behavioural tasks, global knockout (Asic3−/−) and Pv-Cre::Asic3f/f mice produce similar deficits in grid and balance beam walking tasks. We conclude that, at least in mouse, ASIC3 is a molecular determinant contributing to dynamic mechanosensitivity in proprioceptors. PMID:27161260

Rapid construction of a whole-genome transposon insertion collection for Shewanella oneidensis by Knockout Sudoku.

PubMed

Baym, Michael; Shaket, Lev; Anzai, Isao A; Adesina, Oluwakemi; Barstow, Buz

2016-11-10

Whole-genome knockout collections are invaluable for connecting gene sequence to function, yet traditionally, their construction has required an extraordinary technical effort. Here we report a method for the construction and purification of a curated whole-genome collection of single-gene transposon disruption mutants termed Knockout Sudoku. Using simple combinatorial pooling, a highly oversampled collection of mutants is condensed into a next-generation sequencing library in a single day, a 30- to 100-fold improvement over prior methods. The identities of the mutants in the collection are then solved by a probabilistic algorithm that uses internal self-consistency within the sequencing data set, followed by rapid algorithmically guided condensation to a minimal representative set of mutants, validation, and curation. Starting from a progenitor collection of 39,918 mutants, we compile a quality-controlled knockout collection of the electroactive microbe Shewanella oneidensis MR-1 containing representatives for 3,667 genes that is functionally validated by high-throughput kinetic measurements of quinone reduction.

Genetic cathepsin B deficiency reduces beta-amyloid in transgenic mice expressing human wild-type amyloid precursor protein.

PubMed

Hook, Vivian Y H; Kindy, Mark; Reinheckel, Thomas; Peters, Christoph; Hook, Gregory

2009-08-21

Neurotoxic beta-amyloid (Abeta) peptides participate in Alzheimer's disease (AD); therefore, reduction of Abeta generated from APP may provide a therapeutic approach for AD. Gene knockout studies in transgenic mice producing human Abeta may identify targets for reducing Abeta. This study shows that knockout of the cathepsin B gene in mice expressing human wild-type APP (hAPPwt) results in substantial decreases in brain Abeta40 and Abeta42 by 67% and decreases in levels of the C-terminal beta-secretase fragment (CTFbeta) derived from APP. In contrast, knockout of cathepsin B in mice expressing hAPP with the rare Swedish (Swe) and Indiana (Ind) mutations had no effect on Abeta. The difference in reduction of Abeta in hAPPwt mice, but not in hAPPSwe/Ind mice, shows that the transgenic model can affect cathepsin B gene knockout results. Since most AD patients express hAPPwt, these data validate cathepsin B as a target for development of inhibitors to lower Abeta in AD.

A mental retardation gene, motopsin/neurotrypsin/prss12, modulates hippocampal function and social interaction

PubMed Central

Mitsui, Shinichi; Osako, Yoji; Yokoi, Fumiaki; Dang, Mai T.; Yuri, Kazunari; Li, Yuqing; Yamaguchi, Nozomi

2010-01-01

Motopsin is a mosaic serine protease secreted from neuronal cells in various brain regions including the hippocampus. The loss of motopsin function causes nonsyndromic mental retardation in humans and impairs long-term memory formation in Drosophila. To understand motopsin’s function in the mammalian brain, motopsin knockout mice were generated. Motopsin knockout mice did not have significant deficit in memory formation, as was tested using in the Morris water maze, passive avoidance, and Y-maze tests. A social recognition test showed that the motopsin knockout mice had the ability to recognize two stimulator mice, suggesting normal social memory. In a social novelty test, motopsin knockout mice spent a longer time investigating a familiar mouse than wild-type mice did. In a resident-intruder test, motopsin knockout mice showed prolonged social interaction compared to wild-type mice. Consistent with the behavioral deficit, spine density was significantly decreased on apical dendrites, but not on basal dendrites, of hippocampal pyramidal neurons of motopsin knockout mice. In contrast, pyramidal neurons at the cingulate cortex showed normal spine density. Spatial learning and social interaction induced the phosphorylation of cAMP responsive element binding protein (CREB) in hippocampal neurons of wild-type mice, whereas the phosphorylation of CREB was markedly decreased in mutant mouse brains. Our results indicate that an extracellular protease, motopsin, preferentially affects social behaviors, and modulates the functions of hippocampal neurons. PMID:20092579

Abrogation of both short and long forms of latent transforming growth factor-β binding protein-1 causes defective cardiovascular development and is perinatally lethal.

PubMed

Horiguchi, Masahito; Todorovic, Vesna; Hadjiolova, Krassimira; Weiskirchen, Ralf; Rifkin, Daniel B

2015-04-01

Latent transforming growth factor-β binding protein-1 (LTBP-1) is an extracellular protein that is structurally similar to fibrillin and has an important role in controlling transforming growth factor-β (TGF-β) signaling by storing the cytokine in the extracellular matrix and by being involved in the conversion of the latent growth factor to its active form. LTBP-1 is found as both short (LTBP-1S) and long (LTBP-1L) forms, which are derived through the use of separate promoters. There is controversy regarding the importance of LTBP-1L, as Ltbp1L knockout mice showed multiple cardiovascular defects but the complete null mice did not. Here, we describe a third line of Ltbp1 knockout mice generated utilizing a conditional knockout strategy that ablated expression of both L and S forms of LTBP-1. These mice show severe developmental cardiovascular abnormalities and die perinatally; thus these animals display a phenotype similar to previously reported Ltbp1L knockout mice. We reinvestigated the other "complete" knockout line and found that these mice express a splice variant of LTBP-1L and, therefore, are not complete Ltbp1 knockouts. Our results clarify the phenotypes of Ltbp1 null mice and re-emphasize the importance of LTBP-1 in vivo. Copyright © 2015. Published by Elsevier B.V.

Renal sodium transport in renin-deficient Dahl salt-sensitive rats

PubMed Central

Pavlov, Tengis S; Levchenko, Vladislav; Ilatovskaya, Daria V; Moreno, Carol; Staruschenko, Alexander

2016-01-01

Objective: The Dahl salt-sensitive rat is a well-established model of salt-sensitive hypertension. The goal of this study was to assess the expression and activity of renal sodium channels and transporters in the renin-deficient salt-sensitive rat. Methods: Renin knockout (Ren−/−) rats created on the salt-sensitive rat background were used to investigate the role of renin in the regulation of ion transport in salt-sensitive hypertension. Western blotting and patch-clamp analyses were utilized to assess the expression level and activity of Na+ transporters. Results: It has been described previously that Ren−/− rats exhibit severe kidney underdevelopment, polyuria, and lower body weight and blood pressure compared to their wild-type littermates. Here we found that renin deficiency led to decreased expression of sodium-hydrogen antiporter (NHE3), the Na+/H+ exchanger involved in Na+ absorption in the proximal tubules, but did not affect the expression of Na-K-Cl cotransporter (NKCC2), the main transporter in the loop of Henle. In the distal nephron, the expression of sodium chloride cotransporter (NCC) was lower in Ren−/− rats. Single-channel patch clamp analysis detected decreased ENaC activity in Ren−/− rats which was mediated via changes in the channel open probability. Conclusion: These data illustrate that renin deficiency leads to significant dysregulation of ion transporters. PMID:27443990

TRPV2 KNOCKOUT MICE ARE SUSCEPTIBLE TO PERINATAL LETHALITY BUT DISPLAY NORMAL THERMAL AND MECHANICAL NOCICEPTION

PubMed Central

Park, Una; Vastani, Nisha; Guan, Yun; Raja, Srinivasa N.; Koltzenburg, Martin; Caterina, Michael J.

2011-01-01

TRPV2 is a nonselective cation channel expressed prominently in medium- to large-diameter sensory neurons that can be activated by extreme heat (>52°C). These features suggest that TRPV2 might be a transducer of noxious heat in vivo. TRPV2 can also be activated by hypoosmolarity or cell stretch, suggesting potential roles in mechanotransduction. To address the physiological functions of TRPV2 in somatosensation, we generated TRPV2 knockout mice and examined their behavioral and electrophysiological responses to heat and mechanical stimuli. TRPV2 knockout mice showed reduced embryonic weight and perinatal viability. As adults, surviving knockout mice also exhibited a slightly reduced body weight. TRPV2 knockout mice showed normal behavioral responses to noxious heat over a broad range of temperatures and normal responses to punctate mechanical stimuli, both in the basal state and under hyperalgesic conditions such as peripheral inflammation and L5 spinal nerve ligation. Moreover, behavioral assays of TRPV1/TRPV2 double knockout mice or of TRPV2 knockout mice treated with resiniferatoxin to desensitize TRPV1-expressing afferents revealed no thermosensory consequences of TRPV2 absence. In line with behavioral findings, electrophysiological recordings from skin afferents showed that C-fiber responses to heat and C- and Aδ-fiber responses to noxious mechanical stimuli were unimpaired in the absence of TRPV2. The prevalence of thermosensitive Aδ-fibers was too low to permit comparison between genotypes. Thus, TRPV2 is important for perinatal viability but is not essential for heat or mechanical nociception or hypersensitivity in the adult mouse. PMID:21832173

STAT3 and importins are novel mediators of early molecular and cellular responses in experimental duodenal ulceration.

PubMed

Khomenko, Tetyana; Deng, Xiaoming; Ahluwalia, Amrita; Tarnawski, Andrzej; Patel, Khushin N; Sandor, Zsuzsanna; Szabo, Sandor

2014-02-01

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that directly upregulates VEGF, Ref-1, p21, and anti-apoptotic genes such as Bcl-xL. In this study, we hypothesized that STAT3 signaling is activated and provides a critical protective role that is required for enterocyte survival during the early phases of cysteamine-induced duodenal ulcers. We studied the effect of inhibition of STAT3 activity on cysteamine-induced duodenal ulcers in rats and egr-1 knockout mice using STAT3/DNA binding assay, immunohistochemistry, immunoblot, and quantitative reverse transcriptase PCR analyses. We found that G-quartet oligodeoxynucleotides T40214, a specific inhibitor of STAT3/DNA binding, aggravated cysteamine-induced duodenal ulcers in rats 2.8-fold (p < 0.05). In the pre-ulcerogenic stage, cysteamine induced STAT3 tyrosine phosphorylation, its translocation to nuclei, an increased expression and nuclear translocation of importin α and β in the rat duodenal mucosa. Cysteamine enhanced the binding of STAT3 to its DNA consensus sequences at 6, 12, and 24 h after cysteamine by 1.5-, 1.8-, and 3.5-fold, respectively, and activated the expression of STAT3 target genes such as VEGF, Bcl-xL, Ref-1, and STAT3-induced feedback inhibitor, a suppressor of cytokine signaling 3. We also demonstrated that egr-1 knockout mice, which are more susceptible to cysteamine-induced duodenal ulcers, had lower levels of STAT3 expression, its phosphorylation, expression of importin α or β, and STAT3/DNA binding than wild-type mice in response to cysteamine. Thus, STAT3 represents an important new molecular mechanism in experimental duodenal ulceration.

Functions of Tenascin-C and Integrin alpha9beta1 in Mediating Prostate Cancer Bone Metastasis

DTIC Science & Technology

2017-10-01

additional engineered cell lines for verification and we plan to also generate stable knockout cell lines using CRISPR /Cas 9 gene editing technology...addition to the proposed study, we plan to also produce VCaP cells that are null (knockout) for alpha 9 integrin using CRISPR /Cas9 gene editing protocols...We are experienced with CRISPR -Cas knockdown and have successfully engineered cells previously. We do not expect any particular difficulty in

CRISPR/Cas9-Mediated Genomic Deletion of the Beta-1, 4 N-acetylgalactosaminyltransferase 1 Gene in Murine P19 Embryonal Carcinoma Cells Results in Low Sensitivity to Botulinum Neurotoxin Type C.

PubMed

Tsukamoto, Kentaro; Ozeki, Chikako; Kohda, Tomoko; Tsuji, Takao

2015-01-01

Botulinum neurotoxins produced by Clostridium botulinum cause flaccid paralysis by inhibiting neurotransmitter release at peripheral nerve terminals. Previously, we found that neurons derived from the murine P19 embryonal carcinoma cell line exhibited high sensitivity to botulinum neurotoxin type C. In order to prove the utility of P19 cells for the study of the intracellular mechanism of botulinum neurotoxins, ganglioside-knockout neurons were generated by deletion of the gene encoding beta-1,4 N-acetylgalactosaminyltransferase 1 in P19 cells using the clustered regularly interspaced short palindromic repeats combined with Cas9 (CRISPR/Cas9) system. By using this system, knockout cells could be generated more easily than with previous methods. The sensitivity of the generated beta-1,4 N-acetylgalactosaminyltransferase 1-depleted P19 neurons to botulinum neurotoxin type C was decreased considerably, and the exogenous addition of the gangliosides GD1a, GD1b, and GT1b restored the susceptibility of P19 cells to botulinum neurotoxin type C. In particular, addition of a mixture of these three ganglioside more effectively recovered the sensitivity of knockout cells compared to independent addition of GD1a, GD1b, or GT1b. Consequently, the genome-edited P19 cells generated by the CRISPR/Cas9 system were useful for identifying and defining the intracellular molecules involved in the toxic action of botulinum neurotoxins.

Generation of a KLF15 homozygous knockout human embryonic stem cell line using paired CRISPR/Cas9n, and human cardiomyocytes derivation.

PubMed

Noack, Claudia; Haupt, Luis Peter; Zimmermann, Wolfram-Hubertus; Streckfuss-Bömeke, Katrin; Zelarayán, Laura Cecilia

2017-08-01

Krueppel-like factor 15 (KLF15) is abundantly expressed in liver, kidney, and muscle, including myocardium. In the adult heart KLF15 is important to maintain homeostasis and to repress hypertrophic remodeling. We generated a homozygous hESC KLF15 knockout (KO) line using paired CRISPR/Cas9n. KLF15-KO cells maintained full pluripotency and differentiation potential as well as genomic integrity. We demonstrated that KLF15-KO cells can be differentiated into morphologically normal cardiomyocytes turning them into a valuable tool for studying human KLF15-mediated mechanisms resulting in human cardiac dysfunction. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Gene trap and gene inversion methods for conditional gene inactivation in the mouse

PubMed Central

Xin, Hong-Bo; Deng, Ke-Yu; Shui, Bo; Qu, Shimian; Sun, Qi; Lee, Jane; Greene, Kai Su; Wilson, Jason; Yu, Ying; Feldman, Morris; Kotlikoff, Michael I.

2005-01-01

Conditional inactivation of individual genes in mice using site-specific recombinases is an extremely powerful method for determining the complex roles of mammalian genes in developmental and tissue-specific contexts, a major goal of post-genomic research. However, the process of generating mice with recombinase recognition sequences placed at specific locations within a gene, while maintaining a functional allele, is time consuming, expensive and technically challenging. We describe a system that combines gene trap and site-specific DNA inversion to generate mouse embryonic stem (ES) cell clones for the rapid production of conditional knockout mice, and the use of this system in an initial gene trap screen. Gene trapping should allow the selection of thousands of ES cell clones with defined insertions that can be used to generate conditional knockout mice, thereby providing extensive parallelism that eliminates the time-consuming steps of targeting vector construction and homologous recombination for each gene. PMID:15659575

A Reduced Zinc Diet or Zinc Transporter 3 Knockout Attenuate Light Induced Zinc Accumulation and Retinal Degeneration△

PubMed Central

Bai, Shi; Sheline, Carolyn R.; Zhou, Yongdong; Sheline, Christian T.

2013-01-01

Our previous study on retinal light exposure suggests the involvement of zinc (Zn2+) toxicity in the death of RPE and photoreceptors (LD) which could be attenuated by pyruvate and nicotinamide, perhaps through restoration of NAD+ levels. In the present study, we examined Zn2+ toxicity, and the effects of NAD+ restoration in primary retinal cultures. We then reduced Zn2+ levels in rodents by reducing Zn2+ levels in the diet, or by genetics and measured LD. Sprague Dawley albino rats were fed 2, or 61 mg Zn2+/kg of diet for 3 weeks, and exposed to 18 kLux of white light for 4h. We light exposed (70 kLux of white light for 50h) Zn2+ transporter 3 knockout (ZnT3-KO, no synaptic Zn2+), or RPE65 knockout mice (RPE65-KO, lack rhodopsin cycling), or C57/BI6/J controls and determined light damage and Zn2+ staining. Retinal Zn2+ staining was examined at 1h and 4h after light exposure. Retinas were examined after 7d by optical coherence tomography and histology. After LD, rats fed the reduced Zn2+ diet showed less photoreceptor Zn2+ staining and degeneration compared to a normal Zn2+ diet. Similarly, ZnT3-KO and RPE65-KO mice showed less Zn2+ staining, NAD+ loss, and RPE or photoreceptor death than C57/BI6/J control mice. Dietary or ZnT3-dependent Zn2+ stores, and intracellular Zn2+ release from rhodopsin recycling are suggested to be involved in light-induced retinal degeneration. These results implicate novel rhodopsin-mediated mechanisms and therapeutic targets for LD. Our companion manuscript demonstrates that pharmacologic, circadian, or genetic manipulations which maintain NAD+ levels reduce LD. PMID:23274584

Connexin 43 reboots meiosis and reseals blood-testis barrier following toxicant-mediated aspermatogenesis and barrier disruption

PubMed Central

Li, Nan; Mruk, Dolores D.; Mok, Ka-Wai; Li, Michelle W. M.; Wong, Chris K. C.; Lee, Will M.; Han, Daishu; Silvestrini, Bruno; Cheng, C. Yan

2016-01-01

Earlier studies have shown that rats treated with an acute dose of 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide (adjudin, a male contraceptive under development) causes permanent infertility due to irreversible blood-testis barrier (BTB) disruption even though the population of undifferentiated spermatogonia remains similar to normal rat testes, because spermatogonia fail to differentiate into spermatocytes to enter meiosis. Since other studies have illustrated the significance of connexin 43 (Cx43)-based gap junction in maintaining the homeostasis of BTB in the rat testis and the phenotypes of Sertoli cell-conditional Cx43 knockout mice share many of the similarities of the adjudin-treated rats, we sought to examine if overexpression of Cx43 in these adjudin-treated rats would reseal the disrupted BTB and reinitiate spermatogenesis. A full-length Cx43 cloned into mammalian expression vector pCI-neo was used to transfect testes of adjudin-treated rats versus empty vector. It was found that overexpression of Cx43 indeed resealed the Sertoli cell tight junction–permeability barrier based on a functional in vivo assay in tubules displaying signs of meiosis as noted by the presence of round spermatids. Thus, these findings suggest that overexpression of Cx43 reinitiated spermatogenesis at least through the steps of meiosis to generate round spermatids in testes of rats treated with an acute dose of adjudin that led to aspermatogenesis. It was also noted that the round spermatids underwent eventual degeneration with the formation of multinucleated cells following Cx43 overexpression due to the failure of spermiogenesis because no elongating/elongated spermatids were detected in any of the tubules examined. The mechanism by which overexpression of Cx43 reboots meiosis and rescues BTB function was also examined. In summary, overexpression of Cx43 in the testis with aspermatogenesis reboots meiosis and reseals toxicant-induced BTB disruption, even though it fails to support round spermatids to enter spermiogenesis.—Li, N., Mruk, D. D., Mok, K.-W., Li, M. W. M., Wong, C. K. C., Lee, W. M., Han, D., Silvestrini, B., Cheng, C. Y. Connexin 43 reboots meiosis and reseals blood-testis barrier following toxicant-mediated aspermatogenesis and barrier disruption. PMID:26678449

GeneKnockout by Targeted Mutagenesis in a Hemimetabolous Insect, the Two-Spotted Cricket Gryllus bimaculatus, using TALENs.

PubMed

Watanabe, Takahito; Noji, Sumihare; Mito, Taro

2016-01-01

Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically basal. These insects include many deleterious species. The cricket, Gryllus bimaculatus, is an emerging model for hemimetabolous insects, based on the success of RNA interference (RNAi)-based gene-functional analyses and transgenic technology. Taking advantage of genome-editing technologies in this species would greatly promote functional genomics studies. Genome editing using transcription activator-like effector nucleases (TALENs) has proven to be an effective method for site-specific genome manipulation in various species. TALENs are artificial nucleases that are capable of inducing DNA double-strand breaks into specified target sequences. Here, we describe a protocol for TALEN-based gene knockout in G. bimaculatus, including a mutant selection scheme via mutation detection assays, for generating homozygous knockout organisms.

Cells Lacking β-Actin are Genetically Reprogrammed and Maintain Conditional Migratory Capacity*

PubMed Central

Tondeleir, Davina; Lambrechts, Anja; Müller, Matthias; Jonckheere, Veronique; Doll, Thierry; Vandamme, Drieke; Bakkali, Karima; Waterschoot, Davy; Lemaistre, Marianne; Debeir, Olivier; Decaestecker, Christine; Hinz, Boris; Staes, An; Timmerman, Evy; Colaert, Niklaas; Gevaert, Kris; Vandekerckhove, Joël; Ampe, Christophe

2012-01-01

Vertebrate nonmuscle cells express two actin isoforms: cytoplasmic β- and γ-actin. Because of the presence and localized translation of β-actin at the leading edge, this isoform is generally accepted to specifically generate protrusive forces for cell migration. Recent evidence also implicates β-actin in gene regulation. Cell migration without β-actin has remained unstudied until recently and it is unclear whether other actin isoforms can compensate for this cytoplasmic function and/or for its nuclear role. Primary mouse embryonic fibroblasts lacking β-actin display compensatory expression of other actin isoforms. Consistent with this preservation of polymerization capacity, β-actin knockout cells have unchanged lamellipodial protrusion rates despite a severe migration defect. To solve this paradox we applied quantitative proteomics revealing a broad genetic reprogramming of β-actin knockout cells. This also explains why reintroducing β-actin in knockout cells does not restore the affected cell migration. Pathway analysis suggested increased Rho-ROCK signaling, consistent with observed phenotypic changes. We therefore developed and tested a model explaining the phenotypes in β-actin knockout cells based on increased Rho-ROCK signaling and increased TGFβ production resulting in increased adhesion and contractility in the knockout cells. Inhibiting ROCK or myosin restores migration of β-actin knockout cells indicating that other actins compensate for β-actin in this process. Consequently, isoactins act redundantly in providing propulsive forces for cell migration, but β-actin has a unique nuclear function, regulating expression on transcriptional and post-translational levels, thereby preventing myogenic differentiation. PMID:22448045

Spermatogenic Cell-Specific Gene Mutation in Mice via CRISPR-Cas9.

PubMed

Bai, Meizhu; Liang, Dan; Wang, Yinghua; Li, Qing; Wu, Yuxuan; Li, Jinsong

2016-05-20

Tissue-specific knockout technology enables the analysis of the gene function in specific tissues in adult mammals. However, conventional strategy for producing tissue-specific knockout mice is a time- and labor-consuming process, restricting rapid study of the gene function in vivo. CRISPR-Cas9 system from bacteria is a simple and efficient gene-editing technique, which has enabled rapid generation of gene knockout lines in mouse by direct injection of CRISPR-Cas9 into zygotes. Here, we demonstrate CRISPR-Cas9-mediated spermatogenic cell-specific disruption of Scp3 gene in testes in one step. We first generated transgenic mice by pronuclear injection of a plasmid containing Hspa2 promoter driving Cas9 expression and showed Cas9 specific expression in spermatogenic cells. We then produced transgenic mice carrying Hspa2 promoter driven Cas9 and constitutive expressed sgRNA targeting Scp3 gene. Male founders were infertile due to developmental arrest of spermatogenic cells while female founders could produce progeny normally. Consistently, male progeny from female founders were infertile and females could transmit the transgenes to the next generation. Our study establishes a CRISPR-Cas9-based one-step strategy to analyze the gene function in adult tissues by a temporal-spatial pattern. Copyright © 2016 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

Generation of an arrayed CRISPR-Cas9 library targeting epigenetic regulators: from high-content screens to in vivo assays

PubMed Central

2017-01-01

ABSTRACT The CRISPR-Cas9 system has revolutionized genome engineering, allowing precise modification of DNA in various organisms. The most popular method for conducting CRISPR-based functional screens involves the use of pooled lentiviral libraries in selection screens coupled with next-generation sequencing. Screens employing genome-scale pooled small guide RNA (sgRNA) libraries are demanding, particularly when complex assays are used. Furthermore, pooled libraries are not suitable for microscopy-based high-content screens or for systematic interrogation of protein function. To overcome these limitations and exploit CRISPR-based technologies to comprehensively investigate epigenetic mechanisms, we have generated a focused sgRNA library targeting 450 epigenetic regulators with multiple sgRNAs in human cells. The lentiviral library is available both in an arrayed and pooled format and allows temporally-controlled induction of gene knock-out. Characterization of the library showed high editing activity of most sgRNAs and efficient knock-out at the protein level in polyclonal populations. The sgRNA library can be used for both selection and high-content screens, as well as for targeted investigation of selected proteins without requiring isolation of knock-out clones. Using a variety of functional assays we show that the library is suitable for both in vitro and in vivo applications, representing a unique resource to study epigenetic mechanisms in physiological and pathological conditions. PMID:29327641

Comprehensive characterization of glutamine synthetase-mediated selection for the establishment of recombinant CHO cells producing monoclonal antibodies.

PubMed

Noh, Soo Min; Shin, Seunghyeon; Lee, Gyun Min

2018-03-29

To characterize a glutamine synthetase (GS)-based selection system, monoclonal antibody (mAb) producing recombinant CHO cell clones were generated by a single round of selection at various methionine sulfoximine (MSX) concentrations (0, 25, and 50 μM) using two different host cell lines (CHO-K1 and GS-knockout CHO). Regardless of the host cell lines used, the clones selected at 50 μM MSX had the lowest average specific growth rate and the highest average specific production rates of toxic metabolic wastes, lactate and ammonia. Unlike CHO-K1, high producing clones could be generated in the absence of MSX using GS-knockout CHO with an improved selection stringency. Regardless of the host cell lines used, the clones selected at various MSX concentrations showed no significant difference in the GS, heavy chain, and light chain gene copies (P > 0.05). Furthermore, there was no correlation between the specific mAb productivity and these three gene copies (R 2  ≤ 0.012). Taken together, GS-mediated gene amplification does not occur in a single round of selection at a MSX concentration up to 50 μM. The use of the GS-knockout CHO host cell line facilitates the rapid generation of high producing clones with reduced production of lactate and ammonia in the absence of MSX.

Role of Complement in a Rat Model of Paclitaxel-Induced Peripheral Neuropathy.

PubMed

Xu, Jijun; Zhang, Lingjun; Xie, Mian; Li, Yan; Huang, Ping; Saunders, Thomas L; Fox, David A; Rosenquist, Richard; Lin, Feng

2018-06-15

Chemotherapy-induced peripheral neuropathy (CIPN) is a painful and debilitating side effect of cancer chemotherapy with an unclear pathogenesis. Consequently, the available therapies for this neuropathic pain syndrome are inadequate, leading to a significantly reduced quality of life in many patients. Complement, a key component of the innate immune system, has been associated with neuroinflammation, a potentially important trigger of some types of neuropathic pain. However, the role of complement in CIPN remains unclear. To address this issue, we developed a C3 knockout (KO) rat model and induced CIPN in these KO rats and wild-type littermates via the i.p. administration of paclitaxel, a chemotherapeutic agent associated with CIPN. We then compared the severity of mechanical allodynia, complement activation, and intradermal nerve fiber loss between the groups. We found that 1) i.p. paclitaxel administration activated complement in wild-type rats, 2) paclitaxel-induced mechanical allodynia was significantly reduced in C3 KO rats, and 3) the paclitaxel-induced loss of intradermal nerve fibers was markedly attenuated in C3 KO rats. In in vitro studies, we found that paclitaxel-treated rat neuronal cells activated complement, leading to cellular injury. Our findings demonstrate a previously unknown but pivotal role of complement in CIPN and suggest that complement may be a new target for the development of novel therapeutics to manage this painful disease. Copyright © 2018 by The American Association of Immunologists, Inc.

Characterization of a baculovirus lacking the DBP (DNA-binding protein) gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vanarsdall, Adam L.; Mikhailov, Victor S.; N.K. Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Moscow 117808

2007-08-01

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes two proteins that possess properties typical of single-stranded DNA-binding proteins (SSBs), late expression factor-3 (LEF-3), and a protein referred to as DNA-binding protein (DBP). Whereas LEF-3 is a multi-functional protein essential for viral DNA replication, transporting helicase into the nucleus, and forms a stable complex with the baculovirus alkaline nuclease, the role for DBP in baculovirus replication remains unclear. Therefore, to better understand the functional role of DBP in viral replication, a DBP knockout virus was generated from an AcMNPV bacmid and analyzed. The results of a growth curve analysis indicated that the dbpmore » knockout construct was unable to produce budded virus indicating that dbp is essential. The lack of DBP does not cause a general shutdown of the expression of viral genes, as was revealed by accumulation of early (LEF-3), late (VP39), and very late (P10) proteins in cells transfected with the dbp knockout construct. To investigate the role of DBP in DNA replication, a real-time PCR-based assay was employed and showed that, although viral DNA synthesis occurred in cells transfected with the dbp knockout, the levels were less than that of the control virus suggesting that DBP is required for normal levels of DNA synthesis or for stability of nascent viral DNA. In addition, analysis of the viral DNA replicated by the dbp knockout by using field inversion gel electrophoresis failed to detect the presence of genome-length DNA. Furthermore, analysis of DBP from infected cells indicated that similar to LEF-3, DBP was tightly bound to viral chromatin. Assessment of the cellular localization of DBP relative to replicated viral DNA by immunoelectron microscopy indicated that, at 24 h post-infection, DBP co-localized with nascent DNA at distinct electron-dense regions within the nucleus. Finally, immunoelectron microscopic analysis of cells transfected with the dbp knockout revealed that DBP is required for the production of normal-appearing nucleocapsids and for the generation of the virogenic stroma.« less

The Role of ARX in Human Pancreatic Endocrine Specification

PubMed Central

Gage, Blair K.; Asadi, Ali; Baker, Robert K.; Webber, Travis D.; Wang, Rennian; Itoh, Masayuki; Hayashi, Masaharu; Miyata, Rie; Akashi, Takumi; Kieffer, Timothy J.

2015-01-01

The in vitro differentiation of human embryonic stem cells (hESCs) offers a model system to explore human development. Humans with mutations in the transcription factor Aristaless Related Homeobox (ARX) often suffer from the syndrome X-linked lissencephaly with ambiguous genitalia (XLAG), affecting many cell types including those of the pancreas. Indeed, XLAG pancreatic islets lack glucagon and pancreatic polypeptide-positive cells but retain somatostatin, insulin, and ghrelin-positive cells. To further examine the role of ARX in human pancreatic endocrine development, we utilized genomic editing in hESCs to generate deletions in ARX. ARX knockout hESCs retained pancreatic differentiation capacity and ARX knockout endocrine cells were biased toward somatostatin-positive cells (94% of endocrine cells) with reduced pancreatic polypeptide (rarely detected), glucagon (90% reduced) and insulin-positive (65% reduced) lineages. ARX knockout somatostatin-positive cells shared expression patterns with human fetal and adult δ-cells. Differentiated ARX knockout cells upregulated PAX4, NKX2.2, ISL1, HHEX, PCSK1, PCSK2 expression while downregulating PAX6 and IRX2. Re-expression of ARX in ARX knockout pancreatic progenitors reduced HHEX and increased PAX6 and insulin expression following differentiation. Taken together these data suggest that ARX plays a key role in pancreatic endocrine fate specification of pancreatic polypeptide, somatostatin, glucagon and insulin positive cells from hESCs. PMID:26633894

The Role of ARX in Human Pancreatic Endocrine Specification.

PubMed

Gage, Blair K; Asadi, Ali; Baker, Robert K; Webber, Travis D; Wang, Rennian; Itoh, Masayuki; Hayashi, Masaharu; Miyata, Rie; Akashi, Takumi; Kieffer, Timothy J

2015-01-01

The in vitro differentiation of human embryonic stem cells (hESCs) offers a model system to explore human development. Humans with mutations in the transcription factor Aristaless Related Homeobox (ARX) often suffer from the syndrome X-linked lissencephaly with ambiguous genitalia (XLAG), affecting many cell types including those of the pancreas. Indeed, XLAG pancreatic islets lack glucagon and pancreatic polypeptide-positive cells but retain somatostatin, insulin, and ghrelin-positive cells. To further examine the role of ARX in human pancreatic endocrine development, we utilized genomic editing in hESCs to generate deletions in ARX. ARX knockout hESCs retained pancreatic differentiation capacity and ARX knockout endocrine cells were biased toward somatostatin-positive cells (94% of endocrine cells) with reduced pancreatic polypeptide (rarely detected), glucagon (90% reduced) and insulin-positive (65% reduced) lineages. ARX knockout somatostatin-positive cells shared expression patterns with human fetal and adult δ-cells. Differentiated ARX knockout cells upregulated PAX4, NKX2.2, ISL1, HHEX, PCSK1, PCSK2 expression while downregulating PAX6 and IRX2. Re-expression of ARX in ARX knockout pancreatic progenitors reduced HHEX and increased PAX6 and insulin expression following differentiation. Taken together these data suggest that ARX plays a key role in pancreatic endocrine fate specification of pancreatic polypeptide, somatostatin, glucagon and insulin positive cells from hESCs.

Role of AMACR (α-methylacyl-CoA racemase) and MFE-1 (peroxisomal multifunctional enzyme-1) in bile acid synthesis in mice.

PubMed

Autio, Kaija J; Schmitz, Werner; Nair, Remya R; Selkälä, Eija M; Sormunen, Raija T; Miinalainen, Ilkka J; Crick, Peter J; Wang, Yuqin; Griffiths, William J; Reddy, Janardan K; Baes, Myriam; Hiltunen, J Kalervo

2014-07-01

Cholesterol is catabolized to bile acids by peroxisomal β-oxidation in which the side chain of C27-bile acid intermediates is shortened by three carbon atoms to form mature C24-bile acids. Knockout mouse models deficient in AMACR (α-methylacyl-CoA racemase) or MFE-2 (peroxisomal multifunctional enzyme type 2), in which this β-oxidation pathway is prevented, display a residual C24-bile acid pool which, although greatly reduced, implies the existence of alternative pathways of bile acid synthesis. One alternative pathway could involve Mfe-1 (peroxisomal multifunctional enzyme type 1) either with or without Amacr. To test this hypothesis, we generated a double knockout mouse model lacking both Amacr and Mfe-1 activities and studied the bile acid profiles in wild-type, Mfe-1 and Amacr single knockout mouse line and Mfe-1 and Amacr double knockout mouse lines. The total bile acid pool was decreased in Mfe-1-/- mice compared with wild-type and the levels of mature C24-bile acids were reduced in the double knockout mice when compared with Amacr-deficient mice. These results indicate that Mfe-1 can contribute to the synthesis of mature bile acids in both Amacr-dependent and Amacr-independent pathways.

A PET Tracer for Brain α2C Adrenoceptors, (11)C-ORM-13070: Radiosynthesis and Preclinical Evaluation in Rats and Knockout Mice.

PubMed

Arponen, Eveliina; Helin, Semi; Marjamäki, Päivi; Grönroos, Tove; Holm, Patrik; Löyttyniemi, Eliisa; Någren, Kjell; Scheinin, Mika; Haaparanta-Solin, Merja; Sallinen, Jukka; Solin, Olof

2014-07-01

We report the development of a PET tracer for α2C adrenoceptor imaging and its preliminary preclinical evaluation. α2C adrenoceptors in the human brain may be involved in various neuropsychiatric disorders, such as depression, schizophrenia, and neurodegenerative diseases. PET tracers are needed for imaging of this receptor system in vivo. High-specific-activity (11)C-ORM-13070 (1-[(S)-1-(2,3-dihydrobenzo[1,4]dioxin-2-yl)methyl]-4-(3-(11)C-methoxymethylpyridin-2-yl)-piperazine) was synthesized by (11)C-methylation of O-desmethyl-ORM-13070 with (11)C-methyl triflate, which was prepared from cyclotron-produced (11)C-methane via (11)C-methyl iodide. Rats and mice were investigated in vivo with PET and ex vivo with autoradiography. The specificity of (11)C-ORM-13070 binding to α2 adrenoceptors was demonstrated in rats pretreated with atipamezole, an α2 adrenoceptor antagonist. The α2C adrenoceptor selectivity of the tracer was determined by comparing tracer binding in wild-type and α2A- and α2AC adrenoceptor knockout (KO) mice. (11)C-ORM-13070 and its radioactive metabolites in rat plasma and brain tissue were analyzed with radio-high-performance liquid chromatography and mass spectroscopy. Human radiation dose estimates were extrapolated from rat biodistribution data. The radiochemical yield, calculated from initial cyclotron-produced (11)C-methane, was 9.6% ± 2.7% (decay-corrected to end of bombardment). The specific activity of the product was 640 ± 390 GBq/μmol (decay-corrected to end of synthesis). The radiochemical purity exceeded 99% in all syntheses. The highest levels of tracer binding were observed in the striatum and olfactory tubercle of rats and control and α2A KO mice-that is, in the brain regions known to contain the highest densities of α2C adrenoceptors. In rats pretreated with atipamezole and in α2AC KO mice, (11)C tracer binding in the striatum and olfactory tubercle was low, similar to that of the frontal cortex and thalamus, regions with low densities of α2C adrenoceptors. Two radioactive metabolites were found in rat plasma, but only one of them was found in the brain; their identity was not revealed. The estimated effective radiation dose was comparable with the average exposure level in PET studies with (11)C tracers. An efficient method for the radiosynthesis of (11)C-ORM-13070 was developed. (11)C-ORM-13070 emerged as a potential novel radiotracer for in vivo imaging of brain α2C adrenoceptors. © 2014 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Next-generation mammalian genetics toward organism-level systems biology.

PubMed

Susaki, Etsuo A; Ukai, Hideki; Ueda, Hiroki R

2017-01-01

Organism-level systems biology in mammals aims to identify, analyze, control, and design molecular and cellular networks executing various biological functions in mammals. In particular, system-level identification and analysis of molecular and cellular networks can be accelerated by next-generation mammalian genetics. Mammalian genetics without crossing, where all production and phenotyping studies of genome-edited animals are completed within a single generation drastically reduce the time, space, and effort of conducting the systems research. Next-generation mammalian genetics is based on recent technological advancements in genome editing and developmental engineering. The process begins with introduction of double-strand breaks into genomic DNA by using site-specific endonucleases, which results in highly efficient genome editing in mammalian zygotes or embryonic stem cells. By using nuclease-mediated genome editing in zygotes, or ~100% embryonic stem cell-derived mouse technology, whole-body knock-out and knock-in mice can be produced within a single generation. These emerging technologies allow us to produce multiple knock-out or knock-in strains in high-throughput manner. In this review, we discuss the basic concepts and related technologies as well as current challenges and future opportunities for next-generation mammalian genetics in organism-level systems biology.

Characterization of the first knock-out aldh7a1 zebrafish model for pyridoxine-dependent epilepsy using CRISPR-Cas9 technology

PubMed Central

Zabinyakov, Nikita; Bullivant, Garrett; Cao, Feng; Fernandez Ojeda, Matilde; Jia, Zheng Ping; Wen, Xiao-Yan; Dowling, James J.; Salomons, Gajja S.

2017-01-01

Pyridoxine dependent epilepsy (PDE) is caused by likely pathogenic variants in ALDH7A1 (PDE-ALDH7A1) and inherited autosomal recessively. Neurotoxic alpha-amino adipic semialdehyde (alpha-AASA), piperideine 6-carboxylate and pipecolic acid accumulate in body fluids. Neonatal or infantile onset seizures refractory to anti-epileptic medications are clinical features. Treatment with pyridoxine, arginine and lysine-restricted diet does not normalize neurodevelopmental outcome or accumulation of neurotoxic metabolites. There is no animal model for high throughput drug screening. For this reason, we developed and characterized the first knock-out aldh7a1 zebrafish model using CRISPR-Cas9 technology. Zebrafish aldh7a1 mutants were generated by using a vector free method of CRISPR-Cas9 mutagenesis. Genotype analysis of aldh7a1 knock-out zebrafish was performed by high resolution melt analysis, direct sequencing and QIAxcel system. Electroencephalogram was performed. Alpha-AASA, piperideine 6-carboxylate and pipecolic acid, were measured by liquid chromatography-tandem mass spectrometry. Our knock-out aldh7a1 zebrafish has homozygous 5 base pair (bp) mutation in ALDH7A1. Knock-out aldh7a1 embryos have spontaneous rapid increase in locomotion and a rapid circling swim behavior earliest 8-day post fertilization (dpf). Electroencephalogram revealed large amplitude spike discharges compared to wild type. Knock-out aldh7a1 embryos have elevated alpha-AASA, piperideine 6-carboxylate and pipecolic acid compared to wild type embryos at 3 dpf. Knock-out aldh7a1 embryos showed no aldh7a1 protein by western blot compared to wild type. Our knock-out aldh7a1 zebrafish is a well characterized model for large-scale drug screening using behavioral and biochemical features and accurately recapitulates the human PDE-ALDH7A1 disease. PMID:29053735

Characterization of the first knock-out aldh7a1 zebrafish model for pyridoxine-dependent epilepsy using CRISPR-Cas9 technology.

PubMed

Zabinyakov, Nikita; Bullivant, Garrett; Cao, Feng; Fernandez Ojeda, Matilde; Jia, Zheng Ping; Wen, Xiao-Yan; Dowling, James J; Salomons, Gajja S; Mercimek-Andrews, Saadet

2017-01-01

Pyridoxine dependent epilepsy (PDE) is caused by likely pathogenic variants in ALDH7A1 (PDE-ALDH7A1) and inherited autosomal recessively. Neurotoxic alpha-amino adipic semialdehyde (alpha-AASA), piperideine 6-carboxylate and pipecolic acid accumulate in body fluids. Neonatal or infantile onset seizures refractory to anti-epileptic medications are clinical features. Treatment with pyridoxine, arginine and lysine-restricted diet does not normalize neurodevelopmental outcome or accumulation of neurotoxic metabolites. There is no animal model for high throughput drug screening. For this reason, we developed and characterized the first knock-out aldh7a1 zebrafish model using CRISPR-Cas9 technology. Zebrafish aldh7a1 mutants were generated by using a vector free method of CRISPR-Cas9 mutagenesis. Genotype analysis of aldh7a1 knock-out zebrafish was performed by high resolution melt analysis, direct sequencing and QIAxcel system. Electroencephalogram was performed. Alpha-AASA, piperideine 6-carboxylate and pipecolic acid, were measured by liquid chromatography-tandem mass spectrometry. Our knock-out aldh7a1 zebrafish has homozygous 5 base pair (bp) mutation in ALDH7A1. Knock-out aldh7a1 embryos have spontaneous rapid increase in locomotion and a rapid circling swim behavior earliest 8-day post fertilization (dpf). Electroencephalogram revealed large amplitude spike discharges compared to wild type. Knock-out aldh7a1 embryos have elevated alpha-AASA, piperideine 6-carboxylate and pipecolic acid compared to wild type embryos at 3 dpf. Knock-out aldh7a1 embryos showed no aldh7a1 protein by western blot compared to wild type. Our knock-out aldh7a1 zebrafish is a well characterized model for large-scale drug screening using behavioral and biochemical features and accurately recapitulates the human PDE-ALDH7A1 disease.

CYP2F2-generated metabolites, not styrene oxide, are a key event mediating the mode of action of styrene-induced mouse lung tumors.

PubMed

Cruzan, G; Bus, J; Hotchkiss, J; Harkema, J; Banton, M; Sarang, S

2012-02-01

Styrene induces lung tumors in mice but not in rats. Although metabolism of styrene to 7,8-styrene oxide (SO) by CYP2E1 has been suggested as a mediator of styrene toxicity, lung toxicity is not attenuated in CYP2E1 knockout mice. However, styrene and/or SO metabolism by mouse lung Clara cell-localized CYP2F2 to ring-oxidized cytotoxic metabolite(s) has been postulated as a key metabolic gateway responsible for both lung toxicity and possible tumorigenicity. To test this hypothesis, the lung toxicity of styrene and SO was evaluated in C57BL/6 (WT) and CYP2F2⁻/⁻ knockout mice treated with styrene (400 mg/kg/day, gavage, or 200 or 400 mg/kg/day, ip) or S- or R-SO (200 mg/kg/day, ip) for 5 days. Styrene treated WT mice displayed significant necrosis and exfoliation of Clara cells, and cumulative BrdU-labeling index of S-phase cells was markedly increased in terminal bronchioles of WT mice exposed to styrene or S- or RSO. In contrast, Clara and terminal bronchiole cell toxicity was not observed in CYP2F2⁻/⁻ mice exposed to either styrene or SO. This study clearly demonstrates that the mouse lung toxicity of both styrene and SO is critically dependent on metabolism by CYP2F2. Importantly, the human isoform of CYP2F, CYP2F1, is expressed at much lower levels and likely does not catalyze significant styrene metabolism, supporting the hypothesis that styrene-induced mouse lung tumors may not quantitatively, or possibly qualitatively, predict lung tumor potential in humans. Copyright © 2011 Elsevier Inc. All rights reserved.

Studies of styrene, styrene oxide and 4-hydroxystyrene toxicity in CYP2F2 knockout and CYP2F1 humanized mice support lack of human relevance for mouse lung tumors.

PubMed

Cruzan, G; Bus, J; Hotchkiss, J; Sura, R; Moore, C; Yost, G; Banton, M; Sarang, S

2013-06-01

Styrene (S) is lung tumorigenic in mice but not in rats. S and its alkene-oxidized metabolite styrene oxide (SO) were not lung toxic in CYP2F2(-/-) [knockout] mice, indicating S-induced mouse lung tumors are mediated through mouse-specific CYP2F2-generated ring-oxidized metabolite(s) in lung bronchioles. The human relevance of the CYP2F MOA was assessed by insertion of a human CYP2F1, 2A13, 2B6 transgene into CYP2F2(-/-) mice; CYP2F1 expression and activity were confirmed in the transgenic (TG) mice. No evidence of cytotoxicity or increased cell proliferation (BrdU labeling) was seen in TG mice treated with either S or SO (200mg/kg/day ip for 5days). In contrast to S and SO, 4HS (105mg/kg/day ip for 5days) increased BrdU labeling 5-10-fold in WT mice, <3-fold increase in KO mice and 2-4-fold in TG mice. The limited response of 4HS in KO and TG mice may result from intrinsic toxicity or from further metabolism; regardless of the MOA, these findings indicate that the CYP2F-mediated tumorigenic MOA in WT mice is not operative for S, SO, or for 4HS putatively derived from metabolism of S by CYP2F1 in humans, and thus S-induced mouse lung tumors are unlikely to be relevant to human risk. Copyright © 2013. Published by Elsevier Inc.

A homozygous Keap1-knockout human embryonic stem cell line generated using CRISPR/Cas9 mediates gene targeting.

PubMed

Kim, So-Jung; Habib, Omer; Kim, Jin-Soo; Han, Hyo-Won; Koo, Soo Kyung; Kim, Jung-Hyun

2017-03-01

Kelch-like ECH-associated protein 1 (keap1) is a cysteine-rich protein that interacts with transcription factor Nrf2 in a redox-sensitive manner, leading to the degradation of Nrf2 (Kim et al., 2014a). Disruption of Keap1 results in the induction of Nrf2-related signaling pathways involving the expression of a set of anti-oxidant and anti-inflammatory genes. We generated biallelic mutants of the Keap1 gene using a CRISPR-Cas9 genome editing method in the H9 human embryonic stem cell (hESC). The Keap1 homozygous-knockout H9 cell line retained normal morphology, gene expression, and in vivo differentiation potential. Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.

Efficient CRISPR/Cas9-based gene knockout in watermelon.

PubMed

Tian, Shouwei; Jiang, Linjian; Gao, Qiang; Zhang, Jie; Zong, Mei; Zhang, Haiying; Ren, Yi; Guo, Shaogui; Gong, Guoyi; Liu, Fan; Xu, Yong

2017-03-01

CRISPR/Cas9 system can precisely edit genomic sequence and effectively create knockout mutations in T0 generation watermelon plants. Genome editing offers great advantage to reveal gene function and generate agronomically important mutations to crops. Recently, RNA-guided genome editing system using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) has been applied to several plant species, achieving successful targeted mutagenesis. Here, we report the genome of watermelon, an important fruit crop, can also be precisely edited by CRISPR/Cas9 system. ClPDS, phytoene desaturase in watermelon, was selected as the target gene because its mutant bears evident albino phenotype. CRISPR/Cas9 system performed genome editing, such as insertions or deletions at the expected position, in transfected watermelon protoplast cells. More importantly, all transgenic watermelon plants harbored ClPDS mutations and showed clear or mosaic albino phenotype, indicating that CRISPR/Cas9 system has technically 100% of genome editing efficiency in transgenic watermelon lines. Furthermore, there were very likely no off-target mutations, indicated by examining regions that were highly homologous to sgRNA sequences. Our results show that CRISPR/Cas9 system is a powerful tool to effectively create knockout mutations in watermelon.

Porcine Knock-in Fibroblasts Expressing hDAF on α-1,3-Galactosyltransferase (GGTA1) Gene Locus.

PubMed

Kim, Ji Woo; Kim, Hye-Min; Lee, Sang Mi; Kang, Man-Jong

2012-10-01

The Galactose-α1,3-galactose (α1,3Gal) epitope is responsible for hyperacute rejection in pig-to-human xenotransplantation. Human decay-accelerating factor (hDAF) is a cell surface regulatory protein that serves as a complement inhibitor to protect self cells from complement attack. The generation of α1,3-galactosyltransferase (GGTA1) knock-out pigs expressing DAF is a necessary step for their use as organ donors for humans. In this study, we established GGTA1 knock-out cell lines expressing DAF from pig ear fibroblasts for somatic cell nuclear transfer. hDAF expression was detected in hDAF knock-in heterozygous cells, but not in normal pig cells. Expression of the GGTA1 gene was lower in the knock-in heterozygous cell line compared to the normal pig cell. Knock-in heterozygous cells afforded more effective protection against cytotoxicity with human serum than with GGTA1 knock-out heterozygous and control cells. These cell lines may be used in the production of GGTA1 knock-out and DAF expression pigs for xenotransplantation.

GENETIC CATHEPSIN B DEFICIENCY REDUCES β-AMYLOID IN TRANSGENIC MICE EXPRESSING HUMAN WILD-TYPE AMYLOID PRECURSOR PROTEIN

PubMed Central

Hook, Vivian Y. H.; Kindy, Mark; Reinheckel, Thomas; Peters, Christoph; Hook, Gregory

2009-01-01

Neurotoxic β-amyloid (Aβ) peptides participate in Alzheimer’s disease (AD); therefore, reduction of Aβ generated from APP may provide a therapeutic approach for AD. Gene knockout studies in transgenic mice producing human Aβ may identify targets for reducing Aβ. This study shows that knockout of the cathepsin B gene in mice expressing human wild-type APP (hAPPwt) results in substantial decrease of Aβ40 and Aβ42 by 67% in brain, and decreases levels of the C-terminal β-secretase fragment (CTFβ) derived from APP. In contrast, knockout of cathepsin B in mice expressing hAPP with the rare Swedish (Swe) and Indiana (Ind) mutations had no effect on Aβ. The difference in reduction of Aβ in hAPPwt mice, but not in hAPPSwe/Ind mice, shows that the transgenic model can affect cathepsin B gene knockout results. Since most AD patients express hAPPwt, these data validate cathepsin B as a target for development of inhibitors to lower Aβ in AD. PMID:19501042

Gene knockout by targeted mutagenesis in a hemimetabolous insect, the two-spotted cricket Gryllus bimaculatus, using TALENs.

PubMed

Watanabe, Takahito; Noji, Sumihare; Mito, Taro

2014-08-15

Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically basal. These insects include many deleterious species. The cricket, Gryllus bimaculatus, is an emerging model for hemimetabolous insects, based on the success of RNA interference (RNAi)-based gene-functional analyses and transgenic technology. Taking advantage of genome-editing technologies in this species would greatly promote functional genomics studies. Genome editing using transcription activator-like effector nucleases (TALENs) has proven to be an effective method for site-specific genome manipulation in various species. TALENs are artificial nucleases that are capable of inducing DNA double-strand breaks into specified target sequences. Here, we describe a protocol for TALEN-based gene knockout in G. bimaculatus, including a mutant selection scheme via mutation detection assays, for generating homozygous knockout organisms. Copyright © 2014 Elsevier Inc. All rights reserved.

An episomal vector-based CRISPR/Cas9 system for highly efficient gene knockout in human pluripotent stem cells.

PubMed

Xie, Yifang; Wang, Daqi; Lan, Feng; Wei, Gang; Ni, Ting; Chai, Renjie; Liu, Dong; Hu, Shijun; Li, Mingqing; Li, Dajin; Wang, Hongyan; Wang, Yongming

2017-05-24

Human pluripotent stem cells (hPSCs) represent a unique opportunity for understanding the molecular mechanisms underlying complex traits and diseases. CRISPR/Cas9 is a powerful tool to introduce genetic mutations into the hPSCs for loss-of-function studies. Here, we developed an episomal vector-based CRISPR/Cas9 system, which we called epiCRISPR, for highly efficient gene knockout in hPSCs. The epiCRISPR system enables generation of up to 100% Insertion/Deletion (indel) rates. In addition, the epiCRISPR system enables efficient double-gene knockout and genomic deletion. To minimize off-target cleavage, we combined the episomal vector technology with double-nicking strategy and recent developed high fidelity Cas9. Thus the epiCRISPR system offers a highly efficient platform for genetic analysis in hPSCs.

Nrf2 Improves Leptin and Insulin Resistance Provoked by Hypothalamic Oxidative Stress.

PubMed

Yagishita, Yoko; Uruno, Akira; Fukutomi, Toshiaki; Saito, Ritsumi; Saigusa, Daisuke; Pi, Jingbo; Fukamizu, Akiyoshi; Sugiyama, Fumihiro; Takahashi, Satoru; Yamamoto, Masayuki

2017-02-21

The relationship between loss of hypothalamic function and onset of diabetes mellitus remains elusive. Therefore, we generated a targeted oxidative-stress murine model utilizing conditional knockout (KO) of selenocysteine-tRNA (Trsp) using rat-insulin-promoter-driven-Cre (RIP-Cre). These Trsp-KO (Trsp RIP KO) mice exhibit deletion of Trsp in both hypothalamic cells and pancreatic β cells, leading to increased hypothalamic oxidative stress and severe insulin resistance. Leptin signals are suppressed, and numbers of proopiomelanocortin-positive neurons in the hypothalamus are decreased. In contrast, Trsp-KO mice (Trsp Ins1 KO) expressing Cre specifically in pancreatic β cells, but not in the hypothalamus, do not display insulin and leptin resistance, demonstrating a critical role of the hypothalamus in the onset of diabetes mellitus. Nrf2 (NF-E2-related factor 2) regulates antioxidant gene expression. Increased Nrf2 signaling suppresses hypothalamic oxidative stress and improves insulin and leptin resistance in Trsp RIP KO mice. Thus, Nrf2 harbors the potential to prevent the onset of diabetic mellitus by reducing hypothalamic oxidative damage. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Heightened Avidity for Trisodium Pyrophosphate in Mice Lacking Tas1r3

PubMed Central

Aleman, Tiffany R.; McCaughey, Stuart A.

2015-01-01

Laboratory rats and mice prefer some concentrations of tri- and tetrasodium pyrophosphate (Na3HP2O7 and Na4P2O7) to water, but how they detect pyrophosphates is unknown. Here, we assessed whether T1R3 is involved. We found that relative to wild-type littermate controls, Tas1r3 knockout mice had stronger preferences for 5.6–56mM Na3HP2O7 in 2-bottle choice tests, and they licked more 17.8–56mM Na3HP2O7 in brief-access tests. We hypothesize that pyrophosphate taste in the intact mouse involves 2 receptors: T1R3 to produce a hedonically negative signal and an unknown G protein-coupled receptor to produce a hedonically positive signal; in Tas1r3 knockout mice, the hedonically negative signal produced by T1R3 is absent, leading to a heightened avidity for pyrophosphate. PMID:25452580

Severe Intestinal Inflammation in the Small Intestine of Mice Induced by Controllable Deletion of Claudin-7.

PubMed

Li, Wen-Jing; Xu, Chang; Wang, Kun; Li, Teng-Yan; Wang, Xiao-Nan; Yang, Hui; Xing, Tiaosi; Li, Wen-Xia; Chen, Yan-Hua; Gao, Hong; Ding, Lei

2018-05-01

As a potential tumor suppressor gene, Claudin-7 (Cldn7), which is a component of tight junctions, may play an important role in colorectal cancer occurrence and development. To generate a knockout mouse model of inducible conditional Cldn7 in the intestine and analyze the phenotype of the mice after induction with tamoxifen. We constructed Cldn7-flox transgenic mice and crossed them with Villin-CreERT2 mice. The Cldn7 inducible conditional knockout mice appeared normal and were well developed at birth. We induced Cldn7 gene deletion by injecting different dosages of tamoxifen into the mice and then conducted a further phenotypic analysis. After induction for 5 days in succession at a dose of 200 µl tamoxifen in sunflower oil at 10 mg/ml per mouse every time, the mice appeared dehydrated, had a lower temperature, and displayed inactivity or death. The results of hematoxylin-eosin staining showed that the intestines of the Cldn7 inducible conditional knockout mice had severe intestinal defects that included epithelial cell sloughing, necrosis, inflammation and hyperplasia. Owing to the death of ICKO mice, we adjusted the dose of tamoxifen to a dose of 100 µl in sunflower oil at 10 mg/ml per mouse (aged more than 8 weeks old) every 4 days. And we could induce atypical hyperplasia and adenoma in the intestine. Immunofluorescent staining indicated that the intestinal epithelial structure was destroyed. Electron microscopy experimental analysis indicated that the intercellular gap along the basolateral membrane of Cldn7 inducible conditional knockout mice in the intestine was increased and that contact between the cells and matrix was loosened. We generated a model of intestinal Cldn7 inducible conditional knockout mice. Intestinal Cldn7 deletion induced by tamoxifen initiated inflammation and hyperplasia in mice.

Generation of beta-lactoglobulin knock-out goats using CRISPR/Cas9

PubMed Central

Zhou, Wenjun; Wan, Yongjie; Guo, Rihong; Deng, Mingtian; Deng, Kaiping; Wang, Zhen; Zhang, Yanli; Wang, Feng

2017-01-01

Goat’s milk, considered a substitute for cow’s milk, has a high nutritional value. However, goat’s milk contains various allergens, predominantly β-lactoglobulin (BLG). In this study, we employed the CRISPR/Cas9 system to target the BLG locus in goat fibroblasts for sgRNA optimization and generate BLG knock-out goats through co-injection of Cas9 mRNA and small guide RNAs (sgRNAs) into goat embryos at the one-cell stage. We firstly tested sgRNA editing efficiencies in goat fibroblast cells, and approximately 8.00%–9.09% of the cells were modified in single sgRNA-guided targeting experiment. Among the kids, the genome-targeting efficiencies of single sgRNA were 12.5% (10 ng/μL sg1) and 0% (10 ng/μL sg2) and efficiencies of dual sgRNAs were 25.0% (25 ng/μL sg2+sg3 group) and 28.6% (50 ng/μL sg2+sg3 group). Relative expression of BLG in BLG knock-out goat mammary glands significantly (p < 0.01) decreased as well as other milk protein coding genes, such as CSN1S1, CSN1S2, CSN2, CSN3 and LALBA (p < 0.05). As expected, BLG protein had been abolished in the milk of the BLG knock-out goat. In addition, most of the targeted kids were chimeric (3/4), and their various body tissues were edited simultaneously. Our study thus provides a basis for optimizing the quality of goat milk, which can be applied to biomedical and agricultural research. PMID:29016691

Comprehensive phenotypic analysis of knockout mice deficient in cyclin G1 and cyclin G2

PubMed Central

Ohno, Shouichi; Ikeda, Jun-ichiro; Naito, Yoko; Okuzaki, Daisuke; Sasakura, Towa; Fukushima, Kohshiro; Nishikawa, Yukihiro; Ota, Kaori; Kato, Yorika; Wang, Mian; Torigata, Kosuke; Kasama, Takashi; Uchihashi, Toshihiro; Miura, Daisaku; Yabuta, Norikazu; Morii, Eiichi; Nojima, Hiroshi

2016-01-01

Cyclin G1 (CycG1) and Cyclin G2 (CycG2) play similar roles during the DNA damage response (DDR), but their detailed roles remain elusive. To investigate their distinct roles, we generated knockout mice deficient in CycG1 (G1KO) or CycG2 (G2KO), as well as double knockout mice (DKO) deficient in both proteins. All knockouts developed normally and were fertile. Generation of mouse embryonic fibroblasts (MEFs) from these mice revealed that G2KO MEFs, but not G1KO or DKO MEFs, were resistant to DNA damage insults caused by camptothecin and ionizing radiation (IR) and underwent cell cycle arrest. CycG2, but not CycG1, co-localized with γH2AX foci in the nucleus after γ-IR, and γH2AX-mediated DNA repair and dephosphorylation of CHK2 were delayed in G2KO MEFs. H2AX associated with CycG1, CycG2, and protein phosphatase 2A (PP2A), suggesting that γH2AX affects the function of PP2A via direct interaction with its B’γ subunit. Furthermore, expression of CycG2, but not CycG1, was abnormal in various cancer cell lines. Kaplan–Meier curves based on TCGA data disclosed that head and neck cancer patients with reduced CycG2 expression have poorer clinical prognoses. Taken together, our data suggest that reduced CycG2 expression could be useful as a novel prognostic marker of cancer. PMID:27982046

Starch Binding Domain-containing Protein 1 Plays a Dominant Role in Glycogen Transport to Lysosomes in Liver*

PubMed Central

Sun, Tao; Yi, Haiqing; Yang, Chunyu; Kishnani, Priya S.; Sun, Baodong

2016-01-01

A small portion of cellular glycogen is transported to and degraded in lysosomes by acid α-glucosidase (GAA) in mammals, but it is unclear why and how glycogen is transported to the lysosomes. Stbd1 has recently been proposed to participate in glycogen trafficking to lysosomes. However, our previous study demonstrated that knockdown of Stbd1 in GAA knock-out mice did not alter lysosomal glycogen storage in skeletal muscles. To further determine whether Stbd1 participates in glycogen transport to lysosomes, we generated GAA/Stbd1 double knock-out mice. In fasted double knock-out mice, glycogen accumulation in skeletal and cardiac muscles was not affected, but glycogen content in liver was reduced by nearly 73% at 3 months of age and by 60% at 13 months as compared with GAA knock-out mice, indicating that the transport of glycogen to lysosomes was suppressed in liver by the loss of Stbd1. Exogenous expression of human Stbd1 in double knock-out mice restored the liver lysosomal glycogen content to the level of GAA knock-out mice, as did a mutant lacking the Atg8 family interacting motif (AIM) and another mutant that contains only the N-terminal 24 hydrophobic segment and the C-terminal starch binding domain (CBM20) interlinked by an HA tag. Our results demonstrate that Stbd1 plays a dominant role in glycogen transport to lysosomes in liver and that the N-terminal transmembrane region and the C-terminal CBM20 domain are critical for this function. PMID:27358407

Elastase‐2, an angiotensin II‐generating enzyme, contributes to increased angiotensin II in resistance arteries of mice with myocardial infarction

PubMed Central

Silva, Marcondes A B; Durand, Marina T; Prado, Cibele M; Oliveira, Eduardo B; Ribeiro, Mauricio S; Salgado, Helio C; Salgado, Maria Cristina O; Tostes, Rita C

2017-01-01

Background and Purpose Angiotensin II (Ang II), whose generation largely depends on angiotensin‐converting enzyme (ACE) activity, mediates most of the renin‐angiotensin‐system (RAS) effects. Elastase‐2 (ELA‐2), a chymotrypsin‐serine protease elastase family member 2A, alternatively generates Ang II in rat arteries. Myocardial infarction (MI) leads to intense RAS activation, but mechanisms involved in Ang II‐generation in resistance arteries are unknown. We hypothesized that ELA‐2 contributes to vascular Ang II generation and cardiac damage in mice subjected to MI. Experimental Approach Concentration‐effect curves to Ang I and Ang II were performed in mesenteric resistance arteries from male wild type (WT) and ELA‐2 knockout (ELA‐2KO) mice subjected to left anterior descending coronary artery ligation (MI). Key Results MI size was similar in WT and ELA‐2KO mice. Ejection fraction and fractional shortening after MI similarly decreased in both strains. However, MI decreased stroke volume and cardiac output in WT, but not in ELA‐2KO mice. Ang I‐induced contractions increased in WT mice subjected to MI (MI‐WT) compared with sham‐WT mice. No differences were observed in Ang I reactivity between arteries from ELA‐2KO and ELA‐2KO subjected to MI (MI‐ELA‐2KO). Ang I contractions increased in arteries from MI‐WT versus MI‐ELA‐2KO mice. Chymostatin attenuated Ang I‐induced vascular contractions in WT mice, but did not affect Ang I responses in ELA‐2KO arteries. Conclusions and Implications These results provide the first evidence that ELA‐2 contributes to increased Ang II formation in resistance arteries and modulates cardiac function after MI, implicating ELA‐2 as a key player in ACE‐independent dysregulation of the RAS. PMID:28222221

Elastase-2, an angiotensin II-generating enzyme, contributes to increased angiotensin II in resistance arteries of mice with myocardial infarction.

PubMed

Becari, Christiane; Silva, Marcondes A B; Durand, Marina T; Prado, Cibele M; Oliveira, Eduardo B; Ribeiro, Mauricio S; Salgado, Helio C; Salgado, Maria Cristina O; Tostes, Rita C

2017-05-01

Angiotensin II (Ang II), whose generation largely depends on angiotensin-converting enzyme (ACE) activity, mediates most of the renin-angiotensin-system (RAS) effects. Elastase-2 (ELA-2), a chymotrypsin-serine protease elastase family member 2A, alternatively generates Ang II in rat arteries. Myocardial infarction (MI) leads to intense RAS activation, but mechanisms involved in Ang II-generation in resistance arteries are unknown. We hypothesized that ELA-2 contributes to vascular Ang II generation and cardiac damage in mice subjected to MI. Concentration-effect curves to Ang I and Ang II were performed in mesenteric resistance arteries from male wild type (WT) and ELA-2 knockout (ELA-2KO) mice subjected to left anterior descending coronary artery ligation (MI). MI size was similar in WT and ELA-2KO mice. Ejection fraction and fractional shortening after MI similarly decreased in both strains. However, MI decreased stroke volume and cardiac output in WT, but not in ELA-2KO mice. Ang I-induced contractions increased in WT mice subjected to MI (MI-WT) compared with sham-WT mice. No differences were observed in Ang I reactivity between arteries from ELA-2KO and ELA-2KO subjected to MI (MI-ELA-2KO). Ang I contractions increased in arteries from MI-WT versus MI-ELA-2KO mice. Chymostatin attenuated Ang I-induced vascular contractions in WT mice, but did not affect Ang I responses in ELA-2KO arteries. These results provide the first evidence that ELA-2 contributes to increased Ang II formation in resistance arteries and modulates cardiac function after MI, implicating ELA-2 as a key player in ACE-independent dysregulation of the RAS. © 2017 The British Pharmacological Society.

Genome-wide Association Studies Identify Genetic Loci Associated With Albuminuria in Diabetes

PubMed Central

Tin, Adrienne; Sorice, Rossella; Gorski, Mathias; Yeo, Nan Cher; Chu, Audrey Y.; Li, Man; Li, Yong; Mijatovic, Vladan; Ko, Yi-An; Taliun, Daniel; Luciani, Alessandro; Chen, Ming-Huei; Yang, Qiong; Foster, Meredith C.; Olden, Matthias; Hiraki, Linda T.; Tayo, Bamidele O.; Fuchsberger, Christian; Dieffenbach, Aida Karina; Shuldiner, Alan R.; Smith, Albert V.; Zappa, Allison M.; Lupo, Antonio; Kollerits, Barbara; Ponte, Belen; Stengel, Bénédicte; Krämer, Bernhard K.; Paulweber, Bernhard; Mitchell, Braxton D.; Hayward, Caroline; Helmer, Catherine; Meisinger, Christa; Gieger, Christian; Shaffer, Christian M.; Müller, Christian; Langenberg, Claudia; Ackermann, Daniel; Siscovick, David; Boerwinkle, Eric; Kronenberg, Florian; Ehret, Georg B.; Homuth, Georg; Waeber, Gerard; Navis, Gerjan; Gambaro, Giovanni; Malerba, Giovanni; Eiriksdottir, Gudny; Li, Guo; Wichmann, H. Erich; Grallert, Harald; Wallaschofski, Henri; Völzke, Henry; Brenner, Herrmann; Kramer, Holly; Leach, I. Mateo; Rudan, Igor; Hillege, Hans L.; Beckmann, Jacques S.; Lambert, Jean Charles; Luan, Jian'an; Zhao, Jing Hua; Chalmers, John; Coresh, Josef; Denny, Joshua C.; Butterbach, Katja; Launer, Lenore J.; Ferrucci, Luigi; Kedenko, Lyudmyla; Haun, Margot; Metzger, Marie; Woodward, Mark; Hoffman, Matthew J.; Nauck, Matthias; Waldenberger, Melanie; Pruijm, Menno; Bochud, Murielle; Rheinberger, Myriam; Verweij, Niek; Wareham, Nicholas J.; Endlich, Nicole; Soranzo, Nicole; Polasek, Ozren; van der Harst, Pim; Pramstaller, Peter Paul; Vollenweider, Peter; Wild, Philipp S.; Gansevoort, Ron T.; Rettig, Rainer; Biffar, Reiner; Carroll, Robert J.; Katz, Ronit; Loos, Ruth J.F.; Hwang, Shih-Jen; Coassin, Stefan; Bergmann, Sven; Rosas, Sylvia E.; Stracke, Sylvia; Harris, Tamara B.; Corre, Tanguy; Zeller, Tanja; Illig, Thomas; Aspelund, Thor; Tanaka, Toshiko; Lendeckel, Uwe; Völker, Uwe; Gudnason, Vilmundur; Chouraki, Vincent; Koenig, Wolfgang; Kutalik, Zoltan; O'Connell, Jeffrey R.; Parsa, Afshin; Heid, Iris M.; Paterson, Andrew D.; de Boer, Ian H.; Devuyst, Olivier; Lazar, Jozef; Endlich, Karlhans; Susztak, Katalin; Tremblay, Johanne; Hamet, Pavel; Jacob, Howard J.; Böger, Carsten A.

2016-01-01

Elevated concentrations of albumin in the urine, albuminuria, are a hallmark of diabetic kidney disease and are associated with an increased risk for end-stage renal disease and cardiovascular events. To gain insight into the pathophysiological mechanisms underlying albuminuria, we conducted meta-analyses of genome-wide association studies and independent replication in up to 5,825 individuals of European ancestry with diabetes and up to 46,061 without diabetes, followed by functional studies. Known associations of variants in CUBN, encoding cubilin, with the urinary albumin-to-creatinine ratio (UACR) were confirmed in the overall sample (P = 2.4 × 10−10). Gene-by-diabetes interactions were detected and confirmed for variants in HS6ST1 and near RAB38/CTSC. Single nucleotide polymorphisms at these loci demonstrated a genetic effect on UACR in individuals with but not without diabetes. The change in the average UACR per minor allele was 21% for HS6ST1 (P = 6.3 × 10–7) and 13% for RAB38/CTSC (P = 5.8 × 10−7). Experiments using streptozotocin-induced diabetic Rab38 knockout and control rats showed higher urinary albumin concentrations and reduced amounts of megalin and cubilin at the proximal tubule cell surface in Rab38 knockout versus control rats. Relative expression of RAB38 was higher in tubuli of patients with diabetic kidney disease compared with control subjects. The loci identified here confirm known pathways and highlight novel pathways influencing albuminuria. PMID:26631737

Transgenerational effects of adolescent nicotine exposure in rats: Evidence for cognitive deficits in adult female offspring.

PubMed

Renaud, Samantha M; Fountain, Stephen B

2016-01-01

This study investigated whether adolescent nicotine exposure in one generation of rats would impair the cognitive capacity of a subsequent generation. Male and female rats in the parental F0 generation were given twice-daily i.p. injections of either 1.0mg/kg nicotine or an equivalent volume of saline for 35days during adolescence on postnatal days 25-59 (P25-59). After reaching adulthood, male and female nicotine-exposed rats were paired for breeding as were male and female saline control rats. Only female offspring were used in this experiment. Half of the offspring of F0 nicotine-exposed breeders and half of the offspring of F0 saline control rats received twice-daily i.p. injections of 1.0mg/kg nicotine during adolescence on P25-59. The remainder of the rats received twice-daily saline injections for the same period. To evaluate transgenerational effects of nicotine exposure on complex cognitive learning abilities, F1 generation rats were trained to perform a highly structured serial pattern in a serial multiple choice (SMC) task. Beginning on P95, rats in the F1 generation were given either 4days of massed training (20patterns/day) followed by spaced training (10 patterns/day) or only spaced training. Transgenerational effects of adolescent nicotine exposure were observed as greater difficulty in learning a "violation element" of the pattern, which indicated that rats were impaired in the ability to encode and remember multiple sequential elements as compound or configural cues. The results indicated that for rats that received massed training, F1 generation rats with adolescent nicotine exposure whose F0 generation parents also experienced adolescent nicotine exposure showed poorer learning of the violation element than rats that experienced adolescent nicotine exposure only in the F1 generation. Thus, adolescent nicotine exposure in one generation of rats produced a cognitive impairment in the next generation. Copyright © 2016 Elsevier Inc. All rights reserved.

Generation of human endometrial knockout cell lines with the CRISPR/Cas9 system confirms the prostaglandin F2α synthase activity of aldo-ketoreductase 1B1.

PubMed

Lacroix Pépin, Nicolas; Chapdelaine, Pierre; Rodriguez, Yoima; Tremblay, Jacques-P; Fortier, Michel A

2014-07-01

Prostaglandins (PGs) are important regulators of female reproductive function. The primary PGs produced in the endometrium are PGE2 and PGF2α. Relatively little is known about the biosynthetic pathways leading to the formation of PGF2α. We have described the role of aldo-ketoreductase (AKR)1B1 in increased PGF2α production by human endometrial cells following stimulation with interleukin-1β (IL-1β). However, alternate PGF synthases are expressed concurrently in endometrial cells. A definite proof of the role of AKR1B1 would require gene knockout; unfortunately, this gene has no direct equivalent in the mouse. Recently, an efficient genome-editing technology using RNA-guided DNase Cas9 and the clustered regularly interspaced short palindromic repeats (CRISPR) system has been developed. We have adapted this approach to knockout AKR1B1 gene expression in human endometrial cell lines. One clone (16-2) of stromal origin generated by the CRISPR/Cas9 system exhibited a complete loss of AKR1B1 protein and mRNA expression, whereas other clones presented with partial edition. The present report focuses on the characterization of clone 16-2 exhibiting deletion of 68 and 2 nucleotides, respectively, on each of the alleles. Cells from this clone lost their ability to produce PGF2α but maintained their original stromal cell (human endometrial stromal cells-2) phenotype including the capacity to decidualize in the presence of progesterone (medroxyprogesterone acetate) and 8-bromo-cAMP. Knockout cells also maintained their ability to increase PGE2 production in response to IL-1β. In summary, we demonstrate that the new genome editing CRISPR/Cas9 system can be used in human cells to generate stable knockout cell line models. Our results suggest that genome editing of human cell lines can be used to complement mouse KO models to validate the function of genes in differentiated tissues and cells. Our results also confirm that AKR1B1 is involved in the synthesis of PGF2α. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Efficient modification of the myostatin gene in porcine somatic cells and generation of knockout piglets.

PubMed

Rao, Shengbin; Fujimura, Tatsuya; Matsunari, Hitomi; Sakuma, Tetsushi; Nakano, Kazuaki; Watanabe, Masahito; Asano, Yoshinori; Kitagawa, Eri; Yamamoto, Takashi; Nagashima, Hiroshi

2016-01-01

Myostatin (MSTN) is a negative regulator of myogenesis, and disruption of its function causes increased muscle mass in various species. Here, we report the generation of MSTN-knockout (KO) pigs using genome editing technology combined with somatic-cell nuclear transfer (SCNT). Transcription activator-like effector nuclease (TALEN) with non-repeat-variable di-residue variations, called Platinum TALEN, was highly efficient in modifying genes in porcine somatic cells, which were then used for SCNT to create MSTN KO piglets. These piglets exhibited a double-muscled phenotype, possessing a higher body weight and longissimus muscle mass measuring 170% that of wild-type piglets, with double the number of muscle fibers. These results demonstrate that loss of MSTN increases muscle mass in pigs, which may help increase pork production for consumption in the future. © 2015 Wiley Periodicals, Inc.

High-throughput discovery of novel developmental phenotypes.

PubMed

Dickinson, Mary E; Flenniken, Ann M; Ji, Xiao; Teboul, Lydia; Wong, Michael D; White, Jacqueline K; Meehan, Terrence F; Weninger, Wolfgang J; Westerberg, Henrik; Adissu, Hibret; Baker, Candice N; Bower, Lynette; Brown, James M; Caddle, L Brianna; Chiani, Francesco; Clary, Dave; Cleak, James; Daly, Mark J; Denegre, James M; Doe, Brendan; Dolan, Mary E; Edie, Sarah M; Fuchs, Helmut; Gailus-Durner, Valerie; Galli, Antonella; Gambadoro, Alessia; Gallegos, Juan; Guo, Shiying; Horner, Neil R; Hsu, Chih-Wei; Johnson, Sara J; Kalaga, Sowmya; Keith, Lance C; Lanoue, Louise; Lawson, Thomas N; Lek, Monkol; Mark, Manuel; Marschall, Susan; Mason, Jeremy; McElwee, Melissa L; Newbigging, Susan; Nutter, Lauryl M J; Peterson, Kevin A; Ramirez-Solis, Ramiro; Rowland, Douglas J; Ryder, Edward; Samocha, Kaitlin E; Seavitt, John R; Selloum, Mohammed; Szoke-Kovacs, Zsombor; Tamura, Masaru; Trainor, Amanda G; Tudose, Ilinca; Wakana, Shigeharu; Warren, Jonathan; Wendling, Olivia; West, David B; Wong, Leeyean; Yoshiki, Atsushi; MacArthur, Daniel G; Tocchini-Valentini, Glauco P; Gao, Xiang; Flicek, Paul; Bradley, Allan; Skarnes, William C; Justice, Monica J; Parkinson, Helen E; Moore, Mark; Wells, Sara; Braun, Robert E; Svenson, Karen L; de Angelis, Martin Hrabe; Herault, Yann; Mohun, Tim; Mallon, Ann-Marie; Henkelman, R Mark; Brown, Steve D M; Adams, David J; Lloyd, K C Kent; McKerlie, Colin; Beaudet, Arthur L; Bućan, Maja; Murray, Stephen A

2016-09-22

Approximately one-third of all mammalian genes are essential for life. Phenotypes resulting from knockouts of these genes in mice have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5,000 knockout mouse lines, here we identify 410 lethal genes during the production of the first 1,751 unique gene knockouts. Using a standardized phenotyping platform that incorporates high-resolution 3D imaging, we identify phenotypes at multiple time points for previously uncharacterized genes and additional phenotypes for genes with previously reported mutant phenotypes. Unexpectedly, our analysis reveals that incomplete penetrance and variable expressivity are common even on a defined genetic background. In addition, we show that human disease genes are enriched for essential genes, thus providing a dataset that facilitates the prioritization and validation of mutations identified in clinical sequencing efforts.

Analysis of uracil phosphoribosyltransferase expression in Mycobacterium tuberculosis and evaluation of upp knockout strain in infected mice.

PubMed

Villela, Anne Drumond; Pham, Ha; Jones, Victoria; Grzegorzewicz, Anna E; Rodrigues-Junior, Valnês da Silva; Campos, Maria Martha; Basso, Luiz Augusto; Jackson, Mary; Santos, Diógenes Santiago

2017-02-01

The upp (Rv3309c)-encoded uracil phosphoribosyltransferase from Mycobacterium tuberculosis (MtUPRT) converts uracil and 5-phosphoribosyl-α-1-pyrophosphate into pyrophosphate and uridine 5΄-monophosphate, the precursor of all pyrimidine nucleotides. A M. tuberculosis knockout strain for upp gene was generated by allelic replacement. Knockout and complemented strains were validated by a functional assay of uracil incorporation. A basal level of MtUPRT expression is shown to be independent of either growth medium used, addition of bases, or oxygen presence/absence. The upp disruption does not affect M. tuberculosis growth in Middlebrook 7H9 medium, and it is not required for M. tuberculosis virulence in a mouse model of infection. Thus, MtUPRT is unlikely to be a good target for drugs against M. tuberculosis. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

High-throughput discovery of novel developmental phenotypes

PubMed Central

Dickinson, Mary E.; Flenniken, Ann M.; Ji, Xiao; Teboul, Lydia; Wong, Michael D.; White, Jacqueline K.; Meehan, Terrence F.; Weninger, Wolfgang J.; Westerberg, Henrik; Adissu, Hibret; Baker, Candice N.; Bower, Lynette; Brown, James M.; Caddle, L. Brianna; Chiani, Francesco; Clary, Dave; Cleak, James; Daly, Mark J.; Denegre, James M.; Doe, Brendan; Dolan, Mary E.; Edie, Sarah M.; Fuchs, Helmut; Gailus-Durner, Valerie; Galli, Antonella; Gambadoro, Alessia; Gallegos, Juan; Guo, Shiying; Horner, Neil R.; Hsu, Chih-wei; Johnson, Sara J.; Kalaga, Sowmya; Keith, Lance C.; Lanoue, Louise; Lawson, Thomas N.; Lek, Monkol; Mark, Manuel; Marschall, Susan; Mason, Jeremy; McElwee, Melissa L.; Newbigging, Susan; Nutter, Lauryl M.J.; Peterson, Kevin A.; Ramirez-Solis, Ramiro; Rowland, Douglas J.; Ryder, Edward; Samocha, Kaitlin E.; Seavitt, John R.; Selloum, Mohammed; Szoke-Kovacs, Zsombor; Tamura, Masaru; Trainor, Amanda G; Tudose, Ilinca; Wakana, Shigeharu; Warren, Jonathan; Wendling, Olivia; West, David B.; Wong, Leeyean; Yoshiki, Atsushi; MacArthur, Daniel G.; Tocchini-Valentini, Glauco P.; Gao, Xiang; Flicek, Paul; Bradley, Allan; Skarnes, William C.; Justice, Monica J.; Parkinson, Helen E.; Moore, Mark; Wells, Sara; Braun, Robert E.; Svenson, Karen L.; de Angelis, Martin Hrabe; Herault, Yann; Mohun, Tim; Mallon, Ann-Marie; Henkelman, R. Mark; Brown, Steve D.M.; Adams, David J.; Lloyd, K.C. Kent; McKerlie, Colin; Beaudet, Arthur L.; Bucan, Maja; Murray, Stephen A.

2016-01-01

Approximately one third of all mammalian genes are essential for life. Phenotypes resulting from mouse knockouts of these genes have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5000 knockout mouse lines, we have identified 410 lethal genes during the production of the first 1751 unique gene knockouts. Using a standardised phenotyping platform that incorporates high-resolution 3D imaging, we identified novel phenotypes at multiple time points for previously uncharacterized genes and additional phenotypes for genes with previously reported mutant phenotypes. Unexpectedly, our analysis reveals that incomplete penetrance and variable expressivity are common even on a defined genetic background. In addition, we show that human disease genes are enriched for essential genes identified in our screen, thus providing a novel dataset that facilitates prioritization and validation of mutations identified in clinical sequencing efforts. PMID:27626380

Rearing-environment-dependent hippocampal local field potential differences in wild-type and inositol trisphosphate receptor type 2 knockout mice.

PubMed

Tanaka, Mika; Wang, Xiaowen; Mikoshiba, Katsuhiko; Hirase, Hajime; Shinohara, Yoshiaki

2017-10-15

Mice reared in an enriched environment are demonstrated to have larger hippocampal gamma oscillations than those reared in isolation, thereby confirming previous observations in rats. To test whether astrocytic Ca 2+ surges are involved in this experience-dependent LFP pattern modulation, we used inositol trisphosphate receptor type 2 (IP 3 R2)-knockout (KO) mice, in which IP 3 /Ca 2+ signalling in astrocytes is largely diminished. We found that this experience-dependent gamma power alteration persists in the KO mice. Interestingly, hippocampal ripple events, the synchronized events critical for memory consolidation, are reduced in magnitude and frequency by both isolated rearing and IP 3 R2 deficiency. Rearing in an enriched environment (ENR) is known to enhance cognitive and memory abilities in rodents, whereas social isolation (ISO) induces depression-like behaviour. The hippocampus has been documented to undergo morphological and functional changes depending on these rearing environments. For example, rearing condition during juvenility alters CA1 stratum radiatum gamma oscillation power in rats. In the present study, hippocampal CA1 local field potentials (LFP) were recorded from bilateral CA1 in urethane-anaesthetized mice that were reared in either an ENR or ISO condition. Similar to previous findings in rats, gamma oscillation power during theta states was higher in the ENR group. Ripple events that occur during non-theta periods in the CA1 stratum pyramidale also had longer intervals in ISO mice. Because astrocytic Ca 2+ elevations play a key role in synaptic plasticity, we next tested whether these changes in LFP are also expressed in inositol trisphosphate receptor type 2 (IP 3 R2)-knockout (KO) mice, in which astrocytic Ca 2+ elevations are largely diminished. We found that the gamma power was also higher in IP 3 R2-KO-ENR mice compared to IP 3 R2-KO-ISO mice, suggesting that the rearing-environment-dependent gamma power alteration does not necessarily require the astrocytic IP 3 /Ca 2+ pathway. By contrast, ripple events showed genotype-dependent changes, as well as rearing condition-dependent changes: ISO housing and IP 3 R2 deficiency both lead to longer inter-ripple intervals. Moreover, we found that ripple magnitude in the right CA1 tended to be smaller in IP 3 R2-KO. Because IP 3 R2-KO mice have been reported to have depression phenotypes, our results suggest that ripple events and the mood of animals may be broadly correlated. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

An Efficient, Rapid, and Recyclable System for CRISPR-Mediated Genome Editing in Candida albicans.

PubMed

Nguyen, Namkha; Quail, Morgan M F; Hernday, Aaron D

2017-01-01

Candida albicans is the most common fungal pathogen of humans. Historically, molecular genetic analysis of this important pathogen has been hampered by the lack of stable plasmids or meiotic cell division, limited selectable markers, and inefficient methods for generating gene knockouts. The recent development of clustered regularly interspaced short palindromic repeat(s) (CRISPR)-based tools for use with C. albicans has opened the door to more efficient genome editing; however, previously reported systems have specific limitations. We report the development of an optimized CRISPR-based genome editing system for use with C. albicans . Our system is highly efficient, does not require molecular cloning, does not leave permanent markers in the genome, and supports rapid, precise genome editing in C. albicans . We also demonstrate the utility of our system for generating two independent homozygous gene knockouts in a single transformation and present a method for generating homozygous wild-type gene addbacks at the native locus. Furthermore, each step of our protocol is compatible with high-throughput strain engineering approaches, thus opening the door to the generation of a complete C. albicans gene knockout library. IMPORTANCE Candida albicans is the major fungal pathogen of humans and is the subject of intense biomedical and discovery research. Until recently, the pace of research in this field has been hampered by the lack of efficient methods for genome editing. We report the development of a highly efficient and flexible genome editing system for use with C. albicans . This system improves upon previously published C. albicans CRISPR systems and enables rapid, precise genome editing without the use of permanent markers. This new tool kit promises to expedite the pace of research on this important fungal pathogen.

Endoplasmic reticulum stress sensor protein kinase R-like endoplasmic reticulum kinase (PERK) protects against pressure overload-induced heart failure and lung remodeling.

PubMed

Liu, Xiaoyu; Kwak, Dongmin; Lu, Zhongbing; Xu, Xin; Fassett, John; Wang, Huan; Wei, Yidong; Cavener, Douglas R; Hu, Xinli; Hall, Jennifer; Bache, Robert J; Chen, Yingjie

2014-10-01

Studies have reported that development of congestive heart failure is associated with increased endoplasmic reticulum stress. Double stranded RNA-activated protein kinase R-like endoplasmic reticulum kinase (PERK) is a major transducer of the endoplasmic reticulum stress response and directly phosphorylates eukaryotic initiation factor 2α, resulting in translational attenuation. However, the physiological effect of PERK on congestive heart failure development is unknown. To study the effect of PERK on ventricular structure and function, we generated inducible cardiac-specific PERK knockout mice. Under unstressed conditions, cardiac PERK knockout had no effect on left ventricular mass, or its ratio to body weight, cardiomyocyte size, fibrosis, or left ventricular function. However, in response to chronic transverse aortic constriction, PERK knockout mice exhibited decreased ejection fraction, increased left ventricular fibrosis, enhanced cardiomyocyte apoptosis, and exacerbated lung remodeling in comparison with wild-type mice. PERK knockout also dramatically attenuated cardiac sarcoplasmic reticulum Ca(2+)-ATPase expression in response to aortic constriction. Our findings suggest that PERK is required to protect the heart from pressure overload-induced congestive heart failure. © 2014 American Heart Association, Inc.

An efficient method for generation of bi-allelic null mutant mouse embryonic stem cells and its application for investigating epigenetic modifiers

PubMed Central

Cho, Lily Ting-yin; Andrews, Robert; Carroll, Thomas; Iyer, Vivek; Tate, Peri; Rosen, Barry; Stunnenberg, Hendrik G.; Fisher, Amanda G.; Skarnes, William C.

2017-01-01

Abstract Mouse embryonic stem (ES) cells are a popular model system to study biological processes, though uncovering recessive phenotypes requires inactivating both alleles. Building upon resources from the International Knockout Mouse Consortium (IKMC), we developed a targeting vector for second allele inactivation in conditional-ready IKMC ‘knockout-first’ ES cell lines. We applied our technology to several epigenetic regulators, recovering bi-allelic targeted clones with a high efficiency of 60% and used Flp recombinase to restore expression in two null cell lines to demonstrate how our system confirms causality through mutant phenotype reversion. We designed our strategy to select against re-targeting the ‘knockout-first’ allele and identify essential genes in ES cells, including the histone methyltransferase Setdb1. For confirmation, we exploited the flexibility of our system, enabling tamoxifen inducible conditional gene ablation while controlling for genetic background and tamoxifen effects. Setdb1 ablated ES cells exhibit severe growth inhibition, which is not rescued by exogenous Nanog expression or culturing in naive pluripotency ‘2i’ media, suggesting that the self-renewal defect is mediated through pluripotency network independent pathways. Our strategy to generate null mutant mouse ES cells is applicable to thousands of genes and repurposes existing IKMC Intermediate Vectors. PMID:28981838

Mitochondrial pyruvate carrier function determines cell stemness and metabolic reprogramming in cancer cells

PubMed Central

Li, Xiaoran; Kan, Quancheng; Fan, Zhirui; Li, Yaqing; Ji, Yasai; Zhao, Jing; Zhang, Mingzhi; Grigalavicius, Mantas; Berge, Viktor; Goscinski, Mariusz Adam; M. Nesland, Jahn; Suo, Zhenhe

2017-01-01

One of the remarkable features of cancer cells is aerobic glycolysis, a phenomenon known as the “Warburg Effect”, in which cells rely preferentially on glycolysis instead of oxidative phosphorylation (OXPHOS) as the main energy source even in the presence of high oxygen tension. Cells with dysfunctional mitochondria are unable to generate sufficient ATP from mitochondrial OXPHOS, and then are forced to rely on glycolysis for ATP generation. Here we report our results in a prostate cancer cell line in which the mitochondrial pyruvate carrier 1 (MPC1) gene was knockout. It was discovered that the MPC1 gene knockout cells revealed a metabolism reprogramming to aerobic glycolysis with reduced ATP production, and the cells became more migratory and resistant to both chemotherapy and radiotherapy. In addition, the MPC1 knockout cells expressed significantly higher levels of the stemness markers Nanog, Hif1α, Notch1, CD44 and ALDH. To further verify the correlation of MPC gene function and cell stemness/metabolic reprogramming, MPC inhibitor UK5099 was applied in two ovarian cancer cell lines and similar results were obtained. Taken together, our results reveal that functional MPC may determine the fate of metabolic program and the stemness status of cancer cells in vitro. PMID:28624784

Learning and memory depend on fibroblast growth factor receptor 2 functioning in hippocampus.

PubMed

Stevens, Hanna E; Jiang, Ginger Y; Schwartz, Michael L; Vaccarino, Flora M

2012-06-15

Fibroblast growth factor (FGF) signaling controls self-renewal of neural stem cells during embryonic telencephalic development. FGF receptor 2 (FGFR2) has a significant role in the production of cortical neurons during embryogenesis, but its role in the hippocampus during development and in adulthood has not been described. Here we dissociate the role of FGFR2 in the hippocampus during development and during adulthood with the use of embryonic knockout and inducible knockout mice. Embryonic knockout of FGFR2 causes a reduction of hippocampal volume and impairment in adult spatial memory in mice. Spatial reference memory, as assessed by performance on the water maze probe trial, was correlated with reduced hippocampal parvalbumin+ cells, whereas short-term learning was correlated with reduction in immature neurons in the dentate gyrus. Furthermore, short-term learning and newly generated neurons in the dentate gyrus were deficient even when FGFR2 was lacking only in adulthood. Taken together, these findings support a dual role for FGFR2 in hippocampal short-term learning and long-term reference memory, which appear to depend on the abundance of two separate cellular components, parvalbumin interneurons and newly generated granule cells in the hippocampus. Copyright © 2012 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

GLT-1: The elusive presynaptic glutamate transporter

PubMed Central

Rimmele, Theresa S.; Rosenberg, Paul A.

2016-01-01

Historically, glutamate uptake in the CNS was mainly attributed to glial cells for three reasons: 1) none of the glutamate transporters were found to be located in presynaptic terminals of excitatory synapses; 2) the putative glial transporters, GLT-1 and GLAST are expressed at high levels in astrocytes; 3) studies of the constitutive GLT-1 knockout as well as pharmacological studies demonstrated that >90% of glutamate uptake into forebrain synaptosomes is mediated by the operation of GLT-1. Here we summarize the history leading up to the recognition of GLT-1a as a presynaptic glutamate transporter. A major issue now is understanding the physiological and pathophysiologial significance of the expression of GLT-1 in presynaptic terminals. To elucidate the cell-type specific functions of GLT-1, a conditional knockout was generated with which to inactivate the GLT-1 gene in different cell types using Cre/lox technology. Astrocytic knockout led to an 80% reduction of GLT-1 expression, resulting in intractable seizures and early mortality as seen also in the constitutive knockout. Neuronal knockout was associated with no obvious phenotype. Surprisingly, synaptosomal uptake capacity (Vmax) was found to be significantly reduced, by 40%, in the neuronal knockout, indicating that the contribution of neuronal GLT-1 to synaptosomal uptake is disproportionate to its protein expression (5–10%). Conversely, the contribution of astrocytic GLT-1 to synaptosomal uptake was much lower than expected. In contrast, the loss of uptake into liposomes prepared from brain protein from astrocyte and neuronal knockouts was proportionate with the loss of GLT-1 protein, suggesting that a large portion of GLT-1 in astrocytic membranes in synaptosomal preparations is not functional, possibly because of a failure to reseal. These results suggest the need to reinterpret many previous studies using synaptosomal uptake to investigate glutamate transport itself as well as changes in glutamate homeostasis associated with normal functions, neurodegeneration, and response to drugs. PMID:27129805

Severe hypertriglyceridemia, reduced high density lipoprotein, and neonatal death in lipoprotein lipase knockout mice. Mild hypertriglyceridemia with impaired very low density lipoprotein clearance in heterozygotes.

PubMed Central

Weinstock, P H; Bisgaier, C L; Aalto-Setälä, K; Radner, H; Ramakrishnan, R; Levak-Frank, S; Essenburg, A D; Zechner, R; Breslow, J L

1995-01-01

Lipoprotein lipase (LPL)-deficient mice have been created by gene targeting in embryonic stem cells. At birth, homozygous knockout pups have threefold higher triglycerides and sevenfold higher VLDL cholesterol levels than controls. When permitted to suckle, LPL-deficient mice become pale, then cyanotic, and finally die at approximately 18 h of age. Before death, triglyceride levels are severely elevated (15,087 +/- 3,805 vs 188 +/- 71 mg/dl in controls). Capillaries in tissues of homozygous knockout mice are engorged with chylomicrons. This is especially significant in the lung where marginated chylomicrons prevent red cell contact with the endothelium, a phenomenon which is presumably the cause of cyanosis and death in these mice. Homozygous knockout mice also have diminished adipose tissue stores as well as decreased intracellular fat droplets. By crossbreeding with transgenic mice expressing human LPL driven by a muscle-specific promoter, mouse lines were generated that express LPL exclusively in muscle but not in any other tissue. This tissue-specific LPL expression rescued the LPL knockout mice and normalized their lipoprotein pattern. This supports the contention that hypertriglyceridemia caused the death of these mice and that LPL expression in a single tissue was sufficient for rescue. Heterozygous LPL knockout mice survive to adulthood and have mild hypertriglyceridemia, with 1.5-2-fold elevated triglyceride levels compared with controls in both the fed and fasted states on chow, Western-type, or 10% sucrose diets. In vivo turnover studies revealed that heterozygous knockout mice had impaired VLDL clearance (fractional catabolic rate) but no increase in transport rate. In summary, total LPL deficiency in the mouse prevents triglyceride removal from plasma, causing death in the neonatal period, and expression of LPL in a single tissue alleviates this problem. Furthermore, half-normal levels of LPL cause a decrease in VLDL fractional catabolic rate and mild hypertriglyceridemia, implying that partial LPL deficiency has physiological consequences. Images PMID:8675619

Cardiac preconditioning with sphingosine-1-phosphate requires activation of signal transducer and activator of transcription-3

PubMed Central

Kelly-Laubscher, Roisin F; King, Jonathan C; Hacking, Damian; Somers, Sarin; Hastie, Samantha; Stewart, Tessa; Imamdin, Aqeela; Maarman, Gerald; Pedretti, Sarah; Lecour, Sandrine

2014-01-01

Summary Aims Sphingosine-1-phosphate (S1P) is a cardioprotective agent. Signal transducer and activator of transcription 3 (STAT-3) is a key mediator of many cardioprotective agents. We aimed to explore whether STAT-3 is a key mediator in S1P-induced preconditioning. Methods Langendorff-perfused hearts from Wistar rats and wild-type or cardiomyocyte-specific STAT-3 knockout mice were pre-treated with S1P (10 nmol/l), with or without the STAT-3 pathway inhibitor AG490, before an ischaemia–reperfusion insult. Triphenyltetrazolium chloride and Evans blue staining were used for the determination of infarct size. Western blot analysis was carried out on the S1P pre-treated hearts for detection of cytosolic, nuclear and mitochondrial phosphorylated and total STAT-3 proteins. Results Pre-treatment with S1P decreased the infarct size in isolated rat (5 ± 3% vs control 26 ± 8%, p < 0.01) and wild-type mouse hearts (13 ± 1% vs control 33 ± 3%, p < 0.05). This protective effect was abolished in the rat hearts pre-treated with AG490 (30 ± 10%, p = ns vs control) and in the hearts from STAT-3 knockout mice (35 ± 4% vs control 30 ± 3%, p = ns). Levels of phosphorylated STAT-3 were significantly increased in both the nuclear (p < 0.05 vs control) and mitochondrial (p < 0.05 vs control) fractions in the S1P pre-treated hearts, but remained unchanged in the cytosolic fraction (p = ns vs control). Conclusion These novel results demonstrate that pharmacological preconditioning with S1P in the isolated heart is mediated by activation of mitochondrial and nuclear STAT-3, therefore suggesting that S1P may be a novel therapeutic target to modulate mitochondrial and nuclear function in cardiovascular disease in order to protect the heart against ischaemia–reperfusion. PMID:25000441

Inhibition of AAK1 Kinase as a Novel Therapeutic Approach to Treat Neuropathic Pain

PubMed Central

Kostich, Walter; Hamman, Brian D.; Li, Yu-Wen; Naidu, Sreenivasulu; Dandapani, Kumaran; Feng, Jianlin; Easton, Amy; Bourin, Clotilde; Baker, Kevin; Allen, Jason; Savelieva, Katerina; Louis, Justin V.; Dokania, Manoj; Elavazhagan, Saravanan; Vattikundala, Pradeep; Sharma, Vivek; Das, Manish Lal; Shankar, Ganesh; Kumar, Anoop; Holenarsipur, Vinay K.; Gulianello, Michael; Molski, Ted; Brown, Jeffrey M.; Lewis, Martin; Huang, Yanling; Lu, Yifeng; Pieschl, Rick; O’Malley, Kevin; Lippy, Jonathan; Nouraldeen, Amr; Lanthorn, Thomas H.; Ye, Guilan; Wilson, Alan; Balakrishnan, Anand; Denton, Rex; Grace, James E.; Lentz, Kimberley A.; Santone, Kenneth S.; Bi, Yingzhi; Main, Alan; Swaffield, Jon; Carson, Ken; Mandlekar, Sandhya; Vikramadithyan, Reeba K.; Nara, Susheel J.; Dzierba, Carolyn; Bronson, Joanne; Macor, John E.; Zaczek, Robert; Westphal, Ryan; Kiss, Laszlo; Bristow, Linda; Conway, Charles M.

2016-01-01

To identify novel targets for neuropathic pain, 3097 mouse knockout lines were tested in acute and persistent pain behavior assays. One of the lines from this screen, which contained a null allele of the adapter protein-2 associated kinase 1 (AAK1) gene, had a normal response in acute pain assays (hot plate, phase I formalin), but a markedly reduced response to persistent pain in phase II formalin. AAK1 knockout mice also failed to develop tactile allodynia following the Chung procedure of spinal nerve ligation (SNL). Based on these findings, potent, small-molecule inhibitors of AAK1 were identified. Studies in mice showed that one such inhibitor, LP-935509, caused a reduced pain response in phase II formalin and reversed fully established pain behavior following the SNL procedure. Further studies showed that the inhibitor also reduced evoked pain responses in the rat chronic constriction injury (CCI) model and the rat streptozotocin model of diabetic peripheral neuropathy. Using a nonbrain-penetrant AAK1 inhibitor and local administration of an AAK1 inhibitor, the relevant pool of AAK1 for antineuropathic action was found to be in the spinal cord. Consistent with these results, AAK1 inhibitors dose-dependently reduced the increased spontaneous neural activity in the spinal cord caused by CCI and blocked the development of windup induced by repeated electrical stimulation of the paw. The mechanism of AAK1 antinociception was further investigated with inhibitors of α2 adrenergic and opioid receptors. These studies showed that α2 adrenergic receptor inhibitors, but not opioid receptor inhibitors, not only prevented AAK1 inhibitor antineuropathic action in behavioral assays, but also blocked the AAK1 inhibitor–induced reduction in spinal neural activity in the rat CCI model. Hence, AAK1 inhibitors are a novel therapeutic approach to neuropathic pain with activity in animal models that is mechanistically linked (behaviorally and electrophysiologically) to α2 adrenergic signaling, a pathway known to be antinociceptive in humans. PMID:27411717

Type-3 ryanodine receptors mediate hypoxia-, but not neurotransmitter-induced calcium release and contraction in pulmonary artery smooth muscle cells.

PubMed

Zheng, Yun-Min; Wang, Qing-Song; Rathore, Rakesh; Zhang, Wan-Hui; Mazurkiewicz, Joseph E; Sorrentino, Vincenzo; Singer, Harold A; Kotlikoff, Michael I; Wang, Yong-Xiao

2005-04-01

In this study we examined the expression of RyR subtypes and the role of RyRs in neurotransmitter- and hypoxia-induced Ca2+ release and contraction in pulmonary artery smooth muscle cells (PASMCs). Under perforated patch clamp conditions, maximal activation of RyRs with caffeine or inositol triphosphate receptors (IP3Rs) with noradrenaline induced equivalent increases in [Ca2+]i and Ca2+-activated Cl- currents in freshly isolated rat PASMCs. Following maximal IP3-induced Ca2+ release, neither caffeine nor chloro-m-cresol induced a response, whereas prior application of caffeine or chloro-m-cresol blocked IP3-induced Ca2+ release. In cultured human PASMCs, which lack functional expression of RyRs, caffeine failed to affect ATP-induced increases in [Ca2+]i in the presence and absence of extracellular Ca2+. The RyR antagonists ruthenium red, ryanodine, tetracaine, and dantrolene greatly inhibited submaximal noradrenaline- and hypoxia-induced Ca2+ release and contraction in freshly isolated rat PASMCs, but did not affect ATP-induced Ca2+ release in cultured human PASMCs. Real-time quantitative RT-PCR and immunofluorescence staining indicated similar expression of all three RyR subtypes (RyR1, RyR2, and RyR3) in freshly isolated rat PASMCs. In freshly isolated PASMCs from RyR3 knockout (RyR3-/-) mice, hypoxia-induced, but not submaximal noradrenaline-induced, Ca2+ release and contraction were significantly reduced. Ruthenium red and tetracaine can further inhibit hypoxic increase in [Ca2+]i in RyR3-/- mouse PASMCs. Collectively, our data suggest that (a) RyRs play an important role in submaximal noradrenaline- and hypoxia-induced Ca2+ release and contraction; (b) all three subtype RyRs are expressed; and (c) RyR3 gene knockout significantly inhibits hypoxia-, but not submaximal noradrenaline-induced Ca2+ and contractile responses in PASMCs.

A Mutation in the Dmp1 Gene Alters Phosphate Responsiveness in Mice

PubMed Central

Gerard-O'Riley, Rita L.; Acton, Dena; McQueen, Amie K.; Strobel, Isabel E.; Witcher, Phillip C.; Feng, Jian Q.; Econs, Michael J.

2017-01-01

Mutations in the dentin matrix protein 1 (DMP1) gene cause autosomal recessive hypophosphatemic rickets (ARHR). Hypophosphatemia in ARHR results from increased circulating levels of the phosphaturic hormone, fibroblast growth factor 23 (FGF23). Similarly, elevated FGF23, caused by mutations in the PHEX gene, is responsible for the hypophosphatemia in X-linked hypophosphatemic rickets (XLH). Previously, we demonstrated that a Phex mutation in mice creates a lower set point for extracellular phosphate, where an increment in phosphorus further stimulates Fgf23 production to maintain low serum phosphorus levels. To test the presence of the similar set point defect in ARHR, we generated 4- and 12-week-old Dmp1/Galnt3 double knockout mice and controls, including Dmp1 knockout mice (a murine model of ARHR), Galnt3 knockout mice (a murine model of familial tumoral calcinosis), and phenotypically normal double heterozygous mice. Galnt3 knockout mice had increased proteolytic cleavage of Fgf23, leading to low circulating intact Fgf23 levels with consequent hyperphosphatemia. In contrast, Dmp1 knockout mice had little Fgf23 cleavage and increased femoral Fgf23 expression, resulting in hypophosphatemia and low femoral bone mineral density (BMD). However, introduction of the Galnt3 null allele to Dmp1 knockout mice resulted in a significant increase in serum phosphorus and normalization of BMD. This increased serum phosphorus was accompanied by markedly elevated Fgf23 expression and circulating Fgf23 levels, an attempt to reduce serum phosphorus in the face of improving phosphorus levels. These data indicate that a Dmp1 mutation creates a lower set point for extracellular phosphate and maintains it through the regulation of Fgf23 cleavage and expression. PMID:28005411

Dietary phosphate restriction normalizes biochemical and skeletal abnormalities in a murine model of tumoral calcinosis.

PubMed

Ichikawa, Shoji; Austin, Anthony M; Gray, Amie K; Allen, Matthew R; Econs, Michael J

2011-12-01

Mutations in the GALNT3 gene cause tumoral calcinosis characterized by ectopic calcifications due to persistent hyperphosphatemia. We recently developed Galnt3 knockout mice in a mixed background, which had hyperphosphatemia with increased bone mineral density (BMD) and infertility in males. To test the effect of dietary phosphate intake on their phenotype, Galnt3 knockout mice were generated in the C57BL/6J strain and fed various phosphate diets: 0.1% (low), 0.3% (low normal), 0.6% (normal), and 1.65% (high). Sera were analyzed for calcium, phosphorus, alkaline phosphatase, creatinine, blood urine nitrogen, 1,25-dihydroxyvitamin D, osteocalcin, tartrate-resistant acid phosphatase 5b, and fibroblast growth factor 23 (Fgf23). Femurs were evaluated by dual-energy x-ray absorptiometry, dynamic histomorphometry, and/or microcomputed tomography. Galnt3 knockout mice in C57BL/6J had the same biochemical phenotype observed in our previous study: hyperphosphatemia, inappropriately normal 1,25-dihydroxyvitamin D level, decreased alkaline phosphatase activity, and low intact Fgf23 concentration but high Fgf23 fragments. Skeletal analyses of their femurs revealed significantly high BMD with increased cortical bone area and trabecular bone volume. On all four phosphate diets, Galnt3 knockout mice had consistently higher phosphorus levels and lower alkaline phosphatase and intact Fgf23 concentrations than littermate controls. The low-phosphate diet normalized serum phosphorus, alkaline phosphatase, and areal BMD but failed to correct male infertility in Galnt3 knockout mice. The high-phosphate diet did not increase serum phosphorus concentration in either mutant or control mice due to a compensatory increase in circulating intact Fgf23 levels. In conclusion, dietary phosphate restriction normalizes biochemical and skeletal phenotypes of Galnt3 knockout mice and, thus, can be an effective therapy for tumoral calcinosis.

Dietary Phosphate Restriction Normalizes Biochemical and Skeletal Abnormalities in a Murine Model of Tumoral Calcinosis

PubMed Central

Austin, Anthony M.; Gray, Amie K.; Allen, Matthew R.; Econs, Michael J.

2011-01-01

Mutations in the GALNT3 gene cause tumoral calcinosis characterized by ectopic calcifications due to persistent hyperphosphatemia. We recently developed Galnt3 knockout mice in a mixed background, which had hyperphosphatemia with increased bone mineral density (BMD) and infertility in males. To test the effect of dietary phosphate intake on their phenotype, Galnt3 knockout mice were generated in the C57BL/6J strain and fed various phosphate diets: 0.1% (low), 0.3% (low normal), 0.6% (normal), and 1.65% (high). Sera were analyzed for calcium, phosphorus, alkaline phosphatase, creatinine, blood urine nitrogen, 1,25-dihydroxyvitamin D, osteocalcin, tartrate-resistant acid phosphatase 5b, and fibroblast growth factor 23 (Fgf23). Femurs were evaluated by dual-energy x-ray absorptiometry, dynamic histomorphometry, and/or microcomputed tomography. Galnt3 knockout mice in C57BL/6J had the same biochemical phenotype observed in our previous study: hyperphosphatemia, inappropriately normal 1,25-dihydroxyvitamin D level, decreased alkaline phosphatase activity, and low intact Fgf23 concentration but high Fgf23 fragments. Skeletal analyses of their femurs revealed significantly high BMD with increased cortical bone area and trabecular bone volume. On all four phosphate diets, Galnt3 knockout mice had consistently higher phosphorus levels and lower alkaline phosphatase and intact Fgf23 concentrations than littermate controls. The low-phosphate diet normalized serum phosphorus, alkaline phosphatase, and areal BMD but failed to correct male infertility in Galnt3 knockout mice. The high-phosphate diet did not increase serum phosphorus concentration in either mutant or control mice due to a compensatory increase in circulating intact Fgf23 levels. In conclusion, dietary phosphate restriction normalizes biochemical and skeletal phenotypes of Galnt3 knockout mice and, thus, can be an effective therapy for tumoral calcinosis. PMID:22009723

A Mutation in the Dmp1 Gene Alters Phosphate Responsiveness in Mice.

PubMed

Ichikawa, Shoji; Gerard-O'Riley, Rita L; Acton, Dena; McQueen, Amie K; Strobel, Isabel E; Witcher, Phillip C; Feng, Jian Q; Econs, Michael J

2017-03-01

Mutations in the dentin matrix protein 1 (DMP1) gene cause autosomal recessive hypophosphatemic rickets (ARHR). Hypophosphatemia in ARHR results from increased circulating levels of the phosphaturic hormone, fibroblast growth factor 23 (FGF23). Similarly, elevated FGF23, caused by mutations in the PHEX gene, is responsible for the hypophosphatemia in X-linked hypophosphatemic rickets (XLH). Previously, we demonstrated that a Phex mutation in mice creates a lower set point for extracellular phosphate, where an increment in phosphorus further stimulates Fgf23 production to maintain low serum phosphorus levels. To test the presence of the similar set point defect in ARHR, we generated 4- and 12-week-old Dmp1/Galnt3 double knockout mice and controls, including Dmp1 knockout mice (a murine model of ARHR), Galnt3 knockout mice (a murine model of familial tumoral calcinosis), and phenotypically normal double heterozygous mice. Galnt3 knockout mice had increased proteolytic cleavage of Fgf23, leading to low circulating intact Fgf23 levels with consequent hyperphosphatemia. In contrast, Dmp1 knockout mice had little Fgf23 cleavage and increased femoral Fgf23 expression, resulting in hypophosphatemia and low femoral bone mineral density (BMD). However, introduction of the Galnt3 null allele to Dmp1 knockout mice resulted in a significant increase in serum phosphorus and normalization of BMD. This increased serum phosphorus was accompanied by markedly elevated Fgf23 expression and circulating Fgf23 levels, an attempt to reduce serum phosphorus in the face of improving phosphorus levels. These data indicate that a Dmp1 mutation creates a lower set point for extracellular phosphate and maintains it through the regulation of Fgf23 cleavage and expression. Copyright © 2017 by the Endocrine Society.

Starch Binding Domain-containing Protein 1 Plays a Dominant Role in Glycogen Transport to Lysosomes in Liver.

PubMed

Sun, Tao; Yi, Haiqing; Yang, Chunyu; Kishnani, Priya S; Sun, Baodong

2016-08-05

A small portion of cellular glycogen is transported to and degraded in lysosomes by acid α-glucosidase (GAA) in mammals, but it is unclear why and how glycogen is transported to the lysosomes. Stbd1 has recently been proposed to participate in glycogen trafficking to lysosomes. However, our previous study demonstrated that knockdown of Stbd1 in GAA knock-out mice did not alter lysosomal glycogen storage in skeletal muscles. To further determine whether Stbd1 participates in glycogen transport to lysosomes, we generated GAA/Stbd1 double knock-out mice. In fasted double knock-out mice, glycogen accumulation in skeletal and cardiac muscles was not affected, but glycogen content in liver was reduced by nearly 73% at 3 months of age and by 60% at 13 months as compared with GAA knock-out mice, indicating that the transport of glycogen to lysosomes was suppressed in liver by the loss of Stbd1. Exogenous expression of human Stbd1 in double knock-out mice restored the liver lysosomal glycogen content to the level of GAA knock-out mice, as did a mutant lacking the Atg8 family interacting motif (AIM) and another mutant that contains only the N-terminal 24 hydrophobic segment and the C-terminal starch binding domain (CBM20) interlinked by an HA tag. Our results demonstrate that Stbd1 plays a dominant role in glycogen transport to lysosomes in liver and that the N-terminal transmembrane region and the C-terminal CBM20 domain are critical for this function. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Biliary excretion of pravastatin and taurocholate in rats with bile salt export pump (Bsep) impairment.

PubMed

Cheng, Yaofeng; Freeden, Chris; Zhang, Yueping; Abraham, Pamela; Shen, Hong; Wescott, Debra; Humphreys, W Griffith; Gan, Jinping; Lai, Yurong

2016-07-01

The bile salt export pump (BSEP) is expressed on the canalicular membrane of hepatocytes regulating liver bile salt excretion, and impairment of BSEP function may lead to cholestasis in humans. This study explored drug biliary excretion, as well as serum chemistry, individual bile acid concentrations and liver transporter expressions, in the SAGE Bsep knockout (KO) rat model. It was observed that the Bsep protein in KO rats was decreased to 15% of that in the wild type (WT), as quantified using LC-MS/MS. While the levels of Ntcp and Mrp2 were not significantly altered, Mrp3 expression increased and Oatp1a1 decreased in KO animals. Compared with the WT rats, the KO rats had similar serum chemistry and showed normal liver transaminases. Although the total plasma bile salts and bile flow were not significantly changed in Bsep KO rats, individual bile acids in plasma and liver demonstrated variable changes, indicating the impact of Bsep KO. Following an intravenous dose of deuterium labeled taurocholic acid (D4-TCA, 2 mg/kg), the D4-TCA plasma exposure was higher and bile excretion was delayed by approximately 0.5 h in the KO rats. No differences were observed for the pravastatin plasma concentration-time profile or the biliary excretion after intravenous administration (1 mg/kg). Collectively, the results revealed that these rats have significantly lower Bsep expression, therefore affecting the biliary excretion of endogenous bile acids and Bsep substrates. However, these rats are able to maintain a relatively normal liver function through the remaining Bsep protein and via the regulation of other transporters. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Identification of Mom12 and Mom13, two novel modifier loci of Apc (Min) -mediated intestinal tumorigenesis.

PubMed

Crist, Richard C; Roth, Jacquelyn J; Lisanti, Michael P; Siracusa, Linda D; Buchberg, Arthur M

2011-04-01

Colorectal cancer is a heterogeneous disease resulting from a combination of genetic and environmental factors. The C57BL/6J (B6) Apc (Min/+) mouse develops polyps throughout the gastrointestinal tract and has been a valuable model for understanding the genetic basis of intestinal tumorigenesis. Apc (Min/+) mice have been used to study known oncogenes and tumor suppressor genes on a controlled genetic background. These studies often utilize congenic knockout alleles, which can carry an unknown amount of residual donor DNA. The Apc (Min) model has also been used to identify modifer loci, known as Modifier of Min (Mom) loci, which alter Apc (Min) -mediated intestinal tumorigenesis. B6 mice carrying a knockout allele generated in WW6 embryonic stem cells were crossed to B6 Apc (Min/+) mice to determine the effect on polyp multiplicity. The newly generated colony developed significantly more intestinal polyps than Apc (Min/+) controls. Polyp multiplicity did not correlate with inheritance of the knockout allele, suggesting the presence of one or more modifier loci segregating in the colony. Genotyping of simple sequence length polymorphism (SSLP) markers revealed residual 129X1/SvJ genomic DNA within the congenic region of the parental knockout line. An analysis of polyp multiplicity data and SSLP genotyping indicated the presence of two Mom loci in the colony: 1) Mom12, a dominant modifier linked to the congenic region on chromosome 6, and 2) Mom13, which is unlinked to the congenic region and whose effect is masked by Mom12. The identification of Mom12 and Mom13 demonstrates the potential problems resulting from residual heterozygosity present in congenic lines.

The Transient Role for Calcium and Vitamin D during the Developmental Hair Follicle Cycle.

PubMed

Mady, Leila J; Ajibade, Dare V; Hsaio, Connie; Teichert, Arnaud; Fong, Chak; Wang, Yongmei; Christakos, Sylvia; Bikle, Daniel D

2016-07-01

The role for 1,25-dihydroxyvitamin D3 and/or calcium in hair follicle cycling is not clear despite their impact on keratinocyte differentiation. We found that calbindin-D9k null (knockout) pups generated from calbindin-D9k knockout females fed a vitamin D-deficient, low-calcium (0.47%) diet develop transient alopecia. The pups appear phenotypically normal until 13 days of age, after which the hair progressively sheds in a caudocephalic direction, resulting in truncal alopecia totalis by 20-23 days, with spontaneous recovery by 28 days. Histological studies showed markedly dystrophic hair follicles, loss of hair shafts with increased apoptosis, and hyperplastic epidermis during this time. Ha1 expression is lost during catagen in all mice but recovers more slowly in the knockout pups on the vitamin D-deficient, low-calcium diet. Keratin 1 expression is reduced throughout days 19-28. The expressions of involucrin, loricrin, and cathepsin L is initially increased by day 19 but subsequently falls below those of controls by day 23, as does that of desmoglein 3. Feeding the mothers a high-vitamin D/high-calcium (2%)/lactose (20%) diet lessens the phenotype, and knockout pups fostered to mothers fed a normal diet do not develop alopecia. Our results show that in calbindin-D9k knockout pups, a maternal vitamin D-deficient/low-calcium diet leads to transient noncicatricial alopecia. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Mesenchyme-specific knockout of ESET histone methyltransferase causes ectopic hypertrophy and terminal differentiation of articular chondrocytes.

PubMed

Lawson, Kevin A; Teteak, Colin J; Zou, Junhui; Hacquebord, Jacques; Ghatan, Andrew; Zielinska-Kwiatkowska, Anna; Fernandes, Russell J; Chansky, Howard A; Yang, Liu

2013-11-08

The exact molecular mechanisms governing articular chondrocytes remain unknown in skeletal biology. In this study, we have found that ESET (an ERG-associated protein with a SET domain, also called SETDB1) histone methyltransferase is expressed in articular cartilage. To test whether ESET regulates articular chondrocytes, we carried out mesenchyme-specific deletion of the ESET gene in mice. ESET knock-out did not affect generation of articular chondrocytes during embryonic development. Two weeks after birth, there was minimal qualitative difference at the knee joints between wild-type and ESET knock-out animals. At 1 month, ectopic hypertrophy, proliferation, and apoptosis of articular chondrocytes were seen in the articular cartilage of ESET-null animals. At 3 months, additional signs of terminal differentiation such as increased alkaline phosphatase activity and an elevated level of matrix metalloproteinase (MMP)-13 were found in ESET-null cartilage. Staining for type II collagen and proteoglycan revealed that cartilage degeneration became progressively worse from 2 weeks to 12 months at the knee joints of ESET knock-out mutants. Analysis of over 14 pairs of age- and sex-matched wild-type and knock-out mice indicated that the articular chondrocyte phenotype in ESET-null mutants is 100% penetrant. Our results demonstrate that expression of ESET plays an essential role in the maintenance of articular cartilage by preventing articular chondrocytes from terminal differentiation and may have implications in joint diseases such as osteoarthritis.

Novel Therapeutic Targets to Inhibit Tumor Microenvironment Induced Castration-Resistant Prostate Cancer

DTIC Science & Technology

2017-12-01

LNCaP 13 cells with high concentration of Dox led to cell death). We have also tried to use the CRISPR /Cas9 gene editing technology to generate...tried to use the CRISPR /Cas9 gene editing technology to generate MAPK4 knockout LNCaP cells; however, LNCaP cells are in general hard to be cultured at

Genome Editing of Monkey.

PubMed

Liu, Zhen; Cai, Yijun; Sun, Qiang

2017-01-01

Gene-modified monkey models would be particularly valuable in biomedical and neuroscience research. Virus-based transgenic and programmable nucleases-based site-specific gene editing methods (TALEN, CRISPR-cas9) enable the generation of gene-modified monkeys with gain or loss of function of specific genes. Here, we describe the generation of transgenic and knock-out (KO) monkeys with high efficiency by lentivirus and programmable nucleases.

Generation and characterization of a human oral squamous carcinoma cell line SCC-9 with CRISPR/Cas9-mediated deletion of the p75 neurotrophin receptor.

PubMed

Huang, Ping; Tong, Dongdong; Sun, Jing; Li, Qing; Zhang, Fenghe

2017-10-01

To investigate the importance of the p75 neurotrophin receptor (p75 NTR ) in human tongue squamous carcinoma cells, we exploited the CRISPR/Cas9 technology to establish a p75 NTR -knockout SCC-9 cell line and to explore the effect on biological functions. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas9) system was used to generate genomic deletion mutants of p75 NTR in the tongue squamous carcinoma cell lines SCC-9. Single-guide RNA (sgRNA) sequences were designed to target the p75 NTR genomic sequence and were cloned into plasmid pGK1.1. The linearized vector was electroporated into SCC-9 cells and p75 NTR deletion was confirmed using Cruiser™ enzyme digestion and PCR amplification. SCC-9 clones with successful deletion of p75 NTR were identified and verified by sequencing and selected for functional testing in cell proliferation, invasion, migration, and colony-forming assays. Compared with control cells, p75 NTR -knockout SCC-9 cells showed significantly diminished abilities to proliferate, invade, migrate, and form colonies, indicating a reduction in pro-tumorigenic behavior. These data demonstrate, first, that the CRISPR/Cas9 system is a simplified method for generating p75 NTR knockouts with relatively high efficiency, and second, that deletion of p75 NTR suppresses several tumor-promoting properties of SCC-9 cells, suggesting that p75 NTR is a potential target for the development of novel therapies for tongue cancer. Copyright © 2017 Elsevier Ltd. All rights reserved.

Generation of α1,3-galactosyltransferase and cytidine monophospho-N-acetylneuraminic acid hydroxylase gene double-knockout pigs

PubMed Central

MIYAGAWA, Shuji; MATSUNARI, Hitomi; WATANABE, Masahito; NAKANO, Kazuaki; UMEYAMA, Kazuhiro; SAKAI, Rieko; TAKAYANAGI, Shuko; TAKEISHI, Toki; FUKUDA, Tooru; YASHIMA, Sayaka; MAEDA, Akira; EGUCHI, Hiroshi; OKUYAMA, Hiroomi; NAGAYA, Masaki; NAGASHIMA, Hiroshi

2015-01-01

Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are new tools for producing gene knockout (KO) animals. The current study reports produced genetically modified pigs, in which two endogenous genes were knocked out. Porcine fibroblast cell lines were derived from homozygous α1,3-galactosyltransferase (GalT) KO pigs. These cells were subjected to an additional KO for the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene. A pair of ZFN-encoding mRNAs targeting exon 8 of the CMAH gene was used to generate the heterozygous CMAH KO cells, from which cloned pigs were produced by somatic cell nuclear transfer (SCNT). One of the cloned pigs obtained was re-cloned after additional KO of the remaining CMAH allele using the same ZFN-encoding mRNAs to generate GalT/CMAH-double homozygous KO pigs. On the other hand, the use of TALEN-encoding mRNAs targeting exon 7 of the CMAH gene resulted in efficient generation of homozygous CMAH KO cells. These cells were used for SCNT to produce cloned pigs homozygous for a double GalT/CMAH KO. These results demonstrate that the combination of TALEN-encoding mRNA, in vitro selection of the nuclear donor cells and SCNT provides a robust method for generating KO pigs. PMID:26227017

Generation of α1,3-galactosyltransferase and cytidine monophospho-N-acetylneuraminic acid hydroxylase gene double-knockout pigs.

PubMed

Miyagawa, Shuji; Matsunari, Hitomi; Watanabe, Masahito; Nakano, Kazuaki; Umeyama, Kazuhiro; Sakai, Rieko; Takayanagi, Shuko; Takeishi, Toki; Fukuda, Tooru; Yashima, Sayaka; Maeda, Akira; Eguchi, Hiroshi; Okuyama, Hiroomi; Nagaya, Masaki; Nagashima, Hiroshi

2015-01-01

Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are new tools for producing gene knockout (KO) animals. The current study reports produced genetically modified pigs, in which two endogenous genes were knocked out. Porcine fibroblast cell lines were derived from homozygous α1,3-galactosyltransferase (GalT) KO pigs. These cells were subjected to an additional KO for the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene. A pair of ZFN-encoding mRNAs targeting exon 8 of the CMAH gene was used to generate the heterozygous CMAH KO cells, from which cloned pigs were produced by somatic cell nuclear transfer (SCNT). One of the cloned pigs obtained was re-cloned after additional KO of the remaining CMAH allele using the same ZFN-encoding mRNAs to generate GalT/CMAH-double homozygous KO pigs. On the other hand, the use of TALEN-encoding mRNAs targeting exon 7 of the CMAH gene resulted in efficient generation of homozygous CMAH KO cells. These cells were used for SCNT to produce cloned pigs homozygous for a double GalT/CMAH KO. These results demonstrate that the combination of TALEN-encoding mRNA, in vitro selection of the nuclear donor cells and SCNT provides a robust method for generating KO pigs.

Generation and evaluation of Myostatin knock-out rabbits and goats using CRISPR/Cas9 system

PubMed Central

Guo, Rihong; Wan, Yongjie; Xu, Dan; Cui, Libin; Deng, Mingtian; Zhang, Guomin; Jia, Ruoxin; Zhou, Wenjun; Wang, Zhen; Deng, Kaiping; Huang, Mingrui; Wang, Feng; Zhang, Yanli

2016-01-01

Myostatin (Mstn) is a conserved negative regulator of skeletal muscle mass in mammals. However, whether precise disruption of Mstn in livestock can be achieved and safely used to improve meat productivity has not been proven. We applied CRISPR/Cas9 system to generate Mstn knock-out (KO) rabbits and goats and then analyzed the changes in their phenotypes to answer this question. We efficiently generated 24 Mstn KO rabbits out of 32 newborn infants after embryo injection with two sgRNAs targeting rabbit Mstn, and found that the Mstn KO rabbits exhibited increased birthweight and a significantly increase in the weight ratios of the quadriceps and biceps muscles to the whole body. Mstn KO also caused high probability of enlarged tongue phenomenon and severe health problems such as stillbirth and early stage death. Using the same method, one out of four goats was generated with edition at Mstn locus. The early stage growth rate of this goat outperformed the control goats. In conclusion, we efficiently generated Mstn KO rabbits and goats using CRISPR/Cas9 technology. However, Mstn KO causes severe health problems and may also have the same effects on other species. This safety issue must be studied further before applied to animal reproduction processes. PMID:27417210

Loss of Pink1 modulates synaptic mitochondrial bioenergetics in the rat striatum prior to motor symptoms: concomitant complex I respiratory defects and increased complex II-mediated respiration.

PubMed

Stauch, Kelly L; Villeneuve, Lance M; Purnell, Phillip R; Ottemann, Brendan M; Emanuel, Katy; Fox, Howard S

2016-12-01

Mutations in PTEN-induced putative kinase 1 (Pink1), a mitochondrial serine/threonine kinase, cause a recessive inherited form of Parkinson's disease (PD). Pink1 deletion in rats results in a progressive PD-like phenotype, characterized by significant motor deficits starting at 4 months of age. Despite the evidence of mitochondrial dysfunction, the pathogenic mechanism underlying disease due to Pink1-deficiency remains obscure. Striatal synaptic mitochondria from 3-month-old Pink1-deficient rats were characterized using bioenergetic and mass spectroscopy (MS)-based proteomic analyses. Striatal synaptic mitochondria from Pink1-deficient rats exhibit decreased complex I-driven respiration and increased complex II-mediated respiration compared with wild-type rats. MS-based proteomics revealed 69 of the 811 quantified mitochondrial proteins were differentially expressed between Pink1-deficient rats and controls. Down-regulation of several electron carrier proteins, which shuttle electrons to reduce ubiquinone at complex III, in the Pink1-knockouts suggests disruption of the linkage between fatty acid, amino acid, and choline metabolism and the mitochondrial respiratory system. These results suggest that complex II activity is increased to compensate for loss of electron transfer mechanisms due to reduced complex I activity and loss of electron carriers within striatal nerve terminals early during disease progression. This may contribute to the pathogenesis of PD. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effect of Maillard Reacted Peptides on Human Salt Taste and the Amiloride-Insensitive Salt Taste Receptor (TRPV1t)

PubMed Central

Katsumata, Tadayoshi; Nakakuki, Hiroko; Tokunaga, Chikara; Fujii, Noboru; Egi, Makoto; Phan, Tam-Hao T.; Mummalaneni, Shobha; DeSimone, John A.

2008-01-01

Maillard reacted peptides (MRPs) were synthesized by conjugating a peptide fraction (1000–5000 Da) purified from soy protein hydrolyzate with galacturonic acid, glucosamine, xylose, fructose, or glucose. The effect of MRPs was investigated on human salt taste and on the chorda tympani (CT) taste nerve responses to NaCl in Sprague–Dawley rats, wild-type, and transient receptor potential vanilloid 1 (TRPV1) knockout mice. MRPs produced a biphasic effect on human salt taste perception and on the CT responses in rats and wild-type mice in the presence of NaCl + benzamil (Bz, a blocker of epithelial Na+ channels), enhancing the NaCl response at low concentrations and suppressing it at high concentrations. The effectiveness of MRPs as salt taste enhancers varied with the conjugated sugar moiety: galacturonic acid = glucosamine > xylose > fructose > glucose. The concentrations at which MRPs enhanced human salt taste were significantly lower than the concentrations of MRPs that produced increase in the NaCl CT response. Elevated temperature, resiniferatoxin, capsaicin, and ethanol produced additive effects on the NaCl CT responses in the presence of MRPs. Elevated temperature and ethanol also enhanced human salt taste perception. N-(3-methoxyphenyl)-4-chlorocinnamid (a blocker of TRPV1t) inhibited the Bz-insensitive NaCl CT responses in the absence and presence of MRPs. TRPV1 knockout mice demonstrated no Bz-insensitive NaCl CT response in the absence or presence of MRPs. The results suggest that MRPs modulate human salt taste and the NaCl + Bz CT responses by interacting with TRPV1t. PMID:18603652

Opiate physical dependence and N-methyl-D-aspartate receptors.

PubMed

Noda, Yukihiro; Nabeshima, Toshitaka

2004-10-01

The present review focused the involvement of N-methyl-D-aspartate (NMDA) receptors in morphine physical dependence. The increased levels of extracellular glutamate, NMDA receptor zeta subunit (NR1) mRNA, NMDA receptor epsilon 1 subunit (NR2A) protein, phosphorylated Ca(2+)/calmodulin kinase II (p-CaMKII) protein, c-fos mRNA, c-Fos protein, are observed in the specific brain areas of mice and/or rats showing signs of naloxone-precipitated withdrawal. In preclinical and clinical studies, a variety of NMDA receptor antagonists and pretreatment with an antisense oligonucleotide of the NR1 have been reported to inhibit the development, expression and/or maintenance of opiate physical dependence. In contrast to data obtained in adult animals, NMDA receptor antagonists are neither effective in blocking the development of opiate dependence nor the expression of opiate withdrawal in neonatal rats. In the NMDA receptor-deficient mice, the NR2A knockout mice show the marked loss of typical withdrawal abstinence behaviors precipitated by naloxone. The rescue of NR2A protein by electroporation into the nucleus accumbens of NR2A knockout mice reverses the loss of abstinence behaviors. The activation of CaMKII and increased expression of c-Fos protein in the brain of animals with naloxone-precipitated withdrawal syndrome are prevented by NMDA receptor antagonists, whereas the increased levels of extracellular glutamate are not prevented by them. These findings indicate that glutamatergic neurotransmission at the NMDA receptor site contributes to the development, expression and maintenance of opiate dependence, and suggest that NMDA receptor antagonists may be a useful adjunct in the treatment of opiate dependence.

Iron Deficiency with or without Anemia Impairs Prepulse Inhibition of the Startle Reflex

PubMed Central

Pisansky, Marc T.; Wickham, Robert J.; Su, Jianjun; Fretham, Stephanie; Yuan, Li-Lian; Sun, Mu; Gewirtz, Jonathan C.; Georgieff, Michael K.

2013-01-01

Iron deficiency (ID) during early life causes long-lasting detrimental cognitive sequelae, many of which are linked to alterations in hippocampus function, dopamine synthesis, and the modulation of dopaminergic circuitry by the hippocampus. These same features have been implicated in the origins of schizophrenia, a neuropsychiatric disorder with significant cognitive impairments. Deficits in sensorimotor gating represent a reliable endophenotype of schizophrenia that can be measured by prepulse inhibition (PPI) of the acoustic startle reflex. Using two rodent model systems, we investigated the influence of early-life ID on PPI in adulthood. To isolate the role of hippocampal iron in PPI, our mouse model utilized a timed (embryonic day 18.5), hippocampus-specific knockout of Slc11a2, a gene coding an important regulator of cellular iron uptake, the divalent metal transport type 1 protein (DMT-1). Our second model used a classic rat dietary-based global ID during gestation, a condition that closely mimics human gestational ID anemia (IDA). Both models exhibited impaired PPI in adulthood. Furthermore, our DMT-1 knockout model displayed reduced long-term potentiation (LTP) and elevated paired pulse facilitation (PPF), electrophysiological results consistent with previous findings in the IDA rat model. These results, in combination with previous findings demonstrating impaired hippocampus functioning and altered dopaminergic and glutamatergic neurotransmission, suggest that iron availability within the hippocampus is critical for the neurodevelopmental processes underlying sensorimotor gating. Ultimately, evidence of reduced PPI in both of our models may offer insights into the roles of fetal ID and the hippocampus in the pathophysiology of schizophrenia. PMID:23733517

BRN 3.1 Knockouts Affect the Vestibular, Autonomic, and Circadian Rhythm Responses to 2G Exposure

NASA Technical Reports Server (NTRS)

Murakami, D. M.; Erkman, L.; Rosenfeld, M. G.; Fuller, C. A.

1999-01-01

Our previous studies have demonstrated that 2G exposure via centrifugation significantly attenuated the daily mean and circadian rhythm amplitude of rat body temperature (Tb), heart rate, and activity (Act). In addition, 2G exposure activates neural responses in several vestibular, autonomic, and circadian nuclei. Although we have characterized the effect of 2G on an animal's physiological, neuronal, and behavioral responses, it will be important to understand the underlying neural and physiological mechanisms that mediate those responses. For example, the vestibular responses, proprioceptive feedback, or fluid shifts may be the critical factors that mediate the responses to 2G. As a first step to understand the relative importance of these different response pathways to altered gravitational fields, this study examined the contribution of the vestibular system by utilizing an animal model from molecular biology. Brain 3.1 (Bm 3.1) is a POU domain homeobox gene involved in the normal development of the vestibular and auditory system. Brn 3.1 deletion results in a loss of hair cells in the otoliths, semicircular canals, and cochlea. As a result mice with a Brn 3.1 deletion do not have a functioning vestibular or auditory system. The BRN 3.1 knockout mouse could be a very useful animal model for isolating the role of the vestibular system in mediating the physiological responses to 2G exposure. Therefore, this study compared the effect of 2G exposure via centrifugation between Brn 3.1 knockout (KO) versus Wildtype (W) mice.

Induced pluripotent stem cells from goat fibroblasts.

PubMed

Song, Hui; Li, Hui; Huang, Mingrui; Xu, Dan; Gu, Chenghao; Wang, Ziyu; Dong, Fulu; Wang, Feng

2013-12-01

Embryonic stem cells (ESCs) are a powerful model for genetic engineering, studying developmental biology, and modeling disease. To date, ESCs have been established from the mouse (Evans and Kaufman, 1981, Nature 292:154-156), non-human primates (Thomson et al., , Proc Nat Acad Sci USA 92:7844-7848), humans (Thomson et al., 1998, Science 282:1145-1147), and rats (Buehr et al., , Cell 135:1287-1298); however, the derivation of ESCs from domesticated ungulates such as goats, sheep, cattle, and pigs have not been successful. Alternatively, induced pluripotent stem cells (iPSCs) can be generated by reprogramming somatic cells with several combinations of genes encoding transcription factors (OCT3/4, SOX2, KLF4, cMYC, LIN28, and NANOG). To date, iPSCs have been isolated from various species, but only limited information is available regarding goat iPSCs (Ren et al., 2011, Cell Res 21:849-853). The objectives of this study were to generate goat iPSCs from fetal goat primary ear fibroblasts using lentiviral transduction of four human transcription factors: OCT4, SOX2, KLF4, and cMYC. The goat iPSCs were successfully generated by co-culture with mitomycin C-treated mouse embryonic fibroblasts using medium supplemented with knockout serum replacement and human basic fibroblast growth factor. The goat iPSCs colonies are flat, compact, and closely resemble human iPSCs. They have a normal karyotype; stain positive for alkaline phosphatase, OCT4, and NANOG; express endogenous pluripotency genes (OCT4, SOX2, cMYC, and NANOG); and can spontaneously differentiate into three germ layers in vitro and in vivo. © 2013 Wiley Periodicals, Inc.

TRPM7 Is Required for Normal Synapse Density, Learning, and Memory at Different Developmental Stages.

PubMed

Liu, Yuqiang; Chen, Cui; Liu, Yunlong; Li, Wei; Wang, Zhihong; Sun, Qifeng; Zhou, Hang; Chen, Xiangjun; Yu, Yongchun; Wang, Yun; Abumaria, Nashat

2018-06-19

The TRPM7 chanzyme contributes to several biological and pathological processes in different tissues. However, its role in the CNS under physiological conditions remains unclear. Here, we show that TRPM7 knockdown in hippocampal neurons reduces structural synapse density. The synapse density is rescued by the α-kinase domain in the C terminus but not by the ion channel region of TRPM7 or by increasing extracellular concentrations of Mg 2+ or Zn 2+ . Early postnatal conditional knockout of TRPM7 in mice impairs learning and memory and reduces synapse density and plasticity. TRPM7 knockdown in the hippocampus of adult rats also impairs learning and memory and reduces synapse density and synaptic plasticity. In knockout mice, restoring expression of the α-kinase domain in the brain rescues synapse density/plasticity and memory, probably by interacting with and phosphorylating cofilin. These results suggest that brain TRPM7 is important for having normal synaptic and cognitive functions under physiological, non-pathological conditions. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Heightened avidity for trisodium pyrophosphate in mice lacking Tas1r3.

PubMed

Tordoff, Michael G; Aleman, Tiffany R; McCaughey, Stuart A

2015-01-01

Laboratory rats and mice prefer some concentrations of tri- and tetrasodium pyrophosphate (Na3HP2O7 and Na4P2O7) to water, but how they detect pyrophosphates is unknown. Here, we assessed whether T1R3 is involved. We found that relative to wild-type littermate controls, Tas1r3 knockout mice had stronger preferences for 5.6-56mM Na3HP2O7 in 2-bottle choice tests, and they licked more 17.8-56mM Na3HP2O7 in brief-access tests. We hypothesize that pyrophosphate taste in the intact mouse involves 2 receptors: T1R3 to produce a hedonically negative signal and an unknown G protein-coupled receptor to produce a hedonically positive signal; in Tas1r3 knockout mice, the hedonically negative signal produced by T1R3 is absent, leading to a heightened avidity for pyrophosphate. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

HDAC6 regulates thermogenesis of brown adipocytes through activating PKA to induce UCP1 expression.

PubMed

Jung, Suna; Han, Miae; Korm, Sovannarith; Lee, Se-In; Noh, Solhee; Phorl, Sophors; Naskar, Rema; Lee, Kye-Sung; Kim, Geon-Hee; Choi, Yun-Jaie; Lee, Joo Yong

2018-06-08

Mitochondrial uncoupling protein 1 (UCP1) is responsible for nonshivering thermogenesis in brown adipose tissue (BAT). UCP1 increases the conductance of the inner mitochondrial membrane (IMM) for protons to make BAT mitochondria generate heat rather than ATP. HDAC6 is a cytosolic deacetylase for non-histone substrates to regulate various cellular processes, including mitochondrial quality control and dynamics. Here, we showed that the body temperature of HDAC6 knockout mice is slightly decreased in normal hosing condition. Interestingly, UCP1 was downregulated in BAT of HDAC6 knockout mice, which extensively linked mitochondrial thermogenesis. Mechanistically, we showed that cAMP-PKA signaling plays a key role in HDAC6-dependent UCP1 expression. Notably, the size of brown adipocytes and lipid droplets in HDAC6 knockout BAT is increased. Taken together, our findings suggested that HDAC6 contributes to mitochondrial thermogenesis in BAT by increasing UCP1 expression through cAMP-PKA signaling pathway. Copyright © 2018. Published by Elsevier Inc.

Neuronal carbonic anhydrase VII provides GABAergic excitatory drive to exacerbate febrile seizures

PubMed Central

Ruusuvuori, Eva; Huebner, Antje K; Kirilkin, Ilya; Yukin, Alexey Y; Blaesse, Peter; Helmy, Mohamed; Jung Kang, Hyo; El Muayed, Malek; Christopher Hennings, J; Voipio, Juha; Šestan, Nenad; Hübner, Christian A; Kaila, Kai

2013-01-01

Brain carbonic anhydrases (CAs) are known to modulate neuronal signalling. Using a novel CA VII (Car7) knockout (KO) mouse as well as a CA II (Car2) KO and a CA II/VII double KO, we show that mature hippocampal pyramidal neurons are endowed with two cytosolic isoforms. CA VII is predominantly expressed by neurons starting around postnatal day 10 (P10). The ubiquitous isoform II is expressed in neurons at P20. Both isoforms enhance bicarbonate-driven GABAergic excitation during intense GABAA-receptor activation. P13–14 CA VII KO mice show behavioural manifestations atypical of experimental febrile seizures (eFS) and a complete absence of electrographic seizures. A low dose of diazepam promotes eFS in P13–P14 rat pups, whereas seizures are blocked at higher concentrations that suppress breathing. Thus, the respiratory alkalosis-dependent eFS are exacerbated by GABAergic excitation. We found that CA VII mRNA is expressed in the human cerebral cortex before the age when febrile seizures (FS) occur in children. Our data indicate that CA VII is a key molecule in age-dependent neuronal pH regulation with consequent effects on generation of FS. PMID:23881097

FANCA knockout in human embryonic stem cells causes a severe growth disadvantage.

PubMed

Vanuytsel, Kim; Cai, Qing; Nair, Nisha; Khurana, Satish; Shetty, Swati; Vermeesch, Joris R; Ordovas, Laura; Verfaillie, Catherine M

2014-09-01

Fanconi anemia (FA) is an autosomal recessive disorder characterized by progressive bone marrow failure (BMF) during childhood, aside from numerous congenital abnormalities. FA mouse models have been generated; however, they do not fully mimic the hematopoietic phenotype. As there is mounting evidence that the hematopoietic impairment starts already in utero, a human pluripotent stem cell model would constitute a more appropriate system to investigate the mechanisms underlying BMF in FA and its developmental basis. Using zinc finger nuclease (ZFN) technology, we have created a knockout of FANCA in human embryonic stem cells (hESC). We introduced a selection cassette into exon 2 thereby disrupting the FANCA coding sequence and found that whereas mono-allelically targeted cells retain an unaltered proliferation potential, disruption of the second allele causes a severe growth disadvantage. As a result, heterogeneous cultures arise due to the presence of cells still carrying an unaffected FANCA allele, quickly outgrowing the knockout cells. When pure cultures of FANCA knockout hESC are pursued either through selection or single cell cloning, this rapidly results in growth arrest and such cultures cannot be maintained. These data highlight the importance of a functional FA pathway at the pluripotent stem cell stage. Copyright © 2014. Published by Elsevier B.V.

CDK10 Mutations in Humans and Mice Cause Severe Growth Retardation, Spine Malformations, and Developmental Delays.

PubMed

Windpassinger, Christian; Piard, Juliette; Bonnard, Carine; Alfadhel, Majid; Lim, Shuhui; Bisteau, Xavier; Blouin, Stéphane; Ali, Nur'Ain B; Ng, Alvin Yu Jin; Lu, Hao; Tohari, Sumanty; Talib, S Zakiah A; van Hul, Noémi; Caldez, Matias J; Van Maldergem, Lionel; Yigit, Gökhan; Kayserili, Hülya; Youssef, Sameh A; Coppola, Vincenzo; de Bruin, Alain; Tessarollo, Lino; Choi, Hyungwon; Rupp, Verena; Roetzer, Katharina; Roschger, Paul; Klaushofer, Klaus; Altmüller, Janine; Roy, Sudipto; Venkatesh, Byrappa; Ganger, Rudolf; Grill, Franz; Ben Chehida, Farid; Wollnik, Bernd; Altunoglu, Umut; Al Kaissi, Ali; Reversade, Bruno; Kaldis, Philipp

2017-09-07

In five separate families, we identified nine individuals affected by a previously unidentified syndrome characterized by growth retardation, spine malformation, facial dysmorphisms, and developmental delays. Using homozygosity mapping, array CGH, and exome sequencing, we uncovered bi-allelic loss-of-function CDK10 mutations segregating with this disease. CDK10 is a protein kinase that partners with cyclin M to phosphorylate substrates such as ETS2 and PKN2 in order to modulate cellular growth. To validate and model the pathogenicity of these CDK10 germline mutations, we generated conditional-knockout mice. Homozygous Cdk10-knockout mice died postnatally with severe growth retardation, skeletal defects, and kidney and lung abnormalities, symptoms that partly resemble the disease's effect in humans. Fibroblasts derived from affected individuals and Cdk10-knockout mouse embryonic fibroblasts (MEFs) proliferated normally; however, Cdk10-knockout MEFs developed longer cilia. Comparative transcriptomic analysis of mutant and wild-type mouse organs revealed lipid metabolic changes consistent with growth impairment and altered ciliogenesis in the absence of CDK10. Our results document the CDK10 loss-of-function phenotype and point to a function for CDK10 in transducing signals received at the primary cilia to sustain embryonic and postnatal development. Copyright © 2017 American Society of Human Genetics. All rights reserved.

Retinoid-related orphan receptor γ (RORγ) adult induced knockout mice develop lymphoblastic lymphoma.

PubMed

Liljevald, Maria; Rehnberg, Maria; Söderberg, Magnus; Ramnegård, Marie; Börjesson, Jenny; Luciani, Donatella; Krutrök, Nina; Brändén, Lena; Johansson, Camilla; Xu, Xiufeng; Bjursell, Mikael; Sjögren, Anna-Karin; Hornberg, Jorrit; Andersson, Ulf; Keeling, David; Jirholt, Johan

2016-11-01

RORγ is a nuclear hormone receptor which controls polarization of naive CD4 + T-cells into proinflammatory Th17 cells. Pharmacological antagonism of RORγ has therapeutic potential for autoimmune diseases; however, this mechanism may potentially carry target-related safety risks, as mice deficient in Rorc, the gene encoding RORγ, develop T-cell lymphoma with 50% frequency. Due to the requirement of RORγ during development, the Rorc knockout (KO) animals lack secondary lymphoid organs and have a dysregulation in the generation of CD4+ and CD8+ T cells. We wanted to extend the evaluation of RORγ deficiency to address the question whether lymphomas, similar to those observed in the Rorc KO, would develop in an animal with an otherwise intact adult immune system. Accordingly, we designed a conditional RORγ knockout mouse (Rorc CKO) where the Rorc locus could be deleted in adult animals. Based on these studies we can confirm that these animals also develop lymphoma in a similar time frame as embryonic Rorc knockouts. This study also suggests that in animals where the gene deletion is incomplete, the thymus undergoes a rapid selection process replacing Rorc deficient cells with remnant thymocytes carrying a functional Rorc locus and that subsequently, these animals do not develop lymphoblastic lymphoma. Copyright © 2016 Elsevier B.V. All rights reserved.

Conditional knockout of retinal determination genes in differentiating cells in Drosophila.

PubMed

Jin, Meng; Eblimit, Aiden; Pulikkathara, Merlyn; Corr, Stuart; Chen, Rui; Mardon, Graeme

2016-08-01

Conditional gene knockout in postmitotic cells is a valuable technique which allows the study of gene function with spatiotemporal control. Surprisingly, in contrast to its long-term and extensive use in mouse studies, this technology is lacking in Drosophila. Here, we use a novel method for generating complete loss of eyes absent (eya) or sine oculis (so) function in postmitotic cells posterior to the morphogenetic furrow (MF). Specifically, genomic rescue constructs with flippase recognition target (FRT) sequences flanking essential exons are used to generate conditional null alleles. By removing gene function in differentiating cells, we show that eya and so are dispensable for larval photoreceptor differentiation, but are required for differentiation during pupal development. Both eya and so are necessary for photoreceptor survival and the apoptosis caused by loss of eya or so function is likely a secondary consequence of inappropriate differentiation. We also confirm their requirement for cone cell development and reveal a novel role in interommatidial bristle (IOB) formation. In addition, so is required for normal eye disc morphology. This is the first report of a knockout method to study eya and so function in postmitotic cells. This technology will open the door to a large array of new functional studies in virtually any tissue and at any stage of development or in adults. © 2016 Federation of European Biochemical Societies.

An efficient method for generation of bi-allelic null mutant mouse embryonic stem cells and its application for investigating epigenetic modifiers.

PubMed

Fisher, Cynthia L; Marks, Hendrik; Cho, Lily Ting-Yin; Andrews, Robert; Wormald, Sam; Carroll, Thomas; Iyer, Vivek; Tate, Peri; Rosen, Barry; Stunnenberg, Hendrik G; Fisher, Amanda G; Skarnes, William C

2017-12-01

Mouse embryonic stem (ES) cells are a popular model system to study biological processes, though uncovering recessive phenotypes requires inactivating both alleles. Building upon resources from the International Knockout Mouse Consortium (IKMC), we developed a targeting vector for second allele inactivation in conditional-ready IKMC 'knockout-first' ES cell lines. We applied our technology to several epigenetic regulators, recovering bi-allelic targeted clones with a high efficiency of 60% and used Flp recombinase to restore expression in two null cell lines to demonstrate how our system confirms causality through mutant phenotype reversion. We designed our strategy to select against re-targeting the 'knockout-first' allele and identify essential genes in ES cells, including the histone methyltransferase Setdb1. For confirmation, we exploited the flexibility of our system, enabling tamoxifen inducible conditional gene ablation while controlling for genetic background and tamoxifen effects. Setdb1 ablated ES cells exhibit severe growth inhibition, which is not rescued by exogenous Nanog expression or culturing in naive pluripotency '2i' media, suggesting that the self-renewal defect is mediated through pluripotency network independent pathways. Our strategy to generate null mutant mouse ES cells is applicable to thousands of genes and repurposes existing IKMC Intermediate Vectors. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

FAT/CD36: a major regulator of neuronal fatty acid sensing and energy homeostasis in rats and mice.

PubMed

Le Foll, Christelle; Dunn-Meynell, Ambrose; Musatov, Serguei; Magnan, Christophe; Levin, Barry E

2013-08-01

Hypothalamic "metabolic-sensing" neurons sense glucose and fatty acids (FAs) and play an integral role in the regulation of glucose, energy homeostasis, and the development of obesity and diabetes. Using pharmacologic agents, we previously found that ~50% of these neurons responded to oleic acid (OA) by using the FA translocator/receptor FAT/CD36 (CD36). For further elucidation of the role of CD36 in neuronal FA sensing, ventromedial hypothalamus (VMH) CD36 was depleted using adeno-associated viral (AAV) vector expressing CD36 short hairpin RNA (shRNA) in rats. Whereas their neuronal glucosensing was unaffected by CD36 depletion, the percent of neurons that responded to OA was decreased specifically in glucosensing neurons. A similar effect was seen in total-body CD36-knockout mice. Next, weanling rats were injected in the VMH with CD36 AAV shRNA. Despite significant VMH CD36 depletion, there was no effect on food intake, body weight gain, or total carcass adiposity on chow or 45% fat diets. However, VMH CD36-depleted rats did have increased plasma leptin and subcutaneous fat deposition and markedly abnormal glucose tolerance. These results demonstrate that CD36 is a critical factor in both VMH neuronal FA sensing and the regulation of energy and glucose homeostasis.

Different effects of oral administration of synthetic trypsin inhibitor on the pancreas between cholecystokinin-A receptor gene knockout mice and wild type mice.

PubMed

Sato, Norikazu; Suzuki, Shinji; Kanai, Setsuko; Ohta, Minoru; Jimi, Atsuo; Noda, Tetsuo; Takiguchi, Souichi; Funakoshi, Akihiro; Miyasaka, Kyoko

2002-07-01

The synthetic trypsin inhibitor camostat has been used for the treatment of acute and chronic pancreatitis in Japan based on the evidences obtained from a rat experimental model. However, rats differ from other rodents and from humans in terms of lacking a gallbladder and no response of pancreatic bicarbonate secretion to cholecystokinin (CCK). In the present study, we determined whether oral administration of camostat showed a trophic effect in mice as observed in rats and whether the trophic effect, if substantial, was mediated via the CCK-A receptor, using CCK-A receptor gene targeting mice. The chow containing 0.1% camostat was fed to 8-month-old mice. Three- and seven-day treatments with camostat did not affect pancreatic wet weight in CCK-A receptor (+/-) mice. After 14-day treatment, the ratio of pancreatic wet weight/body weight was significantly lower in CCK-A receptor (-/-) than (+/+) mice. The protein and chymotrypsin contents were lower and amylase content was higher in CCK-A receptor (-/-) mice, compared to (+/+) mice. No pathological findings were observed by histological examination. Camostat has a trophic effect on the pancreas in mice and this effect is mediated via the CCK-A receptor, but is less potent than in rats.

The role of nuclear factor E2-Related factor 2 and uncoupling protein 2 in glutathione metabolism: Evidence from an in vivo gene knockout study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Yanyan; The Hamner Institutes for Health Sciences, Research Triangle Park, NC; Xu, Yuanyuan, E-mail: yyxu@cmu.edu.cn

Nuclear factor E2-related factor 2 (NRF2) and uncoupling protein 2 (UCP2) are indicated to protect from oxidative stress. They also play roles in the homeostasis of glutathione. However, the detailed mechanisms are not well understood. In the present study, we found Nrf2-knockout (Nrf2-KO) mice exhibited altered glutathione homeostasis and reduced expression of various genes involved in GSH biosynthesis, regeneration, utilization and transport in the liver. Ucp2-knockout (Ucp2-KO) mice exhibited altered glutathione homeostasis in the liver, spleen and blood, as well as increased transcript of cystic fibrosis transmembrane conductance regulator in the liver, a protein capable of mediating glutathione efflux. Nrf2-Ucp2-doublemore » knockout (DKO) mice showed characteristics of both Nrf2-KO and Ucp2-KO mice. But no significant difference was observed in DKO mice when compared with Nrf2-KO or Ucp2-KO mice, except in blood glutathione levels. These data suggest that ablation of Nrf2 and Ucp2 leads to disrupted GSH balance, which could result from altered expression of genes involved in GSH metabolism. DKO may not evoke more severe oxidative stress than the single gene knockout. - Highlights: • Nrf2/Ucp2 deficiency leads to alteration of glutathione homeostasis. • Nrf2 regulates expression of genes in glutathione generation and utilization. • Ucp2 affects glutathione metabolism by regulating hepatic efflux of glutathione. • Nrf2 deficiency may not aggravate oxidative stress in Ucp2-deficient mice.« less

A specific, nonproliferative role for E2F-5 in choroid plexus function revealed by gene targeting

PubMed Central

Lindeman, Geoffrey J.; Dagnino, Lina; Gaubatz, Stefan; Xu, Yuhui; Bronson, Roderick T.; Warren, Henry B.; Livingston, David M.

1998-01-01

Homozygous E2F-5 knockout embryos and mice have been generated. Although embryonic development appeared normal, newborn mice developed nonobstructive hydrocephalus, suggesting excessive cerebrospinal fluid (CSF) production. Although the CSF-producing choroid plexus displayed normal cellular organization, it contained abundant electron-lucent epithelial cells, consistent with excessive CSF secretory activity. Moreover, E2F-5 CNS expression in normal animals was largely confined to the choroid plexus. Cell cycle kinetics were not perturbed in homozygous knockout embryo fibroblasts. Thus, E2F-5 is not essential for cell proliferation. Rather, it affects the secretory behavior of a differentiated neural tissue. PMID:9553039

Inositol Hexakisphosphate Kinase-3 Regulates the Morphology and Synapse Formation of Cerebellar Purkinje Cells via Spectrin/Adducin

PubMed Central

Fu, Chenglai; Xu, Jing; Li, Ruo-Jing; Crawford, Joshua A.; Khan, A. Basit; Ma, Ting Martin; Cha, Jiyoung Y.; Snowman, Adele M.; Pletnikov, Mikhail V.

2015-01-01

The inositol hexakisphosphate kinases (IP6Ks) are the principal enzymes that generate inositol pyrophosphates. There are three IP6Ks (IP6K1, 2, and 3). Functions of IP6K1 and IP6K2 have been substantially delineated, but little is known of IP6K3's role in normal physiology, especially in the brain. To elucidate functions of IP6K3, we generated mice with targeted deletion of IP6K3. We demonstrate that IP6K3 is highly concentrated in the brain in cerebellar Purkinje cells. IP6K3 physiologically binds to the cytoskeletal proteins adducin and spectrin, whose mutual interactions are perturbed in IP6K3-null mutants. Consequently, IP6K3 knock-out cerebella manifest abnormalities in Purkinje cell structure and synapse number, and the mutant mice display deficits in motor learning and coordination. Thus, IP6K3 is a major determinant of cytoskeletal disposition and function of cerebellar Purkinje cells. SIGNIFICANCE STATEMENT We identified and cloned a family of three inositol hexakisphosphate kinases (IP6Ks) that generate the inositol pyrophosphates, most notably 5-diphosphoinositol pentakisphosphate (IP7). Of these, IP6K3 has been least characterized. In the present study we generated IP6K3 knock-out mice and show that IP6K3 is highly expressed in cerebellar Purkinje cells. IP6K3-deleted mice display defects of motor learning and coordination. IP6K3-null mice manifest aberrations of Purkinje cells with a diminished number of synapses. IP6K3 interacts with the cytoskeletal proteins spectrin and adducin whose altered disposition in IP6K3 knock-out mice may mediate phenotypic features of the mutant mice. These findings afford molecular/cytoskeletal mechanisms by which the inositol polyphosphate system impacts brain function. PMID:26245967

Macrophage migration inhibitory factor (MIF) knockout preserves cardiac homeostasis through alleviating Akt-mediated myocardial autophagy suppression in high-fat diet-induced obesity.

PubMed

Xu, X; Ren, J

2015-03-01

Macrophage migration inhibitory factor (MIF) has a role in the development of obesity and diabetes. However, whether MIF has a role in fat diet-induced obesity and associated cardiac anomalies still remains unknown. The aim of this study was to examine the impact of MIF knockout on high-fat diet-induced obesity, obesity-associated cardiac anomalies and the underlying mechanisms involved with a focus on Akt-mediated autophagy. Adult male wild-type (WT) and MIF knockout (MIF(-/-)) mice were placed on 45% high-fat diet for 5 months. Oxygen consumption, CO2 production, respiratory exchange ratio, locomotor activity and heat generation were measured using energy calorimeter. Echocardiographic, cardiomyocyte mechanical and intracellular Ca2+ properties were assessed. Apoptosis was examined using terminal dUTP nick end labeling staining and western blot analysis. Akt signaling pathway and autophagy markers were evaluated. Cardiomyocytes isolated from WT and MIF(-/-) mice were treated with recombinant mouse MIF (rmMIF). High-fat diet feeding elicited increased body weight gain, insulin resistance and caloric disturbance in WT and MIF(-/-) mice. High-fat diet induced unfavorable geometric, contractile and histological changes in the heart, the effects of which were alleviated by MIF knockout. In addition, fat diet-induced cardiac anomalies were associated with Akt activation and autophagy suppression, which were nullified by MIF deficiency. In cardiomyocytes from WT mice, autophagy was inhibited by exogenous rmMIF through Akt activation. In addition, MIF knockout rescued palmitic acid-induced suppression of cardiomyocyte autophagy, the effect of which was nullified by rmMIF. These results indicate that MIF knockout preserved obesity-associated cardiac anomalies without affecting fat diet-induced obesity, probably through restoring myocardial autophagy in an Akt-dependent manner. Our findings provide new insights for the role of MIF in obesity and associated cardiac anomalies.

Thymosin Beta-4 Induces Mouse Hair Growth

PubMed Central

Hou, Fang; Zhang, Zhipeng; Nuo, Mingtu; Guo, Xudong; Liu, Dongjun

2015-01-01

Thymosin beta-4 (Tβ4) is known to induce hair growth and hair follicle (HF) development; however, its mechanism of action is unknown. We generated mice that overexpressed Tβ4 in the epidermis, as well as Tβ4 global knockout mice, to study the role of Tβ4 in HF development and explore the mechanism of Tβ4 on hair growth. To study Tβ4 function, we depilated control and experimental mice and made tissue sections stained with hematoxylin and eosin (H&E). To explore the effect of Tβ4 on hair growth and HF development, the mRNA and protein levels of Tβ4 and VEGF were detected by real-time PCR and western blotting in control and experimental mice. Protein expression levels and the phosphorylation of P38, ERK and AKT were also examined by western blotting. The results of depilation indicated that hair re-growth was faster in Tβ4-overexpressing mice, but slower in knockout mice. Histological examination revealed that Tβ4-overexpressing mice had a higher number of hair shafts and HFs clustered together to form groups, while the HFs of control mice and knockout mice were separate. Hair shafts in knockout mice were significantly reduced in number compared with control mice. Increased Tβ4 expression at the mRNA and protein levels was confirmed in Tβ4-overexpressing mice, which also had increased VEGF expression. On the other hand, knockout mice had reduced levels of VEGF expression. Mechanistically, Tβ4-overexpressing mice showed increased protein expression levels and phosphorylation of P38, ERK and AKT, whereas knockout mice had decreased levels of both expression and phosphorylation of these proteins. Tβ4 appears to regulate P38/ERK/AKT signaling via its effect on VEGF expression, with a resultant effect on the speed of hair growth, the pattern of HFs and the number of hair shafts. PMID:26083021

Thymosin Beta-4 Induces Mouse Hair Growth.

PubMed

Gao, Xiaoyu; Liang, Hao; Hou, Fang; Zhang, Zhipeng; Nuo, Mingtu; Guo, Xudong; Liu, Dongjun

2015-01-01

Thymosin beta-4 (Tβ4) is known to induce hair growth and hair follicle (HF) development; however, its mechanism of action is unknown. We generated mice that overexpressed Tβ4 in the epidermis, as well as Tβ4 global knockout mice, to study the role of Tβ4 in HF development and explore the mechanism of Tβ4 on hair growth. To study Tβ4 function, we depilated control and experimental mice and made tissue sections stained with hematoxylin and eosin (H&E). To explore the effect of Tβ4 on hair growth and HF development, the mRNA and protein levels of Tβ4 and VEGF were detected by real-time PCR and western blotting in control and experimental mice. Protein expression levels and the phosphorylation of P38, ERK and AKT were also examined by western blotting. The results of depilation indicated that hair re-growth was faster in Tβ4-overexpressing mice, but slower in knockout mice. Histological examination revealed that Tβ4-overexpressing mice had a higher number of hair shafts and HFs clustered together to form groups, while the HFs of control mice and knockout mice were separate. Hair shafts in knockout mice were significantly reduced in number compared with control mice. Increased Tβ4 expression at the mRNA and protein levels was confirmed in Tβ4-overexpressing mice, which also had increased VEGF expression. On the other hand, knockout mice had reduced levels of VEGF expression. Mechanistically, Tβ4-overexpressing mice showed increased protein expression levels and phosphorylation of P38, ERK and AKT, whereas knockout mice had decreased levels of both expression and phosphorylation of these proteins. Tβ4 appears to regulate P38/ERK/AKT signaling via its effect on VEGF expression, with a resultant effect on the speed of hair growth, the pattern of HFs and the number of hair shafts.

A comparative evaluation of the tissue responses associated with polymeric implants in the rat and mouse.

PubMed

Kidd, Kameha R; Dal Ponte, Donny B; Kellar, Robert S; Williams, Stuart K

2002-03-15

End product application is an important consideration when evaluating a material in an in vivo setting (Didisheim, Cardiovasc Pathol 1993;2:1S-2S). Small animal models allow high through-put evaluation of biocompatability. Previous preclinical evaluations have often used a rat subcutaneous model for the characterization of material-tissue interaction. Recent advances in genetic manipulation have provided mouse models with selective expression of a wide range of critical proteins. The rat model does not have many of the resources (i.e., knockouts, SCID, nude) that are present in mouse strains. The availability of these mice provides a resource to delineate the mechanisms regulating the healing associated with implants. However, before the mouse models can be used, they must be validated with respect to their ability to accurately assess tissue responses to materials. In this study the tissue responses after the implantation of expanded polytetrafluoroethylene (ePTFE) were compared between rat and mouse. Discs of ePTFE (30-microm internodal distance) were implanted in subcutaneous and epididymal fat tissue of rats (Sprague-Dawley) and mice (129-SVJ). After 5 weeks the samples were removed and evaluated for vascular density, inflammation, and fibrous encapsulation. No difference in the vessel density was observed within the peri-implant subcutaneous and adipose tissue or within the porous material. However, a significant difference was found in the number of activated macrophages and giant cells between these two species. Implants in the rat exhibited greater numbers of activated inflammatory cells in the peri-implant tissue. The data indicate that the mouse and rat provide a comparable model for evaluating angiogenesis and neovascularization associated with synthetic porous implants. Copyright 2001 John Wiley & Sons, Inc. J Biomed Mater Res 59: 682-689, 2002

Targeting TMPRSS2-ERG in Prostate Cancer

DTIC Science & Technology

2017-11-01

phosphorylated proteins in ERG positive versus negative cell lines following suppression of kinases by shRNA or knockout using CRISPR /Cas9...the cofactor is below detection by silver staining. Future experiments are aimed at generating a stable cell line using CRISPR /Cas9 that has

Conditional Allele Mouse Planner (CAMP): software to facilitate the planning and design of breeding strategies involving mice with conditional alleles.

PubMed

Hoffert, Jason D; Pisitkun, Trairak; Miller, R Lance

2012-06-01

Transgenic and conditional knockout mouse models play an important role in biomedical research and their use has grown exponentially in the last 5-10 years. Generating conditional knockouts often requires breeding multiple alleles onto the background of a single mouse or group of mice. Breeding these mice depends on parental genotype, litter size, transmission frequency, and the number of breeding rounds. Therefore, a well planned breeding strategy is critical for keeping costs to a minimum. However, designing a viable breeding strategy can be challenging. With so many different variables this would be an ideal task for a computer program. To facilitate this process, we created a Java-based program called Conditional Allele Mouse Planner (CAMP). CAMP is designed to provide an estimate of the number of breeders, amount of time, and costs associated with generating mice of a particular genotype. We provide a description of CAMP, how to use it, and offer it freely as an application.

Evaluation of intestinal absorption of amtolmetin guacyl in rats: breast cancer resistant protein as a primary barrier of oral bioavailability.

PubMed

Rong, Zhihui; Xu, Yanjiao; Zhang, Chengliang; Xiang, Daochun; Li, Xiping; Liu, Dong

2013-02-27

The purpose of the present study was to investigate the role of efflux transporters on the intestinal absorption of amtolmetin guacyl (MED-15). The effects of P-glycoprotein (P-gp), multiple resistance-associated protein 2 (MRP2), and breast cancer resistance protein (BCRP) inhibitors on intestinal absorption amount of MED-5 (tolmetin-glycine amide derivative), the metabolite formed from MED-15 in the intestinal epithelial cells were studied in the in vitro everted gut sac experiments. Moreover, the in situ single-pass intestine perfusion was adopted to clarify the role of efflux transporters in excreting MED-5 in knockout mice. The plasma concentration of MED-5 and tolmetin, the metabolite formed from MED-5 was determined in Bcrp1 knockout mice and wild-type mice. BCRP inhibitor Ko143 (50 μM and 100 μM) significantly increased the intestinal absorption amount in jejunum, ileum and colon (p<0.05). However, no effect was observed in the presence of P-gp inhibitor verapamil and MRP2 inhibitor MK571 in each intestinal segment. Furthermore, the plasma concentration MED-5 and tolmetin, metabolites of MED-15, increased 2-fold and 4-fold, respectively, in Bcrp1 knockout mice compared with wild-type mice after the single-pass perfusion of small intestine with MED-15. It may be concluded that BCRP plays an important role in the intestinal efflux of MED-5 and limits the bioavailability after oral administration of MED-15. Copyright © 2013. Published by Elsevier Inc.

Diacylglycerol acyltransferase-1 inhibition enhances intestinal fatty acid oxidation and reduces energy intake in rats[S

PubMed Central

Schober, Gudrun; Arnold, Myrtha; Birtles, Susan; Buckett, Linda K.; Pacheco-López, Gustavo; Turnbull, Andrew V.; Langhans, Wolfgang; Mansouri, Abdelhak

2013-01-01

Acyl CoA:diacylglycerol acyltransferase-1 (DGAT-1) catalyzes the final step in triacylglycerol (TAG) synthesis and is highly expressed in the small intestine. Because DGAT-1 knockout mice are resistant to diet-induced obesity, we investigated the acute effects of intragastric (IG) infusion of a small molecule diacylglycerol acyltransferase-1 inhibitor (DGAT-1i) on eating, circulating fat metabolites, indirect calorimetry, and hepatic and intestinal expression of key fat catabolism enzymes in male rats adapted to an 8 h feeding-16 h deprivation schedule. Also, the DGAT-1i effect on fatty acid oxidation (FAO) was investigated in enterocyte cell culture models. IG DGAT-1i infusions reduced energy intake compared with vehicle in high-fat diet (HFD)-fed rats, but scarcely in chow-fed rats. IG DGAT-1i also blunted the postprandial increase in serum TAG and increased β-hydroxybutyrate levels only in HFD-fed rats, in which it lowered the respiratory quotient and increased intestinal, but not hepatic, protein levels of Complex III of the mitochondrial respiratory chain and of mitochondrial hydroxymethylglutaryl-CoA synthase. Finally, the DGAT-1i enhanced FAO in CaCo2 (EC50 = 0.3494) and HuTu80 (EC50 = 0.00762) cells. Thus, pharmacological DGAT-1 inhibition leads to an increase in intestinal FAO and ketogenesis when dietary fat is available. This may contribute to the observed eating-inhibitory effect. PMID:23449193

The impact of galectin-3 inhibition on aldosterone-induced cardiac and renal injuries.

PubMed

Calvier, Laurent; Martinez-Martinez, Ernesto; Miana, Maria; Cachofeiro, Victoria; Rousseau, Elodie; Sádaba, J Rafael; Zannad, Faiez; Rossignol, Patrick; López-Andrés, Natalia

2015-01-01

This study investigated whether galectin (Gal)-3 inhibition could block aldosterone-induced cardiac and renal fibrosis and improve cardiorenal dysfunction. Aldosterone is involved in cardiac and renal fibrosis that is associated with the development of cardiorenal injury. However, the mechanisms of these interactions remain unclear. Gal-3, a β-galactoside-binding lectin, is increased in heart failure and kidney injury. Rats were treated with aldosterone-salt combined with spironolactone (a mineralocorticoid receptor antagonist) or modified citrus pectin (a Gal-3 inhibitor), for 3 weeks. Wild-type and Gal-3 knockout mice were treated with aldosterone for 3 weeks. Hemodynamic, cardiac, and renal parameters were analyzed. Hypertensive aldosterone-salt-treated rats presented cardiac and renal hypertrophy (at morphometric, cellular, and molecular levels) and dysfunction. Cardiac and renal expressions of Gal-3 as well as levels of molecular markers attesting fibrosis were also augmented by aldosterone-salt treatment. Spironolactone or modified citrus pectin treatment reversed all of these effects. In wild-type mice, aldosterone did not alter blood pressure levels but increased cardiac and renal Gal-3 expression, fibrosis, and renal epithelial-mesenchymal transition. Gal-3 knockout mice were resistant to aldosterone effects. In experimental hyperaldosteronism, the increase in Gal-3 expression was associated with cardiac and renal fibrosis and dysfunction but was prevented by pharmacological inhibition (modified citrus pectin) or genetic disruption of Gal-3. These data suggest a key role for Gal-3 in cardiorenal remodeling and dysfunction induced by aldosterone. Gal-3 could be used as a new biotarget for specific pharmacological interventions. Copyright © 2015 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

Pharmacokinetics of dietary cancer chemopreventive compound dibenzoylmethane in rats and the impact of nanoemulsion and genetic knockout of Nrf2 on its disposition.

PubMed

Lin, Wen; Hong, Jin-Liern; Shen, Guoxiang; Wu, Rachel T; Wang, Yuwen; Huang, Mou-Tuan; Newmark, Harold L; Huang, Qingrong; Khor, Tin Oo; Heimbach, Tycho; Kong, Ah-Ng

2011-03-01

The pharmacokinetic disposition of a dietary cancer chemopreventive compound dibenzoylmethane (DBM) was studied in male Sprague-Dawley rats after intravenous (i.v.) and oral (p.o.) administrations. Following a single i.v. bolus dose, the mean plasma clearance (CL) of DBM was low compared with the hepatic blood flow. DBM displayed a high volume of distribution (Vss). The elimination terminal t1/2 was long. The mean CL, Vss and AUC0-∞/dose were similar between the i.v. 10 and 10 mg/kg doses. After single oral doses (10, 50 and 250 mg/kg), the absolute oral bioavailability (F*) of DBM was 7.4%-13.6%. The increase in AUC was not proportional to the oral doses, suggesting non-linearity. In silico prediction of oral absorption also demonstrated low DBM absorption in vivo. An oil-in-water nanoemulsion containing DBM was formulated to potentially overcome the low F* due to poor water solubility of DBM, with enhanced oral absorption. Finally, to examine the role of Nrf2 on the pharmacokinetics of DBM, since DBM activates the Nrf2-dependent detoxification pathways, Nrf2 wild-type (+/+) mice and Nrf2 knockout (-/-) mice were utilized. There was an increased systemic plasma exposure of DBM in Nrf2 (-/-) mice, suggesting that the Nrf2 genotype could also play a role in the pharmacokinetic disposition of DBM. Taken together, the results show that DBM has low oral bioavailability which could be due in part to poor water solubility and this could be overcome by a nanotechnology-based drug delivery system and furthermore the Nrf2 genotype could also play a role in the pharmacokinetics of DBM. Copyright © 2010 John Wiley & Sons, Ltd.

Bladder pain induced by prolonged peripheral alpha 1A adrenoceptor stimulation involves the enhancement of transient receptor potential vanilloid 1 activity and an increase of urothelial adenosine triphosphate release.

PubMed

Matos, R; Cordeiro, J M; Coelho, A; Ferreira, S; Silva, C; Igawa, Y; Cruz, F; Charrua, A

2016-12-01

Pathophysiological mechanisms of chronic visceral pain (CVP) are unknown. This study explores the association between the sympathetic system and bladder nociceptors activity by testing the effect of a prolonged adrenergic stimulation on transient receptor potential vanilloid 1 (TRPV1) activity and on urothelial adenosine triphosphate (ATP) release. Female Wistar rats received saline, phenylephrine (PHE), PHE + silodosin, PHE + naftopidil or PHE + prazosin. TRPV1 knockout and wild-type mice received saline or PHE. Visceral pain behaviour tests were performed before and after treatment. Cystometry was performed, during saline and capsaicin infusion. Fos immunoreactivity was assessed in L6 spinal cord segment. Human urothelial ATP release induced by mechanical and thermal stimulation was evaluated. Subcutaneous, but not intrathecal, PHE administration induced pain, which was reversed by silodosin, a selective alpha 1A adrenoceptor antagonist, but not by naftopidil, a relatively selective antagonist for alpha 1D adrenoceptor. Silodosin also reversed PHE-induced bladder hyperactivity and L6 spinal cord Fos expression. Thus, in subsequent experiments, only silodosin was used. Wild-type, but not TRPV1 knockout, mice exhibited phenylephrine-induced pain. Capsaicin induced a greater increase in voiding contractions in PHE-treated rats than in control animals, and silodosin reversed this effect. When treated with PHE, ATP release from human urothelial cells was enhanced either by mechanical stimulation or by lowering the thermal threshold of urothelial TRPV1, which becomes abnormally responsive at body temperature. This study suggests that the activation of peripheral alpha 1A adrenoceptors induces CVP, probably through its interaction with TRPV1 and ATP release. © 2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

Genetic inactivation of prohormone convertase (PC1) causes a reduction in cholecystokinin (CCK) levels in the hippocampus, amygdala, pons and medulla in mouse brain that correlates with the degree of colocalization of PC1 and CCK mRNA in these structures in rat brain.

PubMed

Cain, B M; Connolly, K; Blum, A C; Vishnuvardhan, D; Marchand, J E; Zhu, X; Steiner, D F; Beinfeld, M C

2004-04-01

Prohormone convertase (PC1) is found in endocrine cell lines that express cholecystokinin (CCK) mRNA and process pro CCK to biologically active products. Other studies have demonstrated that PC1 may be a one of the enzymes responsible for the endoproteolytic cleavages that occur in pro CCK during its biosynthesis and processing. Prohormone convertase 1 (PC1) has a distribution that is similar to cholecystokinin (CCK) in rat brain. A moderate to high percentage of CCK mRNA-positive neurons express PC1 mRNA. CCK levels were measured in PC1 knockout and control mice to assess the degree to which loss of PC1 changed CCK content. CCK levels were decreased 62% in hippocampus, 53% in amygdala and 57% in pons-medulla in PC1 knockout mice as compared to controls. These results are highly correlated with the colocalization of CCK and PC1. The majority of CCK mRNA-positive neurons in the pyramidal cell layer of the hippocampus express PC1 mRNA and greater than 50% of CCK mRNA-positive neurons in several nuclei of the amygdala also express PC1. These results demonstrate that PC1 is important for CCK processing. PC2 and PC5 are also widely colocalized with CCK. It may be that PC2, PC5 or another non-PC enzyme are able to substitute for PC1 and sustain production of some amidated CCK. Together these enzymes may represent a redundant system to insure the production of CCK.

Titanium biomaterials with complex surfaces induced aberrant peripheral circadian rhythms in bone marrow mesenchymal stromal cells.

PubMed

Hassan, Nathaniel; McCarville, Kirstin; Morinaga, Kenzo; Mengatto, Cristiane M; Langfelder, Peter; Hokugo, Akishige; Tahara, Yu; Colwell, Christopher S; Nishimura, Ichiro

2017-01-01

Circadian rhythms maintain a high level of homeostasis through internal feed-forward and -backward regulation by core molecules. In this study, we report the highly unusual peripheral circadian rhythm of bone marrow mesenchymal stromal cells (BMSCs) induced by titanium-based biomaterials with complex surface modifications (Ti biomaterial) commonly used for dental and orthopedic implants. When cultured on Ti biomaterials, human BMSCs suppressed circadian PER1 expression patterns, while NPAS2 was uniquely upregulated. The Ti biomaterials, which reduced Per1 expression and upregulated Npas2, were further examined with BMSCs harvested from Per1::luc transgenic rats. Next, we addressed the regulatory relationship between Per1 and Npas2 using BMSCs from Npas2 knockout mice. The Npas2 knockout mutation did not rescue the Ti biomaterial-induced Per1 suppression and did not affect Per2, Per3, Bmal1 and Clock expression, suggesting that the Ti biomaterial-induced Npas2 overexpression was likely an independent phenomenon. Previously, vitamin D deficiency was reported to interfere with Ti biomaterial osseointegration. The present study demonstrated that vitamin D supplementation significantly increased Per1::luc expression in BMSCs, though the presence of Ti biomaterials only moderately affected the suppressed Per1::luc expression. Available in vivo microarray data from femurs exposed to Ti biomaterials in vitamin D-deficient rats were evaluated by weighted gene co-expression network analysis. A large co-expression network containing Npas2, Bmal1, and Vdr was observed to form with the Ti biomaterials, which was disintegrated by vitamin D deficiency. Thus, the aberrant BMSC peripheral circadian rhythm may be essential for the integration of Ti biomaterials into bone.

A Novel Nociceptin Receptor Antagonist LY2940094 Inhibits Excessive Feeding Behavior in Rodents: A Possible Mechanism for the Treatment of Binge Eating Disorder.

PubMed

Statnick, Michael A; Chen, Yanyun; Ansonoff, Michael; Witkin, Jeffrey M; Rorick-Kehn, Linda; Suter, Todd M; Song, Min; Hu, Charlie; Lafuente, Celia; Jiménez, Alma; Benito, Ana; Diaz, Nuria; Martínez-Grau, Maria Angeles; Toledo, Miguel A; Pintar, John E

2016-02-01

Nociceptin/orphanin FQ (N/OFQ), a 17 amino acid peptide, is the endogenous ligand of the ORL1/nociceptin-opioid-peptide (NOP) receptor. N/OFQ appears to regulate a variety of physiologic functions including stimulating feeding behavior. Recently, a new class of thienospiro-piperidine-based NOP antagonists was described. One of these molecules, LY2940094 has been identified as a potent and selective NOP antagonist that exhibited activity in the central nervous system. Herein, we examined the effects of LY2940094 on feeding in a variety of behavioral models. Fasting-induced feeding was inhibited by LY2940094 in mice, an effect that was absent in NOP receptor knockout mice. Moreover, NOP receptor knockout mice exhibited a baseline phenotype of reduced fasting-induced feeding, relative to wild-type littermate controls. In lean rats, LY2940094 inhibited the overconsumption of a palatable high-energy diet, reducing caloric intake to control chow levels. In dietary-induced obese rats, LY2940094 inhibited feeding and body weight regain induced by a 30% daily caloric restriction. Last, in dietary-induced obese mice, LY2940094 decreased 24-hour intake of a high-energy diet made freely available. These are the first data demonstrating that a systemically administered NOP receptor antagonist can reduce feeding behavior and body weight in rodents. Moreover, the hypophagic effect of LY2940094 is NOP receptor dependent and not due to off-target or aversive effects. Thus, LY2940094 may be useful in treating disorders of appetitive behavior such as binge eating disorder, food choice, and overeating, which lead to obesity and its associated medical complications and morbidity. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Disrupting the male germ line to find infertility and contraception targets.

PubMed

Archambeault, Denise R; Matzuk, Martin M

2014-05-01

Genetically-manipulated mouse models have become indispensible for broadening our understanding of genes and pathways related to male germ cell development. Until suitable in vitro systems for studying spermatogenesis are perfected, in vivo models will remain the gold standard for inquiry into testicular function. Here, we discuss exciting advances that are allowing researchers faster, easier, and more customizable access to their mouse models of interest. Specifically, the trans-NIH Knockout Mouse Project (KOMP) is working to generate knockout mouse models of every gene in the mouse genome. The related Knockout Mouse Phenotyping Program (KOMP2) is performing systematic phenotypic analysis of this genome-wide collection of knockout mice, including fertility screening. Together, these programs will not only uncover new genes involved in male germ cell development but also provide the research community with the mouse models necessary for further investigations. In addition to KOMP/KOMP2, another promising development in the field of mouse models is the advent of CRISPR (clustered regularly interspaced short palindromic repeat)-Cas technology. Utilizing 20 nucleotide guide sequences, CRISPR/Cas has the potential to introduce sequence-specific insertions, deletions, and point mutations to produce null, conditional, activated, or reporter-tagged alleles. CRISPR/Cas can also successfully target multiple genes in a single experimental step, forgoing the multiple generations of breeding traditionally required to produce mouse models with deletions, insertions, or mutations in multiple genes. In addition, CRISPR/Cas can be used to create mouse models carrying variants identical to those identified in infertile human patients, providing the opportunity to explore the effects of such mutations in an in vivo system. Both the KOMP/KOMP2 projects and the CRISPR/Cas system provide powerful, accessible genetic approaches to the study of male germ cell development in the mouse. A more complete understanding of male germ cell biology is critical for the identification of novel targets for potential non-hormonal contraceptive intervention. Copyright © 2014. Published by Elsevier Masson SAS.

Generating double knockout mice to model genetic intervention for diabetic cardiomyopathy in humans.

PubMed

Chavali, Vishalakshi; Nandi, Shyam Sundar; Singh, Shree Ram; Mishra, Paras Kumar

2014-01-01

Diabetes is a rapidly increasing disease that enhances the chances of heart failure twofold to fourfold (as compared to age and sex matched nondiabetics) and becomes a leading cause of morbidity and mortality. There are two broad classifications of diabetes: type1 diabetes (T1D) and type2 diabetes (T2D). Several mice models mimic both T1D and T2D in humans. However, the genetic intervention to ameliorate diabetic cardiomyopathy in these mice often requires creating double knockout (DKO). In order to assess the therapeutic potential of a gene, that specific gene is either overexpressed (transgenic expression) or abrogated (knockout) in the diabetic mice. If the genetic mice model for diabetes is used, it is necessary to create DKO with transgenic/knockout of the target gene to investigate the specific role of that gene in pathological cardiac remodeling in diabetics. One of the important genes involved in extracellular matrix (ECM) remodeling in diabetes is matrix metalloproteinase-9 (Mmp9). Mmp9 is a collagenase that remains latent in healthy hearts but induced in diabetic hearts. Activated Mmp9 degrades extracellular matrix (ECM) and increases matrix turnover causing cardiac fibrosis that leads to heart failure. Insulin2 mutant (Ins2+/-) Akita is a genetic model for T1D that becomes diabetic spontaneously at the age of 3-4 weeks and show robust hyperglycemia at the age of 10-12 weeks. It is a chronic model of T1D. In Ins2+/- Akita, Mmp9 is induced. To investigate the specific role of Mmp9 in diabetic hearts, it is necessary to create diabetic mice where Mmp9 gene is deleted. Here, we describe the method to generate Ins2+/-/Mmp9-/- (DKO) mice to determine whether the abrogation of Mmp9 ameliorates diabetic cardiomyopathy.

Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function.

PubMed

Yan, C; Wang, P; DeMayo, J; DeMayo, F J; Elvin, J A; Carino, C; Prasad, S V; Skinner, S S; Dunbar, B S; Dube, J L; Celeste, A J; Matzuk, M M

2001-06-01

Knockout mouse technology has been used over the last decade to define the essential roles of ovarian-expressed genes and uncover genetic interactions. In particular, we have used this technology to study the function of multiple members of the transforming growth factor-beta superfamily including inhibins, activins, and growth differentiation factor 9 (GDF-9 or Gdf9). Knockout mice lacking GDF-9 are infertile due to a block in folliculogenesis at the primary follicle stage. In addition, recombinant GDF-9 regulates multiple cumulus granulosa cell functions in the periovulatory period including hyaluronic acid synthesis and cumulus expansion. We have also cloned an oocyte-specific homolog of GDF-9 from mice and humans, which is termed bone morphogenetic protein 15 (BMP-15 or Bmp15). To define the function of BMP-15 in mice, we generated embryonic stem cells and knockout mice, which have a null mutation in this X-linked gene. Male chimeric and Bmp15 null mice are normal and fertile. In contrast to Bmp15 null males and Gdf9 knockout females, Bmp15 null females (Bmp15(-/-)) are subfertile and usually have minimal ovarian histopathological defects, but demonstrate decreased ovulation and fertilization rates. To further decipher possible direct or indirect genetic interactions between GDF-9 and BMP-15, we have generated double mutant mice lacking one or both alleles of these related homologs. Double homozygote females (Bmp15(-/-)Gdf9(-/-)) display oocyte loss and cysts and resemble Gdf9(-/-) mutants. In contrast, Bmp15(-/-)Gdf9(+/-) female mice have more severe fertility defects than Bmp15(-/-) females, which appear to be due to abnormalities in ovarian folliculogenesis, cumulus cell physiology, and fertilization. Thus, the dosage of intact Bmp15 and Gdf9 alleles directly influences the destiny of the oocyte during folliculogenesis and in the periovulatory period. These studies have important implications for human fertility control and the maintenance of fertility and normal ovarian physiology.

Extension to Higher Mass Numbers of an Improved Knockout-Ablation-Coalescence Model for Secondary Neutron and Light Ion Production in Cosmic Ray Interactions

NASA Astrophysics Data System (ADS)

Indi Sriprisan, Sirikul; Townsend, Lawrence; Cucinotta, Francis A.; Miller, Thomas M.

Purpose: An analytical knockout-ablation-coalescence model capable of making quantitative predictions of the neutron spectra from high-energy nucleon-nucleus and nucleus-nucleus collisions is being developed for use in space radiation protection studies. The FORTRAN computer code that implements this model is called UBERNSPEC. The knockout or abrasion stage of the model is based on Glauber multiple scattering theory. The ablation part of the model uses the classical evaporation model of Weisskopf-Ewing. In earlier work, the knockout-ablation model has been extended to incorporate important coalescence effects into the formalism. Recently, alpha coalescence has been incorporated, and the ability to predict light ion spectra with the coalescence model added. The earlier versions were limited to nuclei with mass numbers less than 69. In this work, the UBERNSPEC code has been extended to make predictions of secondary neutrons and light ion production from the interactions of heavy charged particles with higher mass numbers (as large as 238). The predictions are compared with published measurements of neutron spectra and light ion energy for a variety of collision pairs. Furthermore, the predicted spectra from this work are compared with the predictions from the recently-developed heavy ion event generator incorporated in the Monte Carlo radiation transport code HETC-HEDS.

Targeted disruption of glutathione peroxidase 4 (GPx4) in mouse skin epithelial cells impairs postnatal hair follicle morphogenesis that is partially rescued through inhibition of COX-2

PubMed Central

Sengupta, Aniruddha; Lichti, Ulrike F.; Carlson, Bradley A.; Cataisson, Christophe; Ryscavage, Andrew O.; Mikulec, Carol; Conrad, Marcus; Fischer, Susan M.; Hatfield, Dolph L.; Yuspa, Stuart H.

2013-01-01

Selenoproteins are essential molecules for the mammalian antioxidant network. We previously demonstrated that targeted loss of all selenoproteins in mouse epidermis disrupted skin and hair development and caused premature death. In the current study we targeted specific selenoproteins for epidermal deletion to determine whether similar phenotypes developed. Keratinocyte-specific knockout mice lacking either the glutathione peroxidase 4 (GPx4) or thioredoxin reductase 1 (TR1) gene were generated by cre-lox technology using K14-cre. TR1 knockout mice had a normal phenotype in resting skin while GPx4 loss in epidermis caused epidermal hyperplasia, dermal inflammatory infiltrate, dysmorphic hair follicles and alopecia in perinatal mice. Unlike epidermal ablation of all selenoproteins, mice ablated for GPx4 recovered after 5 weeks and had a normal lifespan. GPx1 and TR1 were upregulated in the skin and keratinocytes of GPx4 knockout mice. GPx4 deletion reduces keratinocyte adhesion in culture and increases lipid peroxidation and COX-2 levels in cultured keratinocytes and whole skin. Feeding a COX-2 inhibitor to nursing mothers partially prevents development of the abnormal skin phenotype in knockout pups. These data link the activity of cutaneous GPx4 to the regulation of COX-2 and hair follicle morphogenesis and provide insight into the function of individual selenoprotein activity in maintaining cutaneous homeostasis. PMID:23364477

Generation and phenotypic analysis of mice lacking all urea transporters.

PubMed

Jiang, Tao; Li, Yingjie; Layton, Anita T; Wang, Weiling; Sun, Yi; Li, Min; Zhou, Hong; Yang, Baoxue

2017-02-01

Urea transporters (UT) are a family of transmembrane urea-selective channel proteins expressed in multiple tissues and play an important role in the urine concentrating mechanism of the mammalian kidney. UT inhibitors have diuretic activity and could be developed as novel diuretics. To determine if functional deficiency of all UTs in all tissues causes physiological abnormality, we established a novel mouse model in which all UTs were knocked out by deleting an 87 kb of DNA fragment containing most parts of Slc14a1 and Slc14a2 genes. Western blot analysis and immunofluorescence confirmed that there is no expression of urea transporter in these all-UT-knockout mice. Daily urine output was nearly 3.5-fold higher, with significantly lower urine osmolality in all-UT-knockout mice than that in wild-type mice. All-UT-knockout mice were not able to increase urinary urea concentration and osmolality after water deprivation, acute urea loading, or high protein intake. A computational model that simulated UT-knockout mouse models identified the individual contribution of each UT in urine concentrating mechanism. Knocking out all UTs also decreased the blood pressure and promoted the maturation of the male reproductive system. Thus, functional deficiency of all UTs caused a urea-selective urine-concentrating defect with little physiological abnormality in extrarenal organs. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Generation and phenotypic analysis of mice lacking all urea transporters

PubMed Central

Jiang, Tao; Li, Yingjie; Layton, Anita T.; Wang, Weiling; Sun, Yi; Li, Min; Zhou, Hong; Yang, Baoxue

2017-01-01

Urea transporters (UT) are a family of transmembrane urea-selective channel proteins expressed in multiple tissues and play an important role in the urine concentrating mechanism of the mammalian kidney. UT inhibitors have been identified to have diuretic activity and might be developed as novel diuretics. To determine if functional deficiency of all UTs in all tissues causes physiological abnormality, we established a novel mouse model in which all UTs were knocked out by deleting an 87 kb of DNA fragment containing most parts of Slc14a1 and Slc14a2 genes. Western blot analysis and immunofluorescence confirmed that there is no expression of urea transporter in all-UT-knockout mice. Daily urine output was nearly 3.5-fold higher, with significantly lower urine osmolality, in all-UT-knockout-mice than that in wild-type mice, and urine osmolality was significantly lower. All-UT-knockout mice were not able to increase urinary urea concentration and osmolality after water deprivation, acute urea loading or high protein intake. A computational model that simulated UT knockout mouse models identified the individual contribution of each UT in urine concentrating mechanism. Knocking out all UTs also decreased the blood pressure and promoted the maturation of the male reproductive system. These results revealed that functional deficiency of all UTs caused urea selective urine concentrating defect with little physiological abnormality in extrarenal organs. PMID:27914708

Overexpression of Protein Kinase Mζ in the Hippocampus Enhances Long-Term Potentiation and Long-Term Contextual But Not Cued Fear Memory in Rats.

PubMed

Schuette, Sven R M; Fernández-Fernández, Diego; Lamla, Thorsten; Rosenbrock, Holger; Hobson, Scott

2016-04-13

The persistently active protein kinase Mζ (PKMζ) has been found to be involved in the formation and maintenance of long-term memory. Most of the studies investigating PKMζ, however, have used either putatively unselective inhibitors or conventional knock-out animal models in which compensatory mechanisms may occur. Here, we overexpressed an active form of PKMζ in rat hippocampus, a structure highly involved in memory formation, and embedded in several neural networks. We investigated PKMζ's influence on synaptic plasticity using electrophysiological recordings of basal transmission, paired pulse facilitation, and LTP and combined this with behavioral cognitive experiments addressing formation and retention of both contextual memory during aversive conditioning and spatial memory during spontaneous exploration. We demonstrate that hippocampal slices overexpressing PKMζ show enhanced basal transmission, suggesting a potential role of PKMζ in postsynaptic AMPAR trafficking. Moreover, the PKMζ-overexpressing slices augmented LTP and this effect was not abolished by protein-synthesis blockers, indicating that PKMζ induces enhanced LTP formation in a protein-synthesis-independent manner. In addition, we found selectively enhanced long-term memory for contextual but not cued fear memory, underlining the theory of the hippocampus' involvement in the contextual aspect of aversive reinforced tasks. Memory for spatial orientation during spontaneous exploration remained unaltered, suggesting that PKMζ may not affect the neural circuits underlying spontaneous tasks that are different from aversive tasks. In this study, using an overexpression strategy as opposed to an inhibitor-based approach, we demonstrate an important modulatory role of PKMζ in synaptic plasticity and selective memory processing. Most of the literature investigating protein kinase Mζ (PKMζ) used inhibitors with selectivity that has been called into question or conventional knock-out animal models in which compensatory mechanisms may occur. To avoid these issues, some studies have been done using viral overexpression of PKMζ in different brain structures to show cognitive enhancement. However, electrophysiological experiments were exclusively done in knock-out models or inhibitory studies to show depletion of LTP. There was no study showing the effect of PKMζ overexpression in the hippocampus on behavior and LTP experiments. To our knowledge, this is the first study to combine these aspects with the result of enhanced memory for contextual fear memory and to show enhanced LTP in hippocampal slices overexpressing PKMζ. Copyright © 2016 Schuette et al.

Phenotypic screening of hepatocyte nuclear factor (HNF) 4-{gamma} receptor knockout mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gerdin, Anna Karin; Surve, Vikas V.; Joensson, Marie

2006-10-20

Using the mouse as a model organism in pharmaceutical research presents unique advantages as its physiology in many ways resembles the human physiology, it also has a relatively short generation time, low breeding and maintenance costs, and is available in a wide variety of inbred strains. The ability to genetically modify mouse embryonic stem cells to generate mouse models that better mimic human disease is another advantage. In the present study, a comprehensive phenotypic screening protocol is applied to elucidate the phenotype of a novel mouse knockout model of hepatocyte nuclear factor (HNF) 4-{gamma}. HNF4-{gamma} is expressed in the kidneys,more » gut, pancreas, and testis. First level of the screen is aimed at general health, morphologic appearance, normal cage behaviour, and gross neurological functions. The second level of the screen looks at metabolic characteristics and lung function. The third level of the screen investigates behaviour more in-depth and the fourth level consists of a thorough pathological characterisation, blood chemistry, haematology, and bone marrow analysis. When compared with littermate wild-type mice (HNF4-{gamma}{sup +/+}), the HNF4-{gamma} knockout (HNF4-{gamma}{sup -/-}) mice had lowered energy expenditure and locomotor activity during night time that resulted in a higher body weight despite having reduced intake of food and water. HNF4-{gamma}{sup -/-} mice were less inclined to build nest and were found to spend more time in a passive state during the forced swim test.« less

A Knowledge-Based System for Display and Prediction of O-Glycosylation Network Behaviour in Response to Enzyme Knockouts

PubMed Central

McDonald, Andrew G.; Tipton, Keith F.; Davey, Gavin P.

2016-01-01

O-linked glycosylation is an important post-translational modification of mucin-type protein, changes to which are important biomarkers of cancer. For this study of the enzymes of O-glycosylation, we developed a shorthand notation for representing GalNAc-linked oligosaccharides, a method for their graphical interpretation, and a pattern-matching algorithm that generates networks of enzyme-catalysed reactions. Software for generating glycans from the enzyme activities is presented, and is also available online. The degree distributions of the resulting enzyme-reaction networks were found to be Poisson in nature. Simple graph-theoretic measures were used to characterise the resulting reaction networks. From a study of in-silico single-enzyme knockouts of each of 25 enzymes known to be involved in mucin O-glycan biosynthesis, six of them, β-1,4-galactosyltransferase (β4Gal-T4), four glycosyltransferases and one sulfotransferase, play the dominant role in determining O-glycan heterogeneity. In the absence of β4Gal-T4, all Lewis X, sialyl-Lewis X, Lewis Y and Sda/Cad glycoforms were eliminated, in contrast to knockouts of the N-acetylglucosaminyltransferases, which did not affect the relative abundances of O-glycans expressing these epitopes. A set of 244 experimentally determined mucin-type O-glycans obtained from the literature was used to validate the method, which was able to predict up to 98% of the most common structures obtained from human and engineered CHO cell glycoforms. PMID:27054587

Dendritic Kv3.3 potassium channels in cerebellar purkinje cells regulate generation and spatial dynamics of dendritic Ca2+ spikes.

PubMed

Zagha, Edward; Manita, Satoshi; Ross, William N; Rudy, Bernardo

2010-06-01

Purkinje cell dendrites are excitable structures with intrinsic and synaptic conductances contributing to the generation and propagation of electrical activity. Voltage-gated potassium channel subunit Kv3.3 is expressed in the distal dendrites of Purkinje cells. However, the functional relevance of this dendritic distribution is not understood. Moreover, mutations in Kv3.3 cause movement disorders in mice and cerebellar atrophy and ataxia in humans, emphasizing the importance of understanding the role of these channels. In this study, we explore functional implications of this dendritic channel expression and compare Purkinje cell dendritic excitability in wild-type and Kv3.3 knockout mice. We demonstrate enhanced excitability of Purkinje cell dendrites in Kv3.3 knockout mice, despite normal resting membrane properties. Combined data from local application pharmacology, voltage clamp analysis of ionic currents, and assessment of dendritic Ca(2+) spike threshold in Purkinje cells suggest a role for Kv3.3 channels in opposing Ca(2+) spike initiation. To study the physiological relevance of altered dendritic excitability, we measured [Ca(2+)](i) changes throughout the dendritic tree in response to climbing fiber activation. Ca(2+) signals were specifically enhanced in distal dendrites of Kv3.3 knockout Purkinje cells, suggesting a role for dendritic Kv3.3 channels in regulating propagation of electrical activity and Ca(2+) influx in distal dendrites. These findings characterize unique roles of Kv3.3 channels in dendrites, with implications for synaptic integration, plasticity, and human disease.

Gene targeting in mosquito cells: a demonstration of 'knockout' technology in extrachromosomal gene arrays

PubMed Central

Eggleston, Paul; Zhao, Yuguang

2001-01-01

Background Gene targeting would offer a number of advantages over current transposon-based strategies for insect transformation. These include freedom from both position effects associated with quasi-random integration and concerns over transgene instability mediated by endogenous transposases, independence from phylogenetic restrictions on transposon mobility and the ability to generate gene knockouts. Results We describe here our initial investigations of gene targeting in the mosquito. The target site was a hygromycin resistance gene, stably maintained as part of an extrachromosomal array. Using a promoter-trap strategy to enrich for targeted events, a neomycin resistance gene was integrated into the target site. This resulted in knockout of hygromycin resistance concurrent with the expression of high levels of neomycin resistance from the resident promoter. PCR amplification of the targeted site generated a product that was specific to the targeted cell line and consistent with precise integration of the neomycin resistance gene into the 5' end of the hygromycin resistance gene. Sequencing of the PCR product and Southern analysis of cellular DNA subsequently confirmed this molecular structure. Conclusions These experiments provide the first demonstration of gene targeting in mosquito tissue and show that mosquito cells possess the necessary machinery to bring about precise integration of exogenous sequences through homologous recombination. Further development of these procedures and their extension to chromosomally located targets hold much promise for the exploitation of gene targeting in a wide range of medically and economically important insect species. PMID:11513755

Knockout of the Gnrh genes in zebrafish: effects on reproduction and potential compensation by reproductive and feeding-related neuropeptides.

PubMed

Marvel, Miranda; Spicer, Olivia Smith; Wong, Ten-Tsao; Zmora, Nilli; Zohar, Yonathan

2018-04-04

Gonadotropin-releasing hormone (GnRH) is known as a pivotal upstream regulator of reproduction in vertebrates. However, reproduction is not compromised in the hypophysiotropic Gnrh3 knockout line in zebrafish (gnrh3-/-). In order to determine if Gnrh2, the only other Gnrh isoform in zebrafish brains, is compensating for the loss of Gnrh3, we generated a double Gnrh knockout zebrafish line. Surprisingly, the loss of both Gnrh isoforms resulted in no major impact on reproduction, indicating that a compensatory response, outside of the Gnrh system, was evoked. A plethora of factors acting along the reproductive hypothalamus-pituitary axis were evaluated as possible compensators based on neuroanatomical and differential gene expression studies. In addition, we also examined the involvement of feeding factors in the brain as potential compensators for Gnrh2, which has known anorexigenic effects. We found that the double knockout fish exhibited upregulation of several genes in the brain, specifically gonadotropin-inhibitory hormone (gnih), secretogranin 2 (scg2), tachykinin 3a (tac3a), and pituitary adenylate cyclase-activating peptide 1 (pacap1), and downregulation of agouti-related peptide 1 (agrp1), indicating the compensation occurs outside of Gnrh cells and therefore is a non-cell autonomous response to the loss of Gnrh. While the differential expression of gnih and agrp1 in the double knockout line was confined to the periventricular nucleus and hypothalamus, respectively, the upregulation of scg2 corresponded with a broader neuronal redistribution in the lateral hypothalamus and hindbrain. In conclusion, our results demonstrate the existence of a redundant reproductive regulatory system that comes into play when Gnrh2 and Gnrh3 are lost.

Ascl1 (Mash1) Knockout Perturbs Differentiation of Nonneuronal Cells in Olfactory Epithelium

PubMed Central

Jang, Woochan; Wildner, Hendrik; Schwob, James E.

2012-01-01

The embryonic olfactory epithelium (OE) generates only a very few olfactory sensory neurons when the basic helix-loop-helix transcription factor, ASCL1 (previously known as MASH1) is eliminated by gene mutation. We have closely examined the structure and composition of the OE of knockout mice and found that the absence of neurons dramatically affects the differentiation of multiple other epithelial cell types as well. The most prominent effect is observed within the two known populations of stem and progenitor cells of the epithelium. The emergence of horizontal basal cells, a multipotent progenitor population in the adult epithelium, is anomalous in the Ascl1 knockout mice. The differentiation of globose basal cells, another multipotent progenitor population in the adult OE, is also aberrant. All of the persisting globose basal cells are marked by SOX2 expression, suggesting a prominent role for SOX2 in progenitors upstream of Ascl1. However, NOTCH1-expressing basal cells are absent from the knockout; since NOTCH1 signaling normally acts to suppress Ascl1 via HES1 and drives sustentacular (Sus) cell differentiation during adult epithelial regeneration, its absence suggests reciprocity between neurogenesis and the differentiation of Sus cells. Indeed, the Sus cells of the mutant mice express a markedly lower level of HES1, strengthening that notion of reciprocity. Duct/gland development appears normal. Finally, the expression of cKIT by basal cells is also undetectable, except in those small patches where neurogenesis escapes the effects of Ascl1 knockout and neurons are born. Thus, persistent neurogenic failure distorts the differentiation of multiple other cell types in the olfactory epithelium. PMID:23284756

Role of light and the circadian clock in the rhythmic oscillation of intraocular pressure: Studies in VPAC2 receptor and PACAP deficient mice.

PubMed

Fahrenkrug, Jan; Georg, Birgitte; Hannibal, Jens; Jørgensen, Henrik Løvendahl

2018-04-01

The intraocular pressure of mice displays a daily rhythmicity being highest during the dark period. The present study was performed to elucidate the role of the circadian clock and light in the diurnal and the circadian variations in intraocular pressure in mice, by using animals with disrupted clock function (VPAC2 receptor knockout mice) or impaired light information to the clock (PACAP knockout mice). In wildtype mice, intraocular pressure measured under light/dark conditions showed a statistically significant 24 h sinusoidal rhythm with nadir during the light phase and peak during the dark phase. After transfer of the wildtype mice into constant darkness, the intraocular pressure increased, but the rhythmic changes in intraocular pressure continued with a pattern identical to that obtained during the light/dark cycle. The intraocular pressure in VPAC2 receptor deficient mice during light/dark conditions also showed a sinusoidal pattern with significant changes as a function of a 24 h cycle. However, transfer of the VPAC2 receptor knockout mice into constant darkness completely abolished the rhythmic changes in intraocular pressure. The intraocular pressure in PACAP deficient mice oscillated significantly during both 24 h light and darkness and during constant darkness. During LD conditions, the amplitude of PACAP deficient was significantly lower compared to wildtype mice, resulting in higher daytime and lower nighttime values. In conclusion, by studying the VPAC2 receptor knockout mouse which lacks circadian control and the PACAP knockout mouse which displays impaired light signaling, we provided evidence that the daily intraocular pressure rhythms are primarily generated by the circadian master clock and to a lesser extent by environmental light and darkness. Copyright © 2018 Elsevier Ltd. All rights reserved.

The Xanthomonas oryzae pv. oryzae PhoPQ Two-Component System Is Required for AvrXA21 Activity, hrpG Expression, and Virulence▿ †

PubMed Central

Lee, Sang-Won; Jeong, Kyu-Sik; Han, Sang-Wook; Lee, Seung-Eun; Phee, Bong-Kwan; Hahn, Tae-Ryong; Ronald, Pamela

2008-01-01

The rice pathogen recognition receptor, XA21, confers resistance to Xanthomonas oryzae pv. oryzae strains producing the type one system-secreted molecule, AvrXA21. X. oryzae pv. oryzae requires a regulatory two-component system (TCS) called RaxRH to regulate expression of eight rax (required for AvrXA21 activity) genes and to sense population cell density. To identify other key components in this critical regulatory circuit, we assayed proteins expressed in a raxR gene knockout strain. This survey led to the identification of the phoP gene encoding a response regulator that is up-regulated in the raxR knockout strain. Next we generated a phoP knockout strain and found it to be impaired in X. oryzae pv. oryzae virulence and no longer able to activate the response regulator HrpG (hypersensitive reaction and pathogenicity G) in response to low levels of Ca2+. The impaired virulence of the phoP knockout strain can be partially complemented by constitutive expression of hrpG, indicating that PhoP controls a key aspect of X. oryzae pv. oryzae virulence through regulation of hrpG. A gene encoding the cognate putative histidine protein kinase, phoQ, was also isolated. Growth curve analysis revealed that AvrXA21 activity is impaired in a phoQ knockout strain as reflected by enhanced growth of this strain in rice lines carrying XA21. These results suggest that the X. oryzae pv. oryzae PhoPQ TCS functions in virulence and in the production of AvrXA21 in partnership with RaxRH. PMID:18203830

Direct evidence of iNOS-mediated in vivo free radical production and protein oxidation in acetone-induced ketosis

PubMed Central

Stadler, Krisztian; Bonini, Marcelo G.; Dallas, Shannon; Duma, Danielle; Mason, Ronald P.; Kadiiska, Maria B.

2008-01-01

Diabetic patients frequently encounter ketosis that is characterized by the breakdown of lipids with the consequent accumulation of ketone bodies. Several studies have demonstrated that reactive species are likely to induce tissue damage in diabetes, but the role of the ketone bodies in the process has not been fully investigated. In this study, electron paramagnetic resonance (EPR) spectroscopy combined with novel spin-trapping and immunological techniques has been used to investigate in vivo free radical formation in a murine model of acetone-induced ketosis. A six-line EPR spectrum consistent with the α-(4-pyridyl-1-oxide)-N-t-butylnitrone radical adduct of a carbon-centered lipid-derived radical was detected in the liver extracts. To investigate the possible enzymatic source of these radicals, inducible nitric oxide synthase (iNOS) and NADPH oxidase knockout mice were used. Free radical production was unchanged in the NADPH oxidase knockout but much decreased in the iNOS knockout mice, suggesting a role for iNOS in free radical production. Longer-term exposure to acetone revealed iNOS overexpression in the liver together with protein radical formation, which was detected by confocal microscopy and a novel immunospin-trapping method. Immunohistochemical analysis revealed enhanced lipid peroxidation and protein oxidation as a consequence of persistent free radical generation after 21 days of acetone treatment in control and NADPH oxidase knockout but not in iNOS knockout mice. Taken together, our data demonstrate that acetone administration, a model of ketosis, can lead to protein oxidation and lipid peroxidation through a free radical-dependent mechanism driven mainly by iNOS overexpression. PMID:18559982

Protein phosphatase 2ACα gene knock-out results in cortical atrophy through activating hippo cascade in neuronal progenitor cells.

PubMed

Liu, Bo; Sun, Li-Hua; Huang, Yan-Fei; Guo, Li-Jun; Luo, Li-Shu

2018-02-01

Protein phosphatase 2ACα (PP2ACα), a vital member of the protein phosphatase family, has been studied primarily as a regulator for the development, growth and protein synthesis of a lot of cell types. Dysfunction of PP2ACα protein results in neurodegenerative disease; however, this finding has not been directly confirmed in the mouse model with PP2ACα gene knock-out. Therefore, in this study presented here, we generated the PP2ACα gene knock-out mouse model by the Cre-loxP targeting gene system, with the purpose to directly observe the regulatory role of PP2ACα gene in the development of mouse's cerebral cortex. We observe that knocking-out PP2ACα gene in the central nervous system (CNS) results in cortical neuronal shrinkage, synaptic plasticity impairments, and learning/memory deficits. Further study reveals that PP2ACα gene knock-out initiates Hippo cascade in cortical neuroprogenitor cells (NPCs), which blocks YAP translocation into the nuclei of NPCs. Notably, p73, directly targeted by Hippo cascade, can bind to the promoter of glutaminase2 (GLS2) that plays a dominant role in the enzymatic regulation of glutamate/glutamine cycle. Finally, we find that PP2ACα gene knock-out inhibits the glutamine synthesis through up-regulating the activity of phosphorylated-p73 in cortical NPCs. Taken together, it concludes that PP2ACα critically supports cortical neuronal growth and cognitive function via regulating the signaling transduction of Hippo-p73 cascade. And PP2ACα indirectly modulates the glutamine synthesis of cortical NPCs through targeting p73 that plays a direct transcriptional regulatory role in the gene expression of GLS2. Copyright © 2017 Elsevier Ltd. All rights reserved.

Abnormal cerebellar development and Purkinje cell defects in Lgl1-Pax2 conditional knockout mice.

PubMed

Hou, Congzhe; Ding, Lingcui; Zhang, Jian; Jin, Yecheng; Sun, Chen; Li, Zhenzu; Sun, Xiaoyang; Zhang, Tingting; Zhang, Aizhen; Li, Huashun; Gao, Jiangang

2014-11-01

Lgl1 was initially identified as a tumour suppressor in flies and is characterised as a key regulator of epithelial polarity and asymmetric cell division. A previous study indicated that More-Cre-mediated Lgl1 knockout mice exhibited significant brain dysplasia and died within 24h after birth. To overcome early neonatal lethality, we generated Lgl1 conditional knockout mice mediated by Pax2-Cre, which is expressed in almost all cells in the cerebellum, and we examined the functions of Lgl1 in the cerebellum. Impaired motor coordination was detected in the mutant mice. Consistent with this abnormal behaviour, homozygous mice possessed a smaller cerebellum with fewer lobes, reduced granule precursor cell (GPC) proliferation, decreased Purkinje cell (PC) quantity and dendritic dysplasia. Loss of Lgl1 in the cerebellum led to hyperproliferation and impaired differentiation of neural progenitors in ventricular zone. Based on the TUNEL assay, we observed increased apoptosis in the cerebellum of mutant mice. We proposed that impaired differentiation and increased apoptosis may contribute to decreased PC quantity. To clarify the effect of Lgl1 on cerebellar granule cells, we used Math1-Cre to specifically delete Lgl1 in granule cells. Interestingly, the Lgl1-Math1 conditional knockout mice exhibited normal proliferation of GPCs and cerebellar development. Thus, we speculated that the reduction in the proliferation of GPCs in Lgl1-Pax2 conditional knockout mice may be secondary to the decreased number of PCs, which secrete the mitogenic factor Sonic hedgehog to regulate GPC proliferation. Taken together, these findings suggest that Lgl1 plays a key role in cerebellar development and folia formation by regulating the development of PCs. Copyright © 2014. Published by Elsevier Inc.

Transducin β-Subunit Can Interact with Multiple G-Protein γ-Subunits to Enable Light Detection by Rod Photoreceptors.

PubMed

Dexter, Paige M; Lobanova, Ekaterina S; Finkelstein, Stella; Spencer, William J; Skiba, Nikolai P; Arshavsky, Vadim Y

2018-01-01

The heterotrimeric G-protein transducin mediates visual signaling in vertebrate photoreceptor cells. Many aspects of the function of transducin were learned from knock-out mice lacking its individual subunits. Of particular interest is the knockout of its rod-specific γ-subunit (Gγ 1 ). Two studies using independently generated mice documented that this knockout results in a considerable >60-fold reduction in the light sensitivity of affected rods, but provided different interpretations of how the remaining α-subunit (Gα t ) mediates phototransduction without its cognate Gβ 1 γ 1 -subunit partner. One study found that the light sensitivity reduction matched a corresponding reduction in Gα t content in the light-sensing rod outer segments and proposed that Gα t activation is supported by remaining Gβ 1 associating with other Gγ subunits naturally expressed in photoreceptors. In contrast, the second study reported the same light sensitivity loss but a much lower, only approximately sixfold, reduction of Gα t and proposed that the light responses of these rods do not require Gβγ at all. To resolve this controversy and elucidate the mechanism driving visual signaling in Gγ 1 knock-out rods, we analyzed both mouse lines side by side. We first determined that the outer segments of both mice have identical Gα t content, which is reduced ∼65-fold from the wild-type (WT) level. We further demonstrated that the remaining Gβ 1 is present in a complex with endogenous Gγ 2 and Gγ 3 subunits and that these complexes exist in wild-type rods as well. Together, these results argue against the idea that Gα t alone supports light responses of Gγ 1 knock-out rods and suggest that Gβ 1 γ 1 is not unique in its ability to mediate vertebrate phototransduction.

Generation of muscular dystrophy model rats with a CRISPR/Cas system.

PubMed

Nakamura, Katsuyuki; Fujii, Wataru; Tsuboi, Masaya; Tanihata, Jun; Teramoto, Naomi; Takeuchi, Shiho; Naito, Kunihiko; Yamanouchi, Keitaro; Nishihara, Masugi

2014-07-09

Duchenne muscular dystrophy (DMD) is an X-linked lethal muscle disorder caused by mutations in the Dmd gene encoding Dystrophin. DMD model animals, such as mdx mice and canine X-linked muscular dystrophy dogs, have been widely utilized in the development of a treatment for DMD. Here, we demonstrate the generation of Dmd-mutated rats using a clustered interspaced short palindromic repeats (CRISPR)/Cas system, an RNA-based genome engineering technique that is also adaptive to rats. We simultaneously targeted two exons in the rat Dmd gene, which resulted in the absence of Dystrophin expression in the F0 generation. Dmd-mutated rats exhibited a decline in muscle strength, and the emergence of degenerative/regenerative phenotypes in the skeletal muscle, heart, and diaphragm. These mutations were heritable by the next generation, and F1 male rats exhibited similar phenotypes in their skeletal muscles. These model rats should prove to be useful for developing therapeutic methods to treat DMD.

UHRF2 regulates local 5-methylcytosine and suppresses spontaneous seizures

PubMed Central

Liu, Yidan; Zhang, Bin; Meng, Xiaoyu; Korn, Matthew J.; Parent, Jack M.; Lu, Lin-Yu; Yu, Xiaochun

2017-01-01

ABSTRACT The 5-methylcytosine (5mC) modification regulates multiple cellular processes and is faithfully maintained following DNA replication. In addition to DNA methyltransferase (DNMT) family proteins, ubiquitin-like PHD and ring finger domain-containing protein 1 (UHRF1) plays an important role in the maintenance of 5mC levels. Loss of UHRF1 abolishes 5mC in cells and leads to embryonic lethality in mice. Interestingly, UHRF1 has a paralog, UHRF2, that has similar sequence and domain architecture, but its biologic function is not clear. Here, we have generated Uhrf2 knockout mice and characterized the role of UHRF2 in vivo. Uhrf2 knockout mice are viable, but the adult mice develop frequent spontaneous seizures and display abnormal electrical activities in brain. Despite no global DNA methylation changes, 5mC levels are decreased at certain genomic loci in the brains of Uhrf2 knockout mice. Therefore, our study has revealed a unique role of UHRF2 in the maintenance of local 5mC levels in brain that is distinct from that of its paralog UHRF1. PMID:28402695

Core features of frontotemporal dementia recapitulated in progranulin knockout mice

PubMed Central

Ghoshal, N.; Dearborn, J.T.; Wozniak, D.F.; Cairns, N.J.

2011-01-01

Frontotemporal dementia (FTD) is typified by behavioral and cognitive changes manifested as altered social comportment and impaired memory performance. To investigate the neurodegenerative consequences of progranulin gene (GRN) mutations, which cause an inherited form of FTD, we used previously generated progranulin knockout mice (Grn-/-). Specifically, we characterized two cohorts of early and later middle-age wild type and knockout mice using a battery of tests to assess neurological integrity and behavioral phenotypes analogous to FTD. The Grn-/- mice exhibited reduced social engagement and learning and memory deficits. Immunohistochemical approaches were used to demonstrate the presence of lesions characteristic of frontotemporal lobar degeneration (FTLD) with GRN mutation including ubiquitination, microgliosis, and reactive astrocytosis, the pathological substrate of FTD. Importantly, Grn-/- mice also have decreased overall survival compared to Grn+/+ mice. These data suggest that the Grn-/- mouse reproduces some core features of FTD with respect to behavior, pathology, and survival. This murine model may serve as a valuable in vivo model of FTLD with GRN mutation through which molecular mechanisms underlying the disease can be further dissected. PMID:21933710

Lipid-lowering effects of anti-angiopoietin-like 4 antibody recapitulate the lipid phenotype found in angiopoietin-like 4 knockout mice

PubMed Central

Desai, Urvi; Lee, E-Chiang; Chung, Kyu; Gao, Cuihua; Gay, Jason; Key, Billie; Hansen, Gwenn; Machajewski, Dennis; Platt, Kenneth A.; Sands, Arthur T.; Schneider, Matthias; Van Sligtenhorst, Isaac; Suwanichkul, Adisak; Vogel, Peter; Wilganowski, Nat; Wingert, June; Zambrowicz, Brian P.; Landes, Greg; Powell, David R.

2007-01-01

We used gene knockout mice to explore the role of Angiopoietin-like-4 (Angptl4) in lipid metabolism as well as to generate anti-Angptl4 mAbs with pharmacological activity. Angptl4 −/− mice had lower triglyceride (TG) levels resulting both from increased very low-density lipoprotein (VLDL) clearance and decreased VLDL production and had modestly lower cholesterol levels. Also, both Angptl4 −/− suckling mice and adult mice fed a high-fat diet showed reduced viability associated with lipogranulomatous lesions of the intestines and their draining lymphatics and mesenteric lymph nodes. Treating C57BL/6J, ApoE −/−, LDLr −/−, and db/db mice with the anti-Angptl4 mAb 14D12 recapitulated the lipid and histopathologic phenotypes noted in Angptl4 −/− mice. This demonstrates that the knockout phenotype reflects not only the physiologic function of the Angptl4 gene but also predicts the pharmacologic consequences of Angptl4 protein inhibition with a neutralizing antibody in relevant models of human disease. PMID:17609370

Absence of uncoupling protein-3 leads to greater activation of an adenine nucleotide translocase-mediated proton conductance in skeletal muscle mitochondria from calorie restricted mice.

PubMed

Bevilacqua, Lisa; Seifert, Erin L; Estey, Carmen; Gerrits, Martin F; Harper, Mary-Ellen

2010-08-01

Calorie restriction (CR), without malnutrition, consistently increases lifespan in all species tested, and reduces age-associated pathologies in mammals. Alterations in mitochondrial content and function are thought to underlie some of the effects of CR. Previously, we reported that rats subjected to variable durations of 40% CR demonstrated a rapid and sustained decrease in maximal leak-dependent respiration in skeletal muscle mitochondria. This was accompanied by decreased mitochondrial reactive oxygen species generation and increased uncoupling protein-3 protein (UCP3) expression. The aim of the present study was to determine the contribution of UCP3, as well as the adenine nucleotide translocase to these functional changes in skeletal muscle mitochondria. Consistent with previous findings in rats, short-term CR (2 weeks) in wild-type (Wt) mice resulted in a lowering of the maximal leak-dependent respiration in skeletal muscle mitochondria, without any change in proton conductance. In contrast, skeletal muscle mitochondria from Ucp3-knockout (KO) mice similarly subjected to short-term CR showed no change in maximal leak-dependent respiration, but displayed an increased proton conductance. Determination of ANT activity (by measurement of inhibitor-sensitive leak) and protein expression revealed that the increased proton conductance in mitochondria from CR Ucp3-KO mice could be entirely attributed to a greater acute activation of ANT. These observations implicate UCP3 in CR-induced mitochondrial remodeling. Specifically, they imply the potential for an interaction, or some degree of functional redundancy, between UCP3 and ANT, and also suggest that UCP3 can minimize the induction of the ANT-mediated 'energy-wasting' process during CR. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Involvement of the kinesin family members KIF4A and KIF5C in intellectual disability and synaptic function.

PubMed

Willemsen, Marjolein H; Ba, Wei; Wissink-Lindhout, Willemijn M; de Brouwer, Arjan P M; Haas, Stefan A; Bienek, Melanie; Hu, Hao; Vissers, Lisenka E L M; van Bokhoven, Hans; Kalscheuer, Vera; Nadif Kasri, Nael; Kleefstra, Tjitske

2014-07-01

Kinesin superfamily (KIF) genes encode motor proteins that have fundamental roles in brain functioning, development, survival and plasticity by regulating the transport of cargo along microtubules within axons, dendrites and synapses. Mouse knockout studies support these important functions in the nervous system. The role of KIF genes in intellectual disability (ID) has so far received limited attention, although previous studies have suggested that many ID genes impinge on synaptic function. By applying next-generation sequencing (NGS) in ID patients, we identified likely pathogenic mutations in KIF4A and KIF5C. To further confirm the pathogenicity of these mutations, we performed functional studies at the level of synaptic function in primary rat hippocampal neurons. Four males from a single family with a disruptive mutation in the X-linked KIF4A (c.1489-8_1490delins10; p.?- exon skipping) showed mild to moderate ID and epilepsy. A female patient with a de novo missense mutation in KIF5C (c.11465A>C; p.(Glu237Lys)) presented with severe ID, epilepsy, microcephaly and cortical malformation. Knock-down of Kif4a in rat primary hippocampal neurons altered the balance between excitatory and inhibitory synaptic transmission, whereas the mutation in Kif5c affected its protein function at excitatory synapses. Our results suggest that mutations in KIF4A and KIF5C cause ID by tipping the balance between excitatory and inhibitory synaptic excitability. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Activation and inhibition of mouse muscle and neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes.

PubMed

Papke, Roger L; Wecker, Lynn; Stitzel, Jerry A

2010-05-01

Transgenic mouse models with nicotinic acetylcholine receptor (nAChR) knockouts and knockins have provided important insights into the molecular substrates of addiction and disease. However, most studies of heterologously expressed neuronal nAChR have used clones obtained from other species, usually human or rat. In this work, we use mouse clones expressed in Xenopus oocytes to provide a relatively comprehensive characterization of the three primary classes of nAChR: muscle-type receptors, heteromeric neuronal receptors, and homomeric alpha7-type receptors. We evaluated the activation of these receptor subtypes with acetylcholine and cytisine-related compounds, including varenicline. We also characterized the activity of classic nAChR antagonists, confirming the utility of mecamylamine and dihydro-beta-erythroidine as selective antagonists in mouse models of alpha3beta4 and alpha4beta2 receptors, respectively. We also conducted an in-depth analysis of decamethonium and hexamethonium on muscle and neuronal receptor subtypes. Our data indicate that, as with receptors cloned from other species, pairwise expression of neuronal alpha and beta subunits in oocytes generates heterogeneous populations of receptors, most likely caused by variations in subunit stoichiometry. Coexpression of the mouse alpha5 subunit had varying effects, depending on the other subunits expressed. The properties of cytisine-related compounds are similar for mouse, rat, and human nAChR, except that varenicline produced greater residual inhibition of mouse alpha4beta2 receptors than with human receptors. We confirm that decamethonium is a partial agonist, selective for muscle-type receptors, but also note that it is a nondepolarizing antagonist for neuronal-type receptors. Hexamethonium was a relatively nonselective antagonist with mixed competitive and noncompetitive activity.

Activation and Inhibition of Mouse Muscle and Neuronal Nicotinic Acetylcholine Receptors Expressed in Xenopus Oocytes

PubMed Central

Wecker, Lynn; Stitzel, Jerry A.

2010-01-01

Transgenic mouse models with nicotinic acetylcholine receptor (nAChR) knockouts and knockins have provided important insights into the molecular substrates of addiction and disease. However, most studies of heterologously expressed neuronal nAChR have used clones obtained from other species, usually human or rat. In this work, we use mouse clones expressed in Xenopus oocytes to provide a relatively comprehensive characterization of the three primary classes of nAChR: muscle-type receptors, heteromeric neuronal receptors, and homomeric α7-type receptors. We evaluated the activation of these receptor subtypes with acetylcholine and cytisine-related compounds, including varenicline. We also characterized the activity of classic nAChR antagonists, confirming the utility of mecamylamine and dihydro-β-erythroidine as selective antagonists in mouse models of α3β4 and α4β2 receptors, respectively. We also conducted an in-depth analysis of decamethonium and hexamethonium on muscle and neuronal receptor subtypes. Our data indicate that, as with receptors cloned from other species, pairwise expression of neuronal α and β subunits in oocytes generates heterogeneous populations of receptors, most likely caused by variations in subunit stoichiometry. Coexpression of the mouse α5 subunit had varying effects, depending on the other subunits expressed. The properties of cytisine-related compounds are similar for mouse, rat, and human nAChR, except that varenicline produced greater residual inhibition of mouse α4β2 receptors than with human receptors. We confirm that decamethonium is a partial agonist, selective for muscle-type receptors, but also note that it is a nondepolarizing antagonist for neuronal-type receptors. Hexamethonium was a relatively nonselective antagonist with mixed competitive and noncompetitive activity. PMID:20100906

A conserved role of αA-crystallin in the development of the zebrafish embryonic lens.

PubMed

Zou, Ping; Wu, Shu-Yu; Koteiche, Hanane A; Mishra, Sanjay; Levic, Daniel S; Knapik, Ela; Chen, Wenbiao; Mchaourab, Hassane S

2015-09-01

αA- and αB-crystallins are small heat shock proteins that bind thermodynamically destabilized proteins thereby inhibiting their aggregation. Highly expressed in the mammalian lens, the α-crystallins have been postulated to play a critical role in the maintenance of lens optical properties by sequestering age-damaged proteins prone to aggregation as well as through a multitude of roles in lens epithelial cells. Here, we have examined the role of α-crystallins in the development of the vertebrate zebrafish lens. For this purpose, we have carried out morpholino-mediated knockdown of αA-, αBa- and αBb-crystallin and characterized the gross morphology of the lens. We observed lens abnormalities, including increased reflectance intensity, as a consequence of the interference with expression of these proteins. These abnormalities were less frequent in transgenic zebrafish embryos expressing rat αA-crystallin suggesting a specific role of α-crystallins in embryonic lens development. To extend and confirm these findings, we generated an αA-crystallin knockout zebrafish line. A more consistent and severe lens phenotype was evident in maternal/zygotic αA-crystallin mutants compared to those observed by morpholino knockdown. The penetrance of the lens phenotype was reduced by transgenic expression of rat αA-crystallin and its severity was attenuated by maternal αA-crystallin expression. These findings demonstrate that the role of α-crystallins in lens development is conserved from mammals to zebrafish and set the stage for using the embryonic lens as a model system to test mechanistic aspects of α-crystallin chaperone activity and to develop strategies to fine-tune protein-protein interactions in aging and cataracts. Copyright © 2015 Elsevier Ltd. All rights reserved.

Biotinidase knockout mice show cellular energy deficit and altered carbon metabolism gene expression similar to that of nutritional biotin deprivation: clues for the pathogenesis in the human inherited disorder.

PubMed

Hernández-Vázquez, A; Wolf, B; Pindolia, K; Ortega-Cuellar, D; Hernández-González, R; Heredia-Antúnez, A; Ibarra-González, I; Velázquez-Arellano, A

2013-11-01

Biotin is the prosthetic group of carboxylases that have important roles in the metabolism of glucose, fatty acids and amino acids. Biotinidase has a key role in the reutilization of the biotin, catalyzing the hydrolysis of biocytin (ε-N-biotinyl-l-lysine) and biocytin-containing peptides derived from carboxylase turnover, thus contributing substantially to the bioavailability of this vitamin. Deficient activity of biotinidase causes late-onset multiple carboxylase in humans, whose pathogenic mechanisms are poorly understood. Here we show that a knock-out biotinidase-deficient mouse from a C57BL/6 background that was fed a low biotin diet develops severe ATP deficit with activation of the energy sensor adenosine monophosphate (AMP)-activated protein kinase (AMPK), inhibition of the signaling protein mTOR, driver of protein synthesis and growth, and affecting the expression of central-carbon metabolism genes. In addition, sensitivity to insulin is augmented. These changes are similar to those observed in nutritionally biotin-starved rats. These findings further our understanding of the pathogenesis of human biotinidase deficiency. © 2013 Elsevier Inc. All rights reserved.

Generation of Newly Discovered Resistance Gene mcr-1 Knockout in Escherichia coli Using the CRISPR/Cas9 System.

PubMed

Sun, Lichang; He, Tao; Zhang, Lili; Pang, Maoda; Zhang, Qiaoyan; Zhou, Yan; Bao, Hongduo; Wang, Ran

2017-07-28

The mcr-1 gene is a new "superbug" gene discoverd in China in 2016 that makes bacteria highly resistant to the last-resort class of antibiotics. The mcr-1 gene raised serious concern about its possible global dissemination and spread. Here, we report a potential anti-resistant strategy using the CRISPR/Cas9-mediated approach that can efficiently induce mcr-1 gene knockout in Escherichia coli . Our findings suggested that using the CRISPR/Cas9 system to knock out the resistance gene mcr-1 might be a potential anti-resistant strategy. Bovine myeloid antimicrobial peptide-27 could help deliver plasmid pCas::mcr targeting specific DNA sequences of the mcr-1 gene into microbial populations.

Knockout of an outer membrane protein operon of anaplasma marginale by transposon mutagenesis

USDA-ARS?s Scientific Manuscript database

Large amounts of data generated by genomics, transcriptomics and proteomics technologies have increased our understanding of the biology of Anaplasma marginale. However, these data have also led to new assumptions that require testing, ideally through classic genetic mutation. One example is the def...

Acute and chronic suppression of the central ghrelin signaling system reveals a role in food anticipatory activity.

PubMed

Verhagen, Linda A W; Egecioglu, Emil; Luijendijk, Mieneke C M; Hillebrand, Jacquelien J G; Adan, Roger A H; Dickson, Suzanne L

2011-05-01

Using the rodent activity-based anorexia (ABA) model that mimics clinical features of anorexia nervosa that include food restriction-induced hyperlocomotion, we found that plasma ghrelin levels are highly associated with food anticipatory behaviour, measured by running wheel activity in rats. Furthermore, we showed that ghrelin receptor (GHS-R1A) knockout mice do not anticipate food when exposed to the ABA model, unlike their wild type littermate controls. Likewise, food anticipatory activity in the ABA model was suppressed by a GHS-R1A antagonist administered either by acute central (ICV) injection to rats or by chronic peripheral treatment to mice. Interestingly, the GHS-R1A antagonist did not alter food intake in any of these models. Therefore, we hypothesize that suppression of the central ghrelin signaling system via GHS-R1A provides an interesting therapeutic target to treat hyperactivity in patients suffering from anorexia nervosa. Copyright © 2010. Published by Elsevier B.V.

Critical roles of TRPV2 channels, histamine H1 and adenosine A1 receptors in the initiation of acupoint signals for acupuncture analgesia.

PubMed

Huang, Meng; Wang, Xuezhi; Xing, Beibei; Yang, Hongwei; Sa, Zheyan; Zhang, Di; Yao, Wei; Yin, Na; Xia, Ying; Ding, Guanghong

2018-04-25

Acupuncture is one of the most promising modalities in complimentary medicine. However, the underlying mechanisms are not well understood yet. We found that in TRPV2 knockout male mice, acupuncture-induced analgesia was suppressed with a decreased activation of mast cells in the acupoints stimulated. The mast cell stabilizer sodium cromolyn could suppress the release of adenosine in the acupoints on male rats. A direct injection of adenosine A1 receptor agonist or histamine H1 receptor agonist increased β-endorphin in the cerebral-spinal fluid in the acute adjuvant arthritis male rats and thus replicated the analgesic effect of acupuncture. These observations suggest that the mast cell is the central structure of acupoints and is activated by acupuncture through TRPV2 channels. The mast cell transduces the mechanical stimuli to acupuncture signal by activating either H1 or A1 receptors, therefore triggering the acupuncture effect in the subject. These findings might open new frontiers for acupuncture research.

Generation of insulin-deficient piglets by disrupting INS gene using CRISPR/Cas9 system.

PubMed

Cho, Bumrae; Kim, Su Jin; Lee, Eun-Jin; Ahn, Sun Mi; Lee, Jin Seok; Ji, Dal-Young; Lee, Kiho; Kang, Jung-Taek

2018-06-01

Diabetes mellitus is a chronic disease with accompanying severe complications. Various animal models, mostly rodents due to availability of genetically modified lines, have been used to investigate the pathophysiology of diabetes. Using pigs for diabetic research can be beneficial because of their similarity in size, pathogenesis pathway, physiology, and metabolism with human. However, the use of pigs for diabetes research has been hampered due to only few pig models presenting diabetes symptoms. In this study, we have successfully generated insulin-deficient pigs by generating the indels of the porcine INS gene in somatic cells using CRISPR/Cas9 system followed by somatic cell nuclear transfer. First, somatic cells carrying a modified INS gene were generated using CRISPR/Cas9 system and their genotypes were confirmed by T7E1 assay; targeting efficiency was 40.4% (21/52). After embryo transfer, three live and five stillborn piglets were born. As expected, INS knockout piglets presented high blood glucose levels and glucose was detected in the urine. The level of insulin and c-peptide in the blood serum of INS knockout piglets were constant after feeding and the expression of insulin in the pancreas was absent in those piglets. This study demonstrates effectiveness of CRISPR/Cas9 system in generating novel pig models. We expect that these insulin-deficient pigs can be used in diabetes research to test the efficacy and safety of new drugs and the recipient of islet transplantation to investigate optimal transplantation strategies.

A human relevance investigation of PPARα-mediated key events in the hepatocarcinogenic mode of action of propaquizafop in rats.

PubMed

Strupp, Christian; Bomann, Werner H; Spézia, François; Gervais, Frédéric; Forster, Roy; Richert, Lysiane; Singh, Pramila

2018-06-01

Propaquizafop is an herbicide with demonstrated hepatocarcinogenic activity in rodents. A rodent-specific mode of action (MOA) in the liver via activation of peroxisome proliferator-activated receptor α (PPARα) has been postulated based on existing data. Experience with PPARα-inducing pharmaceuticals indicates a lack of human relevance of this MOA. The objective of the present investigation was to evaluate the dependency of early key events leading to liver tumors on PPARα activation in wildtype (WT) compared to PPARα-knockout (KO) rats following 2 weeks exposure to 75, 500 and 1000 ppm propaquizafop in the diet. In WT rats, both WY-14643 (50 mg/kg bw/day) and propaquizafop (dose-dependently) induced marked increases in liver weights, correlating with liver enlargement and hepatocellular hypertrophy, along with increased CYP4A and acyl-CoA oxidase mRNA expression and enzyme activities versus controls, while in KO rats liver weight was mildly increased only at the high dose with minimal microscopic correlates and without any changes in liver peroxisomal or CYP4A activities. In addition, BrdU labeling resulted in higher numbers and density of positive hepatocytes versus controls in WT but not in KO rats, indicating increased mitotic activity and cell proliferation only in WT rats, thus confirming the PPARα-dependency of the biochemical and histological changes in the liver. Based on an assessment of the results of this investigation, together with existing propaquizafop data according to the MOA-Human Relevance Framework, we conclude that liver tumors observed in rodents after dietary administration of propaquizafop do not pose a relevant health risk to humans. Copyright © 2018 Elsevier Inc. All rights reserved.

Immunological characterization of the aryl hydrocarbon receptor (AHR) knockout rat in the presence and absence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

PubMed

Phadnis-Moghe, Ashwini S; Chen, Weimin; Li, Jinpeng; Crawford, Robert B; Bach, Anthony; D'Ingillo, Shawna; Kovalova, Natalia; Suarez-Martinez, Jose E; Kaplan, Barbara L F; Harrill, Joshua A; Budinsky, Robert; Rowlands, J Craig; Thomas, Russell S; Kaminski, Norbert E

2016-08-10

The aryl hydrocarbon receptor (AHR) has been extensively characterized for the essential role it plays in mediating the toxic responses elicited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Despite similarities across animal species, species-specific differences exist in the profile of toxicity and sensitivity to TCDD owing, in part, to differences in the AHR. Newer reports have implicated the importance of AHR in the development and regulation of the immune system. Our present studies seek to further explore the essential role of AHR in lymphoid tissue composition, B cell function and the immunological responses after TCDD administration using the recently established AHR KO rats. Comprehensive immune cell phenotyping showed a decrease in the CD8 + T cell, CD11c + populations and an increase in NKT cells in 3-week-old AHR KO rats compared to the WT controls. The lipopolysaccharide-induced IgM response and proliferation was markedly suppressed in the WT but not in the AHR KO B cells in the presence of TCDD. However, the percentage of LPS-activated IgM + B cells was significantly higher in the AHR KO B cells as compared to that of WT suggesting the role of AHR in regulating the IgM response. The use of an AHR antagonist further alluded to the endogenous role of AHR in regulating B cell responses in the rat. Overall, the studies report for the first time, comprehensive immune cell phenotyping of the AHR KO rat and the endogenous role of AHR in the regulation of B cell function in the rat. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Generation of Xeroderma Pigmentosum-A Patient-Derived Induced Pluripotent Stem Cell Line for Use As Future Disease Model.

PubMed

Ohnishi, Hiroe; Kawasaki, Takashi; Deguchi, Tomonori; Yuba, Shunsuke

2015-08-01

Xeroderma pigmentosum group A (XP-A) is a genetic disorder in which there is an abnormality in nucleotide excision repair that causes hypersensitivity to sunlight and multiple skin cancers. The development of central and peripheral neurological disorders not correlated to ultraviolet light exposure is associated with XP-A. The genes responsible for XP-A have been identified and a XPA knockout mouse has been generated. These knockout mice exhibit cutaneous symptoms, but they do not show neurological disorders. The mechanism of pathogenesis of neurological disorders is still unclear and therapeutic methods have not been established. Therefore, we generated XP-A patient-derived human induced pluripotent stem cells (XPA-iPSCs) to produce in vitro models of neurological disorders. We obtained iPSC lines from fibroblasts of two patients carrying different mutations. Drugs screened using XPA-iPSC lines can be helpful for treating XP-A patients in Japan. Additionally, we revealed that these iPSCs have the potential to differentiate into neural lineage cells, including dopaminergic neurons, which decrease in XP-A patients. Our results indicate that expression of the normal XPA gene without mutations is not required for generation of iPSCs and differentiation of iPSCs into neural lineage cells. XPA-iPSCs may become useful models that clarify our understanding of neurological pathogenesis and help to establish therapeutic methods.

Tubule-Derived Wnts Are Required for Fibroblast Activation and Kidney Fibrosis.

PubMed

Zhou, Dong; Fu, Haiyan; Zhang, Lu; Zhang, Ke; Min, Yali; Xiao, Liangxiang; Lin, Lin; Bastacky, Sheldon I; Liu, Youhua

2017-08-01

Cell-cell communication via Wnt ligands is necessary in regulating embryonic development and has been implicated in CKD. Because Wnt ligands are ubiquitously expressed, the exact cellular source of the Wnts involved in CKD remains undefined. To address this issue, we generated two conditional knockout mouse lines in which Wntless (Wls), a dedicated cargo receptor that is obligatory for Wnt secretion, was selectively ablated in tubular epithelial cells or interstitial fibroblasts. Blockade of Wnt secretion by genetic deletion of Wls in renal tubules markedly inhibited myofibroblast activation and reduced renal fibrosis after unilateral ureteral obstruction. This effect associated with decreased activation of β -catenin and downstream gene expression and preserved tubular epithelial integrity. In contrast, fibroblast-specific deletion of Wls exhibited little effect on the severity of renal fibrosis after obstructive or ischemia-reperfusion injury. In vitro , incubation of normal rat kidney fibroblasts with tubule-derived Wnts promoted fibroblast proliferation and activation. Furthermore, compared with kidney specimens from patients without CKD, biopsy specimens from patients with CKD also displayed increased expression of multiple Wnt proteins, predominantly in renal tubular epithelium. These results illustrate that tubule-derived Wnts have an essential role in promoting fibroblast activation and kidney fibrosis via epithelial-mesenchymal communication. Copyright © 2017 by the American Society of Nephrology.

Absence of Tec Family Kinases Interleukin-2 Inducible T cell Kinase (Itk) and Bruton's Tyrosine Kinase (Btk) Severely Impairs FcϵRI-dependent Mast Cell Responses*

PubMed Central

Iyer, Archana S.; Morales, J. Luis; Huang, Weishan; Ojo, Folake; Ning, Gang; Wills, Elizabeth; Baines, Joel D.; August, Avery

2011-01-01

Mast cells are critical effector cells in the pathophysiology of allergic asthma and other IgE-mediated diseases. The Tec family of tyrosine kinases Itk and Btk serve as critical signal amplifiers downstream of antigen receptors. Although both kinases are expressed and activated in mast cells following FcϵRI stimulation, their individual contributions are not clear. To determine whether these kinases play unique and/or complementary roles in FcϵRI signaling and mast cell function, we generated Itk and Btk double knock-out mice. Analyses of these mice show decreased mast cell granularity and impaired passive systemic anaphylaxis responses. This impaired response is accompanied by a significant elevation in serum IgE in Itk/Btk double knock-out mice. In vitro analyses of bone marrow-derived mast cells (BMMCs) indicated that Itk/Btk double knock-out BMMCs are defective in degranulation and cytokine secretion responses downstream to FcϵRI activation. These responses were accompanied by a significant reduction in PLCγ2 phosphorylation and severely impaired calcium responses in these cells. This defect also results in altered NFAT1 nuclear localization in double knock-out BMMCs. Network analysis suggests that although they may share substrates, Itk plays both positive and negative roles, while Btk primarily plays a positive role in mast cell FcϵRI-induced cytokine secretion. PMID:21212279

Colocalization of dentin matrix protein 1 and dentin sialoprotein at late stages of rat molar development.

PubMed

Baba, Otto; Qin, Chunlin; Brunn, Jan C; Wygant, James N; McIntyre, Bradley W; Butler, William T

2004-10-01

Dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) are acidic proteins found in the extracellular matrices of bones and teeth. Recent data from gene knockouts, along with those of gene mutations, indicate that these two phosphoproteins are critical for bone and tooth development and/or maintenance. However, the precise functions of the two proteins have not been elucidated. In order to gain insights into their functions in tooth formation, we performed systematic, comparative investigations on the immunolocalization of DMP1 and dentin sialoprotein (DSP, a cleaved fragment of DSPP), using the rat first molar at different developmental stages as a model. Immunohistochemistry (IHC) was performed with specific, monoclonal antibodies against the COOH-terminal fragments of DMP1 and against DSP. In 1-day- and 1-week-old rats, weak immunoreactions for DMP1 were observed in dentinal tubules while stronger reactions for DSP were seen in the tubules and predentin. In rats older than 2 weeks, immunoreactions for DMP1 were found in dentinal tubules, predentin and odontoblasts. In 5-week- and 8-week-old rats, strong immunoreactions for DMP1 were widely distributed in odontoblasts and predentin. The distribution pattern of DSP was strikingly similar to that of DMP1 after 2 weeks and the localization of each was distinctly different from that of bone sialoprotein (BSP). The unique colocalization of DMP1 and DSPP in tooth development suggests that the two proteins play complementary and/or synergistic roles in formation and maintenance of healthy teeth.

Hypoxia-Induced Mitogenic Factor Promotes Cardiac Hypertrophy via Calcium-Dependent and Hypoxia-Inducible Factor-1α Mechanisms.

PubMed

Kumar, Santosh; Wang, Gang; Liu, Wenjuan; Ding, Wenwen; Dong, Ming; Zheng, Na; Ye, Hongyu; Liu, Jie

2018-06-11

HIMF (hypoxia-induced mitogenic factor/found in inflammatory zone 1/resistin like α) is a secretory and cytokine-like protein and serves as a critical stimulator of hypoxia-induced pulmonary hypertension. With a role for HIMF in heart disease unknown, we explored the possible roles for HIMF in cardiac hypertrophy by overexpressing and knocking down HIMF in cardiomyocytes and characterizing HIMF gene ( himf ) knockout mice. We found that HIMF mRNA and protein levels were upregulated in phenylephrine-stimulated cardiomyocyte hypertrophy and our mouse model of transverse aortic constriction-induced cardiac hypertrophy, as well as in human hearts with dilated cardiomyopathy. Furthermore, HIMF overexpression could induce cardiomyocyte hypertrophy, as characterized by elevated protein expression of hypertrophic biomarkers (ANP [atrial natriuretic peptide] and β-MHC [myosin heavy chain-β]) and increased cell-surface area compared with controls. Conversely, HIMF knockdown prevented phenylephrine-induced cardiomyocyte hypertrophy and himf ablation in knockout mice significantly attenuated transverse aortic constriction-induced hypertrophic remodeling and cardiac dysfunction. HIMF overexpression increased the cytosolic Ca 2+ concentration and activated the CaN-NFAT (calcineurin-nuclear factor of activated T cell) and MAPK (mitogen-activated protein kinase) pathways; this effect could be prevented by reducing cytosolic Ca 2+ concentration with L-type Ca 2+ channel blocker nifedipine or inhibiting the CaSR (Ca 2+ sensing receptor) with Calhex 231. Furthermore, HIMF overexpression increased HIF-1α (hypoxia-inducible factor) expression in neonatal rat ventricular myocytes, and HIMF knockout inhibited HIF-1α upregulation in transverse aortic constriction mice. Knockdown of HIF-1α attenuated HIMF-induced cardiomyocyte hypertrophy. In conclusion, HIMF has a critical role in the development of cardiac hypertrophy, and targeting HIMF may represent a potential therapeutic strategy. © 2018 American Heart Association, Inc.

Comparative effects of chlorpyrifos in wild type and cannabinoid Cb1 receptor knockout mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baireddy, Praveena; Liu, Jing; Hinsdale, Myron

2011-11-15

Endocannabinoids (eCBs) modulate neurotransmission by inhibiting the release of a variety of neurotransmitters. The cannabinoid receptor agonist WIN 55.212-2 (WIN) can modulate organophosphorus (OP) anticholinesterase toxicity in rats, presumably by inhibiting acetylcholine (ACh) release. Some OP anticholinesterases also inhibit eCB-degrading enzymes. We studied the effects of the OP insecticide chlorpyrifos (CPF) on cholinergic signs of toxicity, cholinesterase activity and ACh release in tissues from wild type (+/+) and cannabinoid CB1 receptor knockout (-/-) mice. Mice of both genotypes (n = 5-6/treatment group) were challenged with CPF (300 mg/kg, 2 ml/kg in peanut oil, sc) and evaluated for functional and neurochemicalmore » changes. Both genotypes exhibited similar cholinergic signs and cholinesterase inhibition (82-95% at 48 h after dosing) in cortex, cerebellum and heart. WIN reduced depolarization-induced ACh release in vitro in hippocampal slices from wild type mice, but had no effect in hippocampal slices from knockouts or in striatal slices from either genotype. Chlorpyrifos oxon (CPO, 100 {mu}M) reduced release in hippocampal slices from both genotypes in vitro, but with a greater reduction in tissues from wild types (21% vs 12%). CPO had no significant in vitro effect on ACh release in striatum. CPF reduced ACh release in hippocampus from both genotypes ex vivo, but reduction was again significantly greater in tissues from wild types (52% vs 36%). In striatum, CPF led to a similar reduction (20-23%) in tissues from both genotypes. Thus, while CB1 deletion in mice had little influence on the expression of acute toxicity following CPF, CPF- or CPO-induced changes in ACh release appeared sensitive to modulation by CB1-mediated eCB signaling in a brain-regional manner. -- Highlights: Black-Right-Pointing-Pointer C57Bl/6 mice showed dose-related cholinergic toxicity following subcutaneous chlorpyrifos exposure. Black-Right-Pointing-Pointer Wild type and cannabinoid CB1 receptor knockout littermates responded similarly to the toxic effects of chlorpyrifos. Black-Right-Pointing-Pointer OP-induced changes in acetylcholine release appeared sensitive to modulation by CB1-mediated endocannabinoid signaling.« less

ENU mutagenesis to generate genetically modified rat models.

PubMed

van Boxtel, Ruben; Gould, Michael N; Cuppen, Edwin; Smits, Bart M G

2010-01-01

The rat is one of the most preferred model organisms in biomedical research and has been extremely useful for linking physiology and pathology to the genome. However, approaches to genetically modify specific genes in the rat germ line remain relatively scarce. To date, the most efficient approach for generating genetically modified rats has been the target-selected N-ethyl-N-nitrosourea (ENU) mutagenesis-based technology. Here, we describe the detailed protocols for ENU mutagenesis and mutant retrieval in the rat model organism.

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells.

PubMed

Sternburg, Erin L; Dias, Kristen C; Karginov, Fedor V

2017-06-16

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide RNA (sgRNA) that confers specificity, the Cas9 protein cleaves both DNA strands at the targeted locus. The DNA break can trigger either non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ can introduce small deletions or insertions which lead to frame-shift mutations, while HDR allows for larger and more precise perturbations. Here, we present protocols for generating knockout cell lines by coupling established CRISPR/Cas9 methods with two options for downstream selection/screening. The NHEJ approach uses a single sgRNA cut site and selection-independent screening, where protein production is assessed by dot immunoblot in a high-throughput manner. The HDR approach uses two sgRNA cut sites that span the gene of interest. Together with a provided HDR template, this method can achieve deletion of tens of kb, aided by the inserted selectable resistance marker. The appropriate applications and advantages of each method are discussed.

Generation and Characterization of Transgenic Mice Expressing Mouse Ins1 Promoter for Pancreatic β-Cell-Specific Gene Overexpression and Knockout.

PubMed

Cheng, Yulong; Su, Yutong; Shan, Aijing; Jiang, Xiuli; Ma, Qinyun; Wang, Weiqing; Ning, Guang; Cao, Yanan

2015-07-01

The technologies for pancreatic β-cell-specific gene overexpression or knockout are fundamental for investigations of functional genes in vivo. Here we generated the Ins1-Cre-Dsred and Ins1-rtTA mouse models, which expressed the Cre recombinase or reverse tetracycline regulatable transactivator (rtTA) without hGH minigene under the control of mouse Ins1 promoter. Our data showed that the Cre-mediated recombination and rtTA-mediated activation could be efficiently detected at embryonic day 13.5 when these models were crossed with the reporter mice (ROSA(mT/mG) or tetO-HIST1H2BJ/GFP). The Cre and rtTA expression was restricted to β-cells without leakage in the brain and other tissues. Moreover, both the transgenic lines showed normal glucose tolerance and insulin secretion. These results suggested that the Ins1-Cre-Dsred and Ins1-rtTA mice could be used to knock out or overexpress target genes in embryos and adults to facilitate β-cell researches.

Analysis of Foxo1-regulated genes using Foxo1-deficient pancreatic β cells.

PubMed

Miyazaki, Satsuki; Minamida, Rie; Furuyama, Tatsuo; Tashiro, Fumi; Yamato, Eiji; Inagaki, Shinobu; Miyazaki, Jun-ichi

2012-09-01

Several reports have suggested that Foxo1, a key regulator in differentiation, growth and metabolism, is involved in pancreatic β-cell function. However, detailed analyses have been hampered by a lack of Foxo1-deficient β cells. To elucidate Foxo1's function in β cells, we produced a β-cell line with inducible Foxo1 deletion. We generated a conditional knockout mouse line, in which Cre recombinase deletes the Foxo1 gene. We then established a β-cell line from an insulinoma induced in this knockout mouse by the β-cell-specific expression of simian virus 40 T antigen. In this cell line, designated MIN6-Foxo1flox/flox, adenovirus-mediated Cre expression ablates the Foxo1 gene, generating MIN6-Foxo1-KO cells. Using these knockout and floxed cell lines, we found that Foxo1 ablation enhanced the glucose-stimulated insulin secretion (GSIS) at high glucose concentrations and enhanced β-cell proliferation. We also conducted DNA microarray analyses of MIN6-Foxo1-KO cells infected with either an adenovirus vector expressing a constitutively active FOXO1 or a control vector and identified several Foxo1-regulated genes, including some known to be related to β-cell function. These cells should be useful for further studies on Foxo1's roles in β-cells and may lead to novel strategies for treating the impaired insulin secretion in type 2 diabetes mellitus. © 2012 The Authors Journal compilation © 2012 by the Molecular Biology Society of Japan/Wiley Publishing Ltd.

Genecentric: a package to uncover graph-theoretic structure in high-throughput epistasis data.

PubMed

Gallant, Andrew; Leiserson, Mark D M; Kachalov, Maxim; Cowen, Lenore J; Hescott, Benjamin J

2013-01-18

New technology has resulted in high-throughput screens for pairwise genetic interactions in yeast and other model organisms. For each pair in a collection of non-essential genes, an epistasis score is obtained, representing how much sicker (or healthier) the double-knockout organism will be compared to what would be expected from the sickness of the component single knockouts. Recent algorithmic work has identified graph-theoretic patterns in this data that can indicate functional modules, and even sets of genes that may occur in compensatory pathways, such as a BPM-type schema first introduced by Kelley and Ideker. However, to date, any algorithms for finding such patterns in the data were implemented internally, with no software being made publically available. Genecentric is a new package that implements a parallelized version of the Leiserson et al. algorithm (J Comput Biol 18:1399-1409, 2011) for generating generalized BPMs from high-throughput genetic interaction data. Given a matrix of weighted epistasis values for a set of double knock-outs, Genecentric returns a list of generalized BPMs that may represent compensatory pathways. Genecentric also has an extension, GenecentricGO, to query FuncAssociate (Bioinformatics 25:3043-3044, 2009) to retrieve GO enrichment statistics on generated BPMs. Python is the only dependency, and our web site provides working examples and documentation. We find that Genecentric can be used to find coherent functional and perhaps compensatory gene sets from high throughput genetic interaction data. Genecentric is made freely available for download under the GPLv2 from http://bcb.cs.tufts.edu/genecentric.

Knockout of exogenous EGFP gene in porcine somatic cells using zinc-finger nucleases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watanabe, Masahito; Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571; Umeyama, Kazuhiro

2010-11-05

Research highlights: {yields} EGFP gene integrated in porcine somatic cells could be knocked out using the ZFN-KO system. {yields} ZFNs induced targeted mutations in porcine primary cultured cells. {yields} Complete absence of EGFP fluorescence was confirmed in ZFN-treated cells. -- Abstract: Zinc-finger nucleases (ZFNs) are expected as a powerful tool for generating gene knockouts in laboratory and domestic animals. Currently, it is unclear whether this technology can be utilized for knocking-out genes in pigs. Here, we investigated whether knockout (KO) events in which ZFNs recognize and cleave a target sequence occur in porcine primary cultured somatic cells that harbor themore » exogenous enhanced green fluorescent protein (EGFP) gene. ZFN-encoding mRNA designed to target the EGFP gene was introduced by electroporation into the cell. Using the Surveyor nuclease assay and flow cytometric analysis, we confirmed ZFN-induced cleavage of the target sequence and the disappearance of EGFP fluorescence expression in ZFN-treated cells. In addition, sequence analysis revealed that ZFN-induced mutations such as base substitution, deletion, or insertion were generated in the ZFN cleavage site of EGFP-expression negative cells that were cloned from ZFN-treated cells, thereby showing it was possible to disrupt (i.e., knock out) the function of the EGFP gene in porcine somatic cells. To our knowledge, this study provides the first evidence that the ZFN-KO system can be applied to pigs. These findings may open a new avenue to the creation of gene KO pigs using ZFN-treated cells and somatic cell nuclear transfer.« less

Genecentric: a package to uncover graph-theoretic structure in high-throughput epistasis data

PubMed Central

2013-01-01

Background New technology has resulted in high-throughput screens for pairwise genetic interactions in yeast and other model organisms. For each pair in a collection of non-essential genes, an epistasis score is obtained, representing how much sicker (or healthier) the double-knockout organism will be compared to what would be expected from the sickness of the component single knockouts. Recent algorithmic work has identified graph-theoretic patterns in this data that can indicate functional modules, and even sets of genes that may occur in compensatory pathways, such as a BPM-type schema first introduced by Kelley and Ideker. However, to date, any algorithms for finding such patterns in the data were implemented internally, with no software being made publically available. Results Genecentric is a new package that implements a parallelized version of the Leiserson et al. algorithm (J Comput Biol 18:1399-1409, 2011) for generating generalized BPMs from high-throughput genetic interaction data. Given a matrix of weighted epistasis values for a set of double knock-outs, Genecentric returns a list of generalized BPMs that may represent compensatory pathways. Genecentric also has an extension, GenecentricGO, to query FuncAssociate (Bioinformatics 25:3043-3044, 2009) to retrieve GO enrichment statistics on generated BPMs. Python is the only dependency, and our web site provides working examples and documentation. Conclusion We find that Genecentric can be used to find coherent functional and perhaps compensatory gene sets from high throughput genetic interaction data. Genecentric is made freely available for download under the GPLv2 from http://bcb.cs.tufts.edu/genecentric. PMID:23331614

Cytochrome c oxidase subunit 4 isoform 2-knockout mice show reduced enzyme activity, airway hyporeactivity, and lung pathology

PubMed Central

Hüttemann, Maik; Lee, Icksoo; Gao, Xiufeng; Pecina, Petr; Pecinova, Alena; Liu, Jenney; Aras, Siddhesh; Sommer, Natascha; Sanderson, Thomas H.; Tost, Monica; Neff, Frauke; Aguilar-Pimentel, Juan Antonio; Becker, Lore; Naton, Beatrix; Rathkolb, Birgit; Rozman, Jan; Favor, Jack; Hans, Wolfgang; Prehn, Cornelia; Puk, Oliver; Schrewe, Anja; Sun, Minxuan; Höfler, Heinz; Adamski, Jerzy; Bekeredjian, Raffi; Graw, Jochen; Adler, Thure; Busch, Dirk H.; Klingenspor, Martin; Klopstock, Thomas; Ollert, Markus; Wolf, Eckhard; Fuchs, Helmut; Gailus-Durner, Valérie; Hrabě de Angelis, Martin; Weissmann, Norbert; Doan, Jeffrey W.; Bassett, David J. P.; Grossman, Lawrence I.

2012-01-01

Cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial electron transport chain. The purpose of this study was to analyze the function of lung-specific cytochrome c oxidase subunit 4 isoform 2 (COX4i2) in vitro and in COX4i2-knockout mice in vivo. COX was isolated from cow lung and liver as control and functionally analyzed. COX4i2-knockout mice were generated and the effect of the gene knockout was determined, including COX activity, tissue energy levels, noninvasive and invasive lung function, and lung pathology. These studies were complemented by a comprehensive functional screen performed at the German Mouse Clinic (Neuherberg, Germany). We show that isolated cow lung COX containing COX4i2 is about twice as active (88 and 102% increased activity in the presence of allosteric activator ADP and inhibitor ATP, respectively) as liver COX, which lacks COX4i2. In COX4i2-knockout mice, lung COX activity and cellular ATP levels were significantly reduced (−50 and −29%, respectively). Knockout mice showed decreased airway responsiveness (60% reduced Penh and 58% reduced airway resistance upon challenge with 25 and 100 mg methacholine, respectively), and they developed a lung pathology deteriorating with age that included the appearance of Charcot-Leyden crystals. In addition, there was an interesting sex-specific phenotype, in which the knockout females showed reduced lean mass (−12%), reduced total oxygen consumption rate (−8%), improved glucose tolerance, and reduced grip force (−14%) compared to wild-type females. Our data suggest that high activity lung COX is a central determinant of airway function and is required for maximal airway responsiveness and healthy lung function. Since airway constriction requires energy, we propose a model in which reduced tissue ATP levels explain protection from airway hyperresponsiveness, i.e., absence of COX4i2 leads to reduced lung COX activity and ATP levels, which results in impaired airway constriction and thus reduced airway responsiveness; long-term lung pathology develops in the knockout mice due to impairment of energy-costly lung maintenance processes; and therefore, we propose mitochondrial oxidative phosphorylation as a novel target for the treatment of respiratory diseases, such as asthma.—Hüttemann, M., Lee, I., Gao, X., Pecina, P., Pecinova, A., Liu, J., Aras, S., Sommer, N., Sanderson, T. H., Tost, M., Neff, F., Aguilar-Pimentel, J. A., Becker, L., Naton, B., Rathkolb, B., Rozman, J., Favor, J., Hans, W., Prehn, C., Puk, O., Schrewe, A., Sun, M., Höfler, H., Adamski, J., Bekeredjian, R., Graw, J., Adler, T., Busch, D. H., Klingenspor, M., Klopstock, T., Ollert, M., Wolf, E., Fuchs, H., Gailus-Durner, V., Hrabě de Angelis, M., Weissmann, N., Doan, J. W., Bassett, D. J. P., Grossman, L. I. Cytochrome c oxidase subunit 4 isoform 2-knockout mice show reduced enzyme activity, airway hyporeactivity, and lung pathology. PMID:22730437

Isozygous and selectable marker-free MSTN knockout cloned pigs generated by the combined use of CRISPR/Cas9 and Cre/LoxP.

PubMed

Bi, Yanzhen; Hua, Zaidong; Liu, Ximei; Hua, Wenjun; Ren, Hongyan; Xiao, Hongwei; Zhang, Liping; Li, Li; Wang, Zhirui; Laible, Götz; Wang, Yan; Dong, Faming; Zheng, Xinmin

2016-08-17

Predictable, clean genetic modification (GM) in livestock is important for reliable phenotyping and biosafety. Here we reported the generation of isozygous, functional myostatin (MSTN) knockout cloned pigs free of selectable marker gene (SMG) by CRISPR/Cas9 and Cre/LoxP. CRISPR/Cas9-mediated homologous recombination (HR) was exploited to knock out (KO) one allele of MSTN in pig primary cells. Cre recombinase was then used to excise the SMG with an efficiency of 82.7%. The SMG-free non-EGFP cells were isolated by flow cytometery and immediately used as donor nuclei for nuclear transfer. A total of 685 reconstructed embryos were transferred into three surrogates with one delivering two male live piglets. Molecular testing verified the mono-allelic MSTN KO and SMG deletion in these cloned pigs. Western blots showed approximately 50% decrease in MSTN and concurrent increased expression of myogenic genes in muscle. Histological examination revealed the enhanced myofiber quantity but myofiber size remained unaltered. Ultrasonic detection showed the increased longissimus muscle size and decreased backfat thickness. Precision editing of pig MSTN gene has generated isozygous, SMG-free MSTN KO cloned founders, which guaranteed a reliable route for elite livestock production and a strategy to minimize potential biological risks.

Random transposon mutagenesis of the Saccharopolyspora erythraea genome reveals additional genes influencing erythromycin biosynthesis

PubMed Central

Fedashchin, Andrij; Cernota, William H.; Gonzalez, Melissa C.; Leach, Benjamin I.; Kwan, Noelle; Wesley, Roy K.; Weber, J. Mark

2015-01-01

A single cycle of strain improvement was performed in Saccharopolyspora erythraea mutB and 15 genotypes influencing erythromycin production were found. Genotypes generated by transposon mutagenesis appeared in the screen at a frequency of ∼3%. Mutations affecting central metabolism and regulatory genes were found, as well as hydrolases, peptidases, glycosyl transferases and unknown genes. Only one mutant retained high erythromycin production when scaled-up from micro-agar plug fermentations to shake flasks. This mutant had a knockout of the cwh1 gene (SACE_1598), encoding a cell-wall-associated hydrolase. The cwh1 knockout produced visible growth and morphological defects on solid medium. This study demonstrated that random transposon mutagenesis uncovers strain improvement-related genes potentially useful for strain engineering. PMID:26468041

SAMHD1 knockout mice: modeling retrovirus restriction in vivo.

PubMed

Wu, Li

2013-11-20

The host dNTP hydrolase SAMHD1 acts as a viral restriction factor to inhibit the replication of several retroviruses and DNA viruses in non-cycling human immune cells. However, understanding the physiological role of mammalian SAMHD1 has been elusive due to the lack of an animal model. Two recent studies reported the generation of samhd1 knockout mouse models for investigating the restriction of HIV-1 vectors and endogenous retroviruses in vivo. Both studies suggest that SAMHD1 is important for regulating the intracellular dNTP pool and the intrinsic immunity against retroviral infection, despite different outcomes of HIV-1 vector transduction in these mouse models. Here I discuss the significance of these new findings and the future directions in studying SAMHD1-mediated retroviral restriction.

Analysis of Average Telomere Length in Human Telomeric Protein Knockout Cells Generated by CRISPR/Cas9.

PubMed

Xu, Jun; Songyang, Zhou; Liu, Dan; Kim, Hyeung

2017-01-01

Telomeres play an important role in ensuring the integrity of the genome. Telomere shortening can lead to loss of genetic information and trigger DNA damage responses. Cultured mammalian cells have served as critical model systems for studying the function of telomere binding proteins and telomerase. Tremendous heterogeneity can be observed both between species and within a single cell population. Recent advances in genome editing (such as the development of the CRISPR/Cas9 platform) have further enabled researchers to carry out loss-of-function analysis of how disrupting key players in telomere maintenance affects telomere length regulation. Here we describe the steps to be carried out in order to analyze the average length of telomeres in CRISPR-engineered human knockout (KO) cells (TRF analysis).

Genome Editing in Mice Using TALE Nucleases.

PubMed

Wefers, Benedikt; Brandl, Christina; Ortiz, Oskar; Wurst, Wolfgang; Kühn, Ralf

2016-01-01

Gene engineering for generating targeted mouse mutants is a key technology for biomedical research. Using TALENs as sequence-specific nucleases to induce targeted double-strand breaks, the mouse genome can be directly modified in zygotes in a single step without the need for embryonic stem cells. By embryo microinjection of TALEN mRNAs and targeting vectors, knockout and knock-in alleles can be generated fast and efficiently. In this chapter we provide protocols for the application of TALENs in mouse zygotes.

Intrauterine Growth Restricted Rats Exercised at Pregnancy: Maternal-Fetal Repercussions.

PubMed

Corvino, S B; Netto, A O; Sinzato, Y K; Campos, K E; Calderon, I M P; Rudge, M V C; Volpato, G T; Zambrano, E; Damasceno, D C

2015-08-01

To evaluate the effect of swimming in pregnant rats born with intrauterine growth restriction (IUGR) and their offspring, IUGR rats were obtained using the streptozotocin-induced severe diabetic (SD) rats. In this study, the nondiabetic parental generation presented 10 rats and diabetic parental generation presented 116 rats. Of these, the mated nondiabetic female rats were 10 and the number of diabetic rats was 45. In relation to term pregnancy, there were 10 animals in the nondiabetic group and 15 rats in the diabetic group. In the offspring of SD rats (IUGR group), 43 females were classified as small for pregnancy age, 19 rats were classified as appropriate for pregnancy age, and 0 female was classified as large for pregnancy age. The nondiabetic and SD pregnant rats generated offspring with appropriate (control [C]) and small (IUGR) weight for pregnancy age, respectively. At adult life, the C group was maintained as nonexercised C group and IUGR rats were distributed into 2 subgroups, namely, nonexercised (IUGR) and exercised (IUGRex). The rate of mated rats in the IUGR group was reduced compared to the C group. During pregnancy, the IUGR rats presented hyperinsulinemia, impaired reproductive outcomes, decreased body weight, hypertriglyceridemia, and hyperlactacidemia. The IUGRex presented reduced insulin and triglyceride levels. Thus, swimming improved lipid metabolism and increased insulin sensitivity. However, the offspring showed retarded growth, reinforcing the need to stimulate the exercise practice in women under supervision with different professional expertise to promote appropriate gestational conditions and improve perinatal outcomes. © The Author(s) 2015.

Inherent variations in CO-H2S-mediated carotid body O2 sensing mediate hypertension and pulmonary edema

PubMed Central

Peng, Ying-Jie; Makarenko, Vladislav V.; Nanduri, Jayasri; Vasavda, Chirag; Raghuraman, Gayatri; Yuan, Guoxiang; Gadalla, Moataz M.; Kumar, Ganesh K.; Snyder, Solomon H.; Prabhakar, Nanduri R.

2014-01-01

Oxygen (O2) sensing by the carotid body and its chemosensory reflex is critical for homeostatic regulation of breathing and blood pressure. Humans and animals exhibit substantial interindividual variation in this chemosensory reflex response, with profound effects on cardiorespiratory functions. However, the underlying mechanisms are not known. Here, we report that inherent variations in carotid body O2 sensing by carbon monoxide (CO)-sensitive hydrogen sulfide (H2S) signaling contribute to reflex variation in three genetically distinct rat strains. Compared with Sprague-Dawley (SD) rats, Brown-Norway (BN) rats exhibit impaired carotid body O2 sensing and develop pulmonary edema as a consequence of poor ventilatory adaptation to hypobaric hypoxia. Spontaneous Hypertensive (SH) rat carotid bodies display inherent hypersensitivity to hypoxia and develop hypertension. BN rat carotid bodies have naturally higher CO and lower H2S levels than SD rat, whereas SH carotid bodies have reduced CO and greater H2S generation. Higher CO levels in BN rats were associated with higher substrate affinity of the enzyme heme oxygenase 2, whereas SH rats present lower substrate affinity and, thus, reduced CO generation. Reducing CO levels in BN rat carotid bodies increased H2S generation, restoring O2 sensing and preventing hypoxia-induced pulmonary edema. Increasing CO levels in SH carotid bodies reduced H2S generation, preventing hypersensitivity to hypoxia and controlling hypertension in SH rats. PMID:24395806

Investigation of TRPV1 loss-of-function phenotypes in TRPV1 Leu206Stop mice generated by N-ethyl-N-nitrosourea mutagenesis.

PubMed

Christoph, Thomas; Kögel, Babette; Schiene, Klaus; Peters, Thomas; Schröder, Wolfgang

2018-06-02

N-ethyl-N-nitrosourea (ENU) random mutagenesis was used to generate a mouse model for the analysis of the transient receptor potential vanilloid 1 (TRPV1) cation channel. A transversion from T→A in exon 4 led to a Leu206Stop mutation generating a loss-of-function mutant. The TRPV1 agonist capsaicin was used to analyze functional and nociceptive parameters in vitro and in vivo in TRPV1 Leu206Stop mice and congenic C3HeB/FeJ controls. Capsaicin-induced [Ca 2+ ] i changes in small diameter DRG neurons were significantly diminished in TRPV1 Leu206Stop mice and administration of capsaicin induced neither hypothermia nor nocifensive behaviour in vivo. TRPV1 Leu206Stop mice were tested in the spinal nerve ligation of mononeuropathic pain and developed mechanical hypersensitivity two weeks after nerve injury. In the open field test, a significant increase in spontaneous locomotion was detected in TRPV1 Leu206Stop mice as compared to wildtype controls. TRPV1 knockout mice have been reported to carry a similar phenotype regarding capsaicin-evoked responses in vitro and in vivo. However, in contrast to TRPV1 Leu206Stop mice, TRPV1 knockout mice did not differ in spontaneous locomotion as compared to congenic C57BL/6 mice, suggesting subtle ENU-dependent or independent strain differences between TRPV1 Leu206Stop mice and their wildtype controls. In summary, these data revealed a target-related (i.e. capsaicin-evoked) phenotype of TRPV1 Leu206Stop mice closely resembling that of published TRPV1 knockout mice. However, since ENU-mutant mice are congenic with the mouse strain initially used in random mutagenesis, direct phenotypic comparison with the respective wildtype controls is possible, and the time-consuming backcrossing in lines with targeted mutations is avoided. Copyright © 2018 Elsevier Inc. All rights reserved.

Distribution and colocalization of cholecystokinin with the prohormone convertase enzymes PC1, PC2, and PC5 in rat brain.

PubMed

Cain, Brian M; Connolly, Kelly; Blum, Alissa; Vishnuvardhan, Daesety; Marchand, James E; Beinfeld, Margery C; Vishnuvardham, Daesety

2003-12-15

During posttranslational processing to generate CCK 8, pro-cholecystokinin (CCK) undergoes endoproteolytic cleavage at three sites. Several studies using endocrine and neuronal tumor cells in culture and recombinant enzymes and synthetic substrates in vitro have pointed to the subtilisin/kexin-like enzymes prohormone convertase (PC) 1, PC2, and PC5 as potential candidates for these endoproteolytic cleavages. In these experimental models, they all appear to be able to cleave pro-CCK to make the correct products. One rodent model has provided information about the role of PC2. PC2 knockout mouse brains had less CCK 8 than wild-type, although a substantial amount of CCK was still present. The degree to which CCK levels were reduced in these mice was regionally specific. These data indicated that PC2 is important for normal production of CCK but that it is not the only endoprotease that is involved in CCK processing. To evaluate whether PC1 and PC5 are possible candidates for the other enzymes involved in CCK processing, the distribution of PC1, PC2, and PC5 mRNA was studied in rat brain. Their colocalization with CCK mRNA was examined using double-label in situ hybridization. PC2 was the most abundant of these enzymes in terms of the intensity and number of cells labeled. It was widely colocalized with CCK. PC1 and PC5 mRNA-positive cells were less abundant, but they were also widely distributed and strongly colocalized with CCK in the cerebral cortex, hippocampus, amygdala, ventral tegmental area, and substantia nigra zona compacta. The degree of colocalization of the enzymes with CCK was regionally specific. It is clear that PC1 and PC5 are extensively colocalized with CCK and could be participating in CCK processing in the rat brain and may be able to substitute for PC2 in its absence. These three enzymes may represent a redundant system to ensure production of biologically active CCK. Copyright 2003 Wiley-Liss, Inc.

Bm59 is an early gene, but is unessential for the propagation and assembly of Bombyx mori nucleopolyhedrovirus.

PubMed

Hu, Xiaolong; Shen, Yunwang; Zheng, Qin; Wang, Guobao; Wu, Xiaofeng; Gong, Chengliang

2016-02-01

Bombyx mori nucleopolyhedrovirus (BmNPV) is a major pathogen that specifically infects the domestic silkworm and causes serious economic loss to sericulture around the world. The function of BmNPV Bm59 gene in the viral life cycle is inconclusive. To investigate the role of Bm59 during viral infection, the transcription initiation site and temporal expression of Bm59 were analyzed, and Bm59-knockout virus was generated through homologous recombination in Escherichia coli. The results showed that Bm59 is an early transcription gene with an atypia early transcriptional start motif. Budded virion (BV) production and DNA replication in the BmN cells transfected with the Bm59-knockout virus bacmid were similar to those in the cells transfected with the wild-type virus. Electron microscopy revealed that the occlusion-derived virus can be produced in cells infected with the Bm59-knockout virus. These results indicated that Bm59 is an early gene and is not essential for viral replication or assembly of BmNPV. These findings suggested that non-essential gene (Bm59) remained in the viral genome, which may interact with other viral/host genes in a certain situation.

Efficient generation of rat induced pluripotent stem cells using a non-viral inducible vector.

PubMed

Merkl, Claudia; Saalfrank, Anja; Riesen, Nathalie; Kühn, Ralf; Pertek, Anna; Eser, Stefan; Hardt, Markus Sebastian; Kind, Alexander; Saur, Dieter; Wurst, Wolfgang; Iglesias, Antonio; Schnieke, Angelika

2013-01-01

Current methods of generating rat induced pluripotent stem cells are based on viral transduction of pluripotency inducing genes (Oct4, Sox2, c-myc and Klf4) into somatic cells. These activate endogenous pluripotency genes and reprogram the identity of the cell to an undifferentiated state. Epigenetic silencing of exogenous genes has to occur to allow normal iPS cell differentiation. To gain more control over the expression of exogenous reprogramming factors, we used a novel doxycycline-inducible plasmid vector encoding Oct4, Sox2, c-Myc and Klf4. To ensure efficient and controlled generation of iPS cells by plasmid transfection we equipped the reprogramming vector with a bacteriophage φC31 attB site and used a φC31 integrase expression vector to enhance vector integration. A series of doxycycline-independent rat iPS cell lines were established. These were characterized by immunocytochemical detection of Oct4, SSEA1 and SSEA4, alkaline phosphatase staining, methylation analysis of the endogenous Oct4 promoter and RT-PCR analysis of endogenous rat pluripotency genes. We also determined the number of vector integrations and the extent to which reprogramming factor gene expression was controlled. Protocols were developed to generate embryoid bodies and rat iPS cells demonstrated as pluripotent by generating derivatives of all three embryonic germ layers in vitro, and teratoma formation in vivo. All data suggest that our rat iPS cells, generated by plasmid based reprogramming, are similar to rat ES cells. Methods of DNA transfection, protein transduction and feeder-free monolayer culture of rat iPS cells were established to enable future applications.

Stevia and saccharin preferences in rats and mice.

PubMed

Sclafani, Anthony; Bahrani, Mahsa; Zukerman, Steven; Ackroff, Karen

2010-06-01

Use of natural noncaloric sweeteners in commercial foods and beverages has expanded recently to include compounds from the plant Stevia rebaudiana. Little is known about the responses of rodents, the animal models for many studies of taste systems and food intake, to stevia sweeteners. In the present experiments, preferences of female Sprague-Dawley rats and C57BL/6J mice for different stevia products were compared with those for the artificial sweetener saccharin. The stevia component rebaudioside A has the most sweetness and least off-tastes to human raters. In ascending concentration tests (48-h sweetener vs. water), rats and mice preferred a high-rebaudioside, low-stevioside extract as strongly as saccharin, but the extract stimulated less overdrinking and was much less preferred to saccharin in direct choice tests. Relative to the extract, mice drank more pure rebaudioside A and showed stronger preferences but still less than those for saccharin. Mice also preferred a commercial mixture of rebaudioside A and erythritol (Truvia). Similar tests of sweet receptor T1R3 knockout mice and brief-access licking tests with normal mice suggested that the preferences were based on sweet taste rather than post-oral effects. The preference response of rodents to stevia sweeteners is notable in view of their minimal response to some other noncaloric sweeteners (aspartame and cyclamate).

Stevia and Saccharin Preferences in Rats and Mice

PubMed Central

Bahrani, Mahsa; Zukerman, Steven; Ackroff, Karen

2010-01-01

Use of natural noncaloric sweeteners in commercial foods and beverages has expanded recently to include compounds from the plant Stevia rebaudiana. Little is known about the responses of rodents, the animal models for many studies of taste systems and food intake, to stevia sweeteners. In the present experiments, preferences of female Sprague–Dawley rats and C57BL/6J mice for different stevia products were compared with those for the artificial sweetener saccharin. The stevia component rebaudioside A has the most sweetness and least off-tastes to human raters. In ascending concentration tests (48-h sweetener vs. water), rats and mice preferred a high-rebaudioside, low-stevioside extract as strongly as saccharin, but the extract stimulated less overdrinking and was much less preferred to saccharin in direct choice tests. Relative to the extract, mice drank more pure rebaudioside A and showed stronger preferences but still less than those for saccharin. Mice also preferred a commercial mixture of rebaudioside A and erythritol (Truvia). Similar tests of sweet receptor T1R3 knockout mice and brief-access licking tests with normal mice suggested that the preferences were based on sweet taste rather than post-oral effects. The preference response of rodents to stevia sweeteners is notable in view of their minimal response to some other noncaloric sweeteners (aspartame and cyclamate). PMID:20413452

Administration of Luteinizing Hormone Releasing Hormone Agonist for Synchronization of Estrus and Generation of Pseudopregnancy for Embryo Transfer in Rats

PubMed Central

Borjeson, Tiffany M; Pang, Jassia; Fox, James G; García, Alexis

2014-01-01

In the past decade, the use of genetically engineered rats has increased exponentially; therefore, the ability to perform embryo transfer (ET) in rats to rederive, reanimate, or create mutant rat lines is increasingly important. However, the successful generation of pseudopregnant female rats for ET represents a limiting factor. We here evaluated the subcutaneous administration of 40 µg luteinizing hormone releasing hormone agonist (LHRHa) for estrus synchronization during the development and implementation of a rat ET program. Our first experiment assessed endogenous estrus cycling patterns by examining vaginal cytology without administration of LHRHa in 5-wk-old peripubertal Sprague–Dawley female rats. These rats then received LHRHa at approximately 7 wk of age; 57% of the rats were synchronized in proestrus or estrus as assessed by vaginal cytology 96 h later. In a second experiment, 8-wk-old virgin, unmanipulated Sprague–Dawley female rats received LHRHa; 55% were synchronized in proestrus or estrus 96 h later. Copulatory plugs were confirmed in 28% and 82% of the rats that had been synchronized in the first and second experiments, respectively, and mated with vasectomized male rats. Embryo transfer surgery was performed, and live pups were born from both fresh and cryopreserved transgenic rat embryos. Our results indicate that subcutaneous administration of 40 µg LHRHa followed by examination of vaginal cytology 96 h later is an effective technique to generate multiple pseudopregnant recipient rats for use in an ET program. PMID:24827564

Serotonin Reuptake Transporter Deficiency Modulates the Acute Thermoregulatory and Locomotor Activity Response to 3,4-(±)-Methylenedioxymethamphetamine, and Attenuates Depletions in Serotonin Levels in SERT-KO Rats

PubMed Central

Lizarraga, Lucina E.; Phan, Andy V.; Cholanians, Aram B.; Herndon, Joseph M.; Lau, Serrine S.; Monks, Terrence J.

2014-01-01

3,4-(±)-Methylenedioxymethamphetamine (MDMA) is a ring-substituted amphetamine derivative with potent psychostimulant properties. The neuropharmacological effects of MDMA are biphasic in nature, initially causing synaptic monoamine release, primarily of serotonin (5-HT), inducing thermogenesis and hyperactivity (5-HT syndrome). The long-term effects of MDMA manifest as a prolonged depletion in 5-HT, and structural damage to 5-HT nerve terminals. MDMA toxicity is in part mediated by an ability to inhibit the presynaptic 5-HT reuptake transporter (SERT). Using a SERT-knockout (SERT-KO) rat model, we determined the impact of SERT deficiency on thermoregulation, locomotor activity, and neurotoxicity in SERT-KO or Wistar-based wild-type (WT) rats exposed to MDMA. WT and SERT-KO animals exhibited the highest thermogenic responses to MDMA (four times 10 mg/kg, sc at 12 h intervals) during the diurnal (first and third) doses according to peak body temperature and area under the curve (∑°C × h) analysis. Although no differences in peak body temperature were observed between MDMA-treated WT and SERT-KO animals, ∑°C × h following the first MDMA dose was reduced in SERT-KO rats. Exposure to a single dose of MDMA stimulated horizontal velocity in both WT and SERT-KO rats, however, this effect was delayed and attenuated in the KO animals. Finally, SERT-KO rats were insensitive to MDMA-induced long-term (7 days) depletions in 5-HT and its metabolite, 5-hydroxyindole acetic acid, in both cortex and striatum. In conclusion, SERT deficiency modulated MDMA-mediated thermogenesis, hyperactivity and neurotoxicity in KO rats. The data confirm that the SERT is essential for the manifestation of the acute and long-term toxicities of MDMA. PMID:24595820

CaV3.1 isoform of T-type calcium channels supports excitability of rat and mouse ventral tegmental area neurons.

PubMed

Tracy, Matthew E; Tesic, Vesna; Stamenic, Tamara Timic; Joksimovic, Srdjan M; Busquet, Nicolas; Jevtovic-Todorovic, Vesna; Todorovic, Slobodan M

2018-03-23

Recent data have implicated voltage-gated calcium channels in the regulation of the excitability of neurons within the mesolimbic reward system. While the attention of most research has centered on high voltage L-type calcium channel activity, the presence and role of the low voltage-gated T-type calcium channel (T-channels) has not been well explored. Hence, we investigated T-channel properties in the neurons of the ventral tegmental area (VTA) utilizing wild-type (WT) rats and mice, Ca V 3.1 knock-out (KO) mice, and TH-eGFP knock-in (KI) rats in acute horizontal brain slices of adolescent animals. In voltage-clamp experiments, we first assessed T-channel activity in WT rats with characteristic properties of voltage-dependent activation and inactivation, as well as characteristic crisscrossing patterns of macroscopic current kinetics. T-current kinetics were similar in WT mice and WT rats but T-currents were abolished in Ca V 3.1 KO mice. In ensuing current-clamp experiments, we observed the presence of hyperpolarization-induced rebound burst firing in a subset of neurons in WT rats, as well as dopaminergic and non-dopaminergic neurons in TH-eGFP KI rats. Following the application of a pan-selective T-channel blocker TTA-P2, rebound bursting was significantly inhibited in all tested cells. In a behavioral assessment, the acute locomotor increase induced by a MK-801 (Dizocilpine) injection in WT mice was abolished in Ca V 3.1 KO mice, suggesting a tangible role for 3.1 T-type channels in drug response. We conclude that pharmacological targeting of Ca V 3.1 isoform of T-channels may be a novel approach for the treatment of disorders of mesolimbic reward system. Copyright © 2018. Published by Elsevier Ltd.

ADVERSE EFFECTS OF TCDD ON MAMMARY GLAND DEVELOPMENT IN LONG EVANS RATS: A TWO GENERATIONAL STUDY

EPA Science Inventory

Recent studies have demonstrated variable effects on mammary gland development in rat offspring exposed to TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin, 1 ug/kg, gavage) on day 15 of gestation. We have characterized these effects in Long Evans rats, in both one and two-generational...

Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins.

PubMed

Quadros, Rolen M; Miura, Hiromi; Harms, Donald W; Akatsuka, Hisako; Sato, Takehito; Aida, Tomomi; Redder, Ronald; Richardson, Guy P; Inagaki, Yutaka; Sakai, Daisuke; Buckley, Shannon M; Seshacharyulu, Parthasarathy; Batra, Surinder K; Behlke, Mark A; Zeiner, Sarah A; Jacobi, Ashley M; Izu, Yayoi; Thoreson, Wallace B; Urness, Lisa D; Mansour, Suzanne L; Ohtsuka, Masato; Gurumurthy, Channabasavaiah B

2017-05-17

Conditional knockout mice and transgenic mice expressing recombinases, reporters, and inducible transcriptional activators are key for many genetic studies and comprise over 90% of mouse models created. Conditional knockout mice are generated using labor-intensive methods of homologous recombination in embryonic stem cells and are available for only ~25% of all mouse genes. Transgenic mice generated by random genomic insertion approaches pose problems of unreliable expression, and thus there is a need for targeted-insertion models. Although CRISPR-based strategies were reported to create conditional and targeted-insertion alleles via one-step delivery of targeting components directly to zygotes, these strategies are quite inefficient. Here we describe Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR), a targeting strategy in which long single-stranded DNA donors are injected with pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complexes into mouse zygotes. We show for over a dozen loci that Easi-CRISPR generates correctly targeted conditional and insertion alleles in 8.5-100% of the resulting live offspring. Easi-CRISPR solves the major problem of animal genome engineering, namely the inefficiency of targeted DNA cassette insertion. The approach is robust, succeeding for all tested loci. It is versatile, generating both conditional and targeted insertion alleles. Finally, it is highly efficient, as treating an average of only 50 zygotes is sufficient to produce a correctly targeted allele in up to 100% of live offspring. Thus, Easi-CRISPR offers a comprehensive means of building large-scale Cre-LoxP animal resources.

Exposing the Complex III Qo semiquinone radical

PubMed Central

Zhang, Haibo; Osyczka, Artur; Dutton, P. L.; Moser, Christopher C.

2012-01-01

Complex III Qo site semiquinone has been assigned pivotal roles in productive energy-conversion and destructive superoxide generation. After a 30 year search, a genetic heme bH knockout arrests this transient semiquinone EPR radical, revealing the natural engineering balance pitting energy-conserving, short-circuit minimizing, split electron transfer and catalytic speed against damaging oxygen reduction. PMID:17560537

IKKβ inhibition prevents fat-induced beta cell dysfunction in vitro and in vivo in rodents.

PubMed

Ivovic, Aleksandar; Oprescu, Andrei I; Koulajian, Khajag; Mori, Yusaku; Eversley, Judith A; Zhang, Liling; Nino-Fong, Rodolfo; Lewis, Gary F; Donath, Marc Y; Karin, Michael; Wheeler, Michael B; Ehses, Jan; Volchuk, Allen; Chan, Catherine B; Giacca, Adria

2017-10-01

We have previously shown that oxidative stress plays a causal role in beta cell dysfunction induced by fat. Here, we address whether the proinflammatory kinase inhibitor of (nuclear factor) κB kinase β (IKKβ), which is activated by oxidative stress, is also implicated. Fat (oleate or olive oil) was infused intravenously in Wistar rats for 48 h with or without the IKKβ inhibitor salicylate. Thereafter, beta cell function was evaluated in vivo using hyperglycaemic clamps or ex vivo in islets isolated from fat-treated rats. We also exposed rat islets to oleate in culture, with or without salicylate and 4(2'-aminoethyl)amino-1,8-dimethylimidazo(1,2-a)quinoxaline; BMS-345541 (BMS, another inhibitor of IKKβ) and evaluated beta cell function in vitro. Furthermore, oleate was infused in mice treated with BMS and in beta cell-specific Ikkb-null mice. 48 h infusion of fat impaired beta-cell function in vivo, assessed using the disposition index (DI), in rats (saline: 1.41 ± 0.13; oleate: 0.95 ± 0.11; olive oil [OLO]: 0.87 ± 0.15; p < 0.01 for both fats vs saline) and in mice (saline: 2.51 ± 0.39; oleate: 1.20 ± 0.19; p < 0.01 vs saline) and ex vivo (i.e., insulin secretion, units are pmol insulin islet -1  h -1 ) in rat islets (saline: 1.51 ± 0.13; oleate: 1.03 ± 0.10; OLO: 0.91 ± 0.13; p < 0.001 for both fats vs saline) and the dysfunction was prevented by co-infusion of salicylate in rats (oleate + salicylate: 1.30 ± 0.09; OLO + salicylate: 1.33 ± 0.23) or BMS in mice (oleate + BMS: 2.25 ± 0.42) in vivo and by salicylate in rat islets ex vivo (oleate + salicylate: 1.74 ± 0.31; OLO + salicylate: 1.54 ± 0.29). In cultured islets, 48 h exposure to oleate impaired beta-cell function ([in pmol insulin islet -1  h -1 ] control: 0.66 ± 0.12; oleate: 0.23 ± 0.03; p < 0.01 vs saline), an effect prevented by both inhibitors (oleate + salicylate: 0.98 ± 0.08; oleate + BMS: 0.50 ± 0.02). Genetic inhibition of IKKβ also prevented fat-induced beta-cell dysfunction ex vivo ([in pmol insulin islet -1  h -1 ] control saline: 0.16 ± 0.02; control oleate: 0.10 ± 0.02; knockout oleate: 0.17 ± 0.04; p < 0.05 control saline vs. control oleate) and in vivo (DI: control saline: 3.86 ± 0.40; control oleate: 1.95 ± 0.29; knockout oleate: 2.96 ± 0.24; p < 0.01 control saline vs control oleate). Our results demonstrate a causal role for IKKβ in fat-induced beta cell dysfunction in vitro, ex vivo and in vivo.

Thyroid Hormone Receptor α Controls Developmental Timing and Regulates the Rate and Coordination of Tissue-Specific Metamorphosis in Xenopus tropicalis.

PubMed

Wen, Luan; Shibata, Yuki; Su, Dan; Fu, Liezhen; Luu, Nga; Shi, Yun-Bo

2017-06-01

Thyroid hormone (T3) receptors (TRs) mediate the effects of T3 on organ metabolism and animal development. There are two TR genes, TRα and TRβ, in all vertebrates. During animal development, TRα expression is activated earlier than zygotic T3 synthesis and secretion into the plasma, implicating a developmental role of TRα both in the presence and absence of T3. Using T3-dependent amphibian metamorphosis as a model, we previously proposed a dual-function model for TRs, in particular TRα, during development. That is, unliganded TR represses the expression of T3-inducible genes during premetamorphosis to ensure proper animal growth and prevent premature metamorphosis, whereas during metamorphosis, liganded TR activates target gene transcription to promote the transformation of the tadpole into a frog. To determine if TRα has such a dual function, we generated homozygous TRα-knockout animal lines. We show that, indeed, TRα knockout affects both premetamorphic animal development and metamorphosis. Surprisingly, we observed that TRα is not essential for amphibian metamorphosis, given that homozygous knockout animals complete metamorphosis within a similar time period after fertilization as their wild-type siblings. On the other hand, the timing of metamorphosis for different organs is altered by the knockout; limb metamorphosis occurs earlier, whereas intestinal metamorphosis is completed later than in wild-type siblings. Thus, our studies have demonstrated a critical role of endogenous TRα, not only in regulating both the timing and rate of metamorphosis, but also in coordinating temporal metamorphosis of different organs.

Role of alpha-synuclein in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced parkinsonism in mice.

PubMed

Schlüter, O M; Fornai, F; Alessandrí, M G; Takamori, S; Geppert, M; Jahn, R; Südhof, T C

2003-01-01

In humans, mutations in the alpha-synuclein gene or exposure to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produce Parkinson's disease with loss of dopaminergic neurons and depletion of nigrostriatal dopamine. alpha-Synuclein is a vertebrate-specific component of presynaptic nerve terminals that may function in modulating synaptic transmission. To test whether MPTP toxicity involves alpha-synuclein, we generated alpha-synuclein-deficient mice by homologous recombination, and analyzed the effect of deleting alpha-synuclein on MPTP toxicity using these knockout mice. In addition, we examined commercially available mice that contain a spontaneous loss of the alpha-synuclein gene. As described previously, deletion of alpha-synuclein had no significant effects on brain structure or composition. In particular, the levels of synaptic proteins were not altered, and the concentrations of dopamine, dopamine metabolites, and dopaminergic proteins were unchanged. Upon acute MPTP challenge, alpha-synuclein knockout mice were partly protected from chronic depletion of nigrostriatal dopamine when compared with littermates of the same genetic background, whereas mice carrying the spontaneous deletion of the alpha-synuclein gene exhibited no protection. Furthermore, alpha-synuclein knockout mice but not the mice with the alpha-synuclein gene deletion were slightly more sensitive to methamphetamine than littermate control mice. These results demonstrate that alpha-synuclein is not obligatorily coupled to MPTP sensitivity, but can influence MPTP toxicity on some genetic backgrounds, and illustrate the need for extensive controls in studies aimed at describing the effects of mouse knockouts on MPTP sensitivity.

Muscle-Specific Deletion of Rictor Impairs Insulin-Stimulated Glucose Transport and Enhances Basal Glycogen Synthase Activity▿

PubMed Central

Kumar, Anil; Harris, Thurl E.; Keller, Susanna R.; Choi, Kin M.; Magnuson, Mark A.; Lawrence, John C.

2008-01-01

Rictor is an essential component of mTOR (mammalian target of rapamycin) complex 2 (mTORC2), a kinase complex that phosphorylates Akt at Ser473 upon activation of phosphatidylinositol 3-kinase (PI-3 kinase). Since little is known about the role of either rictor or mTORC2 in PI-3 kinase-mediated physiological processes in adult animals, we generated muscle-specific rictor knockout mice. Muscle from male rictor knockout mice exhibited decreased insulin-stimulated glucose uptake, and the mice showed glucose intolerance. In muscle lacking rictor, the phosphorylation of Akt at Ser473 was reduced dramatically in response to insulin. Furthermore, insulin-stimulated phosphorylation of the Akt substrate AS160 at Thr642 was reduced in rictor knockout muscle, indicating a defect in insulin signaling to stimulate glucose transport. However, the phosphorylation of Akt at Thr308 was normal and sufficient to mediate the phosphorylation of glycogen synthase kinase 3 (GSK-3). Basal glycogen synthase activity in muscle lacking rictor was increased to that of insulin-stimulated controls. Consistent with this, we observed a decrease in basal levels of phosphorylated glycogen synthase at a GSK-3/protein phosphatase 1 (PP1)-regulated site in rictor knockout muscle. This change in glycogen synthase phosphorylation was associated with an increase in the catalytic activity of glycogen-associated PP1 but not increased GSK-3 inactivation. Thus, rictor in muscle tissue contributes to glucose homeostasis by positively regulating insulin-stimulated glucose uptake and negatively regulating basal glycogen synthase activity. PMID:17967879

Morphological observation of the stria vascularis in midkine and pleiotrophin knockout mice.

PubMed

Sone, Michihiko; Muramatsu, Hisako; Muramatsu, Takashi; Nakashima, Tsutomu

2011-02-01

Midkine and Pleiotrophin are low molecular weight basic proteins with closely related structures and serve as growth/differentiation factors. They have been reported to be expressed in the cochlea during the embryonic and perinatal periods. In the present study, we focused on the roles of midkine and pleiotrophin in the stria vascularis and investigated morphological changes using mice deficient in these genes. Midkine knockout, pleiotrophin knockout, and double knockout mice were used and compared to wild-type mice. Auditory brain stem responses (ABRs) and cochlear blood flows were measured in each type of mice. Pathological changes in the stria vascularis were examined by light microscopy, including immunohistochemical staining with anti-Kir4.1 antibody, and electron microscopy. Hearing thresholds examined by ABRs were significantly higher in midkine knockout and pleiotrophin knockout mice than in wild-type mice. Double knockout mice showed higher thresholds compared to midkine knockout and pleiotrophin knockout mice. Blood flow in the lateral walls did not significantly differ and light microscopy examination showed an almost normal appearance of the stria vascularis in these knockout mice. However, the expression of Kir4.1 was weak in the knockout mice and severe vacuolar degeneration was observed by electron microscopy in the intermediate cells of the double knockout mice. The present study demonstrates that midkine and pleiotrophin play some roles for the morphological maintenance of intermediate cell in the stria vascularis. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Tagging methyl-CpG-binding domain proteins reveals different spatiotemporal expression and supports distinct functions.

PubMed

Wood, Kathleen H; Johnson, Brian S; Welsh, Sarah A; Lee, Jun Y; Cui, Yue; Krizman, Elizabeth; Brodkin, Edward S; Blendy, Julie A; Robinson, Michael B; Bartolomei, Marisa S; Zhou, Zhaolan

2016-04-01

DNA methylation is recognized by methyl-CpG-binding domain (MBD) proteins. Multiple MBDs are linked to neurodevelopmental disorders in humans and mice. However, the functions of MBD2 are poorly understood. We characterized Mbd2 knockout mice and determined spatiotemporal expression of MBDs and MBD2-NuRD (nucleosome remodeling deacetylase) interactions. We analyzed behavioral phenotypes, generated biotin-tagged MBD1 and MBD2 knockin mice, and performed biochemical studies of MBD2-NuRD. Most behavioral measures are minimally affected in Mbd2 knockout mice. In contrast to other MBDs, MBD2 shows distinct expression patterns. Unlike most MBDs, MBD2 is ubiquitously expressed in all tissues examined and appears dispensable for brain functions measured in this study. We provide novel genetic tools and reveal new directions to investigate MBD2 functions in vivo.

OutKnocker: a web tool for rapid and simple genotyping of designer nuclease edited cell lines.

PubMed

Schmid-Burgk, Jonathan L; Schmidt, Tobias; Gaidt, Moritz M; Pelka, Karin; Latz, Eicke; Ebert, Thomas S; Hornung, Veit

2014-10-01

The application of designer nucleases allows the induction of DNA double-strand breaks (DSBs) at user-defined genomic loci. Due to imperfect DNA repair mechanisms, DSBs can lead to alterations in the genomic architecture, such as the disruption of the reading frame of a critical exon. This can be exploited to generate somatic knockout cell lines. While high genome editing activities can be achieved in various cellular systems, obtaining cell clones that contain all-allelic frameshift mutations at the target locus of interest remains a laborious task. To this end, we have developed an easy-to-follow deep sequencing workflow and the evaluation tool OutKnocker (www.OutKnocker.org), which allows convenient, reliable, and cost-effective identification of knockout cell lines. © 2014 Schmid-Burgk et al.; Published by Cold Spring Harbor Laboratory Press.

Differential cardiac effects in rats exposed to atmospheric ...

EPA Pesticide Factsheets

The results of this study demonstrate that atmospheric smog generated from both isoprene and toluene cause cardiac effects in rats. In addition, it appears that smog from toluene is more toxic in terms of cardiac arrhythmogenicity. Smog, which is a complex mixture of particulate matter and gaseous irritants (ozone, sulfur dioxide, reactive aldehydes), as well as components which react with sunlight to form secondary pollutants, has recently been linked to increased risk of adverse cardiac responses. The components, and therefore health effects, of atmospheric smog are determined by the fuel used to generate them. In this study we examined the difference between isoprene- and toluene-generated smog in causing cardiac effects in rats and hypothesized that both atmospheres would cause cardiac electrical and functional changes in rats. Male Wistar-Kyoto rats were exposed to either atmospheric smog generated by the USEPA’s mobile reaction chamber using either isoprene or toluene, or filtered air for four hours. One day later, rats were anesthetized and left ventricular functional responses to dobutamine were measured using a Millar probe and arrhythmia sensitivity to aconitine. Baseline left ventricular pressure (LVP) was lower in toluene-exposed animals but not isoprene when compared to air. Increases in LVP with increasing doses of dobutamine were impaired only in toluene-exposed rats. Both isoprene and toluene impaired the rate of ventri

Screening for main components associated with the idiosyncratic hepatotoxicity of a tonic herb, Polygonum multiflorum.

PubMed

Li, Chunyu; Niu, Ming; Bai, Zhaofang; Zhang, Congen; Zhao, Yanling; Li, Ruiyu; Tu, Can; Li, Huifang; Jing, Jing; Meng, Yakun; Ma, Zhijie; Feng, Wuwen; Tang, Jinfa; Zhu, Yun; Li, Jinjie; Shang, Xiaoya; Zou, Zhengsheng; Xiao, Xiaohe; Wang, Jiabo

2017-06-01

The main constituents of a typical medicinal herb, Polygonum multiflorum (Heshouwu in Chinese), that induces idiosyncratic liver injury remain unclear. Our previous work has shown that cotreatment with a nontoxic dose of lipopolysaccharide (LPS) and therapeutic dose of Heshouwu can induce liver injury in rats, whereas the solo treatment cannot induce observable injury. In the present work, using the constituent "knock-out" and "knock-in" strategy, we found that the ethyl acetate (EA) extract of Heshouwu displayed comparable idiosyncratic hepatotoxicity to the whole extract in LPS-treated rats. Results indicated a significant elevation of plasma alanine aminotransferase, aspartate aminotransferase, and liver histologic changes, whereas other separated fractions failed to induce liver injury. The mixture of EA extract with other separated fractions induced comparable idiosyncratic hepatotoxicity to the whole extract in LPS-treated rats. Chemical analysis further revealed that 2,3,5,4'-tetrahydroxy trans-stilbene-2-O-β-glucoside (trans-SG) and its cis-isomer were the two major compounds in EA extract. Furthermore, the isolated cis-, and not its trans-isomer, displayed comparable idiosyncratic hepatotoxicity to EA extract in LPS-treated rats. Higher contents of cis-SG were detected in Heshouwu liquor or preparations from actual liver intoxication patients associated with Heshouwu compared with general collected samples. In addition, plasma metabolomics analysis showed that cis-SG-disturbing enriched pathways remarkably differed from trans-SG ones in LPS-treated rats. All these results suggested that cis-SG was closely associated with the idiosyncratic hepatotoxicity of Heshouwu. Considering that the cis-trans isomerization of trans-SG was mediated by ultraviolet light or sunlight, our findings serve as reference for controlling photoisomerization in drug discovery and for the clinical use of Heshouwu and stilbene-related medications.

NCKX3 was compensated by calcium transporting genes and bone resorption in a NCKX3 KO mouse model.

PubMed

Yang, Hyun; Ahn, Changhwan; Shin, Eun-Kyeong; Lee, Ji-Sun; An, Beum-Soo; Jeung, Eui-Bae

2017-10-15

Gene knockout is the most powerful tool for determination of gene function or permanent modification of the phenotypic characteristics of an animal. Existing methods for gene disruption are limited by their efficiency, time required for completion and potential for confounding off-target effects. In this study, a rapid single-step approach to knockout of a targeted gene in mice using zinc-finger nucleases (ZFNs) was demonstrated for generation of mutant (knockout; KO) alleles. Specifically, ZFNs to target the sodium/calcium/potassium exchanger3 (NCKX3) gene in C57bl/6j were designed using the concept of this approach. NCKX3 KO mice were generated and the phenotypic characterization and molecular regulation of active calcium transporting genes was assessed when mice were fed different calcium diets during growth. General phenotypes such as body weight and plasma ion level showed no distinct abnormalities. Thus, the potassium/sodium/calcium exchanger of NCKX3 KO mice proceeded normally in this study. As a result, the compensatory molecular regulation of this mechanism was elucidated. Renal TRPV5 mRNA of NCKX3 KO mice increased in both male and female mice. Expression of TRPV6 mRNA was only down-regulated in the duodenum of male KO mice. Renal- and duodenal expression of PTHR and VDR were not changed; however, GR mRNA expression was increased in the kidney of NCKX3 KO mice. Depletion of the NCKX3 gene in a KO mouse model showed loss of bone mineral contents and increased plasma parathyroid hormone, suggesting that NCKX3 may play a role in regulating calcium homeostasis. Copyright © 2017 Elsevier B.V. All rights reserved.

Progressive hearing loss and degeneration of hair cell stereocilia in taperin gene knockout mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Mo; Wang, Qin; Zhu, Gang-Hua

The TPRN gene encodes taperin, which is prominently present at the taper region of hair cell stereocilia. Mutations in TPRN have been reported to cause autosomal recessive nonsyndromic deafness 79(DFNB 79). To investigate the role of taperin in pathogenesis of hearing loss, we generated TPRN knockout mice using TALEN technique. Sanger sequencing confirmed an 11 bp deletion at nucleotide 177–187 in exon 1 of TPRN, which results in a truncated form of taperin protein. Heterozygous TPRN{sup +/−} mice showed apparently normal auditory phenotypes to their wide-type (WT) littermates. Homozygous TPRN{sup −/−} mice exhibited progressive sensorineural hearing loss as reflected bymore » auditory brainstem response to both click and tone burst stimuli at postnatal days 15 (P15), 30 (P30), and 60 (P60). Alex Fluor-594 phalloidin labeling showed no obvious difference in hair cell numbers in the cochlea between TPRN{sup −/−} mice and WT mice under light microscope. However, scanning electronic microscopy revealed progressive degeneration of inner hair cell stereocilia, from apparently normal at postnatal days 3 (P3) to scattered absence at P15 and further to substantial loss at P30. The outer hair cell stereocilia also showed progressive degeneration, though much less severe, Collectively, we conclude that taperin plays an important role in maintenance of hair cell stereocilia. Establishment of TPRN knockout mice enables further investigation into the function of this gene. - Highlights: • TPRN{sup −/−} mice were generated using TALEN technique. • TPRN{sup −/−} mice presented progressive hearing loss. • WT and TPRN{sup −/−} mice showed no difference in hair cell numbers. • TPRN{sup −/−} mice showed progressive degeneration of hair cell stereocilia.« less

Neuronal Nitric Oxide Synthase and NADPH Oxidase Interact to Affect Cognitive, Affective, and Social Behaviors in Mice

PubMed Central

Walton, James C.; Selvakumar, Balakrishnan; Weil, Zachary M.; Snyder, Solomon H.; Nelson, Randy J.

2013-01-01

Both nitric oxide (NO) and reactive oxygen species (ROS) generated by nNOS and NADPH oxidase (NOX), respectively, in the brain have been implicated in an array of behaviors ranging from learning and memory to social interactions. Although recent work has elucidated how these separate redox pathways regulate neural function and behavior, the interaction of these two pathways in the regulation of neural function and behavior remains unspecified. Toward this end, the p47phox subunit of NOX, and nNOS were deleted to generate double knockout mice that were used to characterize the behavioral outcomes of concurrent impairment of the NO and ROS pathways in the brain. Mice were tested in a battery of behavioral tasks to evaluate learning and memory, as well as social, affective, and cognitive behaviors. p47phox deletion did not affect depressive-like behavior, whereas nNOS deletion abolished it. Both p47phox and nNOS deletion singly reduced anxiety-like behavior, increased general locomotor activity, impaired spatial learning and memory, and impaired preference for social novelty. Deletion of both genes concurrently had synergistic effects to elevate locomotor activity, impair spatial learning and memory, and disrupt prepulse inhibition of acoustic startle. Although preference for social novelty was impaired in single knockouts, double knockout mice displayed elevated levels of preference for social novelty above that of wild type littermates. These data demonstrate that, depending upon modality, deletion of p47phox and nNOS genes have dissimilar, similar, or additive effects. The current findings provide evidence that the NOX and nNOS redox signaling cascades interact in the brain to affect both cognitive function and social behavior. PMID:23948215

Angiotensin II stimulates superoxide production in the thick ascending limb by activating NOX4

PubMed Central

Hong, Nancy J.; Garvin, Jeffrey L.

2012-01-01

Angiotensin II (ANG II) stimulates production of superoxide (O2−) by NADPH oxidase (NOX) in medullary thick ascending limbs (TALs). There are three isoforms of the catalytic subunit (NOX1, 2, and 4) known to be expressed in the kidney. We hypothesized that NOX2 mediates ANG II-induced O2− production by TALs. To test this, we measured NOX1, 2, and 4 mRNA and protein by RT-PCR and Western blot in TAL suspensions from rats and found three catalytic subunits expressed in the TAL. We measured O2− production using a lucigenin-based assay. To assess the contribution of NOX2, we measured ANG II-induced O2− production in wild-type and NOX2 knockout mice (KO). ANG II increased O2− production by 346 relative light units (RLU)/mg protein in the wild-type mice (n = 9; P < 0.0007 vs. control). In the knockout mice, ANG II increased O2− production by 290 RLU/mg protein (n = 9; P < 0.007 vs. control). This suggests that NOX2 does not contribute to ANG II-induced O2− production (P < 0.6 WT vs. KO). To test whether NOX4 mediates the effect of ANG II, we selectively decreased NOX4 expression in rats using an adenovirus that expresses NOX4 short hairpin (sh)RNA. Six to seven days after in vivo transduction of the kidney outer medulla, NOX4 mRNA was reduced by 77%, while NOX1 and NOX2 mRNA was unaffected. In control TALs, ANG II stimulated O2− production by 96%. In TALs transduced with NOX4 shRNA, ANG II-stimulated O2− production was not significantly different from the baseline. We concluded that NOX4 is the main catalytic isoform of NADPH oxidase that contributes to ANG II-stimulated O2− production by TALs. PMID:22875785

Lack of toxic effect of technical azadirachtin during postnatal development of rats.

PubMed

Srivastava, M K; Raizada, R B

2007-03-01

Azadirachtin, a biopesticide has been evaluated for its possible toxic effects during postnatal development of rats over two generations. Rats were fed 100, 500 and 1000ppm technical azadirachtin through diet which is equivalent to 5, 25 and 50mg/kg body weight of rats. Technical azadirachtin has not produced any adverse effects on reproductive function and data were comparable to control animals over two generations. There were no toxicological effect in parent rats as evidenced by clinical signs of toxicity, enzymatic parameters like AST, ALT, ALP, S. bilirubin, S. cholesterol, total protein and histopathology of liver, brain, kidney and testes/ovary. The litters of F(1B) and F(2B) generations were devoid of any morphological, visceral and teratological changes. The percent cumulative loss and growth index of pups were also comparable to respective controls in successive growth period of 0, 4, 7, 14 and 21 days in two generations. There were no major malformations in fetuses while some insignificant minor skeletal variations like missing 5th sternebrae and bipartite thoracic centre found were not compound or dose related. No significant pathomorphological changes were observed in liver, kidney, brain and gonads of F(2B) pups. In conclusion rats fed technical azadirachtin showed no evidence of cumulative effects on postnatal development and reproductive performance over two generations. Absence of any major adverse reproductive effects in adults as well as in 21 days old pups of F(2B) generation suggest the safe use of technical azadirachtin as a biopesticide.

Histone deacetylase inhibition reduces hypothyroidism-induced neurodevelopmental defects in rats.

PubMed

Kumar, Praveen; Mohan, Vishwa; Sinha, Rohit Anthony; Chagtoo, Megha; Godbole, Madan M

2015-11-01

Thyroid hormone (TH) through its receptor (TRα/β) influences spatio-temporal regulation of its target gene repertoire during brain development. Though hypothyroidism in WT rodent models of perinatal hypothyroidism severely impairs neurodevelopment, its effect on TRα/β knockout mice is less severe. An explanation to this paradox is attributed to a possible repressive action of unliganded TRs during development. Since unliganded TRs suppress gene expression through the recruitment of histone deacetylase (HDACs) via co-repressor complexes, we tested whether pharmacological inhibition of HDACs may prevent the effects of hypothyroidism on brain development. Using valproate, an HDAC inhibitor, we show that HDAC inhibition significantly blocks the deleterious effects of hypothyroidism on rat cerebellum, evident by recovery of TH target genes like Bdnf, Pcp2 and Mbp as well as improved dendritic structure of cerebellar Purkinje neurons. Together with this, HDAC inhibition also rescues hypothyroidism-induced motor and cognitive defects. This study therefore provides an insight into the role of HDACs in TH insufficiency during neurodevelopment and their inhibition as a possible therapeutics for treatment. © 2015 Society for Endocrinology.

RAGE deficiency attenuates the protective effect of Lidocaine against sepsis-induced acute lung injury.

PubMed

Zhang, Zhuo; Zhou, Jie; Liao, Changli; Li, Xiaobing; Liu, Minghua; Song, Daqiang; Jiang, Xian

2017-04-01

Lidocaine (Lido) is reported to suppress inflammatory responses and exhibit a therapeutic effect in models of cecal ligation and puncture (CLP)-induced acute lung injury (ALI). The receptor for advanced glycation end product (RAGE) exerts pro-inflammatory effects by enhancing pro-inflammatory cytokine production. However, the precise mechanism by which Lido confers protection against ALI is not clear. ALI was induced in RAGE WT and RAGE knockout (KO) rats using cecal ligation and puncture (CLP) operations for 24 h. The results showed that Lido significantly inhibited CLP-induced lung inflammation and histopathological lung injury. Furthermore, Lido significantly reduced CLP-induced upregulation of HMGB1 and RAGE expression and activation of the NF-κB and MAPK signaling pathways. With the use of RAGE KO rats, we demonstrate here that RAGE deficiency attenuates the protective effect of Lido against CLP-induced lung inflammatory cell infiltration and histopathological lung injury. These results suggest that RAGE deficiency attenuates the protective effect of Lido against CLP-induced ALI by attenuating the pro-inflammatory cytokines production.

Expression of VGF mRNA in developing neuroendocrine and endocrine tissues.

PubMed

Snyder, S E; Peng, B; Pintar, J E; Salton, S R J

2003-11-01

Analysis of knockout mice suggests that the neurotropin-inducible secreted polypeptide VGF (non-acronymic) plays an important role in the regulation of energy balance. VGF is synthesized by neurons in the central and peripheral nervous systems (CNS, PNS), as well as in the adult pituitary, adrenal medulla, endocrine cells of the stomach and pancreatic beta cells. Thus VGF, like cholecystokinin, leptin, ghrelin and other peptide hormones that have been shown to regulate feeding and energy expenditure, is synthesized in both the gut and the brain. Although detailed developmental studies of VGF localization in the CNS and PNS have been completed, little is known about the ontogeny of VGF expression in endocrine and neuroendocrine tIssues. Here, we report that VGF mRNA is detectable as early as embryonic day 15.5 in the developing rat gastrointestinal and esophageal lumen, pancreas, adrenal, and pituitary, and we further demonstrate that VGF mRNA is synthesized in the gravid rat uterus, together supporting possible functional roles for this polypeptide outside the nervous system and in the enteric plexus.

A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids

PubMed Central

Beneke, Tom; Makin, Laura; Valli, Jessica; Sunter, Jack

2017-01-01

Clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR-associated gene 9 (Cas9) genome editing is set to revolutionize genetic manipulation of pathogens, including kinetoplastids. CRISPR technology provides the opportunity to develop scalable methods for high-throughput production of mutant phenotypes. Here, we report development of a CRISPR-Cas9 toolkit that allows rapid tagging and gene knockout in diverse kinetoplastid species without requiring the user to perform any DNA cloning. We developed a new protocol for single-guide RNA (sgRNA) delivery using PCR-generated DNA templates which are transcribed in vivo by T7 RNA polymerase and an online resource (LeishGEdit.net) for automated primer design. We produced a set of plasmids that allows easy and scalable generation of DNA constructs for transfections in just a few hours. We show how these tools allow knock-in of fluorescent protein tags, modified biotin ligase BirA*, luciferase, HaloTag and small epitope tags, which can be fused to proteins at the N- or C-terminus, for functional studies of proteins and localization screening. These tools enabled generation of null mutants in a single round of transfection in promastigote form Leishmania major, Leishmania mexicana and bloodstream form Trypanosoma brucei; deleted genes were undetectable in non-clonal populations, enabling for the first time rapid and large-scale knockout screens. PMID:28573017

Cathepsin H Functions as an Aminopeptidase in Secretory Vesicles for Production of Enkephalin and Galanin Peptide Neurotransmitters

PubMed Central

Lu, W. Douglas; Funkelstein, Lydiane; Toneff, Thomas; Reinheckel, Thomas; Peters, Christoph; Hook, Vivian

2012-01-01

Peptide neurotransmitters function as key intercellular signaling molecules in the nervous system. These peptides are generated in secretory vesicles from proneuropeptides by proteolytic processing at dibasic residues, followed by removal of N- and/or C-terminal basic residues to form active peptides. Enkephalin biosynthesis from proenkephalin utilizes the cysteine protease cathepsin L and the subtilisin-like prohormone convertase 2 (PC2). Cathepsin L generates peptide intermediates with N-terminal basic residue extensions, which must be removed by an aminopeptidase. In this study, we identified cathepsin H as an aminopeptidase in secretory vesicles that produces (Met)enkephalin (ME) by sequential removal of basic residues from KR-ME and KK-ME, supported by in vivo knockout of the cathepsin H gene. Localization of cathepsin H in secretory vesicles was demonstrated by immunoelectron microscopy and confocal immunofluorescence microscopy. Purified human cathepsin H sequentially removes N-terminal basic residues to generate ME, with peptide products characterized by nano-LC-MS/MS tandem mass spectrometry. Cathepsin H shows highest activities for cleaving N-terminal basic residues (Arg and Lys) among amino acid fluorogenic substrates. Notably, knockout of the cathepsin H gene results in reduction of ME in mouse brain. Cathepsin H deficient mice also show a substantial decrease in galanin peptide neurotransmitter levels in brain. These results illustrate a role for cathepsin H as an aminopeptidase for enkephalin and galanin peptide neurotransmitter production. PMID:22582844

Putative free radical-scavenging activity of an extract of Cineraria maritima in preventing selenite-induced cataractogenesis in Wistar rat pups

PubMed Central

Anitha, Thirugnanasambandhar Sivasubramanian; Muralidharan, Arumugam Ramachandran; Annadurai, Thangaraj; Jesudasan, Christdas Arul Nelson; Thomas, Philip Aloysius

2013-01-01

Purpose To investigate the possible free radical-scavenging activity of an extract of Cineraria maritima on selenite-induced cataractous lenses in Wistar rat pups. Methods In the present study, Wistar rat pups were divided into three experimental groups. On P10, Group I (control) rat pups received an intraperitoneal injection of 0.89% saline. Rats in groups II (selenite-challenged, untreated) and III (selenite-challenged, C. maritima treated) received a subcutaneous injection of sodium selenite (19 μmol/kg bodyweight); Group III rat pups also received an intraperitoneal injection of the extract of C. maritima (350 mg/kg bodyweight) once daily P9–14. Both eyes of each pup were examined from P16 until P30. Cytochemical localization of nitroblue tetrazolium salts and generation of superoxide, hydroxyl, and nitric oxide levels were measured. The expression of the inducible nitric oxide synthase gene was evaluated with reverse transcription-PCR. Immunoblot analysis was also performed to confirm the differential expression of the inducible nitric oxide synthase protein. Results Subcutaneous injection of sodium selenite led to severe oxidative damage in the lenticular tissues, shown by increased formation of formazan crystals, elevated generation of superoxide, hydroxyl, and nitric oxide radicals, and elevated inducible nitric oxide synthase gene and protein expression that possibly contributed to the opacification of the lens and thus cataract formation. When rat pups were treated with intraperitoneal administration of the extract of C. maritima, the generation of free radicals as well as the messenger ribonucleic acid and protein expression of inducible nitric oxide synthase were maintained at near normal levels. Conclusions The data generated by this study suggest that an ethanolic extract of C. maritima possibly prevents cataractogenesis in a rat model by minimizing free radical generation. PMID:24357923

Deletion of Suppressor of Cytokine Signaling 3 from Forebrain Neurons Delays Infertility and Onset of Hypothalamic Leptin Resistance in Response to a High Caloric Diet

PubMed Central

McEwen, Hayden J. L.; Inglis, Megan A.; Quennell, Janette H.; Grattan, David R.

2016-01-01

The cellular processes that cause high caloric diet (HCD)-induced infertility are poorly understood but may involve upregulation of suppressor of cytokine signaling (SOCS-3) proteins that are associated with hypothalamic leptin resistance. Deletion of SOCS-3 from brain cells is known to protect mice from diet-induced obesity, but the effects on HCD-induced infertility are unknown. We used neuron-specific SOCS3 knock-out mice to elucidate this and the effects on regional hypothalamic leptin resistance. As expected, male and female neuron-specific SOCS3 knock-out mice were protected from HCD-induced obesity. While female wild-type mice became infertile after 4 months of HCD feeding, infertility onset in knock-out females was delayed by 4 weeks. Similarly, knock-out mice had delayed leptin resistance development in the medial preoptic area and anteroventral periventricular nucleus, regions important for generation of the surge of GnRH and LH that induces ovulation. We therefore tested whether the suppressive effects of HCD on the estradiol-induced GnRH/LH surge were overcome by neuron-specific SOCS3 knock-out. Although only 20% of control HCD-mice experienced a preovulatory-like LH surge, LH surges could be induced in almost all neuron-specific SOCS3 knock-out mice on this diet. In contrast to females, HCD-fed male mice did not exhibit any fertility decline compared with low caloric diet-fed males despite their resistance to the satiety effects of leptin. These data show that deletion of SOCS3 delays the onset of leptin resistance and infertility in HCD-fed female mice, but given continued HCD feeding this state does eventually occur, presumably in response to other mechanisms inhibiting leptin signal transduction. SIGNIFICANCE STATEMENT Obesity is commonly associated with infertility in humans and other animals. Treatments for human infertility show a decreased success rate with increasing body mass index. A hallmark of obesity is an increase in circulating leptin levels; despite this, the brain responds as if there were low levels of leptin, leading to increased appetite and suppressed fertility. Here we show that leptin resistant infertility is caused in part by the leptin signaling molecule SOCS3. Deletion of SOCS3 from brain neurons delays the onset of diet-induced infertility. PMID:27383590

Deletion of Suppressor of Cytokine Signaling 3 from Forebrain Neurons Delays Infertility and Onset of Hypothalamic Leptin Resistance in Response to a High Caloric Diet.

PubMed

McEwen, Hayden J L; Inglis, Megan A; Quennell, Janette H; Grattan, David R; Anderson, Greg M

2016-07-06

The cellular processes that cause high caloric diet (HCD)-induced infertility are poorly understood but may involve upregulation of suppressor of cytokine signaling (SOCS-3) proteins that are associated with hypothalamic leptin resistance. Deletion of SOCS-3 from brain cells is known to protect mice from diet-induced obesity, but the effects on HCD-induced infertility are unknown. We used neuron-specific SOCS3 knock-out mice to elucidate this and the effects on regional hypothalamic leptin resistance. As expected, male and female neuron-specific SOCS3 knock-out mice were protected from HCD-induced obesity. While female wild-type mice became infertile after 4 months of HCD feeding, infertility onset in knock-out females was delayed by 4 weeks. Similarly, knock-out mice had delayed leptin resistance development in the medial preoptic area and anteroventral periventricular nucleus, regions important for generation of the surge of GnRH and LH that induces ovulation. We therefore tested whether the suppressive effects of HCD on the estradiol-induced GnRH/LH surge were overcome by neuron-specific SOCS3 knock-out. Although only 20% of control HCD-mice experienced a preovulatory-like LH surge, LH surges could be induced in almost all neuron-specific SOCS3 knock-out mice on this diet. In contrast to females, HCD-fed male mice did not exhibit any fertility decline compared with low caloric diet-fed males despite their resistance to the satiety effects of leptin. These data show that deletion of SOCS3 delays the onset of leptin resistance and infertility in HCD-fed female mice, but given continued HCD feeding this state does eventually occur, presumably in response to other mechanisms inhibiting leptin signal transduction. Obesity is commonly associated with infertility in humans and other animals. Treatments for human infertility show a decreased success rate with increasing body mass index. A hallmark of obesity is an increase in circulating leptin levels; despite this, the brain responds as if there were low levels of leptin, leading to increased appetite and suppressed fertility. Here we show that leptin resistant infertility is caused in part by the leptin signaling molecule SOCS3. Deletion of SOCS3 from brain neurons delays the onset of diet-induced infertility. Copyright © 2016 the authors 0270-6474/16/367142-12$15.00/0.

Genetic Regulation of Development of Thymic Lymphomas Induced by N‐Propyl‐N‐nitrosourea in the Rat

PubMed Central

Fukami, Hiroko; Nishimura, Mayumi; Matsuyama, Mutsushi

1995-01-01

To clarify the linkage between Hbb and Tls‐1 (thymic lymphoma susceptible‐1) loci and to investigate other loci concerned in thymic lymphomagenesis, the BUF/Mna rat, which is highly sensitive to the lymphomagenic activity of N‐propyl‐N‐nitrosourea (PNU), the WKY/NCrj rat, reported to be resistant, and their cross offspring were subjected to genetic analysis. F1 hybrid and backcross generations were raised from the 2 strains, and 6 genetic markers including Hbb were analyzed in individuals of the backcross generation. However, no linkage between Hbb and Tls‐1 loci could be demonstrated since WKY rats also developed a high incidence of thymic lymphomas in response to PNU. Nevertheless, thymic lymphomas developed more rapidly and reached a larger size in the BUF rats. F1 rats expressed a rather rapid and large tumor growth phenotype, while the [(WKY × BUF) × WKY] backcross generation consisted of rats with either rapidly growing or slowly growing tumors. It was thus concluded that rapid development of thymic lymphomas is determined by a gene, provisionally designated Tls‐3. Analysis of the relationship between 6 genetic markers and development of thymic lymphoma in the backcross generation demonstrated that the Tls‐3 locus is loosely linked to the Gc locus, suggesting a possible location on rat chromosome 14. Tls‐3 may not be identical with Tls‐1 and other genes known to be relevant to thymic tumors, but its relationship with Tls‐2 remains obscure. PMID:7559080

Prediction of reaction knockouts to maximize succinate production by Actinobacillus succinogenes

PubMed Central

Nag, Ambarish; St. John, Peter C.; Crowley, Michael F.

2018-01-01

Succinate is a precursor of multiple commodity chemicals and bio-based succinate production is an active area of industrial bioengineering research. One of the most important microbial strains for bio-based production of succinate is the capnophilic gram-negative bacterium Actinobacillus succinogenes, which naturally produces succinate by a mixed-acid fermentative pathway. To engineer A. succinogenes to improve succinate yields during mixed acid fermentation, it is important to have a detailed understanding of the metabolic flux distribution in A. succinogenes when grown in suitable media. To this end, we have developed a detailed stoichiometric model of the A. succinogenes central metabolism that includes the biosynthetic pathways for the main components of biomass—namely glycogen, amino acids, DNA, RNA, lipids and UDP-N-Acetyl-α-D-glucosamine. We have validated our model by comparing model predictions generated via flux balance analysis with experimental results on mixed acid fermentation. Moreover, we have used the model to predict single and double reaction knockouts to maximize succinate production while maintaining growth viability. According to our model, succinate production can be maximized by knocking out either of the reactions catalyzed by the PTA (phosphate acetyltransferase) and ACK (acetyl kinase) enzymes, whereas the double knockouts of PEPCK (phosphoenolpyruvate carboxykinase) and PTA or PEPCK and ACK enzymes are the most effective in increasing succinate production. PMID:29381705

Prediction of reaction knockouts to maximize succinate production by Actinobacillus succinogenes.

PubMed

Nag, Ambarish; St John, Peter C; Crowley, Michael F; Bomble, Yannick J

2018-01-01

Succinate is a precursor of multiple commodity chemicals and bio-based succinate production is an active area of industrial bioengineering research. One of the most important microbial strains for bio-based production of succinate is the capnophilic gram-negative bacterium Actinobacillus succinogenes, which naturally produces succinate by a mixed-acid fermentative pathway. To engineer A. succinogenes to improve succinate yields during mixed acid fermentation, it is important to have a detailed understanding of the metabolic flux distribution in A. succinogenes when grown in suitable media. To this end, we have developed a detailed stoichiometric model of the A. succinogenes central metabolism that includes the biosynthetic pathways for the main components of biomass-namely glycogen, amino acids, DNA, RNA, lipids and UDP-N-Acetyl-α-D-glucosamine. We have validated our model by comparing model predictions generated via flux balance analysis with experimental results on mixed acid fermentation. Moreover, we have used the model to predict single and double reaction knockouts to maximize succinate production while maintaining growth viability. According to our model, succinate production can be maximized by knocking out either of the reactions catalyzed by the PTA (phosphate acetyltransferase) and ACK (acetyl kinase) enzymes, whereas the double knockouts of PEPCK (phosphoenolpyruvate carboxykinase) and PTA or PEPCK and ACK enzymes are the most effective in increasing succinate production.

Contributions of β2-microglobulin–dependent molecules and lymphocytes to iron regulation: insights from HfeRag1−/− and β2mRag1−/− double knock-out mice

PubMed Central

Miranda, Carlos J.; Makui, Hortence; Andrews, Nancy C.; Santos, Manuela M.

2010-01-01

Genetic causes of hereditary hemochromatosis (HH) include mutations in the HFE gene, coding for a β2-microglobulin (β2m)–associated major histocompatibility complex class I-like protein. However, iron accumulation in patients with HH can be highly variable. Previously, analysis of β2mRag1−/− double-deficient mice, lacking all β2m-dependent molecules and lymphocytes, demonstrated increased iron accumulation in the pancreas and heart compared with β2m single knock-out mice. To evaluate whether the observed phenotype in β2mRag1−/− mice was due solely to the absence of Hfe or to other β2m-dependent molecules, we generated HfeRag1−/− double-deficient mice. Our studies revealed that introduction of Rag1 deficiency in Hfe knock-out mice leads to heightened iron overload, mainly in the liver, whereas the heart and pancreas are relatively spared compared with β2mRag1−/− mice. These results suggest that other β2m-interacting protein(s) may be involved in iron regulation and that in the absence of functional Hfe molecules lymphocyte numbers may influence iron overload severity. PMID:14656877

Epistatic interaction between the lipase-encoding genes Pnpla2 and Lipe causes liposarcoma in mice

PubMed Central

Wang, Shu Pei; Yang, Hao; Ji, Bo; Gladdy, Rebecca; Andelfinger, Gregor; Mitchell, Grant A.

2017-01-01

Liposarcoma is an often fatal cancer of fat cells. Mechanisms of liposarcoma development are incompletely understood. The cleavage of fatty acids from acylglycerols (lipolysis) has been implicated in cancer. We generated mice with adipose tissue deficiency of two major enzymes of lipolysis, adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL), encoded respectively by Pnpla2 and Lipe. Adipocytes from double adipose knockout (DAKO) mice, deficient in both ATGL and HSL, showed near-complete deficiency of lipolysis. All DAKO mice developed liposarcoma between 11 and 14 months of age. No tumors occurred in single knockout or control mice. The transcriptome of DAKO adipose tissue showed marked differences from single knockout and normal controls as early as 3 months. Gpnmb and G0s2 were among the most highly dysregulated genes in premalignant and malignant DAKO adipose tissue, suggesting a potential utility as early markers of the disease. Similar changes of GPNMB and G0S2 expression were present in a human liposarcoma database. These results show that a previously-unknown, fully penetrant epistatic interaction between Pnpla2 and Lipe can cause liposarcoma in mice. DAKO mice provide a promising model for studying early premalignant changes that lead to late-onset malignant disease. PMID:28459858

Random Splicing of Several Exons Caused by a Single Base Change in the Target Exon of CRISPR/Cas9 Mediated Gene Knockout.

PubMed

Kapahnke, Marcel; Banning, Antje; Tikkanen, Ritva

2016-12-14

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated sequence 9 (CRISPR/Cas9) system is widely used for genome editing purposes as it facilitates an efficient knockout of a specific gene in, e.g. cultured cells. Targeted double-strand breaks are introduced to the target sequence of the guide RNAs, which activates the cellular DNA repair mechanism for non-homologous-end-joining, resulting in unprecise repair and introduction of small deletions or insertions. Due to this, sequence alterations in the coding region of the target gene frequently cause frame-shift mutations, facilitating degradation of the mRNA. We here show that such CRISPR/Cas9-mediated alterations in the target exon may also result in altered splicing of the respective pre-mRNA, most likely due to mutations of splice-regulatory sequences. Using the human FLOT-1 gene as an example, we demonstrate that such altered splicing products also give rise to aberrant protein products. These may potentially function as dominant-negative proteins and thus interfere with the interpretation of the data generated with these cell lines. Since most researchers only control the consequences of CRISPR knockout at genomic and protein level, our data should encourage to also check the alterations at the mRNA level.

Differential Requirements for c-Myc in Chronic Hematopoietic Hyperplasia and Acute Hematopoietic Malignancies in Pten-null Mice

PubMed Central

Zhang, Jun; Xiao, Yechen; Guo, Yinshi; Breslin, Peter; Zhang, Shubin; Wei, Wei; Zhang, Zhou; Zhang, Jiwang

2011-01-01

Myeloproliferative disorders (MPDs), lymphoproliferative disorders (LPDs), acute T-lymphocytic or myeloid leukemia and T-lymphocytic lymphoma were developed in inducible Pten-knockout (Pten−/−) mice. The appearance of these multiple diseases in one animal model provides an opportunity to study the pathogenesis of multiple diseases simultaneously. To study whether Myc function is required for the development of these hematopoietic disorders in Pten−/− mice, we generated inducible Pten/Myc double-knockout mice (Pten−/−/Myc−/−). By comparing the hematopoietic phenotypes of these double-knockout mice with those of Pten−/− mice, we found that both sets of animals developed MPDs and LPDs. However, none of the compound-mutant mice developed acute leukemia or lymphoma. Interestingly, in contrast to the MPDs which developed in Pten−/− mice which are dominated by granulocytes, megakaryocytes predominate in the MPDs of Pten−/−/Myc−/− mice. Our study suggests that the deregulation of PI3K/Akt signaling in Pten−/− hematopoietic cells protects these cells from apoptotic cell death, resulting in chronic proliferative disorders. But due to the differential requirement for Myc in granulocyte as compared to megakaryocyte proliferation, Myc deletion converts Pten−/− MPDs from granulocyte-dominated to megakaryocyte-dominated conditions. Myc is absolutely required for the development of acute hematopoietic malignancies. PMID:21926961

Analysis of BmNPV orf101 disruption: orf101 is essential for mediating budded virus production.

PubMed

Chen, Huiqing; Li, Mei; Mai, Weijun; Tang, Qi; Li, Guohui; Chen, Keping; Zhou, Yajing

2014-12-01

In our previous study, Orf101 (Bm101) of Bombyx mori nucleopolyhedrovirus (BmNPV) was identified as a component of the budded virions important for viral late gene expression. In this study we demonstrate that Bm101 is actually a previously unrecognized core gene and that it is essential for mediating budded virus production. To determine the role of Bm101 in the baculovirus life cycle, a Bm101 knockout bacmid containing the BmNPV genome was generated through homologous recombination in Escherichia coli. Furthermore, a Bm101 repair bacmid was constructed by transposing the Bm101 open reading frame with its native promoter region into the polyhedrin locus of the Bm101 knockout bacmid. Bacmid DNA transfection assay revealed that the Bm101 knockout bacmid was unable to produce the infectious budded virus, while the Bm101 repair bacmid rescued this defect, allowing budded-virus titers to reach wild-type levels. Real time PCR analysis indicated that the viral DNA genome in the absence of Bm101 was unaffected in the first 24 h p.t. Thus, studies of a Bm101-null BACmid indicate that Bm101 is required for viral DNA replication during the infection cycle.

Selective role for DNMT3a in learning and memory.

PubMed

Morris, Michael J; Adachi, Megumi; Na, Elisa S; Monteggia, Lisa M

2014-11-01

Methylation of cytosine nucleotides is governed by DNA methyltransferases (DNMTs) that establish de novo DNA methylation patterns in early embryonic development (e.g., DNMT3a and DNMT3b) or maintain those patterns on hemimethylated DNA in dividing cells (e.g., DNMT1). DNMTs continue to be expressed at high levels in mature neurons, however their impact on neuronal function and behavior are unclear. To address this issue we examined DNMT1 and DNMT3a expression following associative learning. We also generated forebrain specific conditional Dnmt1 or Dnmt3a knockout mice and characterized them in learning and memory paradigms as well as for alterations in long-term potentiation (LTP) and synaptic plasticity. Here, we report that experience in an associative learning task impacts expression of Dnmt3a, but not Dnmt1, in brain areas that mediate learning of this task. We also found that Dnmt3a knockout mice, and not Dnmt1 knockouts have synaptic alterations as well as learning deficits on several associative and episodic memory tasks. These findings indicate that the de novo DNA methylating enzyme DNMT3a in postmitotic neurons is necessary for normal memory formation and its function cannot be substituted by the maintenance DNA methylating enzyme DNMT1. Copyright © 2014 Elsevier Inc. All rights reserved.

Regulatory functions of limbic Y1 receptors in body weight and anxiety uncovered by conditional knockout and maternal care

PubMed Central

Bertocchi, Ilaria; Oberto, Alessandra; Longo, Angela; Mele, Paolo; Sabetta, Marianna; Bartolomucci, Alessandro; Palanza, Paola; Sprengel, Rolf; Eva, Carola

2011-01-01

Neuropeptide Y (NPY) plays an important role in stress, anxiety, obesity, and energy homeostasis via activation of NPY-Y1 receptors (Y1Rs) in the brain. However, global knockout of the Npy1r gene has low or no impact on anxiety and body weight. To uncover the role of limbic Y1Rs, we generated conditional knockout mice in which the inactivation of the Npy1r gene was restricted to excitatory neurons of the forebrain, starting from juvenile stages (Npy1rrfb). Npy1rrfb mice exhibited increased anxiety and reduced body weight, less adipose tissue, and lower serum leptin levels. Npy1rrfb mutants also had a hyperactive hypothalamic–pituitary–adrenocortical axis, as indicated by higher peripheral corticosterone and higher density of NPY immunoreactive fibers and corticotropin releasing hormone immunoreactive cell bodies in the paraventricular hypothalamic nucleus. Importantly, through fostering experiments, we determined that differences in phenotype between Npy1rrfb and Npy1r2lox mice became apparent when both genotypes were raised by FVB/J but not by C57BL/6J dams, suggesting that limbic Y1Rs are key targets of maternal care-induced programming of anxiety and energy homeostasis. PMID:22084082

Serotonin controls initiation of locomotion and afferent modulation of coordination via 5-HT7 receptors in adult rats.

PubMed

Cabaj, Anna M; Majczyński, Henryk; Couto, Erika; Gardiner, Phillip F; Stecina, Katinka; Sławińska, Urszula; Jordan, Larry M

2017-01-01

Experiments on neonatal rodent spinal cord showed that serotonin (5-HT), acting via 5-HT 7 receptors, is required for initiation of locomotion and for controlling the action of interneurons responsible for inter- and intralimb coordination, but the importance of the 5-HT system in adult locomotion is not clear. Blockade of spinal 5-HT 7 receptors interfered with voluntary locomotion in adult rats and fictive locomotion in paralysed decerebrate rats with no afferent feedback, consistent with a requirement for activation of descending 5-HT neurons for production of locomotion. The direct control of coordinating interneurons by 5-HT 7 receptors observed in neonatal animals was not found during fictive locomotion, revealing a developmental shift from direct control of locomotor interneurons in neonates to control of afferent input from the moving limb in adults. An understanding of the afferents controlled by 5-HT during locomotion is required for optimal use of rehabilitation therapies involving the use of serotonergic drugs. Serotonergic pathways to the spinal cord are implicated in the control of locomotion based on studies using serotonin type 7 (5-HT 7 ) receptor agonists and antagonists and 5-HT 7 receptor knockout mice. Blockade of these receptors is thought to interfere with the activity of coordinating interneurons, a conclusion derived primarily from in vitro studies on isolated spinal cord of neonatal rats and mice. Developmental changes in the effects of serotonin (5-HT) on spinal neurons have recently been described, and there is increasing data on control of sensory input by 5-HT 7 receptors on dorsal root ganglion cells and/or dorsal horn neurons, leading us to determine the effects of 5-HT 7 receptor blockade on voluntary overground locomotion and on locomotion without afferent input from the moving limb (fictive locomotion) in adult animals. Intrathecal injections of the selective 5-HT 7 antagonist SB269970 in adult intact rats suppressed locomotion by partial paralysis of hindlimbs. This occurred without a direct effect on motoneurons as revealed by an investigation of reflex activity. The antagonist disrupted intra- and interlimb coordination during locomotion in all intact animals but not during fictive locomotion induced by stimulation of the mesencephalic locomotor region (MLR). MLR-evoked fictive locomotion was transiently blocked, then the amplitude and frequency of rhythmic activity were reduced by SB269970, consistent with the notion that the MLR activates 5-HT neurons, leading to excitation of central pattern generator neurons with 5-HT 7 receptors. Effects on coordination in adults required the presence of afferent input, suggesting a switch to 5-HT 7 receptor-mediated control of sensory pathways during development. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

Serotonin controls initiation of locomotion and afferent modulation of coordination via 5‐HT7 receptors in adult rats

PubMed Central

Majczyński, Henryk; Couto, Erika; Gardiner, Phillip F.; Stecina, Katinka; Sławińska, Urszula

2016-01-01

Key points Experiments on neonatal rodent spinal cord showed that serotonin (5‐HT), acting via 5‐HT7 receptors, is required for initiation of locomotion and for controlling the action of interneurons responsible for inter‐ and intralimb coordination, but the importance of the 5‐HT system in adult locomotion is not clear.Blockade of spinal 5‐HT7 receptors interfered with voluntary locomotion in adult rats and fictive locomotion in paralysed decerebrate rats with no afferent feedback, consistent with a requirement for activation of descending 5‐HT neurons for production of locomotion.The direct control of coordinating interneurons by 5‐HT7 receptors observed in neonatal animals was not found during fictive locomotion, revealing a developmental shift from direct control of locomotor interneurons in neonates to control of afferent input from the moving limb in adults.An understanding of the afferents controlled by 5‐HT during locomotion is required for optimal use of rehabilitation therapies involving the use of serotonergic drugs. Abstract Serotonergic pathways to the spinal cord are implicated in the control of locomotion based on studies using serotonin type 7 (5‐HT7) receptor agonists and antagonists and 5‐HT7 receptor knockout mice. Blockade of these receptors is thought to interfere with the activity of coordinating interneurons, a conclusion derived primarily from in vitro studies on isolated spinal cord of neonatal rats and mice. Developmental changes in the effects of serotonin (5‐HT) on spinal neurons have recently been described, and there is increasing data on control of sensory input by 5‐HT7 receptors on dorsal root ganglion cells and/or dorsal horn neurons, leading us to determine the effects of 5‐HT7 receptor blockade on voluntary overground locomotion and on locomotion without afferent input from the moving limb (fictive locomotion) in adult animals. Intrathecal injections of the selective 5‐HT7 antagonist SB269970 in adult intact rats suppressed locomotion by partial paralysis of hindlimbs. This occurred without a direct effect on motoneurons as revealed by an investigation of reflex activity. The antagonist disrupted intra‐ and interlimb coordination during locomotion in all intact animals but not during fictive locomotion induced by stimulation of the mesencephalic locomotor region (MLR). MLR‐evoked fictive locomotion was transiently blocked, then the amplitude and frequency of rhythmic activity were reduced by SB269970, consistent with the notion that the MLR activates 5‐HT neurons, leading to excitation of central pattern generator neurons with 5‐HT7 receptors. Effects on coordination in adults required the presence of afferent input, suggesting a switch to 5‐HT7 receptor‐mediated control of sensory pathways during development. PMID:27393215

Characterization of the nociceptin receptor (ORL-1) agonist, Ro64-6198, in tests of anxiety across multiple species.

PubMed

Varty, G B; Hyde, L A; Hodgson, R A; Lu, S X; McCool, M F; Kazdoba, T M; Del Vecchio, R A; Guthrie, D H; Pond, A J; Grzelak, M E; Xu, X; Korfmacher, W A; Tulshian, D; Parker, E M; Higgins, G A

2005-10-01

Previous studies have demonstrated behaviors indicative of anxiolysis in rats pretreated with the nociceptin receptor (opioid receptor like-1, ORL-1) agonist, Ro64-6198. The aim of this study was to examine the effects of Ro64-6198 in anxiety models across three species: rat, guinea pig, and mouse. In addition, the receptor specificity of Ro64-6198 was studied, using the ORL-1 receptor antagonist, J-113397, and ORL-1 receptor knockout (KO) mice. Finally, neurological studies examined potential side effects of Ro64-6198 in the rat and mouse. Ro64-6198 (3-10 mg/kg) increased punished responding in a rat conditioned lick suppression test similarly to chlordiazepoxide (6 mg/kg). This effect of Ro64-6198 was attenuated by J-113397 (10 mg/kg), but not the mu opioid antagonist, naltrexone (3 mg/kg). In addition, Ro64-6198 (1-3 mg/kg) reduced isolation-induced vocalizations in rat and guinea pig pups. Ro64-6198 (3 mg/kg) increased the proportion of punished responding in a mouse Geller-Seifter test in wild-type (WT) but not ORL-1 KO mice, whereas diazepam (1-5.6 mg/kg) was effective in both genotypes. In rats, Ro64-6198 reduced locomotor activity (LMA) and body temperature and impaired rotarod, beam walking, and fixed-ratio (FR) performance at doses of 10-30 mg/kg, i.e., three to ten times higher than an anxiolytic dose. In WT mice, Ro64-6198 (3-10 mg/kg) reduced LMA and rotarod performance, body temperature, and FR responding, but these same measures were unaffected in ORL-1 KO mice. Haloperidol (0.3-3 mg/kg) reduced these measures to a similar extent in both genotypes. These studies confirm the potent, ORL-1 receptor-mediated, anxiolytic-like effects of Ro64-6198, extending the findings across three species. Ro64-6198 has target-based side effects, although the magnitude of these effects varies across species.

Robust Reductions of Excess Weight and Hyperphagia by Beloranib in Rat Models of Genetic and Hypothalamic Obesity.

PubMed

Elfers, Clinton T; Roth, Christian L

2017-01-01

Hypothalamic lesions or deficient melanocortin (MC) signaling via MC4 receptor (MC4r) mutations often lead to hyperphagia and severe treatment-resistant obesity. We tested the methionine aminopeptidase 2-inhibitor beloranib (ZGN-440) in 2 male rat models of obesity, one modeling hypothalamic obesity with a combined medial hypothalamic lesion (CMHL) and the other modeling a monogenic form of obesity with MC4r mutations (MC4r knockout [MC4rKO]). In CMHL rats (age 3 months), postsurgery excess weight gain was significantly inhibited (ZGN-440, 0.2 ± 0.7 g/d; vehicle, 3.8 ± 0.6 g/d; P < 0.001) during 12 days of ZGN-440 treatment (0.1 mg/kg daily subcutaneously) together with a 30% reduction of daily food intake vs vehicle injection. In addition, ZGN-440 treatment improved glucose tolerance and reduced plasma insulin, and circulating levels of α-melanocyte stimulating hormone were increased. Serum lipid levels did not differ significantly in ZGN-440-treated vs vehicle-treated rats. Similar results were found in MC4rKO rats: ZGN-440 treatment (14-21 d) was associated with significant reductions of body weight gain (MC4rKO, -1.7 ± 0.6 vs 2.8 ± 0.4 g/d; lean wild-type controls, -0.7 ± 0.2 vs 1.7 ± 0.7 g/d; ZGN-440 vs vehicle, respectively), reduction of food intake (MC4rKO, -28%; lean controls, -7.5%), and insulin resistance, whereas circulating levels of interleukin-1β did not change. In both obesity models, body temperature and locomotor activity were not affected by ZGN-440 treatment. In conclusion, the robust reduction of body weight in response to ZGN-440 observed in rats with severe obesity is related to a strong reduction of food intake that is likely related to changes in the central regulation of feeding. Copyright © 2017 by the Endocrine Society.

Pituitary-adrenal responses to oxotremorine and acute stress in male and female M1 muscarinic receptor knockout mice: comparisons to M2 muscarinic receptor knockout mice.

PubMed

Rhodes, M E; Rubin, R T; McKlveen, J M; Karwoski, T E; Fulton, B A; Czambel, R K

2008-05-01

Both within the brain and in the periphery, M(1) muscarinic receptors function primarily as postsynaptic receptors and M(2) muscarinic receptors function primarily as presynaptic autoreceptors. In addition to classical parasympathetic effectors, cholinergic stimulation of central muscarinic receptors influences the release of adrenocorticotrophic hormone (ACTH) and corticosterone. We previously reported that oxotremorine administration to male and female M(2) receptor knockout and wild-type mice increased ACTH to a significantly greater degree in knockout males compared to all other groups, and that M(2) knockout mice of both sexes were significantly more responsive to the mild stress of saline injection than were wild-type mice. These results accord with the primary function of M(2) receptors as presynaptic autoreceptors. In the present study, we explored the role of the M(1) receptor in pituitary-adrenal responses to oxotremorine and saline in male and female M(1) knockout and wild-type mice. Because these mice responded differently to the mild stress of saline injection than did the M(2) knockout and wild-type mice, we also determined hormone responses to restraint stress in both M(1) and M(2) knockout and wild-type mice. Male and female M(1) knockout and wild-type mice were equally unresponsive to the stress of saline injection. Oxotremorine increased both ACTH and corticosterone in M(1) wild-type mice to a significantly greater degree than in knockout mice. In both M(1) knockout and wild-type animals, ACTH responses were greater in males compared to females, and corticosterone responses were greater in females compared to males. Hormone responses to restraint stress were increased in M(2) knockout mice and decreased in M(1) knockout mice compared to their wild-type counterparts. These findings suggest that M(1) and M(2) muscarinic receptor subtypes differentially influence male and female pituitary-adrenal responses to cholinergic stimulation and stress. The decreased pituitary-adrenal sensitivity to oxotremorine and restraint stress noted in M(1) knockout mice is consistent with M(1) being primarily a postsynaptic receptor. Conversely, the increased pituitary-adrenal sensitivity to these challenges noted in M(2) knockout mice is consistent with M(2) being primarily a presynaptic autoreceptor.

Rac1 Is Required for Cardiomyocyte Apoptosis During Hyperglycemia

PubMed Central

Shen, E.; Li, Yanwen; Li, Ying; Shan, Limei; Zhu, Huaqing; Feng, Qingping; Arnold, J. Malcolm O.; Peng, Tianqing

2009-01-01

OBJECTIVE Hyperglycemia induces reactive oxygen species (ROS) and apoptosis in cardiomyocytes, which contributes to diabetic cardiomyopathy. The present study was to investigate the role of Rac1 in ROS production and cardiomyocyte apoptosis during hyperglycemia. RESEARCH DESIGN AND METHODS Mice with cardiomyocyte-specific Rac1 knockout (Rac1-ko) were generated. Hyperglycemia was induced in Rac1-ko mice and their wild-type littermates by injection of streptozotocin (STZ). In cultured adult rat cardiomyocytes, apoptosis was induced by high glucose. RESULTS The results showed a mouse model of STZ-induced diabetes, 7 days of hyperglycemia-upregulated Rac1 and NADPH oxidase activation, elevated ROS production, and induced apoptosis in the heart. These effects of hyperglycemia were significantly decreased in Rac1-ko mice or wild-type mice treated with apocynin. Interestingly, deficiency of Rac1 or apocynin treatment significantly reduced hyperglycemia-induced mitochondrial ROS production in the heart. Deficiency of Rac1 also attenuated myocardial dysfunction after 2 months of STZ injection. In cultured cardiomyocytes, high glucose upregulated Rac1 and NADPH oxidase activity and induced apoptotic cell death, which were blocked by overexpression of a dominant negative mutant of Rac1, knockdown of gp91phox or p47phox, or NADPH oxidase inhibitor. In type 2 diabetic db/db mice, administration of Rac1 inhibitor, NSC23766, significantly inhibited NADPH oxidase activity and apoptosis and slightly improved myocardial function. CONCLUSIONS Rac1 is pivotal in hyperglycemia-induced apoptosis in cardiomyocytes. The role of Rac1 is mediated through NADPH oxidase activation and associated with mitochondrial ROS generation. Our study suggests that Rac1 may serve as a potential therapeutic target for cardiac complications of diabetes. PMID:19592621

Aucubin protects against pressure overload-induced cardiac remodelling via the β3 -adrenoceptor-neuronal NOS cascades.

PubMed

Wu, Qing-Qing; Xiao, Yang; Duan, Ming-Xia; Yuan, Yuan; Jiang, Xiao-Han; Yang, Zheng; Liao, Hai-Han; Deng, Wei; Tang, Qi-Zhu

2018-05-01

Aucubin, the predominant component of Eucommia ulmoides Oliv., has been shown to have profound effects on oxidative stress. As oxidative stress has previously been demonstrated to contribute to acute and chronic myocardial injury, we tested the effects of aucubin on cardiac remodelling and heart failure. Initially, H9c2 cardiomyocytes and neonatal rat cardiomyocytes pretreated with aucubin (1, 3, 10, 25 and 50 μM) were challenged with phenylephrine. Secondly, the transverse aorta was constricted in C57/B6 and neuronal NOS (nNOS)-knockout mice, then aucubin (1 or 5 mg·kg -1 body weight day -1 ) was injected i.p. for 25 days. Hypertrophy was evaluated by assessing morphological changes, echocardiographic parameters, histological analyses and hypertrophic markers. Oxidative stress was evaluated by examining ROS generation, oxidase activity and NO generation. NOS expression was determined by Western blotting. Aucubin effectively suppressed cardiac remodelling; in mice, aucubin substantially inhibited pressure overload-induced cardiac hypertrophy, fibrosis and inflammation, whereas knocking out nNOS abolished these cardioprotective effects of aucubin. Blocking or knocking down the β 3 -adrenoceptor abolished the protective effects of aucubin in vitro. Furthermore, aucubin enhanced the protective effects of a β 3 -adrenoceptor agonist in vitro by increasing cellular cAMP levels, whereas treatment with an adenylate cyclase (AC) inhibitor abolished the cardioprotective effects of aucubin. Aucubin suppresses oxidative stress during cardiac remodelling by increasing the expression of nNOS in a process that requires activation of the β 3 -adrenoceptor/AC/cAMP pathway. These findings suggest that aucubin could have potential as a treatment for cardiac remodelling and heart failure. © 2018 The British Pharmacological Society.

Deletion of the transforming growth factor β receptor type II gene in articular chondrocytes leads to a progressive osteoarthritis-like phenotype in mice.

PubMed

Shen, Jie; Li, Jia; Wang, Baoli; Jin, Hongting; Wang, Meina; Zhang, Yejia; Yang, Yunzhi; Im, Hee-Jeong; O'Keefe, Regis; Chen, Di

2013-12-01

While transforming growth factor β (TGFβ) signaling plays a critical role in chondrocyte metabolism, the TGFβ signaling pathways and target genes involved in cartilage homeostasis and the development of osteoarthritis (OA) remain unclear. Using an in vitro cell culture method and an in vivo mouse genetic approach, we undertook this study to investigate TGFβ signaling in chondrocytes and to determine whether Mmp13 and Adamts5 are critical downstream target genes of TGFβ signaling. TGFβ receptor type II (TGFβRII)-conditional knockout (KO) (TGFβRII(Col2ER)) mice were generated by breeding TGFβRII(flox/flox) mice with Col2-CreER-transgenic mice. Histologic, histomorphometric, and gene expression analyses were performed. In vitro TGFβ signaling studies were performed using chondrogenic rat chondrosarcoma cells. To determine whether Mmp13 and Adamts5 are critical downstream target genes of TGFβ signaling, TGFβRII/matrix metalloproteinase 13 (MMP-13)- and TGFβRII/ADAMTS-5-double-KO mice were generated and analyzed. Inhibition of TGFβ signaling (deletion of the Tgfbr2 gene in chondrocytes) resulted in up-regulation of Runx2, Mmp13, and Adamts5 expression in articular cartilage tissue and progressive OA development in TGFβRII(Col2ER) mice. Deletion of the Mmp13 or Adamts5 gene significantly ameliorated the OA-like phenotype induced by the loss of TGFβ signaling. Treatment of TGFβRII(Col2ER) mice with an MMP-13 inhibitor also slowed OA progression. Mmp13 and Adamts5 are critical downstream target genes involved in the TGFβ signaling pathway during the development of OA. Copyright © 2013 by the American College of Rheumatology.

Identification of genetic elements in metabolism by high-throughput mouse phenotyping.

PubMed

Rozman, Jan; Rathkolb, Birgit; Oestereicher, Manuela A; Schütt, Christine; Ravindranath, Aakash Chavan; Leuchtenberger, Stefanie; Sharma, Sapna; Kistler, Martin; Willershäuser, Monja; Brommage, Robert; Meehan, Terrence F; Mason, Jeremy; Haselimashhadi, Hamed; Hough, Tertius; Mallon, Ann-Marie; Wells, Sara; Santos, Luis; Lelliott, Christopher J; White, Jacqueline K; Sorg, Tania; Champy, Marie-France; Bower, Lynette R; Reynolds, Corey L; Flenniken, Ann M; Murray, Stephen A; Nutter, Lauryl M J; Svenson, Karen L; West, David; Tocchini-Valentini, Glauco P; Beaudet, Arthur L; Bosch, Fatima; Braun, Robert B; Dobbie, Michael S; Gao, Xiang; Herault, Yann; Moshiri, Ala; Moore, Bret A; Kent Lloyd, K C; McKerlie, Colin; Masuya, Hiroshi; Tanaka, Nobuhiko; Flicek, Paul; Parkinson, Helen E; Sedlacek, Radislav; Seong, Je Kyung; Wang, Chi-Kuang Leo; Moore, Mark; Brown, Steve D; Tschöp, Matthias H; Wurst, Wolfgang; Klingenspor, Martin; Wolf, Eckhard; Beckers, Johannes; Machicao, Fausto; Peter, Andreas; Staiger, Harald; Häring, Hans-Ulrich; Grallert, Harald; Campillos, Monica; Maier, Holger; Fuchs, Helmut; Gailus-Durner, Valerie; Werner, Thomas; Hrabe de Angelis, Martin

2018-01-18

Metabolic diseases are a worldwide problem but the underlying genetic factors and their relevance to metabolic disease remain incompletely understood. Genome-wide research is needed to characterize so-far unannotated mammalian metabolic genes. Here, we generate and analyze metabolic phenotypic data of 2016 knockout mouse strains under the aegis of the International Mouse Phenotyping Consortium (IMPC) and find 974 gene knockouts with strong metabolic phenotypes. 429 of those had no previous link to metabolism and 51 genes remain functionally completely unannotated. We compared human orthologues of these uncharacterized genes in five GWAS consortia and indeed 23 candidate genes are associated with metabolic disease. We further identify common regulatory elements in promoters of candidate genes. As each regulatory element is composed of several transcription factor binding sites, our data reveal an extensive metabolic phenotype-associated network of co-regulated genes. Our systematic mouse phenotype analysis thus paves the way for full functional annotation of the genome.

Translating human genetics into mouse: the impact of ultra-rapid in vivo genome editing.

PubMed

Aida, Tomomi; Imahashi, Risa; Tanaka, Kohichi

2014-01-01

Gene-targeted mutant animals, such as knockout or knockin mice, have dramatically improved our understanding of the functions of genes in vivo and the genetic diversity that characterizes health and disease. However, the generation of targeted mice relies on gene targeting in embryonic stem (ES) cells, which is a time-consuming, laborious, and expensive process. The recent groundbreaking development of several genome editing technologies has enabled the targeted alteration of almost any sequence in any cell or organism. These technologies have now been applied to mouse zygotes (in vivo genome editing), thereby providing new avenues for simple, convenient, and ultra-rapid production of knockout or knockin mice without the need for ES cells. Here, we review recent achievements in the production of gene-targeted mice by in vivo genome editing. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

Human androgen deficiency: insights gained from androgen receptor knockout mouse models

PubMed Central

Rana, Kesha; Davey, Rachel A; Zajac, Jeffrey D

2014-01-01

The mechanism of androgen action is complex. Recently, significant advances have been made into our understanding of how androgens act via the androgen receptor (AR) through the use of genetically modified mouse models. A number of global and tissue-specific AR knockout (ARKO) models have been generated using the Cre-loxP system which allows tissue- and/or cell-specific deletion. These ARKO models have examined a number of sites of androgen action including the cardiovascular system, the immune and hemopoetic system, bone, muscle, adipose tissue, the prostate and the brain. This review focuses on the insights that have been gained into human androgen deficiency through the use of ARKO mouse models at each of these sites of action, and highlights the strengths and limitations of these Cre-loxP mouse models that should be considered to ensure accurate interpretation of the phenotype. PMID:24480924

Translation of Genotype to Phenotype by a Hierarchy of Cell Subsystems.

PubMed

Yu, Michael Ku; Kramer, Michael; Dutkowski, Janusz; Srivas, Rohith; Licon, Katherine; Kreisberg, Jason; Ng, Cherie T; Krogan, Nevan; Sharan, Roded; Ideker, Trey

2016-02-24

Accurately translating genotype to phenotype requires accounting for the functional impact of genetic variation at many biological scales. Here we present a strategy for genotype-phenotype reasoning based on existing knowledge of cellular subsystems. These subsystems and their hierarchical organization are defined by the Gene Ontology or a complementary ontology inferred directly from previously published datasets. Guided by the ontology's hierarchical structure, we organize genotype data into an "ontotype," that is, a hierarchy of perturbations representing the effects of genetic variation at multiple cellular scales. The ontotype is then interpreted using logical rules generated by machine learning to predict phenotype. This approach substantially outperforms previous, non-hierarchical methods for translating yeast genotype to cell growth phenotype, and it accurately predicts the growth outcomes of two new screens of 2,503 double gene knockouts impacting DNA repair or nuclear lumen. Ontotypes also generalize to larger knockout combinations, setting the stage for interpreting the complex genetics of disease.

The microRNA-processing enzyme Dicer is essential for thyroid function.

PubMed

Frezzetti, Daniela; Reale, Carla; Calì, Gaetano; Nitsch, Lucio; Fagman, Henrik; Nilsson, Ola; Scarfò, Marzia; De Vita, Gabriella; Di Lauro, Roberto

2011-01-01

Dicer is a type III ribonuclease required for the biogenesis of microRNAs (miRNAs), a class of small non-coding RNAs regulating gene expression at the post-transcriptional level. To explore the functional role of miRNAs in thyroid gland function, we generated a thyrocyte-specific Dicer conditional knockout mouse. Here we show that development and early differentiation of the thyroid gland are not affected by the absence of Dicer, while severe hypothyroidism gradually develops after birth, leading to reduced body weight and shortened life span. Histological and molecular characterization of knockout mice reveals a dramatic loss of the thyroid gland follicular architecture associated with functional aberrations and down-regulation of several differentiation markers. The data presented in this study show for the first time that an intact miRNAs processing machinery is essential for thyroid physiology, suggesting that deregulation of specific miRNAs could be also involved in human thyroid dysfunctions.

Armc5 deletion causes developmental defects and compromises T-cell immune responses

PubMed Central

Hu, Yan; Lao, Linjiang; Mao, Jianning; Jin, Wei; Luo, Hongyu; Charpentier, Tania; Qi, Shijie; Peng, Junzheng; Hu, Bing; Marcinkiewicz, Mieczyslaw Martin; Lamarre, Alain; Wu, Jiangping

2017-01-01

Armadillo repeat containing 5 (ARMC5) is a cytosolic protein with no enzymatic activities. Little is known about its function and mechanisms of action, except that gene mutations are associated with risks of primary macronodular adrenal gland hyperplasia. Here we map Armc5 expression by in situ hybridization, and generate Armc5 knockout mice, which are small in body size. Armc5 knockout mice have compromised T-cell proliferation and differentiation into Th1 and Th17 cells, increased T-cell apoptosis, reduced severity of experimental autoimmune encephalitis, and defective immune responses to lymphocytic choriomeningitis virus infection. These mice also develop adrenal gland hyperplasia in old age. Yeast 2-hybrid assays identify 16 ARMC5-binding partners. Together these data indicate that ARMC5 is crucial in fetal development, T-cell function and adrenal gland growth homeostasis, and that the functions of ARMC5 probably depend on interaction with multiple signalling pathways. PMID:28169274

Analysis of mammalian gene function through broad based phenotypic screens across a consortium of mouse clinics

PubMed Central

Adams, David J; Adams, Niels C; Adler, Thure; Aguilar-Pimentel, Antonio; Ali-Hadji, Dalila; Amann, Gregory; André, Philippe; Atkins, Sarah; Auburtin, Aurelie; Ayadi, Abdel; Becker, Julien; Becker, Lore; Bedu, Elodie; Bekeredjian, Raffi; Birling, Marie-Christine; Blake, Andrew; Bottomley, Joanna; Bowl, Mike; Brault, Véronique; Busch, Dirk H; Bussell, James N; Calzada-Wack, Julia; Cater, Heather; Champy, Marie-France; Charles, Philippe; Chevalier, Claire; Chiani, Francesco; Codner, Gemma F; Combe, Roy; Cox, Roger; Dalloneau, Emilie; Dierich, André; Di Fenza, Armida; Doe, Brendan; Duchon, Arnaud; Eickelberg, Oliver; Esapa, Chris T; El Fertak, Lahcen; Feigel, Tanja; Emelyanova, Irina; Estabel, Jeanne; Favor, Jack; Flenniken, Ann; Gambadoro, Alessia; Garrett, Lilian; Gates, Hilary; Gerdin, Anna-Karin; Gkoutos, George; Greenaway, Simon; Glasl, Lisa; Goetz, Patrice; Da Cruz, Isabelle Goncalves; Götz, Alexander; Graw, Jochen; Guimond, Alain; Hans, Wolfgang; Hicks, Geoff; Hölter, Sabine M; Höfler, Heinz; Hancock, John M; Hoehndorf, Robert; Hough, Tertius; Houghton, Richard; Hurt, Anja; Ivandic, Boris; Jacobs, Hughes; Jacquot, Sylvie; Jones, Nora; Karp, Natasha A; Katus, Hugo A; Kitchen, Sharon; Klein-Rodewald, Tanja; Klingenspor, Martin; Klopstock, Thomas; Lalanne, Valerie; Leblanc, Sophie; Lengger, Christoph; le Marchand, Elise; Ludwig, Tonia; Lux, Aline; McKerlie, Colin; Maier, Holger; Mandel, Jean-Louis; Marschall, Susan; Mark, Manuel; Melvin, David G; Meziane, Hamid; Micklich, Kateryna; Mittelhauser, Christophe; Monassier, Laurent; Moulaert, David; Muller, Stéphanie; Naton, Beatrix; Neff, Frauke; Nolan, Patrick M; Nutter, Lauryl MJ; Ollert, Markus; Pavlovic, Guillaume; Pellegata, Natalia S; Peter, Emilie; Petit-Demoulière, Benoit; Pickard, Amanda; Podrini, Christine; Potter, Paul; Pouilly, Laurent; Puk, Oliver; Richardson, David; Rousseau, Stephane; Quintanilla-Fend, Leticia; Quwailid, Mohamed M; Racz, Ildiko; Rathkolb, Birgit; Riet, Fabrice; Rossant, Janet; Roux, Michel; Rozman, Jan; Ryder, Ed; Salisbury, Jennifer; Santos, Luis; Schäble, Karl-Heinz; Schiller, Evelyn; Schrewe, Anja; Schulz, Holger; Steinkamp, Ralf; Simon, Michelle; Stewart, Michelle; Stöger, Claudia; Stöger, Tobias; Sun, Minxuan; Sunter, David; Teboul, Lydia; Tilly, Isabelle; Tocchini-Valentini, Glauco P; Tost, Monica; Treise, Irina; Vasseur, Laurent; Velot, Emilie; Vogt-Weisenhorn, Daniela; Wagner, Christelle; Walling, Alison; Weber, Bruno; Wendling, Olivia; Westerberg, Henrik; Willershäuser, Monja; Wolf, Eckhard; Wolter, Anne; Wood, Joe; Wurst, Wolfgang; Yildirim, Ali Önder; Zeh, Ramona; Zimmer, Andreas; Zimprich, Annemarie

2015-01-01

The function of the majority of genes in the mouse and human genomes remains unknown. The mouse ES cell knockout resource provides a basis for characterisation of relationships between gene and phenotype. The EUMODIC consortium developed and validated robust methodologies for broad-based phenotyping of knockouts through a pipeline comprising 20 disease-orientated platforms. We developed novel statistical methods for pipeline design and data analysis aimed at detecting reproducible phenotypes with high power. We acquired phenotype data from 449 mutant alleles, representing 320 unique genes, of which half had no prior functional annotation. We captured data from over 27,000 mice finding that 83% of the mutant lines are phenodeviant, with 65% demonstrating pleiotropy. Surprisingly, we found significant differences in phenotype annotation according to zygosity. Novel phenotypes were uncovered for many genes with unknown function providing a powerful basis for hypothesis generation and further investigation in diverse systems. PMID:26214591

Reduced osteoblast activity in the mice lacking TR4 nuclear receptor leads to osteoporosis.

PubMed

Lin, Shin-Jen; Ho, Hsin-Chiu; Lee, Yi-Fen; Liu, Ning-Chun; Liu, Su; Li, Gonghui; Shyr, Chih-Rong; Chang, Chawnshang

2012-06-07

Early studies suggested that TR4 nuclear receptor might play important roles in the skeletal development, yet its detailed mechanism remains unclear. We generated TR4 knockout mice and compared skeletal development with their wild type littermates. Primary bone marrow cells were cultured and we assayed bone differentiation by alkaline phosphatase and alizarin red staining. Primary calvaria were cultured and osteoblastic marker genes were detected by quantitative PCR. Luciferase reporter assays, chromatin immunoprecipitation (ChIP) assays, and electrophoretic mobility shift assays (EMSA) were performed to demonstrate TR4 can directly regulate bone differentiation marker osteocalcin. We first found mice lacking TR4 might develop osteoporosis. We then found that osteoblast progenitor cells isolated from bone marrow of TR4 knockout mice displayed reduced osteoblast differentiation capacity and calcification. Osteoblast primary cultures from TR4 knockout mice calvaria also showed higher proliferation rates indicating lower osteoblast differentiation ability in mice after loss of TR4. Mechanism dissection found the expression of osteoblast markers genes, such as ALP, type I collagen alpha 1, osteocalcin, PTH, and PTHR was dramatically reduced in osteoblasts from TR4 knockout mice as compared to those from TR4 wild type mice. In vitro cell line studies with luciferase reporter assay, ChIP assay, and EMSA further demonstrated TR4 could bind directly to the promoter region of osteocalcin gene and induce its gene expression at the transcriptional level in a dose dependent manner. Together, these results demonstrate TR4 may function as a novel transcriptional factor to play pathophysiological roles in maintaining normal osteoblast activity during the bone development and remodeling, and disruption of TR4 function may result in multiple skeletal abnormalities.

Bloomsbury report on mouse embryo phenotyping: recommendations from the IMPC workshop on embryonic lethal screening.

PubMed

Adams, David; Baldock, Richard; Bhattacharya, Shoumo; Copp, Andrew J; Dickinson, Mary; Greene, Nicholas D E; Henkelman, Mark; Justice, Monica; Mohun, Timothy; Murray, Stephen A; Pauws, Erwin; Raess, Michael; Rossant, Janet; Weaver, Tom; West, David

2013-05-01

Identifying genes that are important for embryo development is a crucial first step towards understanding their many functions in driving the ordered growth, differentiation and organogenesis of embryos. It can also shed light on the origins of developmental disease and congenital abnormalities. Current international efforts to examine gene function in the mouse provide a unique opportunity to pinpoint genes that are involved in embryogenesis, owing to the emergence of embryonic lethal knockout mutants. Through internationally coordinated efforts, the International Knockout Mouse Consortium (IKMC) has generated a public resource of mouse knockout strains and, in April 2012, the International Mouse Phenotyping Consortium (IMPC), supported by the EU InfraCoMP programme, convened a workshop to discuss developing a phenotyping pipeline for the investigation of embryonic lethal knockout lines. This workshop brought together over 100 scientists, from 13 countries, who are working in the academic and commercial research sectors, including experts and opinion leaders in the fields of embryology, animal imaging, data capture, quality control and annotation, high-throughput mouse production, phenotyping, and reporter gene analysis. This article summarises the outcome of the workshop, including (1) the vital scientific importance of phenotyping embryonic lethal mouse strains for basic and translational research; (2) a common framework to harmonise international efforts within this context; (3) the types of phenotyping that are likely to be most appropriate for systematic use, with a focus on 3D embryo imaging; (4) the importance of centralising data in a standardised form to facilitate data mining; and (5) the development of online tools to allow open access to and dissemination of the phenotyping data.

Double Knockout of the Na+-Driven Cl−/HCO3− Exchanger and Na+/Cl− Cotransporter Induces Hypokalemia and Volume Depletion

PubMed Central

Sinning, Anne; Radionov, Nikita; Trepiccione, Francesco; López-Cayuqueo, Karen I.; Jayat, Maximilien; Baron, Stéphanie; Cornière, Nicolas; Alexander, R. Todd; Hadchouel, Juliette; Eladari, Dominique; Hübner, Christian A.

2017-01-01

We recently described a novel thiazide–sensitive electroneutral NaCl transport mechanism resulting from the parallel operation of the Cl−/HCO3− exchanger pendrin and the Na+–driven Cl−/2HCO3− exchanger (NDCBE) in β-intercalated cells of the collecting duct. Although a role for pendrin in maintaining Na+ balance, intravascular volume, and BP is well supported, there is no in vivo evidence for the role of NDCBE in maintaining Na+ balance. Here, we show that deletion of NDCBE in mice caused only subtle perturbations of Na+ homeostasis and provide evidence that the Na+/Cl− cotransporter (NCC) compensated for the inactivation of NDCBE. To unmask the role of NDCBE, we generated Ndcbe/Ncc double–knockout (dKO) mice. On a normal salt diet, dKO and single-knockout mice exhibited similar activation of the renin-angiotensin-aldosterone system, whereas only dKO mice displayed a lower blood K+ concentration. Furthermore, dKO mice displayed upregulation of the epithelial sodium channel (ENaC) and the Ca2+–activated K+ channel BKCa. During NaCl depletion, only dKO mice developed marked intravascular volume contraction, despite dramatically increased renin activity. Notably, the increase in aldosterone levels expected on NaCl depletion was attenuated in dKO mice, and single-knockout and dKO mice had similar blood K+ concentrations under this condition. In conclusion, NDCBE is necessary for maintaining sodium balance and intravascular volume during salt depletion or NCC inactivation in mice. Furthermore, NDCBE has an important role in the prevention of hypokalemia. Because NCC and NDCBE are both thiazide targets, the combined inhibition of NCC and the NDCBE/pendrin system may explain thiazide-induced hypokalemia in some patients. PMID:27151921

Loss of Adult Cardiac Myocyte GSK-3 Leads to Mitotic Catastrophe Resulting in Fatal Dilated Cardiomyopathy.

PubMed

Zhou, Jibin; Ahmad, Firdos; Parikh, Shan; Hoffman, Nichole E; Rajan, Sudarsan; Verma, Vipin K; Song, Jianliang; Yuan, Ancai; Shanmughapriya, Santhanam; Guo, Yuanjun; Gao, Erhe; Koch, Walter; Woodgett, James R; Madesh, Muniswamy; Kishore, Raj; Lal, Hind; Force, Thomas

2016-04-15

Cardiac myocyte-specific deletion of either glycogen synthase kinase (GSK)-3α and GSK-3β leads to cardiac protection after myocardial infarction, suggesting that deletion of both isoforms may provide synergistic protection. This is an important consideration because of the fact that all GSK-3-targeted drugs, including the drugs already in clinical trial target both isoforms of GSK-3, and none are isoform specific. To identify the consequences of combined deletion of cardiac myocyte GSK-3α and GSK-3β in heart function. We generated tamoxifen-inducible cardiac myocyte-specific mice lacking both GSK-3 isoforms (double knockout). We unexpectedly found that cardiac myocyte GSK-3 is essential for cardiac homeostasis and overall survival. Serial echocardiographic analysis reveals that within 2 weeks of tamoxifen treatment, double-knockout hearts leads to excessive dilatative remodeling and ventricular dysfunction. Further experimentation with isolated adult cardiac myocytes and fibroblasts from double-knockout implicated cardiac myocytes intrinsic factors responsible for observed phenotype. Mechanistically, loss of GSK-3 in adult cardiac myocytes resulted in induction of mitotic catastrophe, a previously unreported event in cardiac myocytes. Double-knockout cardiac myocytes showed cell cycle progression resulting in increased DNA content and multinucleation. However, increased cell cycle activity was rivaled by marked activation of DNA damage, cell cycle checkpoint activation, and mitotic catastrophe-induced apoptotic cell death. Importantly, mitotic catastrophe was also confirmed in isolated adult cardiac myocytes. Together, our findings suggest that cardiac myocyte GSK-3 is required to maintain normal cardiac homeostasis, and its loss is incompatible with life because of cell cycle dysregulation that ultimately results in a severe fatal dilated cardiomyopathy. © 2016 American Heart Association, Inc.

Morphologic and Histologic Comparison of Hypertrophic Scar in Nude Mice, T-Cell Receptor, and Recombination Activating Gene Knockout Mice.

PubMed

Momtazi, Moein; Ding, Jie; Kwan, Peter; Anderson, Colin C; Honardoust, Dariush; Goekjian, Serge; Tredget, Edward E

2015-12-01

Proliferative scars in nude mice have demonstrated morphologic and histologic similarities to human hypertrophic scar. Gene knockout technology provides the opportunity to study the effect of deleting immune cells in various disease processes. The authors' objective was to test whether grafting human skin onto T-cell receptor (TCR) αβ-/-γδ-/-, recombination activating gene (RAG)-1-/-, and RAG-2γ-/-c-/- mice results in proliferative scars consistent with human hypertrophic scar and to characterize the morphologic, histologic, and cellular changes that occur after removing immune cells. Nude TCRαβ-/-γδ-/-, RAG-1-/-, and RAG-2-/-γc-/- mice (n = 20 per strain) were grafted with human skin and euthanized at 30, 60, 120, and 180 days. Controls (n = 5 per strain) were autografted with mouse skin. Scars and normal skin were harvested at each time point. Sections were stained with hematoxylin and eosin, Masson's trichrome, and immunohistochemistry for anti-human leukocyte antigen-ABC, α-smooth muscle actin, decorin, and biglycan. TCRαβ-/-γδ-/-, RAG-1-/-, and RAG-2-/-γc-/- mice grafted with human skin developed firm, elevated scars with histologic and immunohistochemical similarities to human hypertrophic scar. Autografted controls showed no evidence of pathologic scarring. Knockout animals demonstrated a capacity for scar remodeling not observed in nude mice where reductions in α-smooth muscle actin staining pattern and scar thickness occurred over time. Human skin transplanted onto TCRαβ-/-γδ-/-, RAG-1-/-, and RAG-2-/-γc-/- mice results in proliferative scars with morphologic and histologic features of human hypertrophic scar. Remodeling of proliferative scars generated in knockout animals is analogous to changes in human hypertrophic scar. These animal models may better represent the natural history of human hypertrophic scar.

Double Knockout of the Na+-Driven Cl-/HCO3- Exchanger and Na+/Cl- Cotransporter Induces Hypokalemia and Volume Depletion.

PubMed

Sinning, Anne; Radionov, Nikita; Trepiccione, Francesco; López-Cayuqueo, Karen I; Jayat, Maximilien; Baron, Stéphanie; Cornière, Nicolas; Alexander, R Todd; Hadchouel, Juliette; Eladari, Dominique; Hübner, Christian A; Chambrey, Régine

2017-01-01

We recently described a novel thiazide-sensitive electroneutral NaCl transport mechanism resulting from the parallel operation of the Cl - /HCO 3 - exchanger pendrin and the Na + -driven Cl - /2HCO 3 - exchanger (NDCBE) in β-intercalated cells of the collecting duct. Although a role for pendrin in maintaining Na + balance, intravascular volume, and BP is well supported, there is no in vivo evidence for the role of NDCBE in maintaining Na + balance. Here, we show that deletion of NDCBE in mice caused only subtle perturbations of Na + homeostasis and provide evidence that the Na + /Cl - cotransporter (NCC) compensated for the inactivation of NDCBE. To unmask the role of NDCBE, we generated Ndcbe/Ncc double-knockout (dKO) mice. On a normal salt diet, dKO and single-knockout mice exhibited similar activation of the renin-angiotensin-aldosterone system, whereas only dKO mice displayed a lower blood K + concentration. Furthermore, dKO mice displayed upregulation of the epithelial sodium channel (ENaC) and the Ca 2+ -activated K + channel BKCa. During NaCl depletion, only dKO mice developed marked intravascular volume contraction, despite dramatically increased renin activity. Notably, the increase in aldosterone levels expected on NaCl depletion was attenuated in dKO mice, and single-knockout and dKO mice had similar blood K + concentrations under this condition. In conclusion, NDCBE is necessary for maintaining sodium balance and intravascular volume during salt depletion or NCC inactivation in mice. Furthermore, NDCBE has an important role in the prevention of hypokalemia. Because NCC and NDCBE are both thiazide targets, the combined inhibition of NCC and the NDCBE/pendrin system may explain thiazide-induced hypokalemia in some patients. Copyright © 2016 by the American Society of Nephrology.

Distinct Roles of the DmNav and DSC1 Channels in the Action of DDT and Pyrethroids

PubMed Central

Rinkevich, Frank D.; Du, Yuzhe; Tolinski, Josh; Ueda, Atsushi; Wu, Chun-Fang; Zhorov, Boris S.; Dong, Ke

2015-01-01

Voltage-gated sodium channels (Nav channels) are critical for electrical signaling in the nervous system and are the primary targets of the insecticides DDT and pyrethroids. In Drosophila melanogaster, besides the canonical Nav channel, Para (also called DmNav), there is a sodium channel-like cation channel called DSC1 (Drosophila sodium channel 1). Temperature-sensitive paralytic mutations in DmNav (parats) confer resistance to DDT and pyrethroids, whereas DSC1 knockout flies exhibit enhanced sensitivity to pyrethroids. To further define the roles and interaction of DmNav and DSC1 channels in DDT and pyrethroid neurotoxicology, we generated a DmNav/DSC1 double mutant line by introducing a parats1 allele (carrying the I265N mutation) into a DSC1 knockout line. We confirmed that the I265N mutation reduced the sensitivity to two pyrethroids, permethrin and deltamethrin of a DmNav variant expressed in Xenopus oocytes. Computer modeling predicts that the I265N mutation confers pyrethroid resistance by allosterically altering the second pyrethroid receptor site on the DmNav channel. Furthermore, we found that I265N-mediated pyrethroid resistance in parats1 mutant flies was almost completely abolished in parats1;DSC1−/− double mutant flies. Unexpectedly, however, the DSC1 knockout flies were less sensitive to DDT, compared to the control flies (w1118A), and the parats1;DSC1−/− double mutant flies were even more resistant to DDT compared to the DSC1 knockout or parats1 mutant. Our findings revealed distinct roles of the DmNav and DSC1 channels in the neurotoxicology of DDT vs. pyrethroids and implicate the exciting possibility of using DSC1 channel blockers or modifiers in the management of pyrethroid resistance. PMID:25687544

Reducing Ventral Tegmental Dopamine D2 Receptor Expression Selectively Boosts Incentive Motivation

PubMed Central

de Jong, Johannes W; Roelofs, Theresia J M; Mol, Frédérique M U; Hillen, Anne E J; Meijboom, Katharina E; Luijendijk, Mieneke C M; van der Eerden, Harrie A M; Garner, Keith M; Vanderschuren, Louk J M J; Adan, Roger A H

2015-01-01

Altered mesolimbic dopamine signaling has been widely implicated in addictive behavior. For the most part, this work has focused on dopamine within the striatum, but there is emerging evidence for a role of the auto-inhibitory, somatodendritic dopamine D2 receptor (D2R) in the ventral tegmental area (VTA) in addiction. Thus, decreased midbrain D2R expression has been implicated in addiction in humans. Moreover, knockout of the gene encoding the D2R receptor (Drd2) in dopamine neurons has been shown to enhance the locomotor response to cocaine in mice. Therefore, we here tested the hypothesis that decreasing D2R expression in the VTA of adult rats, using shRNA knockdown, promotes addiction-like behavior in rats responding for cocaine or palatable food. Rats with decreased VTA D2R expression showed markedly increased motivation for both sucrose and cocaine under a progressive ratio schedule of reinforcement, but the acquisition or maintenance of cocaine self-administration were not affected. They also displayed enhanced cocaine-induced locomotor activity, but no change in basal locomotion. This robust increase in incentive motivation was behaviorally specific, as we did not observe any differences in fixed ratio responding, extinction responding, reinstatement or conditioned suppression of cocaine, and sucrose seeking. We conclude that VTA D2R knockdown results in increased incentive motivation, but does not directly promote other aspects of addiction-like behavior. PMID:25735756

Leptin actions on food intake and body temperature are mediated by IL-1.

PubMed

Luheshi, G N; Gardner, J D; Rushforth, D A; Loudon, A S; Rothwell, N J

1999-06-08

Leptin regulates energy balance through its actions in the brain on appetite and energy expenditure and also shares properties with cytokines such as IL-1. We report here that leptin, injected into rats intracerebroventricularly or peripherally, induces significant dose-dependent increases in core body temperature as well as suppression of appetite. Leptin failed to affect food intake or body temperature in obese (fa/fa) Zucker rats, which posses a defective leptin receptor. Furthermore, injection of leptin increased levels of the proinflammatory cytokine IL-1beta in the hypothalamus of normal Sprague-Dawley rats. Central injection of IL-1 receptor antagonist (IL-1ra) inhibited the suppression of food intake caused by central or peripheral injection of leptin (60 and 84%, respectively) and abolished the leptin-induced increase in body temperature in both cases. Mice lacking (gene knockout) the main IL-1 receptor (80 kDa, R1) responsible for IL-1 actions showed no reduction in food intake in response to leptin. These data indicate that leptin actions in the brain depend on IL-1, and we show further that the effect of leptin on fever, but not food intake, is abolished by a cyclooxygenase inhibitor. Thus, we propose that in addition to its role in body weight regulation, leptin may mediate neuroimmune responses via actions in the brain dependent on release of IL-1 and prostaglandins.

Loss of quinone reductase 2 function selectively facilitates learning behaviors.

PubMed

Benoit, Charles-Etienne; Bastianetto, Stephane; Brouillette, Jonathan; Tse, YiuChung; Boutin, Jean A; Delagrange, Philippe; Wong, TakPan; Sarret, Philippe; Quirion, Rémi

2010-09-22

High levels of reactive oxygen species (ROS) are associated with deficits in learning and memory with age as well as in Alzheimer's disease. Using DNA microarray, we demonstrated the overexpression of quinone reductase 2 (QR2) in the hippocampus in two models of learning deficits, namely the aged memory impaired rats and the scopolamine-induced amnesia model. QR2 is a cytosolic flavoprotein that catalyzes the reduction of its substrate and enhances the production of damaging activated quinone and ROS. QR2-like immunostaining is enriched in cerebral structures associated with learning behaviors, such as the hippocampal formation and the temporofrontal cortex of rat, mouse, and human brains. In cultured rat embryonic hippocampal neurons, selective inhibitors of QR2, namely S26695 and S29434, protected against menadione-induced cell death by reversing its proapoptotic action. S26695 (8 mg/kg) also significantly inhibited scopolamine-induced amnesia. Interestingly, adult QR2 knock-out mice demonstrated enhanced learning abilities in various tasks, including Morris water maze, object recognition, and rotarod performance test. Other behaviors related to anxiety (elevated plus maze), depression (forced swim), and schizophrenia (prepulse inhibition) were not affected in QR2-deficient mice. Together, these data suggest a role for QR2 in cognitive behaviors with QR2 inhibitors possibly representing a novel therapeutic strategy toward the treatment of learning deficits especially observed in the aged brain.

Evidence that central pathways that mediate defecation utilize ghrelin receptors but do not require endogenous ghrelin.

PubMed

Pustovit, Ruslan V; Callaghan, Brid; Ringuet, Mitchell T; Kerr, Nicole F; Hunne, Billie; Smyth, Ian M; Pietra, Claudio; Furness, John B

2017-08-01

In laboratory animals and in human, centrally penetrant ghrelin receptor agonists, given systemically or orally, cause defecation. Animal studies show that the effect is due to activation of ghrelin receptors in the spinal lumbosacral defecation centers. However, it is not known whether there is a physiological role of ghrelin or the ghrelin receptor in the control of defecation. Using immunohistochemistry and immunoassay, we detected and measured ghrelin in the stomach, but were unable to detect ghrelin by either method in the lumbosacral spinal cord, or other regions of the CNS In rats in which the thoracic spinal cord was transected 5 weeks before, the effects of a ghrelin agonist on colorectal propulsion were significantly enhanced, but defecation caused by water avoidance stress (WAS) was reduced. In knockout rats that expressed no ghrelin and in wild-type rats, WAS-induced defecation was reduced by a ghrelin receptor antagonist, to similar extents. We conclude that the ghrelin receptors of the lumbosacral defecation centers have a physiological role in the control of defecation, but that their role is not dependent on ghrelin. This implies that a transmitter other than ghrelin engages the ghrelin receptor or a ghrelin receptor complex. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Physiologic regulation of atrial natriuretic peptide receptors in rat renal glomeruli.

PubMed Central

Ballermann, B J; Hoover, R L; Karnovsky, M J; Brenner, B M

1985-01-01

Isolated rat renal glomeruli and cultured glomerular mesangial and epithelial cells were examined for atrial natriuretic peptide (ANP) receptors, and for ANP-stimulated cyclic guanosine monophosphate (cGMP) generation. In glomeruli from normal rats, human (1-28) 125I-ANP bound to a single population of high affinity receptors with a mean equilibrium dissociation constant of 0.46 nM. Human (1-28) ANP markedly stimulated cGMP generation, but not cAMP generation in normal rat glomeruli. Analogues of ANP that bound to the glomerular ANP receptor with high affinity stimulated cGMP accumulation, whereas the (13-28) ANP fragment, which failed to bind to the receptor, was devoid of functional activity. Cell surface receptors for ANP were expressed on cultured glomerular mesangial but not epithelial cells, and appreciable ANP-stimulated cGMP accumulation was elicited only in mesangial cells. Approximately 12,000 ANP receptor sites were present per mesangial cell, with an average value for the equilibrium dissociation constant of 0.22 nM. Feeding of a low-salt diet to rats for 2 wk resulted in marked up regulation of the glomerular ANP receptor density to a mean of 426 fmol/mg protein, compared with 116 fmol/mg in rats given a high-salt diet. A modest reduction in the affinity of glomerular ANP receptors was also observed in rats fed the low-salt diet. ANP-stimulated cGMP generation in glomeruli did not change with alterations in salt intake. We conclude that high salt feeding in the rat results in reduced glomerular ANP receptor density relative to values in salt restricted rats. Furthermore, the mesangial cell is a principal target for ANP binding in the glomerulus. Images PMID:3001139

Biasing genome-editing events toward precise length deletions with an RNA-guided TevCas9 dual nuclease.

PubMed

Wolfs, Jason M; Hamilton, Thomas A; Lant, Jeremy T; Laforet, Marcon; Zhang, Jenny; Salemi, Louisa M; Gloor, Gregory B; Schild-Poulter, Caroline; Edgell, David R

2016-12-27

The CRISPR/Cas9 nuclease is commonly used to make gene knockouts. The blunt DNA ends generated by cleavage can be efficiently ligated by the classical nonhomologous end-joining repair pathway (c-NHEJ), regenerating the target site. This repair creates a cycle of cleavage, ligation, and target site regeneration that persists until sufficient modification of the DNA break by alternative NHEJ prevents further Cas9 cutting, generating a heterogeneous population of insertions and deletions typical of gene knockouts. Here, we develop a strategy to escape this cycle and bias events toward defined length deletions by creating an RNA-guided dual active site nuclease that generates two noncompatible DNA breaks at a target site, effectively deleting the majority of the target site such that it cannot be regenerated. The TevCas9 nuclease, a fusion of the I-TevI nuclease domain to Cas9, functions robustly in HEK293 cells and generates 33- to 36-bp deletions at frequencies up to 40%. Deep sequencing revealed minimal processing of TevCas9 products, consistent with protection of the DNA ends from exonucleolytic degradation and repair by the c-NHEJ pathway. Directed evolution experiments identified I-TevI variants with broadened targeting range, making TevCas9 an easy-to-use reagent. Our results highlight how the sequence-tolerant cleavage properties of the I-TevI homing endonuclease can be harnessed to enhance Cas9 applications, circumventing the cleavage and ligation cycle and biasing genome-editing events toward defined length deletions.

Hyperphagia and obesity in OLETF rats lacking CCK-1 receptors

PubMed Central

Moran, Timothy H; Bi, Sheng

2006-01-01

The brain–gut peptide cholecystokinin (CCK) inhibits food intake following peripheral or site directed central administration. Peripheral exogenous CCK inhibits food intake by reducing the size and duration of a meal. Antagonist studies have demonstrated that the actions of the exogenous peptide mimic those of endogenous CCK. Antagonist administration results in increased meal size and meal duration. The feeding inhibitory actions of CCK are mediated through interactions with CCK-1 receptors. The recent identification of the Otsuka–Long–Evans–Tokushima Fatty (OLETF) rat as a spontaneous CCK-1 receptor knockout model has allowed a more comprehensive evaluation of the feeding actions of CCK. OLETF rats become obese and develop non-insulin dependent diabetes mellitus (NIDDM). Consistent with the absence of CCK-1 receptors, OLETF rats do not respond to exogenous CCK. OLETF rats are hyperphagic and their increased food intake is characterized by a large increase in meal size with a decrease in meal frequency that is not sufficient to compensate for the meal size increase. Deficits in meal size control are evident in OLETF rats as young as 2 days of age. OLETF obesity is secondary to the increased food intake. Pair feeding to amounts consumed by intact control rats normalizes body weight, body fat and elevated insulin and glucose levels. Hypothalamic arcuate nucleus peptide mRNA expression in OLETF rats is appropriate to their obesity and is normalized by pair feeding. In contrast, pair fed and young pre-obese OLETF rats have greatly elevated dorsomedial hypothalamic (DMH) neuropeptide Y (NPY) mRNA expression. Elevated DMH NPY in OLETF rats appears to be a consequence of the absence of CCK-1 receptors. In intact rats NPY and CCK-1 receptors colocalize to neurons within the compact subregion of the DMH and local CCK administration reduces food intake and decreases DMH NPY mRNA expression. We have proposed that the absence of DMH CCK-1 receptors significantly contributes to the OLETF's inability to compensate for their meal size control deficit leading to their overall hyperphagia. Access to a running wheel and the resulting exercise normalizes food intake and body weight in OLETF rats. When given access to running wheels for 6 weeks shortly after weaning, OLETF rats do not gain weight to the same degree as sedentary OLETF rats and do not develop NIDDM. Exercise also prevents elevated levels of DMH NPY mRNA expression, suggesting that exercise exerts an alternative, non-CCK mediated, control on DMH NPY. The OLETF rat is a valuable model for characterizing actions of CCK in energy balance and has provided novel insights into interactions between exercise and food intake. PMID:16815799

VINCLOZOLIN (V) TREATMENT INDUCES REPRODUCTIVE MALFORMATIONS AND INFERTILITY IN F1 MALE RATS WHEN ADMINISTERED DURING SEXUAL BUT NOT GONADAL DIFFERENTIATION. THE EFFECTS ARE NOT TRANSMITTED TO THE SUBSEQUENT GENERATIONS.

EPA Science Inventory

V produces adverse reproductive effects in male rats when administered during sexual differentiation by acting as an androgen-antagonist. It was recently reported that four generations of SD rats, derived from dams dosed via ip injection GD8-15 with 100 mg V/kg/day, displayed pro...

Nucleus accumbens neuronal maturation differences in young rats bred for low versus high voluntary running behaviour

PubMed Central

Roberts, Michael D; Toedebusch, Ryan G; Wells, Kevin D; Company, Joseph M; Brown, Jacob D; Cruthirds, Clayton L; Heese, Alexander J; Zhu, Conan; Rottinghaus, George E; Childs, Thomas E; Booth, Frank W

2014-01-01

We compared the nucleus accumbens (NAc) transcriptomes of generation 8 (G8), 34-day-old rats selectively bred for low (LVR) versus high voluntary running (HVR) behaviours in rats that never ran (LVRnon-run and HVRnon-run), as well as in rats after 6 days of voluntary wheel running (LVRrun and HVRrun). In addition, the NAc transcriptome of wild-type Wistar rats was compared. The purpose of this transcriptomics approach was to generate testable hypotheses as to possible NAc features that may be contributing to running motivation differences between lines. Ingenuity Pathway Analysis and Gene Ontology analyses suggested that ‘cell cycle’-related transcripts and the running-induced plasticity of dopamine-related transcripts were lower in LVR versus HVR rats. From these data, a hypothesis was generated that LVR rats might have less NAc neuron maturation than HVR rats. Follow-up immunohistochemistry in G9–10 LVRnon-run rats suggested that the LVR line inherently possessed fewer mature medium spiny (Darpp-32-positive) neurons (P < 0.001) and fewer immature (Dcx-positive) neurons (P < 0.001) than their G9–10 HVR counterparts. However, voluntary running wheel access in our G9–10 LVRs uniquely increased their Darpp-32-positive and Dcx-positive neuron densities. In summary, NAc cellularity differences and/or the lack of running-induced plasticity in dopamine signalling-related transcripts may contribute to low voluntary running motivation in LVR rats. PMID:24665095

Structural and Functional Analysis of HIV-1 Coreceptors: Roles of Charged Residues and Posttranslational Modifications on Coreceptor Activity

DTIC Science & Technology

2000-01-01

various organs and to sites of inflammation. They may have additional functions. For example analysis of CXCR4 knockout mice show that CXCR4, which...SDF-1 knockout mice had similar phenotypes (195). Homozygous knockout of CXCR4 or SDF-1 results in embyonic lethality. Though CCR5 appears to be...dispensable, other chemokine receptors have vital functions. CXCR5 knockout mice have B-cell homing defects (118), and CXCR2 knockout mice

HCN channels segregate stimulation‐evoked movement responses in neocortex and allow for coordinated forelimb movements in rodents

PubMed Central

Farrell, Jordan S.; Palmer, Laura A.; Singleton, Anna C.; Pittman, Quentin J.; Teskey, G. Campbell

2016-01-01

Key points The present study tested whether HCN channels contribute to the organization of motor cortex and to skilled motor behaviour during a forelimb reaching task.Experimental reductions in HCN channel signalling increase the representation of complex multiple forelimb movements in motor cortex as assessed by intracortical microstimulation.Global HCN1KO mice exhibit reduced reaching accuracy and atypical movements during a single‐pellet reaching task relative to wild‐type controls.Acute pharmacological inhibition of HCN channels in forelimb motor cortex decreases reaching accuracy and increases atypical movements during forelimb reaching. Abstract The mechanisms by which distinct movements of a forelimb are generated from the same area of motor cortex have remained elusive. Here we examined a role for HCN channels, given their ability to alter synaptic integration, in the expression of forelimb movement responses during intracortical microstimulation (ICMS) and movements of the forelimb on a skilled reaching task. We used short‐duration high‐resolution ICMS to evoke forelimb movements following pharmacological (ZD7288), experimental (electrically induced cortical seizures) or genetic approaches that we confirmed with whole‐cell patch clamp to substantially reduce I h current. We observed significant increases in the number of multiple movement responses evoked at single sites in motor maps to all three experimental manipulations in rats or mice. Global HCN1 knockout mice were less successful and exhibited atypical movements on a skilled‐motor learning task relative to wild‐type controls. Furthermore, in reaching‐proficient rats, reaching accuracy was reduced and forelimb movements were altered during infusion of ZD7288 within motor cortex. Thus, HCN channels play a critical role in the separation of overlapping movement responses and allow for successful reaching behaviours. These data provide a novel mechanism for the encoding of multiple movement responses within shared networks of motor cortex. This mechanism supports a viewpoint of primary motor cortex as a site of dynamic integration for behavioural output. PMID:27568501

Improved Antibodies Against ERBB4/HER4 | NCI Technology Transfer Center | TTC

Cancer.gov

The Eunice Kennedy Shriver National Institute of Child Health and Human Development, Section on Molecular Neurobiology seeks parties interested in licensing or collaborative research to further evaluate or commercialize specific rabbit monoclonal antibodies generated against the ErbB4 receptor (also known as HER4) that have been validated for specificity using tissue sections and extracts from ErbB4 knockout mice.

Quorum-sensing contributes to virulence, twitching motility, seed attachment and biofilm formation in the wild type strain Aac-5 of Acidovorax citrulli

USDA-ARS?s Scientific Manuscript database

Acidovorax citrulli is a seed-borne pathogen that causes bacterial fruit blotch of cucurbits including melon and watermelon. We investigated the roles of quorum sensing in the wild-type group II strain Aac-5 of A. citrulli by generating aacR and aacI knockout mutants and their complementation strain...

New Hippocampal Neurons Are Not Obligatory for Memory Formation; Cyclin D2 Knockout Mice with No Adult Brain Neurogenesis Show Learning

ERIC Educational Resources Information Center

Jaholkowski, Piotr; Kiryk, Anna; Jedynak, Paulina; Abdallah, Nada M. Ben; Knapska, Ewelina; Kowalczyk, Anna; Piechal, Agnieszka; Blecharz-Klin, Kamilla; Figiel, Izabela; Lioudyno, Victoria; Widy-Tyszkiewicz, Ewa; Wilczynski, Grzegorz M.; Lipp, Hans-Peter; Kaczmarek, Leszek; Filipkowski, Robert K.

2009-01-01

The role of adult brain neurogenesis (generating new neurons) in learning and memory appears to be quite firmly established in spite of some criticism and lack of understanding of what the new neurons serve the brain for. Also, the few experiments showing that blocking adult neurogenesis causes learning deficits used irradiation and various drugs…

Peptidomic analysis of the neurolysin-knockout mouse brain.

PubMed

Castro, Leandro M; Cavalcanti, Diogo M L P; Araujo, Christiane B; Rioli, Vanessa; Icimoto, Marcelo Y; Gozzo, Fábio C; Juliano, Maria; Juliano, Luiz; Oliveira, Vitor; Ferro, Emer S

2014-12-05

A large number of intracellular peptides are constantly produced following protein degradation by the proteasome. A few of these peptides function in cell signaling and regulate protein-protein interactions. Neurolysin (Nln) is a structurally defined and biochemically well-characterized endooligopeptidase, and its subcellular distribution and biological activity in the vertebrate brain have been previously investigated. However, the contribution of Nln to peptide metabolism in vivo is poorly understood. In this study, we used quantitative mass spectrometry to investigate the brain peptidome of Nln-knockout mice. An additional in vitro digestion assay with recombinant Nln was also performed to confirm the identification of the substrates and/or products of Nln. Altogether, the data presented suggest that Nln is a key enzyme in the in vivo degradation of only a few peptides derived from proenkephalin, such as Met-enkephalin and octapeptide. Nln was found to have only a minor contribution to the intracellular peptide metabolism in the entire mouse brain. However, further studies appear necessary to investigate the contribution of Nln to the peptide metabolism in specific areas of the murine brain. Neurolysin was first identified in the synaptic membranes of the rat brain in the middle 80's by Frederic Checler and colleagues. Neurolysin was well characterized biochemically, and its brain distribution has been confirmed by immunohistochemical methods. The neurolysin contribution to the central and peripheral neurotensin-mediated functions in vivo has been delineated through inhibitor-based pharmacological approaches, but its genuine contribution to the physiological inactivation of neuropeptides remains to be firmly established. As a result, the main significance of this work is the first characterization of the brain peptidome of the neurolysin-knockout mouse. This article is part of a Special Issue entitled: Proteomics, mass spectrometry and peptidomics, Cancun 2013. Guest Editors: César López-Camarillo, Victoria Pando-Robles and Bronwyn Jane Barkla. Copyright © 2014. Published by Elsevier B.V.

The modA10 phasevarion of nontypeable Haemophilus influenzae R2866 regulates multiple virulence-associated traits.

PubMed

VanWagoner, Timothy M; Atack, John M; Nelson, Kevin L; Smith, Hannah K; Fox, Kate L; Jennings, Michael P; Stull, Terrence L; Smith, Arnold L

2016-03-01

Non-typeable Haemophilus influenzae (NTHi) is a human restricted commensal and pathogen that elicits inflammation by adhering to and invading airway epithelia cells: transcytosis across these cells can result in systemic infection. NTHi strain R2866 was isolated from the blood of a normal 30-month old infant with meningitis, and is unusual for NTHi in that it is able to cause systemic infection. Strain R2866 is able to replicate in normal human serum due to expression of lgtC which mimics human blood group p(k). R2866 contains a phase-variable DNA methyltransferase, modA10 which switches ON and OFF randomly and reversibly due to polymerase slippage over a long tetrameric repeat tract located in its open reading frame. Random gain or loss of repeats during replication can results in expressed (ON), or not expressed (OFF) states, the latter due to a frameshift or transcriptional termination at a premature stop codon. We sought to determine if the unusual virulence of R2866 was modified by modA10 phase-variation. A modA10 knockout mutant was found to have increased adherence to, and invasion of, human ear and airway monolayers in culture, and increased invasion and transcytosis of polarized human bronchial epithelial cells. Intriguingly, the rate of bacteremia was lower in the infant rat model of infection than a wild-type R2866 strain, but the fatality rate was greater. Transcriptional analysis comparing the modA10 knockout to the R2866 wild-type parent strain showed increased expression of genes in the modA10 knockout whose products mediate cellular adherence. We conclude that loss of ModA10 function in strain R2866 enhances colonization and invasion by increasing expression of genes that allow for increased adherence, which can contribute to the increased virulence of this strain. Copyright © 2015 Elsevier Ltd. All rights reserved.

Steroid withdrawal in the mouse results in anxiogenic effects of 3alpha,5beta-THP: a possible model of premenstrual dysphoric disorder.

PubMed

Smith, Sheryl S; Ruderman, Yevgeniy; Frye, Cheryl; Homanics, Gregg; Yuan, Maoli

2006-06-01

3alpha-OH-5alpha[beta]-pregnan-20-one (THP) is a positive modulator of the GABAA receptor (GABAR), which underlies its reported anxiolytic effect. However, there are conditions such as premenstrual dysphoric disorder (PMDD) where increases in THP levels can be associated with adverse mood. In order to test for conditions where THP might be anxiogenic, we developed a mouse model of THP withdrawal. Because delta-containing GABAR are highly sensitive to THP modulation, results were compared in wild-type and delta knockout mice. Finasteride, a 5alpha-reductase blocker, was administered for 3 days to female wild-type or delta knockout mice. Then, animals were tested in the elevated plus maze, following acute administration of THP, lorazepam, flumazenil, or 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), and results compared to vehicle-injected controls. CA1 hippocampal GABAR alpha4 subunit levels were assessed by Western blot. After THP withdrawal, THP produced anxiogenic effects, decreasing open arm entries on the elevated plus maze, following a brief shock, in contrast to its expected anxiolytic effects. As we have shown in rats, THP withdrawal also resulted in increased expression of the alpha4 subunit in mouse CA1 hippocampus. As expected for increases in alpha4-containing GABAR, THP withdrawn mice were relatively insensitive to the benzodiazepine (BDZ) lorazepam and had atypical responses to the BDZ antagonist flumazenil when tested on the plus maze. In contrast, they showed a greater anxiolytic response to THIP, which has greater efficacy at alpha4betadelta than other GABAR. Although THP withdrawal in delta knockout mice also increased the alpha4 GABAR subunit, the anxiogenic effects of THP and the anxiolytic effects of THIP were not observed, implicating alpha4betadelta GABAR in these effects. Based on these behavioral and pharmacological findings, we suggest that THP withdrawal in the mouse may serve as a rodent model of PMDD.

Role of fructose and fructokinase in acute dehydration-induced vasopressin gene expression and secretion in mice

PubMed Central

Roncal-Jimenez, Carlos A.; Lanaspa-Garcia, Miguel A.; Oppelt, Sarah A.; Kuwabara, Masanari; Jensen, Thomas; Milagres, Tamara; Andres-Hernando, Ana; Ishimoto, Takuji; Garcia, Gabriela E.; Johnson, Ginger; MacLean, Paul S.; Sanchez-Lozada, Laura-Gabriela; Tolan, Dean R.; Johnson, Richard J.

2016-01-01

Fructose stimulates vasopressin in humans and can be generated endogenously by activation of the polyol pathway with hyperosmolarity. We hypothesized that fructose metabolism in the hypothalamus might partly control vasopressin responses after acute dehydration. Wild-type and fructokinase-knockout mice were deprived of water for 24 h. The supraoptic nucleus was evaluated for vasopressin and markers of the aldose reductase-fructokinase pathway. The posterior pituitary vasopressin and serum copeptin levels were examined. Hypothalamic explants were evaluated for vasopressin secretion in response to exogenous fructose. Water restriction increased serum and urine osmolality and serum copeptin in both groups of mice, although the increase in copeptin in wild-type mice was larger than that in fructokinase-knockout mice. Water-restricted, wild-type mice showed an increase in vasopressin and aldose reductase mRNA, sorbitol, fructose and uric acid in the supraoptic nucleus. In contrast, fructokinase-knockout mice showed no change in vasopressin or aldose reductase mRNA, and no changes in sorbitol or uric acid, although fructose levels increased. With water restriction, vasopressin in the pituitary of wild-type mice was significantly less than that of fructokinase-knockout mice, indicating that fructokinase-driven vasopressin secretion overrode synthesis. Fructose increased vasopressin release in hypothalamic explants that was not observed in fructokinase-knockout mice. In situ hybridization documented fructokinase mRNA in the supraoptic nucleus, paraventricular nucleus and suprachiasmatic nucleus. Acute dehydration activates the aldose reductase-fructokinase pathway in the hypothalamus and partly drives the vasopressin response. Exogenous fructose increases vasopressin release in hypothalamic explants dependent on fructokinase. Nevertheless, circulating vasopressin is maintained and urinary concentrating is not impaired. NEW & NOTEWORTHY This study increases our understanding of the mechanisms leading to vasopressin release under conditions of water restriction (acute dehydration). Specifically, these studies suggest that the aldose reductase-fructokinase pathways may be involved in vasopressin synthesis in the hypothalamus and secretion by the pituitary in response to acute dehydration. Nevertheless, mice undergoing water restriction remain capable of maintaining sufficient vasopressin (copeptin) levels to allow normal urinary concentration. Further studies of the aldose reductase-fructokinase system in vasopressin regulation appear indicated. PMID:27852737

Role of fructose and fructokinase in acute dehydration-induced vasopressin gene expression and secretion in mice.

PubMed

Song 宋志林, Zhilin; Roncal-Jimenez, Carlos A; Lanaspa-Garcia, Miguel A; Oppelt, Sarah A; Kuwabara, Masanari; Jensen, Thomas; Milagres, Tamara; Andres-Hernando, Ana; Ishimoto, Takuji; Garcia, Gabriela E; Johnson, Ginger; MacLean, Paul S; Sanchez-Lozada, Laura-Gabriela; Tolan, Dean R; Johnson, Richard J

2017-02-01

Fructose stimulates vasopressin in humans and can be generated endogenously by activation of the polyol pathway with hyperosmolarity. We hypothesized that fructose metabolism in the hypothalamus might partly control vasopressin responses after acute dehydration. Wild-type and fructokinase-knockout mice were deprived of water for 24 h. The supraoptic nucleus was evaluated for vasopressin and markers of the aldose reductase-fructokinase pathway. The posterior pituitary vasopressin and serum copeptin levels were examined. Hypothalamic explants were evaluated for vasopressin secretion in response to exogenous fructose. Water restriction increased serum and urine osmolality and serum copeptin in both groups of mice, although the increase in copeptin in wild-type mice was larger than that in fructokinase-knockout mice. Water-restricted, wild-type mice showed an increase in vasopressin and aldose reductase mRNA, sorbitol, fructose and uric acid in the supraoptic nucleus. In contrast, fructokinase-knockout mice showed no change in vasopressin or aldose reductase mRNA, and no changes in sorbitol or uric acid, although fructose levels increased. With water restriction, vasopressin in the pituitary of wild-type mice was significantly less than that of fructokinase-knockout mice, indicating that fructokinase-driven vasopressin secretion overrode synthesis. Fructose increased vasopressin release in hypothalamic explants that was not observed in fructokinase-knockout mice. In situ hybridization documented fructokinase mRNA in the supraoptic nucleus, paraventricular nucleus and suprachiasmatic nucleus. Acute dehydration activates the aldose reductase-fructokinase pathway in the hypothalamus and partly drives the vasopressin response. Exogenous fructose increases vasopressin release in hypothalamic explants dependent on fructokinase. Nevertheless, circulating vasopressin is maintained and urinary concentrating is not impaired. This study increases our understanding of the mechanisms leading to vasopressin release under conditions of water restriction (acute dehydration). Specifically, these studies suggest that the aldose reductase-fructokinase pathways may be involved in vasopressin synthesis in the hypothalamus and secretion by the pituitary in response to acute dehydration. Nevertheless, mice undergoing water restriction remain capable of maintaining sufficient vasopressin (copeptin) levels to allow normal urinary concentration. Further studies of the aldose reductase-fructokinase system in vasopressin regulation appear indicated. Copyright © 2017 the American Physiological Society.

Genetic reduction of mitochondrial complex I function does not lead to loss of dopamine neurons in vivo.

PubMed

Kim, Hyung-Wook; Choi, Won-Seok; Sorscher, Noah; Park, Hyung Joon; Tronche, François; Palmiter, Richard D; Xia, Zhengui

2015-09-01

Inhibition of mitochondrial complex I activity is hypothesized to be one of the major mechanisms responsible for dopaminergic neuron death in Parkinson's disease. However, loss of complex I activity by systemic deletion of the Ndufs4 gene, one of the subunits comprising complex I, does not cause dopaminergic neuron death in culture. Here, we generated mice with conditional Ndufs4 knockout in dopaminergic neurons (Ndufs4 conditional knockout mice [cKO]) to examine the effect of complex I inhibition on dopaminergic neuron function and survival during aging and on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment in vivo. Ndufs4 cKO mice did not show enhanced dopaminergic neuron loss in the substantia nigra pars compacta or dopamine-dependent motor deficits over the 24-month life span. These mice were just as susceptible to MPTP as control mice. However, compared with control mice, Ndufs4 cKO mice exhibited an age-dependent reduction of dopamine in the striatum and increased α-synuclein phosphorylation in dopaminergic neurons of the substantia nigra pars compacta. We also used an inducible Ndufs4 knockout mouse strain (Ndufs4 inducible knockout) in which Ndufs4 is conditionally deleted in all cells in adult to examine the effect of adult onset, complex I inhibition on MPTP sensitivity of dopaminergic neurons. The Ndufs4 inducible knockout mice exhibited similar sensitivity to MPTP as control littermates. These data suggest that mitochondrial complex I inhibition in dopaminergic neurons does contribute to dopamine loss and the development of α-synuclein pathology. However, it is not sufficient to cause cell-autonomous dopaminergic neuron death during the normal life span of mice. Furthermore, mitochondrial complex I inhibition does not underlie MPTP toxicity in vivo in either cell autonomous or nonautonomous manner. These results provide strong evidence that inhibition of mitochondrial complex I activity is not sufficient to cause dopaminergic neuron death during aging nor does it contribute to dopamine neuron toxicity in the MPTP model of Parkinson's disease. These findings suggest the existence of alternative mechanisms of dopaminergic neuron death independent of mitochondrial complex I inhibition. Copyright © 2015 Elsevier Inc. All rights reserved.

Knockout Mice for Dyslexia Susceptibility Gene Homologs KIAA0319 and KIAA0319L have Unaffected Neuronal Migration but Display Abnormal Auditory Processing

PubMed Central

Guidi, Luiz G; Mattley, Jane; Martinez-Garay, Isabel; Monaco, Anthony P; Linden, Jennifer F; Velayos-Baeza, Antonio

2017-01-01

Abstract Developmental dyslexia is a neurodevelopmental disorder that affects reading ability caused by genetic and non-genetic factors. Amongst the susceptibility genes identified to date, KIAA0319 is a prime candidate. RNA-interference experiments in rats suggested its involvement in cortical migration but we could not confirm these findings in Kiaa0319-mutant mice. Given its homologous gene Kiaa0319L (AU040320) has also been proposed to play a role in neuronal migration, we interrogated whether absence of AU040320 alone or together with KIAA0319 affects migration in the developing brain. Analyses of AU040320 and double Kiaa0319;AU040320 knockouts (dKO) revealed no evidence for impaired cortical lamination, neuronal migration, neurogenesis or other anatomical abnormalities. However, dKO mice displayed an auditory deficit in a behavioral gap-in-noise detection task. In addition, recordings of click-evoked auditory brainstem responses revealed suprathreshold deficits in wave III amplitude in AU040320-KO mice, and more general deficits in dKOs. These findings suggest that absence of AU040320 disrupts firing and/or synchrony of activity in the auditory brainstem, while loss of both proteins might affect both peripheral and central auditory function. Overall, these results stand against the proposed role of KIAA0319 and AU040320 in neuronal migration and outline their relationship with deficits in the auditory system. PMID:29045729

Lactobacillus acidophilus ATCC 4356 Prevents Atherosclerosis via Inhibition of Intestinal Cholesterol Absorption in Apolipoprotein E-Knockout Mice

PubMed Central

Wang, Jinfeng; Quan, Guihua; Wang, Xiaojun; Yang, Longfei; Zhong, Lili

2014-01-01

The objective of this study was to investigate the effect of Lactobacillus acidophilus ATCC 4356 on the development of atherosclerosis in apolipoprotein E-knockout (ApoE−/−) mice. Eight-week-old ApoE−/− mice were fed a Western diet with or without L. acidophilus ATCC 4356 daily for 16 weeks. L. acidophilus ATCC 4356 protected ApoE−/− mice from atherosclerosis by reducing their plasma cholesterol levels from 923 ± 44 to 581 ± 18 mg/dl, likely via a marked decrease in cholesterol absorption caused by modulation of Niemann-Pick C1-like 1 (NPC1L1). In addition, suppression of cholesterol absorption induced reverse cholesterol transport (RCT) in macrophages through the peroxisome proliferator-activated receptor/liver X receptor (PPAR/LXR) pathway. Fecal lactobacillus and bifidobacterium counts were significantly (P < 0.05) higher in the L. acidophilus ATCC 4356 treatment groups than in the control groups. Furthermore, L. acidophilus ATCC 4356 was detected in the rat small intestine, colon, and feces during the feeding trial. The bacterial levels remained high even after the administration of lactic acid bacteria had been stopped for 2 weeks. These results suggest that administration of L. acidophilus ATCC 4356 can protect against atherosclerosis through the inhibition of intestinal cholesterol absorption. Therefore, L. acidophilus ATCC 4356 may be a potential therapeutic material for preventing the progression of atherosclerosis. PMID:25261526

Production of genome-edited pluripotent stem cells and mice by CRISPR/Cas.

PubMed

Horii, Takuro; Hatada, Izuho

2016-01-01

Clustered regularly at interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) nucleases, so-called CRISPR/Cas, was recently developed as an epoch-making genome engineering technology. This system only requires Cas9 nuclease and single-guide RNA complementary to a target locus. CRISPR/Cas enables the generation of knockout cells and animals in a single step. This system can also be used to generate multiple mutations and knockin in a single step, which is not possible using other methods. In this review, we provide an overview of genome editing by CRISPR/Cas in pluripotent stem cells and mice.

Effect of Gestational Intake of Fisetin (3,3',4',7-Tetrahydroxyflavone) on Developmental Methyl Mercury Neurotoxicity in F1 Generation Rats.

PubMed

Jacob, Sherin; Thangarajan, Sumathi

2017-06-01

Methyl mercury (MeHg) is a developmental neurotoxin that causes irreversible cognitive damage in offspring of gestationally exposed mothers. Currently, no preventive drugs are established against MeHg developmental neurotoxicity. The neuroprotective effect of gestational administration of a flavanoid against in utero toxicity of MeHg is not explored much. Hence, the present study validated the effect of a bioactive flavanoid, fisetin, on MeHg developmental neurotoxicity outcomes in rat offspring at postnatal weaning age. Pregnant Wistar rats were simultaneously given MeHg (1.5 mg/kg b.w.) and two doses of fisetin (10 and 50 mg/kg b.w. in two separate groups) orally from gestational day (GD) 5 till parturition. Accordingly, after parturition, on postnatal day (PND) 24, weaning F 1 generation rats were studied for motor and cognitive behavioural changes. Biochemical and histopathological changes were also studied in the cerebral cortex, cerebellum and hippocampus on PND 25. Administration of fisetin during pregnancy prevented behavioural impairment due to transplacental MeHg exposure in weaning rats. Fisetin decreased the levels of oxidative stress markers, increased enzymatic and non-enzymatic antioxidant levels and increased the activity of membrane-bound ATPases and cholinergic function in F 1 generation rats. In light microscopic studies, fisetin treatment protected the specific offspring brain regions from significant morphological aberrations. Between the two doses of fisetin studied, 10 mg/kg b.w. was found to be more satisfactory and effective than 50 mg/kg b.w. The present study shows that intake of fisetin during pregnancy in rats ameliorated in utero MeHg exposure-induced neurotoxicity outcomes in postnatal weaning F 1 generation rats.

Generation and characterization of rat liver stem cell lines and their engraftment in a rat model of liver failure

PubMed Central

Kuijk, Ewart W.; Rasmussen, Shauna; Blokzijl, Francis; Huch, Meritxell; Gehart, Helmuth; Toonen, Pim; Begthel, Harry; Clevers, Hans; Geurts, Aron M.; Cuppen, Edwin

2016-01-01

The rat is an important model for liver regeneration. However, there is no in vitro culture system that can capture the massive proliferation that can be observed after partial hepatectomy in rats. We here describe the generation of rat liver stem cell lines. Rat liver stem cells, which grow as cystic organoids, were characterized by high expression of the stem cell marker Lgr5, by the expression of liver progenitor and duct markers, and by low expression of hepatocyte markers, oval cell markers, and stellate cell markers. Prolonged cultures of rat liver organoids depended on high levels of WNT-signalling and the inhibition of BMP-signaling. Upon transplantation of clonal lines to a Fah−/− Il2rg−/− rat model of liver failure, the rat liver stem cells engrafted into the host liver where they differentiated into areas with FAH and Albumin positive hepatocytes. Rat liver stem cell lines hold potential as consistent reliable cell sources for pharmacological, toxicological or metabolic studies. In addition, rat liver stem cell lines may contribute to the development of regenerative medicine in liver disease. To our knowledge, the here described liver stem cell lines represent the first organoid culture system in the rat. PMID:26915950

Efficient Generation of Myostatin Knock-Out Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes.

PubMed

Crispo, M; Mulet, A P; Tesson, L; Barrera, N; Cuadro, F; dos Santos-Neto, P C; Nguyen, T H; Crénéguy, A; Brusselle, L; Anegón, I; Menchaca, A

2015-01-01

While CRISPR/Cas9 technology has proven to be a valuable system to generate gene-targeted modified animals in several species, this tool has been scarcely reported in farm animals. Myostatin is encoded by MSTN gene involved in the inhibition of muscle differentiation and growth. We determined the efficiency of the CRISPR/Cas9 system to edit MSTN in sheep and generate knock-out (KO) animals with the aim to promote muscle development and body growth. We generated CRISPR/Cas9 mRNAs specific for ovine MSTN and microinjected them into the cytoplasm of ovine zygotes. When embryo development of CRISPR/Cas9 microinjected zygotes (n = 216) was compared with buffer injected embryos (n = 183) and non microinjected embryos (n = 173), cleavage rate was lower for both microinjected groups (P<0.05) and neither was affected by CRISPR/Cas9 content in the injected medium. Embryo development to blastocyst was not affected by microinjection and was similar among the experimental groups. From 20 embryos analyzed by Sanger sequencing, ten were mutant (heterozygous or mosaic; 50% efficiency). To obtain live MSTN KO lambs, 53 blastocysts produced after zygote CRISPR/Cas9 microinjection were transferred to 29 recipient females resulting in 65.5% (19/29) of pregnant ewes and 41.5% (22/53) of newborns. From 22 born lambs analyzed by T7EI and Sanger sequencing, ten showed indel mutations at MSTN gene. Eight showed mutations in both alleles and five of them were homozygous for indels generating out-of frame mutations that resulted in premature stop codons. Western blot analysis of homozygous KO founders confirmed the absence of myostatin, showing heavier body weight than wild type counterparts. In conclusion, our results demonstrate that CRISPR/Cas9 system was a very efficient tool to generate gene KO sheep. This technology is quick and easy to perform and less expensive than previous techniques, and can be applied to obtain genetically modified animal models of interest for biomedicine and livestock.

Activation of PPARγ Ameliorates Spatial Cognitive Deficits through Restoring Expression of AMPA Receptors in Seipin Knock-Out Mice.

PubMed

Zhou, Libin; Chen, Tingting; Li, Guoxi; Wu, Chaoming; Wang, Conghui; Li, Lin; Sha, Sha; Chen, Lei; Liu, George; Chen, Ling

2016-01-27

A characteristic phenotype of congenital generalized lipodystrophy 2 (CGL2) that is caused by loss-of-function of seipin gene is mental retardation. Here, we show that seipin deficiency in hippocampal CA1 pyramidal cells caused the reduction of peroxisome proliferator-activated receptor gamma (PPARγ). Twelve-week-old systemic seipin knock-out mice and neuronal seipin knock-out (seipin-nKO) mice, but not adipose seipin knock-out mice, exhibited spatial cognitive deficits as assessed by the Morris water maze and Y-maze, which were ameliorated by the treatment with the PPARγ agonist rosiglitazone (rosi). In addition, seipin-nKO mice showed the synaptic dysfunction and the impairment of NMDA receptor-dependent LTP in hippocampal CA1 regions. The density of AMPA-induced current (IAMPA) in CA1 pyramidal cells and GluR1/GluR2 expression were significantly reduced in seipin-nKO mice, whereas the NMDA-induced current (INMDA) and NR1/NR2 expression were not altered. Rosi treatment in seipin-nKO mice could correct the decrease in expression and activity of AMPA receptor (AMPAR) and was accompanied by recovered synaptic function and LTP induction. Furthermore, hippocampal ERK2 and CREB phosphorylation in seipin-nKO mice were reduced and this could be rescued by rosi treatment. Rosi treatment in seipin-nKO mice elevated BDNF concentration. The MEK inhibitor U0126 blocked rosi-restored AMPAR expression and LTP induction in seipin-nKO mice, but the Trk family inhibitor K252a did not. These findings indicate that the neuronal seipin deficiency selectively suppresses AMPAR expression through reducing ERK-CREB activities, leading to the impairment of LTP and spatial memory, which can be rescued by PPARγ activation. Congenital generalized lipodystrophy 2 (CGL2), caused by loss-of-function mutation of seipin gene, is characterized by mental retardation. By the generation of systemic or neuronal seipin knock-out mice, the present study provides in vivo evidence that neuronal seipin deficiency causes deficits in spatial memory and hippocampal LTP induction. Neuronal seipin deficiency selectively suppresses AMPA receptor expression, ERK-CREB phosphorylation with the decline of PPARγ. The PPARγ agonist rosiglitazone can ameliorate spatial cognitive deficits and rescue the LTP induction in seipin knock-out mice by restoring AMPA receptor expression and ERK-CREB activities. Copyright © 2016 the authors 0270-6474/16/361242-12$15.00/0.

Differential cardiac effects in rats exposed to atmospheric smog generated from isoprene versus toluene

EPA Science Inventory

The results of this study demonstrate that atmospheric smog generated from both isoprene and toluene cause cardiac effects in rats. In addition, it appears that smog from toluene is more toxic in terms of cardiac arrhythmogenicity. Smog, which is a comple...

aP2-Cre-mediated inactivation of acetyl-CoA carboxylase 1 causes growth retardation and reduced lipid accumulation in adipose tissues

USDA-ARS?s Scientific Manuscript database

Adipose tissue is one of the major sites for fatty acid synthesis and lipid storage. We generated adipose (fat)-specific ACC1 knockout (FACC1KO) mice using the aP2-Cre/loxP system. FACC1KO mice showed prenatal growth retardation; after weaning, however, their weight gain was comparable to that of wi...

Functional Characterization of CENP-A Post-Translational Modifications in Chromosome Segregation

DTIC Science & Technology

2016-09-01

our overall findings in discussion part, and finally we will explain major materials and methods we used. Results CENP-A α-amino methylation...centromere and kinetochore and accurate segregation of the genetic materials . Moreover, we established that centromere/kinetochore defects in the absence...developed. Materials and methods: Creation of CENP-A complete replacement RPE cells: RPE CENP-A knockout cell line generated by Don Cleaveland Lab7 used

Synergistic Action of FOXP3 and TSC1 Pathways During Tumor Progression

DTIC Science & Technology

2015-10-01

invasive carcinoma and, ultimately, metastatic disease [1-3]. Mouse models of PIN (mPIN) generated by a single- mutant gene in prostate do not progress...downstream target) is sufficient to significantly reduce the initiation of prostate cancer in the Pten conditional knockout mouse model [19-21...the possibility that these two genetic hits cooperate to promote tumor progression, and mouse models show that this cooperation accelerates

Copper transporters and chaperones CTR1, CTR2, ATOX1, and CCS as determinants of cisplatin sensitivity.

PubMed

Bompiani, Kristin M; Tsai, Cheng-Yu; Achatz, Felix P; Liebig, Janika K; Howell, Stephen B

2016-09-01

The development of resistance to cisplatin (cDDP) is commonly accompanied by reduced drug uptake or increased efflux. Previous studies in yeast and murine embryonic fibroblasts have reported that the copper (Cu) transporters and chaperones participate in the uptake, efflux, and intracellular distribution of cDDP. However, there is conflicting data from studies in human cells. We used CRISPR-Cas9 genome editing to individually knock out the human copper transporters CTR1 and CTR2 and the copper chaperones ATOX1 and CCS. Isogenic knockout cell lines were generated in both human HEK-293T and ovarian carcinoma OVCAR8 cells. All knockout cell lines had slowed growth compared to parental cells, small changes in basal Cu levels, and varying sensitivities to Cu depending on the gene targeted. However, all of the knockouts demonstrated only modest 2 to 5-fold changes in cDDP sensitivity that did not differ from the range of sensitivities of 10 wild type clones grown from the same parental cell population. We conclude that, under basal conditions, loss of CTR1, CTR2, ATOX1, or CCS does not produce a change in cisplatin sensitivity that exceeds the variance found within the parental population, suggesting that they are not essential to the mechanism by which cDDP enters these cell lines and is transported to the nucleus.

Chronic AMPK activation via loss of FLCN induces functional beige adipose tissue through PGC-1α/ERRα

PubMed Central

Yan, Ming; Audet-Walsh, Étienne; Manteghi, Sanaz; Dufour, Catherine Rosa; Walker, Benjamin; Baba, Masaya; St-Pierre, Julie; Giguère, Vincent; Pause, Arnim

2016-01-01

The tumor suppressor folliculin (FLCN) forms a repressor complex with AMP-activated protein kinase (AMPK). Given that AMPK is a master regulator of cellular energy homeostasis, we generated an adipose-specific Flcn (Adipoq-FLCN) knockout mouse model to investigate the role of FLCN in energy metabolism. We show that loss of FLCN results in a complete metabolic reprogramming of adipose tissues, resulting in enhanced oxidative metabolism. Adipoq-FLCN knockout mice exhibit increased energy expenditure and are protected from high-fat diet (HFD)-induced obesity. Importantly, FLCN ablation leads to chronic hyperactivation of AMPK, which in turns induces and activates two key transcriptional regulators of cellular metabolism, proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α) and estrogen-related receptor α (ERRα). Together, the AMPK/PGC-1α/ERRα molecular axis positively modulates the expression of metabolic genes to promote mitochondrial biogenesis and activity. In addition, mitochondrial uncoupling proteins as well as other markers of brown fat are up-regulated in both white and brown FLCN-null adipose tissues, underlying the increased resistance of Adipoq-FLCN knockout mice to cold exposure. These findings identify a key role of FLCN as a negative regulator of mitochondrial function and identify a novel molecular pathway involved in the browning of white adipocytes and the activity of brown fat. PMID:27151976

Abrogated Freud-1/CC2D1A repression of 5-HT1A autoreceptors induces fluoxetine-resistant anxiety/depression-like behavior

PubMed Central

Vahid-Ansari, Faranak; Daigle, Mireille; Manzini, M. Chiara; Tanaka, Kenji F.; Hen, René; Geddes, Sean D.; Béïque, Jean-Claude; James, Jonathan; Merali, Zul; Albert, Paul R.

2017-01-01

Freud-1/CC2D1A represses the gene transcription of serotonin-1A (5-HT1A) autoreceptors, which negatively regulate 5-HT tone. To test the role of Freud-1 in vivo, we generated mice with adulthood conditional knockout of Freud-1 in 5-HT neurons (cF1ko). In cF1ko mice, 5-HT1A autoreceptor protein, binding and hypothermia response were increased, with reduced 5-HT content and neuronal activity in the dorsal raphe. The cF1ko mice displayed increased anxiety- and depression-like behavior that was resistant to chronic antidepressant (fluoxetine) treatment. Using conditional Freud-1/5-HT1A double knockout (cF1/1A dko) to disrupt both Freud-1 and 5-HT1A genes in 5-HT neurons, no increase in anxiety- or depression-like behaviour was seen upon knockout of Freud-1 on the 5-HT1A autoreceptor-negative background, rather a reduction in depression-like behaviour emerged. These studies implicate transcriptional dys-regulation of 5-HT1A autoreceptors by the repressor Freud-1 in anxiety and depression and provide a clinically relevant genetic model of antidepressant resistance. Targeting specific transcription factors like Freud-1 to restore transcriptional balance may augment response to antidepressant treatment. PMID:29101244

TALEN mediated targeted editing of GM2/GD2-synthase gene modulates anchorage independent growth by reducing anoikis resistance in mouse tumor cells

PubMed Central

Mahata, Barun; Banerjee, Avisek; Kundu, Manjari; Bandyopadhyay, Uday; Biswas, Kaushik

2015-01-01

Complex ganglioside expression is highly deregulated in several tumors which is further dependent on specific ganglioside synthase genes. Here, we designed and constructed a pair of highly specific transcription-activator like effector endonuclease (TALENs) to disrupt a particular genomic locus of mouse GM2-synthase, a region conserved in coding sequence of all four transcript variants of mouse GM2-synthase. Our designed TALENs effectively work in different mouse cell lines and TALEN induced mutation rate is over 45%. Clonal selection strategy is undertaken to generate stable GM2-synthase knockout cell line. We have also demonstrated non-homologous end joining (NHEJ) mediated integration of neomycin cassette into the TALEN targeted GM2-synthase locus. Functionally, clonally selected GM2-synthase knockout clones show reduced anchorage-independent growth (AIG), reduction in tumor growth and higher cellular adhesion as compared to wild type Renca-v cells. Insight into the mechanism shows that, reduced AIG is due to loss in anoikis resistance, as both knockout clones show increased sensitivity to detachment induced apoptosis. Therefore, TALEN mediated precise genome editing at GM2-synthase locus not only helps us in understanding the function of GM2-synthase gene and complex gangliosides in tumorigenicity but also holds tremendous potential to use TALENs in translational cancer research and therapeutics. PMID:25762467

TALEN mediated targeted editing of GM2/GD2-synthase gene modulates anchorage independent growth by reducing anoikis resistance in mouse tumor cells.

PubMed

Mahata, Barun; Banerjee, Avisek; Kundu, Manjari; Bandyopadhyay, Uday; Biswas, Kaushik

2015-03-12

Complex ganglioside expression is highly deregulated in several tumors which is further dependent on specific ganglioside synthase genes. Here, we designed and constructed a pair of highly specific transcription-activator like effector endonuclease (TALENs) to disrupt a particular genomic locus of mouse GM2-synthase, a region conserved in coding sequence of all four transcript variants of mouse GM2-synthase. Our designed TALENs effectively work in different mouse cell lines and TALEN induced mutation rate is over 45%. Clonal selection strategy is undertaken to generate stable GM2-synthase knockout cell line. We have also demonstrated non-homologous end joining (NHEJ) mediated integration of neomycin cassette into the TALEN targeted GM2-synthase locus. Functionally, clonally selected GM2-synthase knockout clones show reduced anchorage-independent growth (AIG), reduction in tumor growth and higher cellular adhesion as compared to wild type Renca-v cells. Insight into the mechanism shows that, reduced AIG is due to loss in anoikis resistance, as both knockout clones show increased sensitivity to detachment induced apoptosis. Therefore, TALEN mediated precise genome editing at GM2-synthase locus not only helps us in understanding the function of GM2-synthase gene and complex gangliosides in tumorigenicity but also holds tremendous potential to use TALENs in translational cancer research and therapeutics.

Prediction of reaction knockouts to maximize succinate production by Actinobacillus succinogenes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nag, Ambarish; St. John, Peter C.; Crowley, Michael F.

Succinate is a precursor of multiple commodity chemicals and bio-based succinate production is an active area of industrial bioengineering research. One of the most important microbial strains for bio-based production of succinate is the capnophilic gram-negative bacterium Actinobacillus succinogenes, which naturally produces succinate by a mixed-acid fermentative pathway. To engineer A. succinogenes to improve succinate yields during mixed acid fermentation, it is important to have a detailed understanding of the metabolic flux distribution in A. succinogenes when grown in suitable media. To this end, we have developed a detailed stoichiometric model of the A. succinogenes central metabolism that includes themore » biosynthetic pathways for the main components of biomass - namely glycogen, amino acids, DNA, RNA, lipids and UDP-N-Acetyl-a-D-glucosamine. We have validated our model by comparing model predictions generated via flux balance analysis with experimental results on mixed acid fermentation. Moreover, we have used the model to predict single and double reaction knockouts to maximize succinate production while maintaining growth viability. According to our model, succinate production can be maximized by knocking out either of the reactions catalyzed by the PTA (phosphate acetyltransferase) and ACK (acetyl kinase) enzymes, whereas the double knockouts of PEPCK (phosphoenolpyruvate carboxykinase) and PTA or PEPCK and ACK enzymes are the most effective in increasing succinate production.« less

Prediction of reaction knockouts to maximize succinate production by Actinobacillus succinogenes

DOE PAGES

Nag, Ambarish; St. John, Peter C.; Crowley, Michael F.; ...

2018-01-30

Succinate is a precursor of multiple commodity chemicals and bio-based succinate production is an active area of industrial bioengineering research. One of the most important microbial strains for bio-based production of succinate is the capnophilic gram-negative bacterium Actinobacillus succinogenes, which naturally produces succinate by a mixed-acid fermentative pathway. To engineer A. succinogenes to improve succinate yields during mixed acid fermentation, it is important to have a detailed understanding of the metabolic flux distribution in A. succinogenes when grown in suitable media. To this end, we have developed a detailed stoichiometric model of the A. succinogenes central metabolism that includes themore » biosynthetic pathways for the main components of biomass - namely glycogen, amino acids, DNA, RNA, lipids and UDP-N-Acetyl-a-D-glucosamine. We have validated our model by comparing model predictions generated via flux balance analysis with experimental results on mixed acid fermentation. Moreover, we have used the model to predict single and double reaction knockouts to maximize succinate production while maintaining growth viability. According to our model, succinate production can be maximized by knocking out either of the reactions catalyzed by the PTA (phosphate acetyltransferase) and ACK (acetyl kinase) enzymes, whereas the double knockouts of PEPCK (phosphoenolpyruvate carboxykinase) and PTA or PEPCK and ACK enzymes are the most effective in increasing succinate production.« less

Parathyroid hormone-related protein is required for tooth eruption

PubMed Central

Philbrick, William M.; Dreyer, Barbara E.; Nakchbandi, Inaam A.; Karaplis, Andrew C.

1998-01-01

Parathyroid hormone (PTH)-related protein (PTHrP)-knockout mice die at birth with a chondrodystrophic phenotype characterized by premature chondrocyte differentiation and accelerated bone formation, whereas overexpression of PTHrP in the chondrocytes of transgenic mice produces a delay in chondrocyte maturation and endochondral ossification. Replacement of PTHrP expression in the chondrocytes of PTHrP-knockout mice using a procollagen II-driven transgene results in the correction of the lethal skeletal abnormalities and generates animals that are effectively PTHrP-null in all sites other than cartilage. These rescued PTHrP-knockout mice survive to at least 6 months of age but are small in stature and display a number of developmental defects, including cranial chondrodystrophy and a failure of tooth eruption. Teeth appear to develop normally but become trapped by the surrounding bone and undergo progressive impaction. Localization of PTHrP mRNA during normal tooth development by in situ hybridization reveals increasing levels of expression in the enamel epithelium before the formation of the eruption pathway. The type I PTH/PTHrP receptor is expressed in both the adjacent dental mesenchyme and in the alveolar bone. The replacement of PTHrP expression in the enamel epithelium with a keratin 14-driven transgene corrects the defect in bone resorption and restores the normal program of tooth eruption. PTHrP therefore represents an essential signal in the formation of the eruption pathway. PMID:9751753

Growth and physiology of Clostridium perfringens wild-type and ΔazoC knockout: an azo dye exposure study.

PubMed

Morrison, Jessica M; John, Gilbert H

2016-02-01

Clostridium perfringens, a strictly anaerobic micro-organism and inhabitant of the human intestine, has been shown to produce the azoreductase enzyme AzoC, an NAD(P)H-dependent flavin oxidoreductase. This enzyme reduces azo dyes to aromatic amines, which are carcinogenic in nature. A significant amount of work has been completed that focuses on the activity of this enzyme; however, few studies have been completed that focus on the physiology of azo dye reduction. Dye reduction studies coupled with C. perfringens growth studies in the presence of ten different azo dyes and in media of varying complexities were completed to compare the growth rates and dye-reducing activity of C. perfringens WT cells, a C. perfringens ΔazoC knockout, and Bifidobacterium infantis, a non-azoreductase-producing control bacterium. The presence of azo dyes significantly increased the generation time of C. perfringens in rich medium, an effect that was not seen in minimal medium. In addition, azo dye reduction studies with the ΔazoC knockout suggested the presence of additional functional azoreductases in this medically important bacterium. Overall, this study addresses a major gap in the literature by providing the first look, to our knowledge, at the complex physiology of C. perfringens upon azo dye exposure and the effect that both azo dyes and the azoreductase enzyme have on growth.

Dual CRISPR-Cas9 Cleavage Mediated Gene Excision and Targeted Integration in Yarrowia lipolytica.

PubMed

Gao, Difeng; Smith, Spencer; Spagnuolo, Michael; Rodriguez, Gabriel; Blenner, Mark

2018-05-29

CRISPR-Cas9 technology has been successfully applied in Yarrowia lipolytica for targeted genomic editing including gene disruption and integration; however, disruptions by existing methods typically result from small frameshift mutations caused by indels within the coding region, which usually resulted in unnatural protein. In this study, a dual cleavage strategy directed by paired sgRNAs is developed for gene knockout. This method allows fast and robust gene excision, demonstrated on six genes of interest. The targeted regions for excision vary in length from 0.3 kb up to 3.5 kb and contain both non-coding and coding regions. The majority of the gene excisions are repaired by perfect nonhomologous end-joining without indel. Based on this dual cleavage system, two targeted markerless integration methods are developed by providing repair templates. While both strategies are effective, homology mediated end joining (HMEJ) based method are twice as efficient as homology recombination (HR) based method. In both cases, dual cleavage leads to similar or improved gene integration efficiencies compared to gene excision without integration. This dual cleavage strategy will be useful for not only generating more predictable and robust gene knockout, but also for efficient targeted markerless integration, and simultaneous knockout and integration in Y. lipolytica. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Strong morphological defects in conditional Arabidopsis abp1 knock-down mutants generated in absence of functional ABP1 protein.

PubMed

Michalko, Jaroslav; Glanc, Matouš; Perrot-Rechenmann, Catherine; Friml, Jiří

2016-01-01

The Auxin Binding Protein 1 (ABP1) is one of the most studied proteins in plants. Since decades ago, it has been the prime receptor candidate for the plant hormone auxin with a plethora of described functions in auxin signaling and development. The developmental importance of ABP1 has recently been questioned by identification of Arabidopsis thaliana abp1 knock-out alleles that show no obvious phenotypes under normal growth conditions. In this study, we examined the contradiction between the normal growth and development of the abp1 knock-outs and the strong morphological defects observed in three different ethanol-inducible abp1 knock-down mutants ( abp1-AS, SS12K, SS12S). By analyzing segregating populations of abp1 knock-out vs. abp1 knock-down crosses we show that the strong morphological defects that were believed to be the result of conditional down-regulation of ABP1 can be reproduced also in the absence of the functional ABP1 protein. This data suggests that the phenotypes in  abp1 knock-down lines are due to the off-target effects and asks for further reflections on the biological function of ABP1 or alternative explanations for the missing phenotypic defects in the abp1 loss-of-function alleles.

Animal models of female pelvic organ prolapse: lessons learned

PubMed Central

Couri, Bruna M; Lenis, Andrew T; Borazjani, Ali; Paraiso, Marie Fidela R; Damaser, Margot S

2012-01-01

Pelvic organ prolapse is a vaginal protrusion of female pelvic organs. It has high prevalence worldwide and represents a great burden to the economy. The pathophysiology of pelvic organ prolapse is multifactorial and includes genetic predisposition, aberrant connective tissue, obesity, advancing age, vaginal delivery and other risk factors. Owing to the long course prior to patients becoming symptomatic and ethical questions surrounding human studies, animal models are necessary and useful. These models can mimic different human characteristics – histological, anatomical or hormonal, but none present all of the characteristics at the same time. Major animal models include knockout mice, rats, sheep, rabbits and nonhuman primates. In this article we discuss different animal models and their utility for investigating the natural progression of pelvic organ prolapse pathophysiology and novel treatment approaches. PMID:22707980

ROLE OF NMDA, NICOTINIC, AND GABA RECEPTORS IN THE STEADY STATE VISUAL EVOKED POTENTIAL IN RATS.

EPA Science Inventory

This manuscript characterizes the receptor pathways involved in pattern-evoked potential generation in rats

" NMDA and nicotinic acetylcholine receptors appear to be involved in the generation of the steady-state pattern evoked response in vivo.

" The pattern evok...

Involvement of arterial baroreflex in the protective effect of dietary restriction against stroke

PubMed Central

Liu, Ai-Jun; Guo, Jin-Min; Liu, Wei; Su, Feng-Yun; Zhai, Qi-Wei; Mehta, Jawahar L; Wang, Wei-Zhong; Su, Ding-Feng

2013-01-01

Dietary restriction (DR) protects against neuronal dysfunction and degeneration, and reduces the risk of ischemic stroke. This study examined the role of silent information regulator T1 (SIRT1) and arterial baroreflex in the beneficial effects of DR against stroke, using two distinct stroke models: stroke-prone spontaneously hypertensive rats (SP-SHRs) and Sprague-Dawley (SD) rats with middle cerebral artery occlusion (MCAO). Sirt1 knockout (KO) mice were used to examine the involvement of sirt1. Sinoaortic denervation was used to inactivate arterial baroreflex. Dietary restriction was defined as 40% reduction of dietary intake. Briefly, DR prolonged the life span of SP-SHRs and reduced the infarct size induced by MCAO. Dietary restriction also improved the function arterial baroreflex, decreased the release of proinflammatory cytokines, and reduced end-organ damage. The beneficial effect of DR on stroke was markedly attenuated by blunting arterial baroreflex. Lastly, the infarct area in sirt1 KO mice was significantly larger than in the wild-type mice. However, the beneficial effect of DR against ischemic injury was still apparent in sirt1 KO mice. Accordingly, arterial baroreflex, but not sirt1, is important in the protective effect of DR against stroke. PMID:23443169

A Novel Mouse Model of Penetrating Brain Injury

PubMed Central

Cernak, Ibolja; Wing, Ian D.; Davidsson, Johan; Plantman, Stefan

2014-01-01

Penetrating traumatic brain injury (pTBI) has been difficult to model in small laboratory animals, such as rats or mice. Previously, we have established a non-fatal, rat model for pTBI using a modified air-rifle that accelerates a pellet, which hits a small probe that then penetrates the experimental animal’s brain. Knockout and transgenic strains of mice offer attractive tools to study biological reactions induced by TBI. Hence, in the present study, we adapted and modified our model to be used with mice. The technical characterization of the impact device included depth and speed of impact, as well as dimensions of the temporary cavity formed in a brain surrogate material after impact. Biologically, we have focused on three distinct levels of severity (mild, moderate, and severe), and characterized the acute phase response to injury in terms of tissue destruction, neural degeneration, and gliosis. Functional outcome was assessed by measuring bodyweight and motor performance on rotarod. The results showed that this model is capable of reproducing major morphological and neurological changes of pTBI; as such, we recommend its utilization in research studies aiming to unravel the biological events underlying injury and regeneration after pTBI. PMID:25374559

Transepithelial ultrafiltration and fractal power diffusion of D-glucose in the perfused rat intestine.

PubMed

Kochak, Gregory M; Mangat, Surinder

2002-12-23

Despite an enormous body of research investigating the mass transfer of D-glucose through biological membranes, carrier-mediated and first-order models have remained the prevalent models describing glucose's quantitative behavior even though they have proven to be inadequate over extended concentration ranges. Recent evidence from GLUT2 knockout studies further questions our understanding of molecular models, especially those employing Michaelis-Menten (MM)-type kinetic models. In this report, evidence is provided that D-glucose is absorbed by rat intestinal epithelium by a combination of convective ultrafiltration and nonlinear diffusion. The diffusive component of mass transfer is described by a concentration-dependent permeability coefficient, modeled as a fractal power function. Glucose and sodium chloride-dependent-induced aqueous convection currents are the result of prevailing oncotic and osmotic pressure effects, and a direct effect of glucose and sodium chloride on intestinal epithelium resulting in enhanced glucose, sodium ion, and water mobility. The fractal power model of glucose diffusion was superior to the conventional MM description. A convection-diffusion model of mass transfer adequately characterized glucose mass transfer over a 105-fold glucose concentration range in the presence and absence of sodium ion.

Animal models for human T-lymphotropic virus type 1 (HTLV-1) infection and transformation

PubMed Central

Lairmore, Michael D; Silverman, Lee; Ratner, Lee

2009-01-01

Over the past 25 years, animal models of human T-lymphotropic virus type 1 (HTLV-1) infection and transformation have provided critical knowledge about viral and host factors in adult T-cell leukemia/lymphoma (ATL). The virus consistently infects rabbits, some non-human primates, and to a lesser extent rats. In addition to providing fundamental concepts in viral transmission and immune responses against HTLV-1 infection, these models have provided new information about the role of viral proteins in carcinogenesis. Mice and rats, in particular immunodeficient strains, are useful models to assess immunologic parameters mediating tumor outgrowth and therapeutic invention strategies against lymphoma. Genetically altered mice including both transgenic and knockout mice offer important models to test the role of specific viral and host genes in the development of HTLV-1-associated lymphoma. Novel approaches in genetic manipulation of both HTLV-1 and animal models are available to address the complex questions that remain about viral-mediated mechanisms of cell transformation and disease. Current progress in the understanding of the molecular events of HTLV-1 infection and transformation suggests that answers to these questions are approachable using animal models of HTLV-1-associated lymphoma PMID:16155607

MicroRNAs Are Mediators of Androgen Action in Prostate and Muscle

PubMed Central

Narayanan, Ramesh; Jiang, Jinmai; Gusev, Yuriy; Jones, Amanda; Kearbey, Jeffrey D.; Miller, Duane D.; Schmittgen, Thomas D.; Dalton, James T.

2010-01-01

Androgen receptor (AR) function is critical for the development of male reproductive organs, muscle, bone and other tissues. Functionally impaired AR results in androgen insensitivity syndrome (AIS). The interaction between AR and microRNA (miR) signaling pathways was examined to understand the role of miRs in AR function. Reduction of androgen levels in Sprague-Dawley rats by castration inhibited the expression of a large set of miRs in prostate and muscle, which was reversed by treatment of castrated rats with 3 mg/day dihydrotestosterone (DHT) or selective androgen receptor modulators. Knockout of the miR processing enzyme, DICER, in LNCaP prostate cancer cells or tissue specifically in mice inhibited AR function leading to AIS. Since the only function of miRs is to bind to 3′ UTR and inhibit translation of target genes, androgens might induce miRs to inhibit repressors of AR function. In concordance, knock-down of DICER in LNCaP cells and in tissues in mice induced the expression of corepressors, NCoR and SMRT. These studies demonstrate a feedback loop between miRs, corepressors and AR and the imperative role of miRs in AR function in non-cancerous androgen-responsive tissues. PMID:21048966

Lack of specificity of antibodies raised against CLN3, the lysosomal/endosomal transmembrane protein mutated in juvenile Batten disease

PubMed Central

Nelson, Tarah

2017-01-01

Juvenile CLN3 (Batten) disease, a fatal, childhood neurodegenerative disorder, results from mutations in the CLN3 gene encoding a lysosomal/endosomal transmembrane protein. The exact physiological function of CLN3 is still unknown and it is unclear how CLN3 mutations lead to selective neurodegeneration. To study the tissue expression and subcellular localization of the CLN3 protein, a number of anti-CLN3 antibodies have been generated using either the whole CLN3 protein or short peptides from CLN3 for immunization. The specificity of these antibodies, however, has never been tested properly. Using immunoblot experiments, we show that commercially available or researcher-generated anti-CLN3 antibodies lack specificity: they detect the same protein bands in wild-type (WT) and Cln3−/− mouse brain and kidney extracts prepared with different detergents, in membrane proteins isolated from the cerebellum, cerebral hemisphere and kidney of WT and Cln3−/− mice, in cell extracts of WT and Cln3−/− mouse embryonic fibroblast cultures, and in lysates of BHK cells lacking or overexpressing human CLN3. Protein BLAST searches with sequences from peptides used to generate anti-CLN3 antibodies identified short motifs present in a number of different mouse and human proteins, providing a plausible explanation for the lack of specificity of anti-CLN3 antibodies. Our data provide evidence that immunization against a transmembrane protein with low to medium expression level does not necessarily generate specific antibodies. Because of the possible cross-reactivity to other proteins, the specificity of an antibody should always be checked using tissue samples from an appropriate knock-out animal or using knock-out cells. PMID:29089465

Beneficial Effects of Myo-Inositol Oxygenase Deficiency in Cisplatin-Induced AKI

PubMed Central

Dutta, Rajesh K.; Kondeti, Vinay K.; Sharma, Isha; Chandel, Navdeep S.; Quaggin, Susan E.

2017-01-01

Overexpression of the proximal tubular enzyme myo-inositol oxygenase (MIOX) induces oxidant stress in vitro. However, the relevance of MIOX to tubular pathobiology remains enigmatic. To investigate the role of MIOX in cisplatin-induced tubular AKI, we generated conditional MIOX-overexpressing transgenic (MIOX-TG) mice and MIOX-knockout (MIOX−/−) mice with tubule-specific MIOX overexpression or knockout, respectively. Compared with cisplatin-treated wild-type (WT) mice, cisplatin-treated MIOX-TG mice had even greater increases in urea, creatinine, and KIM-1 levels and more tubular injury and apoptosis, but these effects were attenuated in cisplatin-treated MIOX−/− mice. Similarly, MIOX-TG mice had the highest and MIOX−/− mice had the lowest renal levels of Bax, cleaved caspase-3, and NADPH oxidase-4 expression and reactive oxygen species (ROS) generation after cisplatin treatment. In vitro, cisplatin dose-dependently increased ROS generation in LLC-PK1 cells. Furthermore, MIOX overexpression in these cells accentuated cisplatin-induced ROS generation and perturbations in the ratio of GSH to oxidized GSH, whereas MIOX-siRNA or N-acetyl cysteine treatment attenuated these effects. Additionally, the cisplatin-induced enhancement of p53 activation, NF-κB binding to DNA, and NF-κB nuclear translocation in WT mice was exacerbated in MIOX-TG mice but absent in MIOX−/− mice. In vitro, MIOX-siRNA or NAC treatment reduced the dose-dependent increase in p53 expression induced by cisplatin. We also observed a remarkable influx of inflammatory cells and upregulation of cytokines in kidneys of cisplatin-treated MIOX-TG mice. Finally, analysis of genomic DNA in WT mice revealed cisplatin-induced hypomethylation of the MIOX promoter. These data suggest that MIOX overexpression exacerbates, whereas MIOX gene disruption protects against, cisplatin-induced AKI. PMID:27895157

Evidence of reactive astrocytes but not peripheral immune system activation in a mouse model of Fragile X Syndrome

PubMed Central

Yuskaitis, Christopher J.; Beurel, Eleonore; Jope, Richard S.

2010-01-01

Fragile X syndrome (FXS) is the most common form of inherited mental retardation and is one of the few known genetic causes of autism. FXS results from the loss of Fmr1 gene function, thus Fmr1 knockout mice provide a model to study impairments associated with FXS and autism and to test potential therapeutic interventions. The inhibitory serine-phosphorylation of glycogen synthase kinase-3 (GSK3) is lower in brain regions of Fmr1 knockout mice than wild-type mice and the GSK3 inhibitor lithium rescues several behavioral impairments in Fmr1 knockout mice. Therefore, we examined if the serine-phosphorylation of GSK3 in Fmr1 knockout mice also was altered outside the brain and if administration of lithium ameliorated the macroorchidism phenotype. Additionally, since GSK3 regulates numerous functions of the immune system and immune alterations have been associated with autism, we tested if immune function is altered in Fmr1 knockout mice. The inhibitory serine-phosphorylation of GSK3 was significantly lower in the testis and liver of Fmr1 knockout mice than wild-type mice, and chronic lithium treatment reduced macroorchidism in Fmr1 knockout mice. No alterations in peripheral immune function were identified in Fmr1 knockout mice. However, examination of glia, the immune cells of the brain, revealed reactive astrocytes in several brain regions of Fmr1 knockout mice and treatment with lithium reduced this in the striatum and cerebellum. These results provide further evidence of the involvement of dysregulated GSK3 in FXS, and demonstrate that lithium administration reduces macroorchidism and reactive astrocytes in Fmr1 knockout mice. PMID:20600866

Toxicological Significance of Renal Bcrp: Another Potential Transporter in the Elimination of Mercuric Ions from Proximal Tubular Cells

PubMed Central

Bridges, Christy C.; Zalups, Rudolfs K.; Joshee, Lucy

2015-01-01

Secretion of inorganic mercury (Hg2+) from proximal tubular cells into the tubular lumen has been shown to involve the multidrug resistance-associated protein 2 (Mrp2). Considering similarities in localization and substrate specificity between Mrp2 and the breast cancer resistance protein (Bcrp), we hypothesize that Bcrp may also play a role in the proximal tubular secretion of mercuric species. In order to test this hypothesis, the uptake of Hg2+ was examined initially using inside-out membrane vesicles containing Bcrp. The results of these studies suggest that Bcrp may be capable of transporting certain conjugates of Hg2+. To further characterize the role of Bcrp in the handling of mercuric ions and in the induction of Hg2+-induced nephropathy, Sprague-Dawley and Bcrp knockout (bcrp−/−) rats were exposed intravenously to a non-nephrotoxic (0.5 μmol • kg−1), a moderately nephrotoxic (1.5 μmol • kg−1) or a significantly nephrotoxic (2.0 μmol • kg−1) dose of HgCl2. In general, the accumulation of Hg2+ was greater in organs of bcrp−/− rats than in Sprague-Dawley rats, suggesting that Bcrp may play a role in the export of Hg2+ from target cells. Within the kidney, cellular injury and necrosis was more severe in bcrp−/− rats than in controls. The pattern of necrosis, which was localized in the inner cortex and the outer stripe of the outer medulla was significantly different from that observed in Mrp2-deficient animals. These findings suggest that Bcrp may be involved in the cellular export of select mercuric species and that its role in this export may differ from that of Mrp2. PMID:25868844

Angiotensin-converting enzyme 2 activation improves endothelial function.

PubMed

Fraga-Silva, Rodrigo A; Costa-Fraga, Fabiana P; Murça, Tatiane M; Moraes, Patrícia L; Martins Lima, Augusto; Lautner, Roberto Q; Castro, Carlos H; Soares, Célia Maria A; Borges, Clayton L; Nadu, Ana Paula; Oliveira, Marilene L; Shenoy, Vinayak; Katovich, Michael J; Santos, Robson A S; Raizada, Mohan K; Ferreira, Anderson J

2013-06-01

Diminished release and function of endothelium-derived nitric oxide coupled with increases in reactive oxygen species production is critical in endothelial dysfunction. Recent evidences have shown that activation of the protective axis of the renin-angiotensin system composed by angiotensin-converting enzyme 2, angiotensin-(1-7), and Mas receptor promotes many beneficial vascular effects. This has led us to postulate that activation of intrinsic angiotensin-converting enzyme 2 would improve endothelial function by decreasing the reactive oxygen species production. In the present study, we tested 1-[[2-(dimetilamino)etil]amino]-4-(hidroximetil)-7-[[(4-metilfenil)sulfonil]oxi]-9H-xantona-9 (XNT), a small molecule angiotensin-converting enzyme 2 activator, on endothelial function to validate this hypothesis. In vivo treatment with XNT (1 mg/kg per day for 4 weeks) improved the endothelial function of spontaneously hypertensive rats and of streptozotocin-induced diabetic rats when evaluated through the vasorelaxant responses to acetylcholine/sodium nitroprusside. Acute in vitro incubation with XNT caused endothelial-dependent vasorelaxation in aortic rings of rats. This vasorelaxation effect was attenuated by the Mas antagonist D-pro7-Ang-(1-7), and it was reduced in Mas knockout mice. These effects were associated with reduction in reactive oxygen species production. In addition, Ang II-induced reactive oxygen species production in human aortic endothelial cells was attenuated by preincubation with XNT. These results showed that chronic XNT administration improves the endothelial function of hypertensive and diabetic rat vessels by attenuation of the oxidative stress. Moreover, XNT elicits an endothelial-dependent vasorelaxation response, which was mediated by Mas. Thus, this study indicated that angiotensin-converting enzyme 2 activation promotes beneficial effects on the endothelial function and it is a potential target for treating cardiovascular disease.

Satb2-Independent Acquisition of the Cholinergic Sudomotor Phenotype in Rodents

PubMed Central

Schütz, Burkhard; Schaäfer, Martin K.-H.; Gördes, Markus; Eiden, Lee E.; Weihe, Eberhard

2014-01-01

Expression of Satb2 (Special AT-rich sequence-binding protein-2) elicits expression of the vesicular acetylcholine transporter (VAChT) and choline acetyltransferase (ChAT) in cultured rat sympathetic neurons exposed to soluble differentiation factors. Here, we determined whether or not Satb2 plays a similar role in cholinergic differentiation in vivo, by comparing the postnatal profile of Satb2 expression in the rodent stellate ganglion to that of VAChT and ChAT. Throughout postnatal development, VAChT and ChAT were found to be co-expressed in a numerically stable subpopulation of rat stellate ganglion neurons. Nerve fibers innervating rat forepaw sweat glands on P1 were VAChT immunoreactive, while ChAT was detectable at this target only after P5. The postnatal abundance of VAChT transcripts in the stellate ganglion was at maximum already on P1, whereas ChAT mRNA levels increased from low levels on P1 to reach maximum levels between P5 and P21. Satb2 mRNA was detected in cholinergic neurons in the stellate ganglion beginning with P8, thus coincident with the onset of unequivocal detection of ChAT immunoreactivity in forepaw sweat gland endings. Satb2 knockout mice exhibited no change in the P1 cholinergic VAChT/ChAT co-phenotype in stellate ganglion neurons. Thus, cholinergic phenotype maturation involves first, early target (sweat-gland)-independent expression and trafficking of VAChT, and later, potentially target- and Satb2-dependent elevation of ChAT mRNA and protein transport into sweat gland endings. In rat sudomotor neurons that, unlike mouse sudomotor neurons, co-express calcitonin gene-related peptide (CGRP), Satb2 may also be related to the establishment of species-specific neuropeptide co-phenotypes during postnatal development. PMID:25239161

The aryl hydrocarbon receptor is indispensable for dioxin-induced defects in sexually-dimorphic behaviors due to the reduction in fetal steroidogenesis of the pituitary-gonadal axis in rats.

PubMed

Hattori, Yukiko; Takeda, Tomoki; Nakamura, Arisa; Nishida, Kyoko; Shioji, Yuko; Fukumitsu, Haruki; Yamada, Hideyuki; Ishii, Yuji

2018-05-16

Many forms of the toxic effects produced by dioxins and related chemicals take place following activation of the aryl hydrocarbon receptor (AHR). Our previous studies have demonstrated that treating pregnant rats with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a highly toxic dioxin, attenuates the pituitary expression of gonadotropins to reduce testicular steroidogenesis during the fetal stage, resulting in the impairment of sexually-dimorphic behaviors after the offspring reach maturity. To investigate the contribution of AHR to these disorders, we examined the effects of TCDD on AHR-knockout (AHR-KO) Wistar rats. When pregnant AHR-heterozygous rats were given an oral dose of 1 µg/kg TCDD at gestational day (GD) 15, TCDD reduced the expression of pituitary gonadotropins and testicular steroidogenic proteins in male wild-type fetuses at GD20 without affecting body weight, sex ratio and litter size. However, the same defect did not occur in AHR-KO fetuses. Further, fetal exposure to TCDD impaired the activity of masculine sexual behavior after reaching adulthood only in the wild-type offspring. Also, in female offspring, not only the fetal gonadotropins production but also sexual dimorphism, such as saccharin preference, after growing up were suppressed by TCDD only in the wild-type. Interestingly, in the absence of TCDD, deleting AHR reduced masculine sexual behavior, as well as fetal steroidogenesis of the pituitary-gonadal axis. These results provide novel evidence that 1) AHR is required for TCDD-produced defects in sexually-dimorphic behaviors of the offspring, and 2) AHR signaling plays a role in gonadotropin synthesis during the developmental stage to acquire sexual dimorphism after reaching adulthood. Copyright © 2018 Elsevier Inc. All rights reserved.

ANGIOTENSIN-CONVERTING ENZYME 2 ACTIVATION IMPROVES ENDOTHELIAL FUNCTION

PubMed Central

Fraga-Silva, Rodrigo A.; Costa-Fraga, Fabiana P.; Murça, Tatiane M.; Moraes, Patrícia L.; Lima, Augusto Martins; Lautner, Roberto Q.; Castro, Carlos H.; Soares, Célia Maria A.; Borges, Clayton L.; Nadu, Ana Paula; Oliveira, Marilene L.; Shenoy, Vinayak; Katovich, Michael J.; Santos, Robson A.S.; Raizada, Mohan K.; Ferreira, Anderson J.

2013-01-01

Diminished release and function of endothelium-derived nitric oxide (NO) coupled with increases in reactive oxygen species (ROS) production is critical in endothelial dysfunction. Recent evidences have shown that activation of the protective axis of the renin-angiotensin system composed by angiotensin-converting enzyme2 (ACE2), Angiotensin-(1-7) [Ang-(1-7)] and Mas receptor promotes many beneficial vascular effects. This has led us to postulate that activation of intrinsic ACE2 would improve endothelial function by decreasing the ROS production. In the present study, we tested 1-[[2-(dimetilamino)etil]amino]-4-(hidroximetil)-7-[[(4-metilfenil)sulfonil]oxi]-9H-xantona-9 (XNT), a small molecule ACE2 activator, on endothelial function to validate this hypothesis. In vivo treatment with XNT (1mg/kg/day for 4 weeks) improved the endothelial function of spontaneously hypertensive rats and of streptozotocin-induced diabetic rats when evaluated through the vasorelaxant responses to acetylcholine/sodium nitroprusside. Acute in vitro incubation with XNT caused endothelial-dependent vasorelaxation in aortic rings of rats. This vasorelaxation effect was attenuated by the Mas antagonist D-pro7-Ang-(1-7) and it was reduced in Mas knockout mice. These effects were associated with reduction in ROS production. In addition, Ang II-induced ROS production in human aortic endothelial cells was attenuated by pre-incubation with XNT. These results showed that chronic XNT administration improves the endothelial function of hypertensive and diabetic rat vessels by attenuation of the oxidative stress. Moreover, XNT elicits an endothelial-dependent vasorelaxation response, which was mediated by Mas. Thus, this study indicated that ACE2 activation promotes beneficial effects on the endothelial function and it is a potential target for treating cardiovascular disease. PMID:23608648

Evidence against a critical role of CB1 receptors in adaptation of the hypothalamic-pituitary-adrenal axis and other consequences of daily repeated stress.

PubMed

Rabasa, Cristina; Pastor-Ciurana, Jordi; Delgado-Morales, Raúl; Gómez-Román, Almudena; Carrasco, Javier; Gagliano, Humberto; García-Gutiérrez, María S; Manzanares, Jorge; Armario, Antonio

2015-08-01

There is evidence that endogenous cannabinoids (eCBs) play a role in the control of the hypothalamic-pituitary-adrenal (HPA) axis, although they appear to have dual, stimulatory and inhibitory, effects. Recent data in rats suggest that eCBs, acting through CB1 receptors (CB1R), may be involved in adaptation of the HPA axis to daily repeated stress. In the present study we analyze this issue in male mice and rats. Using a knock-out mice for the CB1 receptor (CB1-/-) we showed that mutant mice presented similar adrenocorticotropic hormone (ACTH) response to the first IMO as wild-type mice. Daily repeated exposure to 1h of immobilization reduced the ACTH response to the stressor, regardless of the genotype, demonstrating that adaptation occurred to the same extent in absence of CB1R. Prototypical changes observed after repeated stress such as enhanced corticotropin releasing factor (CRH) gene expression in the paraventricular nucleus of the hypothalamus, impaired body weight gain and reduced thymus weight were similarly observed in both genotypes. The lack of effect of CB1R in the expression of HPA adaptation to another similar stressor (restraint) was confirmed in wild-type CD1 mice by the lack of effect of the CB1R antagonist AM251 just before the last exposure to stress. Finally, the latter drug did not blunt the HPA, glucose and behavioral adaptation to daily repeated forced swim in rats. Thus, the present results indicate that CB1R is not critical for overall effects of daily repeated stress or proper adaptation of the HPA axis in mice and rats. Copyright © 2015 Elsevier B.V. and ECNP. All rights reserved.

Change of Rin1 and Stathmin in the Animal Model of Traumatic Stresses

PubMed Central

Han, Fang; Jiang, Jingzhi; Ding, Jinlan; Liu, Hong; Xiao, Bing; Shi, Yuxiu

2017-01-01

The molecular mechanism of fear memory is poorly understood. Therefore, the pathogenesis of post-traumatic stress disorder (PTSD), whose symptom presentation can enhance fear memory, remains largely unclear. Recent studies with knockout animals have reported that Rin1 and stathmin regulate fear memory. Rin1 inhibits acquisition and promotes memory extinction, whereas stathmin regulates innate and basal fear. The aim of our study was to examine changes in the expression of Rin1 and stathmin in different animal models of stress, particluarly traumatic stress. We used three animal traumatic stresses: single prolonged stress (SPS, which is a rodent model of PTSD), an immobilization-stress (IM) and a Loud sound stress (LSS), to examine the change and uniqueness in Rin1/stathmin expression. Behavioral tests of SPS rats demonstrated increased anxiety and contextual fear-conditioning. They showed decreased long-term potentiation (LTP), as well as decreased stathmin and increased Rin1 expression in the hippocampus and the amygdala. Expression of the stathmin effector, tubulin, and downstream molecules Rin1, Rab5, and Abl, appeared to increase. Rin1 and EphA4 were endogenously coexpressed in primary neurons after SPS stimulation. IM rats exhibited increased anxiety behavior and enhanced fear-conditioning to contextual and auditory stimuli. Similar changes in expression of Rin1/stathmin were observed in IM rats whereas no changes were observed in rats exposed to a loud sound. These data suggest that changes in expression of the Rin1 and stathmin genes may be involved in rodents with SPS and IM stresses, which provide valuable insight into fear memories under abnormal conditions, particularly in PTSD. PMID:28491025

Selenoprotein P: an extracellular protein with unique physical characteristics and a role in selenium homeostasis.

PubMed

Burk, Raymond F; Hill, Kristina E

2005-01-01

Selenoprotein P is an abundant extracellular glycoprotein that is rich in selenocysteine. It has two domains with respect to selenium content. The N-terminal domain of the rat protein contains one selenocysteine residue in a UxxC redox motif. This domain also has a pH-sensitive heparin-binding site and two histidine-rich amino acid stretches. The smaller C-terminal domain contains nine selenocysteine and ten cysteine residues. Four isoforms of selenoprotein P are present in rat plasma. They share the same N terminus and amino acid sequence. One isoform is full length and the three others terminate at the positions of the second, third, and seventh selenocysteine residues. Selenoprotein P turns over rapidly in rat plasma with the consequence that approximately 25% of the amount of whole-body selenium passes through it each day. Evidence supports functions of the protein in selenium homeostasis and oxidant defense. Selenoprotein P knockout mice have very low selenium concentrations in the brain, the testis, and the fetus, with severe pathophysiological consequences in each tissue. In addition, those mice waste moderate amounts of selenium in the urine. Selenoprotein P binds to endothelial cells in the rat, and plasma levels of the protein correlate with prevention of diquat-induced lipid peroxidation and hepatic endothelial cell injury. The mechanisms of these apparent functions remain speculative and much work on the mechanism of selenoprotein P function lies ahead. Measurement of selenoprotein P in human plasma has shown that it is depressed by selenium deficiency and by cirrhosis. Selenium supplementation of selenium-deficient human subjects showed that glutathione peroxidase activity was optimized before selenoprotein P concentration was optimized, indicating that plasma selenoprotein P is the better index of human selenium nutritional status.

Sigma-1 receptor mediates cocaine-induced transcriptional regulation by recruiting chromatin-remodeling factors at the nuclear envelope.

PubMed

Tsai, Shang-Yi A; Chuang, Jian-Ying; Tsai, Meng-Shan; Wang, Xiao-Fei; Xi, Zheng-Xiong; Hung, Jan-Jong; Chang, Wen-Chang; Bonci, Antonello; Su, Tsung-Ping

2015-11-24

The sigma-1 receptor (Sig-1R) chaperone at the endoplasmic reticulum (ER) plays important roles in cellular regulation. Here we found a new function of Sig-1R, in that it translocates from the ER to the nuclear envelope (NE) to recruit chromatin-remodeling molecules and regulate the gene transcription thereof. Sig-1Rs mainly reside at the ER-mitochondrion interface. However, on stimulation by agonists such as cocaine, Sig-1Rs translocate from ER to the NE, where Sig-1Rs bind NE protein emerin and recruit chromatin-remodeling molecules, including lamin A/C, barrier-to-autointegration factor (BAF), and histone deacetylase (HDAC), to form a complex with the gene repressor specific protein 3 (Sp3). Knockdown of Sig-1Rs attenuates the complex formation. Cocaine was found to suppress the gene expression of monoamine oxidase B (MAOB) in the brain of wild-type but not Sig-1R knockout mouse. A single dose of cocaine (20 mg/kg) in rats suppresses the level of MAOB at nuclear accumbens without affecting the level of dopamine transporter. Daily injections of cocaine in rats caused behavioral sensitization. Withdrawal from cocaine in cocaine-sensitized rats induced an apparent time-dependent rebound of the MAOB protein level to about 200% over control on day 14 after withdrawal. Treatment of cocaine-withdrawn rats with the MAOB inhibitor deprenyl completely alleviated the behavioral sensitization to cocaine. Our results demonstrate a role of Sig-1R in transcriptional regulation and suggest cocaine may work through this newly discovered genomic action to achieve its addictive action. Results also suggest the MAOB inhibitor deprenyl as a therapeutic agent to block certain actions of cocaine during withdrawal.

Sigma-1 receptor mediates cocaine-induced transcriptional regulation by recruiting chromatin-remodeling factors at the nuclear envelope

PubMed Central

Tsai, Shang-Yi A.; Chuang, Jian-Ying; Tsai, Meng-Shan; Wang, Xiao-fei; Hung, Jan-Jong; Chang, Wen-Chang; Bonci, Antonello; Su, Tsung-Ping

2015-01-01

The sigma-1 receptor (Sig-1R) chaperone at the endoplasmic reticulum (ER) plays important roles in cellular regulation. Here we found a new function of Sig-1R, in that it translocates from the ER to the nuclear envelope (NE) to recruit chromatin-remodeling molecules and regulate the gene transcription thereof. Sig-1Rs mainly reside at the ER–mitochondrion interface. However, on stimulation by agonists such as cocaine, Sig-1Rs translocate from ER to the NE, where Sig-1Rs bind NE protein emerin and recruit chromatin-remodeling molecules, including lamin A/C, barrier-to-autointegration factor (BAF), and histone deacetylase (HDAC), to form a complex with the gene repressor specific protein 3 (Sp3). Knockdown of Sig-1Rs attenuates the complex formation. Cocaine was found to suppress the gene expression of monoamine oxidase B (MAOB) in the brain of wild-type but not Sig-1R knockout mouse. A single dose of cocaine (20 mg/kg) in rats suppresses the level of MAOB at nuclear accumbens without affecting the level of dopamine transporter. Daily injections of cocaine in rats caused behavioral sensitization. Withdrawal from cocaine in cocaine-sensitized rats induced an apparent time-dependent rebound of the MAOB protein level to about 200% over control on day 14 after withdrawal. Treatment of cocaine-withdrawn rats with the MAOB inhibitor deprenyl completely alleviated the behavioral sensitization to cocaine. Our results demonstrate a role of Sig-1R in transcriptional regulation and suggest cocaine may work through this newly discovered genomic action to achieve its addictive action. Results also suggest the MAOB inhibitor deprenyl as a therapeutic agent to block certain actions of cocaine during withdrawal. PMID:26554014

A novel component of cannabis extract potentiates excitatory synaptic transmission in rat olfactory cortex in vitro.

PubMed

Whalley, Benjamin J; Wilkinson, Jonathan D; Williamson, Elizabeth M; Constanti, Andrew

2004-07-15

Cannabis is a potential treatment for epilepsy, although the few human studies supporting this use have proved inconclusive. Previously, we showed that a standardized cannabis extract (SCE), isolated Delta9-tetrahydrocannabinol (Delta9-THC), and even Delta9-THC-free SCE inhibited muscarinic agonist-induced epileptiform bursting in rat olfactory cortical brain slices, acting via CB1 receptors. The present work demonstrates that although Delta9-THC (1 microM) significantly depressed evoked depolarizing postsynaptic potentials (PSPs) in rat olfactory cortex neurones, both SCE and Delta9-THC-free SCE significantly potentiated evoked PSPs (all results were fully reversed by the CB1 receptor antagonist SR141716A, 1 microM); interestingly, the potentiation by Delta9-THC-free SCE was greater than that produced by SCE. On comparing the effects of Delta9-THC-free SCE upon evoked PSPs and artificial PSPs (aPSPs; evoked electrotonically following brief intracellular current injection), PSPs were enhanced, whereas aPSPs were unaffected, suggesting that the effect was not due to changes in background input resistance. Similar recordings made using CB1 receptor-deficient knockout mice (CB1-/-) and wild-type littermate controls revealed cannabinoid or extract-induced changes in membrane resistance, cell excitability and synaptic transmission in wild-type mice that were similar to those seen in rat neurones, but no effect on these properties were seen in CB1-/- cells. It appears that the unknown extract constituent(s) effects over-rode the suppressive effects of Delta9-THC on excitatory neurotransmitter release, which may explain some patients' preference for herbal cannabis rather than isolated Delta9-THC (due to attenuation of some of the central Delta9-THC side effects) and possibly account for the rare incidence of seizures in some individuals taking cannabis recreationally.

Antagonism of V1b receptors promotes maternal motivation to retrieve pups in the MPOA and impairs pup-directed behavior during maternal defense in the mpBNST of lactating rats.

PubMed

Bayerl, Doris S; Kaczmarek, Veronika; Jurek, Benjamin; van den Burg, Erwin H; Neumann, Inga D; Gaßner, Barbara M; Klampfl, Stefanie M; Bosch, Oliver J

2016-03-01

Recent studies using V1b receptor (V1bR) knockout mice or central pharmacological manipulations in lactating rats highlighted the influence of this receptor for maternal behavior. However, its role in specific brain sites known to be important for maternal behavior has not been investigated to date. In the present study, we reveal that V1bR mRNA (qPCR) and protein levels (Western blot) within either the medial preoptic area (MPOA) or the medial-posterior part of the bed nucleus of the stria terminalis (mpBNST) did not differ between virgin and lactating rats. Furthermore, we characterized the effects of V1bR blockade via bilateral injections of the receptor subtype-specific antagonist SSR149415 within the MPOA or the mpBNST on maternal behavior (maternal care under non-stress and stress conditions, maternal motivation to retrieve pups in a novel environment, maternal aggression) and anxiety-related behavior in lactating rats. Blocking V1bR within the MPOA increased pup retrieval, whereas within the mpBNST it decreased pup-directed behavior, specifically licking/grooming the pups, during the maternal defense test. In addition, immediately after termination of the maternal defense test, V1bR antagonism in both brain regions reduced nursing, particularly arched back nursing. Anxiety-related behavior was not affected by V1bR antagonism in either brain region. In conclusion our data indicate that V1bR antagonism significantly modulates different aspects of maternal behavior in a brain region-dependent manner. Copyright © 2015 Elsevier Inc. All rights reserved.

Centrally Administered Ghrelin Acutely Influences Food Choice in Rodents

PubMed Central

Schéle, Erik; Bake, Tina; Rabasa, Cristina; Dickson, Suzanne L.

2016-01-01

We sought to determine whether the orexigenic hormone, ghrelin, is involved in the intrinsic regulation of food choice in rats. Ghrelin would seem suited to serve such a role given that it signals hunger information from the stomach to brain areas important for feeding control, including the hypothalamus and reward system (e.g. ventral tegmental area, VTA). Thus, in rats offered a choice of palatable foods (sucrose pellets and lard) superimposed on regular chow for 2 weeks, we explored whether acute central delivery of ghrelin (intracerebroventricular (ICV) or intra-VTA) is able to redirect their dietary choice. The major unexpected finding is that, in rats with high baseline lard intake, acute ICV ghrelin injection increased their chow intake over 3-fold, relative to vehicle-injected controls, measured at both 3 hr and 6 hr after injection. Similar effects were observed when ghrelin was delivered to the VTA, thereby identifying the VTA as a likely contributing neurobiological substrate for these effects. We also explored food choice after an overnight fast, when endogenous ghrelin levels are elevated, and found similar effects of dietary choice to those described for ghrelin. These effects of fasting on food choice were suppressed in models of suppressed ghrelin signaling (i.e. peripheral injection of a ghrelin receptor antagonist to rats and ghrelin receptor (GHSR) knock-out mice), implicating a role for endogenous ghrelin in the changes in food choice that occur after an overnight fast. Thus, in line with its role as a gut-brain hunger hormone, ghrelin appears to be able to acutely alter food choice, with notable effects to promote “healthy” chow intake, and identify the VTA as a likely contributing neurobiological substrate for these effects. PMID:26925974

Demonstration of vesicular glutamate transporter-1 in corticotroph cells in the anterior pituitary of the rat.

PubMed

Kocsis, Zsuzsa S; Molnár, Csilla S; Watanabe, Masahiko; Daneels, Guy; Moechars, Dieder; Liposits, Zsolt; Hrabovszky, Erik

2010-02-01

Recent immunohistochemical studies of the rat adenohypophysis identified type-2 vesicular glutamate transporter (VGLUT2), a marker for glutamatergic neuronal phenotype, in high percentages of adenohypophysial gonadotrophs and thyrotrophs. The presence and molecular identity of amino acid neurotransmitters in the remaining hormone producing cell types are unknown. In the present study we addressed the putative synthesis of another glutamatergic marker, VGLUT1 by adenohypophysial cells. Immunohistochemical studies revealed VGLUT1 immunoreactivity in a small subset of polygonal medium-sized cells in the anterior lobe. Western blot analysis revealed a single major 60 kDa protein band in the adenohypophysis. Furthermore, the expression of VGLUT1 mRNA was confirmed by reverse transcription-polymerase chain reaction followed by sequence analysis of the amplicon. In contrast with rats which only showed VGLUT1 signal in the anterior lobe of the pituitary, mice contained high levels of VGLUT1 immunoreactivity in the intermediate, in addition to the anterior lobe. No signal was present in VGLUT1-knockout mice, providing evidence for specificity. In rats, results of colocalization studies with dual-immunofluorescent labeling provided evidence for VGLUT1 immunoreactivity in 45.9% of corticotrophs and 7.7% of luteinizing hormone beta-immunopositive gonadotrophs. Cells of the other peptide hormone phenotypes were devoid of VGLUT1 signal. A few cells in the adenohypophysis expressed both VGLUT1 and VGLUT2 immunoreactivities. The presence of the glutamate markers VGLUT1 and VGLUT2 in distinct populations of peptide hormone-secreting hypophysial cells highly indicates the involvement of endogenous glutamate release in autocrine/paracrine regulatory mechanisms. The biological function of adenohypophysial glutamate will require clarification. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

A two-generation reproduction study to assess the effects of cows' milk on reproductive development in male and female rats.

PubMed

Ganmaa, Davaasambuu; Qin, Li-Qiang; Wang, Pei-Yu; Tezuka, Hideo; Teramoto, Shoji; Sato, Akio

2004-10-01

To examine whether the considerable quantities of increased female sex hormone levels found in modern milk as a result of modern dairy farming practices are safe for human consumption. Males and females of the P generation were maintained on a diet containing milk for 10 weeks before mating. Exposure to milk was continued up to the end of weaning of the F2b offspring. Two-generation reproduction study. Male and female Wistar Galas rats. P- and F1-generation rats were mated. Fertility, fecundity, and morphology and function of reproductive organs. Although milk had growth-promoting effects in both parents and offspring, it caused no impairments in fertility, fecundity, or reproductive organ development in either generation. However, a whole litter from a dam of the P generation was born dead, three litters in total had a pup with skeletal abnormalities, and the AGD of F2a female pups was reduced. These events occurred only in the milk-treated rats. It is unknown whether these issues had any relevance to milk or only happened by chance. Further study is required to determine whether milk from pregnant cows is completely free from adverse effects on reproductive health.

The role of periodontal ASIC3 in orofacial pain induced by experimental tooth movement in rats.

PubMed

Gao, Meiya; Long, Hu; Ma, Wenqiang; Liao, Lina; Yang, Xin; Zhou, Yang; Shan, Di; Huang, Renhuan; Jian, Fan; Wang, Yan; Lai, Wenli

2016-12-01

This study aimed to clarify the roles of Acid-sensing ion channel 3 (ASIC3) in orofacial pain following experimental tooth movement. Sixty male Sprague-Dawley rats were divided into the experimental group (40g, n = 30) and the sham group (0g, n = 30). Closed coil springs were ligated between maxillary incisor and molars to achieve experimental tooth movement. Rat grimace scale (RGS) scores were assessed at 0, 1, 3, 5, 7, and 14 days after the placement of the springs. ASIC3 immunostaining was performed and the expression levels of ASIC3 were measured through integrated optical density/area in Image-Pro Plus 6.0. Moreover, 18 rats were divided into APETx2 group (n = 6), amiloride group (n = 6), and vehicle group (n = 6), and RGS scores were obtained compared among them to verify the roles of ASIC3 in orofacial pain following tooth movement. ASIC3 expression levels became significantly higher in the experimental group than in sham group on 1, 3, and 5 days and became similar on 7 and 14 days. Pain levels (RGS scores) increased in both groups and were significantly higher in the experimental group on 1, 3, 5, and 7 days and were similar on 14 days. Periodontal ASIC3 expression levels were correlated with orofacial pain levels following experimental tooth movement. Periodontal administrations of ASIC3 antagonists (APETx2 and amiloride) could alleviate pain. This study needs to be better evidenced by RNA interference of ASIC3 in periodontal tissues in rats following experimental tooth movement. Moreover, we hope further studies would concentrate on the pain perception of ASIC3 knockout (ASIC3 -/- ) mice. Our results suggest that periodontal ASIC3 plays an important role in orofacial pain induced by experimental tooth movement. © The Author 2015. Published by Oxford University Press on behalf of the European Orthodontic Society. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Heterogeneous Stock Rat: A Unique Animal Model for Mapping Genes Influencing Bone Fragility

PubMed Central

Alam, Imranul; Koller, Daniel L.; Sun, Qiwei; Roeder, Ryan K.; Cañete, Toni; Blázquez, Gloria; López-Aumatell, Regina; Martínez-Membrives, Esther; Vicens-Costa, Elia; Mont, Carme; Díaz, Sira; Tobeña, Adolf; Fernández-Teruel, Alberto; Whitley, Adam; Strid, Pernilla; Diez, Margarita; Johannesson, Martina; Flint, Jonathan; Econs, Michael J.; Turner, Charles H.; Foroud, Tatiana

2011-01-01

Previously, we demonstrated that skeletal mass, structure and biomechanical properties vary considerably among 11 different inbred rat strains. Subsequently, we performed quantitative trait loci (QTL) analysis in 4 inbred rat strains (F344, LEW, COP and DA) for different bone phenotypes and identified several candidate genes influencing various bone traits. The standard approach to narrowing QTL intervals down to a few candidate genes typically employs the generation of congenic lines, which is time consuming and often not successful. A potential alternative approach is to use a highly genetically informative animal model resource capable of delivering very high-resolution gene mapping such as Heterogeneous stock (HS) rat. HS rat was derived from eight inbred progenitors: ACI/N, BN/SsN, BUF/N, F344/N, M520/N, MR/N, WKY/N and WN/N. The genetic recombination pattern generated across 50 generations in these rats has been shown to deliver ultra-high even gene-level resolution for complex genetic studies. The purpose of this study is to investigate the usefulness of the HS rat model for fine mapping and identification of genes underlying bone fragility phenotypes. We compared bone geometry, density and strength phenotypes at multiple skeletal sites in HS rats with those obtained from 5 of the 8 progenitor inbred strains. In addition, we estimated the heritability for different bone phenotypes in these rats and employed principal component analysis to explore relationships among bone phenotypes in the HS rats. Our study demonstrates that significant variability exists for different skeletal phenotypes in HS rats compared with their inbred progenitors. In addition, we estimated high heritability for several bone phenotypes and biologically interpretable factors explaining significant overall variability, suggesting that the HS rat model could be a unique genetic resource for rapid and efficient discovery of the genetic determinants of bone fragility. PMID:21334473

Heterogeneous stock rat: a unique animal model for mapping genes influencing bone fragility.

PubMed

Alam, Imranul; Koller, Daniel L; Sun, Qiwei; Roeder, Ryan K; Cañete, Toni; Blázquez, Gloria; López-Aumatell, Regina; Martínez-Membrives, Esther; Vicens-Costa, Elia; Mont, Carme; Díaz, Sira; Tobeña, Adolf; Fernández-Teruel, Alberto; Whitley, Adam; Strid, Pernilla; Diez, Margarita; Johannesson, Martina; Flint, Jonathan; Econs, Michael J; Turner, Charles H; Foroud, Tatiana

2011-05-01

Previously, we demonstrated that skeletal mass, structure and biomechanical properties vary considerably among 11 different inbred rat strains. Subsequently, we performed quantitative trait loci (QTL) analysis in four inbred rat strains (F344, LEW, COP and DA) for different bone phenotypes and identified several candidate genes influencing various bone traits. The standard approach to narrowing QTL intervals down to a few candidate genes typically employs the generation of congenic lines, which is time consuming and often not successful. A potential alternative approach is to use a highly genetically informative animal model resource capable of delivering very high resolution gene mapping such as Heterogeneous stock (HS) rat. HS rat was derived from eight inbred progenitors: ACI/N, BN/SsN, BUF/N, F344/N, M520/N, MR/N, WKY/N and WN/N. The genetic recombination pattern generated across 50 generations in these rats has been shown to deliver ultra-high even gene-level resolution for complex genetic studies. The purpose of this study is to investigate the usefulness of the HS rat model for fine mapping and identification of genes underlying bone fragility phenotypes. We compared bone geometry, density and strength phenotypes at multiple skeletal sites in HS rats with those obtained from five of the eight progenitor inbred strains. In addition, we estimated the heritability for different bone phenotypes in these rats and employed principal component analysis to explore relationships among bone phenotypes in the HS rats. Our study demonstrates that significant variability exists for different skeletal phenotypes in HS rats compared with their inbred progenitors. In addition, we estimated high heritability for several bone phenotypes and biologically interpretable factors explaining significant overall variability, suggesting that the HS rat model could be a unique genetic resource for rapid and efficient discovery of the genetic determinants of bone fragility. Copyright © 2010 Elsevier Inc. All rights reserved.

Mouse model of fragile X syndrome: behavioral and hormonal response to stressors.

PubMed

Nielsen, Darci M; Evans, Jeffrey J; Derber, William J; Johnston, Kenzie A; Laudenslager, Mark L; Crnic, Linda S; Maclean, Kenneth N

2009-06-01

Fragile X syndrome, a form of mental retardation caused by inadequate levels of fragile X mental retardation protein (FMRP), is characterized by extreme sensitivity to sensory stimuli and increased behavioral and hormonal reactivity to stressors. Fmr1 knockout mice lack FMRP and exhibit abnormal responses to auditory stimuli. This study sought to determine whether Fmr1 knockout mice on an F1 hybrid background are normal in their response to footshock. Knockout mice were also examined for signs of hyperexcitation across an extended trial range, and serum corticosterone levels were evaluated in response to various stressors. The ability to acquire conditioned taste aversion was also assessed. Knockout mice exhibited no impairment in associative aversive learning or memory, since they successfully expressed conditioned taste aversion. Footshock-sensitivity, freezing behavior, and corticosterone response to various stressors did not differ between knockout and wild-type mice. However, knockout mice exhibited significantly increased responses during the extended test. The knockout mice's increased responsiveness to footshock in the extended test may be an indication of increased vulnerability to stress or enhanced emotional reactivity. Copyright (c) 2009 APA, all rights reserved.

New mechanistic explanation for the localization of ulcers in the rat duodenum: role of iron and selective uptake of cysteamine.

PubMed

Khomenko, Tetyana; Kolodney, Joanna; Pinto, John T; McLaren, Gordon D; Deng, Xiaoming; Chen, Longchuan; Tolstanova, Ganna; Paunovic, Brankica; Krasnikov, Boris F; Hoa, Neil; Cooper, Arthur J L; Szabo, Sandor

2012-09-01

Cysteamine, a coenzyme A metabolite, induces duodenal ulcers in rodents. Our recent studies showed that ulcer formation was aggravated by iron overload and diminished in iron deficiency. We hypothesized that cysteamine is selectively taken up in the duodenal mucosa, where iron absorption primarily occurs, and is transported by a carrier-mediated process. Here we report that cysteamine administration in rats leads to cysteamine accumulation in the proximal duodenum, where the highest concentration of iron in the gastrointestinal tract is found. In vitro, iron loading of intestinal epithelial cells (IEC-6) accelerated reactive oxygen species (ROS) production and increased [(14)C]cysteamine uptake. [(14)C]Cysteamine uptake by isolated gastrointestinal mucosal cells and by IEC-6 was pH-dependent and inhibited by unlabeled cysteamine. The uptake of [(14)C]cysteamine by IEC-6 was Na(+)-independent, saturable, inhibited by structural analogs, H(2)-histamine receptor antagonists, and organic cation transporter (OCT) inhibitors. OCT1 mRNA was markedly expressed in the rat duodenum and in IEC-6, and transfection of IEC-6 with OCT1 siRNA decreased OCT1 mRNA expression and inhibited [(14)C]cysteamine uptake. Cysteamine-induced duodenal ulcers were decreased in OCT1/2 knockout mice. These studies provide new insights into the mechanism of cysteamine absorption and demonstrate that intracellular iron plays a critical role in cysteamine uptake and in experimental duodenal ulcerogenesis. Copyright © 2012 Elsevier Inc. All rights reserved.

Galectin-3 Negatively Regulates Hippocampus-Dependent Memory Formation through Inhibition of Integrin Signaling and Galectin-3 Phosphorylation

PubMed Central

Chen, Yan-Chu; Ma, Yun-Li; Lin, Cheng-Hsiung; Cheng, Sin-Jhong; Hsu, Wei-Lun; Lee, Eminy H.-Y.

2017-01-01

Galectin-3, a member of the galectin protein family, has been found to regulate cell proliferation, inhibit apoptosis and promote inflammatory responses. Galectin-3 is also expressed in the adult rat hippocampus, but its role in learning and memory function is not known. Here, we found that contextual fear-conditioning training, spatial training or injection of NMDA into the rat CA1 area each dramatically decreased the level of endogenous galectin-3 expression. Overexpression of galectin-3 impaired fear memory, whereas galectin-3 knockout (KO) enhanced fear retention, spatial memory and hippocampal long-term potentiation. Galectin-3 was further found to associate with integrin α3, an association that was decreased after fear-conditioning training. Transfection of the rat CA1 area with small interfering RNA against galectin-3 facilitated fear memory and increased phosphorylated focal adhesion kinase (FAK) levels, effects that were blocked by co-transfection of the FAK phosphorylation-defective mutant Flag-FAKY397F. Notably, levels of serine-phosphorylated galectin-3 were decreased by fear conditioning training. In addition, blockade of galectin-3 phosphorylation at Ser-6 facilitated fear memory, whereas constitutive activation of galectin-3 at Ser-6 impaired fear memory. Interestingly galectin-1 plays a role in fear-memory formation similar to that of galectin-3. Collectively, our data provide the first demonstration that galectin-3 is a novel negative regulator of memory formation that exerts its effects through both extracellular and intracellular mechanisms. PMID:28744198

Circadian dynamics of the cone-rod homeobox (CRX) transcription factor in the rat pineal gland and its role in regulation of arylalkylamine N-acetyltransferase (AANAT).

PubMed

Rohde, Kristian; Rovsing, Louise; Ho, Anthony K; Møller, Morten; Rath, Martin F

2014-08-01

The cone-rod homeobox (Crx) gene encodes a transcription factor in the retina and pineal gland. Crx deficiency influences the pineal transcriptome, including a reduced expression of arylalkylamine N-acetyltransferase (Aanat), a key enzyme in nocturnal pineal melatonin production. However, previous functional studies on pineal Crx have been performed in melatonin-deficient mice. In this study, we have investigated the role of Crx in the melatonin-proficient rat pineal gland. The current study shows that pineal Crx transcript levels exhibit a circadian rhythm with a peak in the middle of the night, which is transferred into daily changes in CRX protein. The study further shows that the sympathetic innervation of the pineal gland controls the Crx rhythm. By use of adenovirus-mediated short hairpin RNA gene knockdown targeting Crx mRNA in primary rat pinealocyte cell culture, we here show that intact levels of Crx mRNA are required to obtain high levels of Aanat expression, whereas overexpression of Crx induces Aanat transcription in vitro. This regulatory function of Crx is further supported by circadian analysis of Aanat in the pineal gland of the Crx-knockout mouse. Our data indicate that the rhythmic nature of pineal CRX protein may directly modulate the daily profile of Aanat expression by inducing nighttime expression of this enzyme, thus facilitating nocturnal melatonin synthesis in addition to its role in ensuring a correct tissue distribution of Aanat expression.

PPAR-α, a lipid-sensing transcription factor, regulates blood–brain barrier efflux transporter expression

PubMed Central

More, Vijay R; Campos, Christopher R; Evans, Rebecca A; Oliver, Keith D; Chan, Gary NY; Miller, David S

2016-01-01

Lipid sensor peroxisome proliferator-activated receptor alpha (PPAR-α) is the master regulator of lipid metabolism. Dietary release of endogenous free fatty acids, fibrates, and certain persistent environmental pollutants, e.g. perfluoroalkyl fire-fighting foam components, are peroxisome proliferator-activated receptor alpha ligands. Here, we define a role for peroxisome proliferator-activated receptor alpha in regulating the expression of three ATP-driven drug efflux transporters at the rat and mouse blood–brain barriers: P-glycoprotein (Abcb1), breast cancer resistance protein (Bcrp/Abcg2), and multidrug resistance-associated protein 2 (Mrp2/Abcc2). Exposing isolated rat brain capillaries to linoleic acid, clofibrate, or PKAs increased the transport activity and protein expression of the three ABC transporters. These effects were blocked by the PPAR-α antagonist, GW6471. Dosing rats with 20 mg/kg or 200 mg/kg of clofibrate decreased the brain accumulation of the P-glycoprotein substrate, verapamil, by 50% (in situ brain perfusion; effects blocked by GW6471) and increased P-glycoprotein expression and activity in capillaries ex vivo. Fasting C57Bl/6 wild-type mice for 24 h increased both serum lipids and brain capillary P-glycoprotein transport activity. Fasting did not alter P-glycoprotein activity in PPAR-α knockout mice. These results indicate that hyperlipidemia, lipid-lowering fibrates and exposure to certain fire-fighting foam components activate blood–brain barrier peroxisome proliferator-activated receptor alpha, increase drug efflux transporter expression and reduce drug delivery to the brain. PMID:27193034

A novel small-molecule thienoquinolin urea transporter inhibitor acts as a potential diuretic.

PubMed

Li, Fei; Lei, Tianluo; Zhu, Juanjuan; Wang, Weiling; Sun, Yi; Chen, Jihui; Dong, Zixun; Zhou, Hong; Yang, Baoxue

2013-06-01

Urea transporters (UTs) are a family of membrane channel proteins that are specifically permeable to urea and play an important role in intrarenal urea recycling and in urine concentration. Using an erythrocyte osmotic lysis assay, we screened a small-molecule library for inhibitors of UT-facilitated urea transport. A novel class of thienoquinolin UT-B inhibitors were identified, of which PU-14 had potent inhibition activity on human, rabbit, rat, and mouse UT-B. The half-maximal inhibitory concentration of PU-14 on rat UT-B-mediated urea transport was ∼0.8 μmol/l, and it did not affect urea transport in mouse erythrocytes lacking UT-B but inhibited UT-A-type urea transporters, with 36% inhibition at 4 μmol/l. PU-14 showed no significant cellular toxicity at concentrations up to its solubility limit of 80 μmol/l. Subcutaneous delivery of PU-14 (at 12.5, 50, and 100 mg/kg) to rats caused an increase of urine output and a decrease of the urine urea concentration and subsequent osmolality without electrolyte disturbances and liver or renal damages. This suggests that PU-14 has a diuretic effect by urea-selective diuresis. Thus, PU-14 or its analogs might be developed as a new diuretic to increase renal fluid clearance in diseases associated with water retention without causing electrolyte imbalance. PU-14 may establish 'chemical knockout' animal models to study the physiological functions of UTs.

SPAK and OSR1 play essential roles in potassium homeostasis through actions on the distal convoluted tubule

PubMed Central

Ferdaus, Mohammed Z.; Barber, Karl W.; López‐Cayuqueo, Karen I.; Terker, Andrew S.; Argaiz, Eduardo R.; Gassaway, Brandon M.; Chambrey, Régine; Gamba, Gerardo; Rinehart, Jesse

2016-01-01

Key points STE20 (Sterile 20)/SPS‐1 related proline/alanine‐rich kinase (SPAK) and oxidative stress‐response kinase‐1 (OSR1) phosphorylate and activate the renal Na+–K+–2Cl− cotransporter 2 (NKCC2) and Na+Cl− cotransporter (NCC).Mouse models suggest that OSR1 mainly activates NKCC2‐mediated sodium transport along the thick ascending limb, while SPAK mainly activates NCC along the distal convoluted tubule, but the kinases may compensate for each other. We hypothesized that disruption of both kinases would lead to polyuria and severe salt‐wasting, and generated SPAK/OSR1 double knockout mice to test this.Despite a lack of SPAK and OSR1, phosphorylated NKCC2 abundance was still high, suggesting the existence of an alternative activating kinase.Compensatory changes in SPAK/OSR1‐independent phosphorylation sites on both NKCC2 and NCC and changes in sodium transport along the collecting duct were also observed.Potassium restriction revealed that SPAK and OSR1 play essential roles in the emerging model that NCC activation is central to sensing changes in plasma [K+]. Abstract STE20 (Sterile 20)/SPS‐1 related proline/alanine‐rich kinase (SPAK) and oxidative stress‐response kinase‐1 (OSR1) activate the renal cation cotransporters Na+–K+–2Cl− cotransporter (NKCC2) and Na+–Cl− cotransporter (NCC) via phosphorylation. Knockout mouse models suggest that OSR1 mainly activates NKCC2, while SPAK mainly activates NCC, with possible cross‐compensation. We tested the hypothesis that disrupting both kinases causes severe polyuria and salt‐wasting by generating SPAK/OSR1 double knockout (DKO) mice. DKO mice displayed lower systolic blood pressure compared with SPAK knockout (SPAK‐KO) mice, but displayed no severe phenotype even after dietary salt restriction. Phosphorylation of NKCC2 at SPAK/OSR1‐dependent sites was lower than in SPAK‐KO mice, but still significantly greater than in wild type mice. In the renal medulla, there was significant phosphorylation of NKCC2 at SPAK/OSR1‐dependent sites despite a complete absence of SPAK and OSR1, suggesting the existence of an alternative activating kinase. The distal convoluted tubule has been proposed to sense plasma [K+], with NCC activation serving as the primary effector pathway that modulates K+ secretion, by metering sodium delivery to the collecting duct. Abundance of phosphorylated NCC (pNCC) is dramatically lower in SPAK‐KO mice than in wild type mice, and the additional disruption of OSR1 further reduced pNCC. SPAK‐KO and kidney‐specific OSR1 single knockout mice maintained plasma [K+] following dietary potassium restriction, but DKO mice developed severe hypokalaemia. Unlike mice lacking SPAK or OSR1 alone, DKO mice displayed an inability to phosphorylate NCC under these conditions. These data suggest that SPAK and OSR1 are essential components of the effector pathway that maintains plasma [K+]. PMID:27068441

Comparative photodynamic therapy cytotoxicity of mannose-conjugated chlorin and talaporfin sodium in cultured human and rat cells.

PubMed

Shinoda, Yo; Takahashi, Tsutomu; Akimoto, Jiro; Ichikawa, Megumi; Yamazaki, Hiromi; Narumi, Atsushi; Yano, Shigenobu; Fujiwara, Yasuyuki

2017-01-01

Photodynamic therapy (PDT) is a Food and Drug Administration authorized method for cancer treatment, which uses photosensitizer and laser photo-irradiation to generate reactive oxygen species to induce cell death in tumors. Photosensitizers have been progressively developed, from first to third generation, with improvements in cell specificity, reduced side effects and toxicity, increased sensitivity for irradiation and reduced persistence of photosensitizer in healthy cells. These improvements have been achieved by basic comparative experiments between current and novel photosensitizers using cell lines; however, photosensitizers should be carefully evaluated because they may have cell type specificity. In the present study, we compared a third-generation photosensitizer, β-mannose-conjugated chlorin (β-M-chlorin), with the second generation, talaporfin sodium (NPe6), using seven different rat and human cell lines and a neuronal/glial primary culture prepared from rat embryos. NPe6 was more effective than β-M-chlorin in human-derived cell lines, and β-M-chlorin was more effective than NPe6 in rat primary cultures and rat-derived cell lines, except for the rat pheochromocytoma cell line, PC12. These differences of phototoxicity in different cell types are not because of differences in photosensitivity between the photosensitizers, but rather are associated with different distribution and accumulation rates in the different cell types. These data suggest that evaluation of photosensitizers for PDT should be carried out using as large a variety of cell types as possible because each photosensitizer may have cell type specificity.

Few effects of multi-generational dietary exposure to genistein or nonylphenol on sodium solution intake in male and female Sprague-Dawley rats.

PubMed

Ferguson, Sherry A; Delclos, K Barry; Newbold, Retha R; Flynn, Katherine M

2009-01-01

Previous work in our laboratory indicated that lifelong dietary exposure to estrogen-like endocrine disrupters increased sodium solution intake in adult male and female rats. Here, we sought to discern the critical periods necessary for this alteration as well as establish the effects of lower dietary concentrations of genistein and nonylphenol. Male and female Sprague-Dawley rats (F0) consumed phytoestrogen-free chow containing 0, 5, 100, or 500 ppm genistein (approximately equal to 0.0, 0.4, 8.0, and 40.0 mg/kg/day) or 0, 25, 200, or 750 ppm nonylphenol (approximately equal to 0.0, 2.0, 16.0, and 60.0 mg/kg/day). Rats were mated within treatment groups and offspring (F1) maintained on the same diets. Mating for the F1, F2, and F3 (genistein only) was within treatment groups. At postnatal day (PND) 21, the F3 generation began to consume unadulterated phytoestrogen-free chow such that genistein exposure occurred only in utero and preweaning. The F4 generation was never directly exposed to genistein. On PNDs 65-68, intake of regular water and a 3.0% sodium chloride solution was measured for F1-F4 generations (genistein portion) or F1-F2 (nonylphenol portion). Although body weights were decreased by the highest dietary concentrations of genistein and nonylphenol, there were only minimal effects of exposure on sodium solution intake. As expected, intake was highest in female rats. With previous data, these results indicate that the dietary concentrations necessary to increase adult sodium solution intake in rats are greater than 500 ppm genistein and 750 ppm nonylphenol and such effects do not appear to increase across generations.

Sexual dimorphism in hybrids rats.

PubMed

Garcia-Falgueras, Alicia; Pinos, Helena; Fernández, Rosa; Collado, Paloma; Pasaro, Eduardo; Segovia, Santiago; Guillamon, Antonio

2006-12-06

Laboratory rat strains descend from Wistar rats as a consequence of artificial selection. Previously we reported that the medial posterior division of the bed nucleus of the stria terminalis (BSTMP) was sexually dimorphic in Wistar and Long-Evans strains while the medial anterior division (BSTMA) and the locus coeruleus (LC) only showed sex differences in the ancestor Wistar strain. The lateral posterior division (BSTLP) was isomorphic in both strains. The present work studies the number of neurons in the BSTMP, BSTMA, BSTLP and LC of male and female Wistar and Long-Evans rats (F(0)) and their hybrid F(1) and F(2) generations. The BSTMP is sexually dimorphic in the F(0), F(1) and F(2) generations while sex differences in the LC are only seen in F(0) Wistar rats but not in the F(0) Long-Evans or the F(1) and F(2) hybrid generations. Sex differences in the BSTMA are seen in F(0) Wistar but not in F(0) Long-Evans rats and completely disappear in the F(2) generations. The number of neurons in the LC of both males and females decreased in heterozygotic individuals (F(1)) but increased in homozygotic (F(2)). However, the number of neurons in the BSTMP changes significantly over the generations, although the ratio of neurons (female/male) is stable and unaffected in homo- or heterozygosis. Thus, the mechanism that regulates the neuronal female/male ratio would be different from the one that controls the number of neurons. The facts that sex differences in the BSTMP are not affected by homo- or heterozygosis and that they are seen in several mammalian orders suggest the existence of a "fixed" type of brain sex differences in the Mammalia Class.

[Long-term effect of iodine deficiency on growth and food utilization rate in second filial generation rats].

PubMed

Muyeseer, Ainiwaer; Zhang, G X; Wang, J; Liu, Y; Meng, X H; Liu, Q

2017-02-06

Objective: To study the effect of iodine deficiency on body weight, food consumption, and food utilization rate of second filial generation Wistar rats. Methods: According to the food pattern of a high-iodine deficient population, two types of low-iodine food have been produced using the main crops grown in this area (iodine levels of 50 and 20 μg/kg, respectively). Wistar rats were randomly divided into three groups, normal iodine group (NI group), low-iodine group one (LI group) and low-iodine group two (LII group), using the random number table method and fed diets containing 300, 50, and 20 μg/kg of iodine, respectively. Parental generation rats were fed until they reached reproductive age; first filial generation rats were allocated to the same diet as their mothers. After 3 months of feeding, first filial generation rats gave birth to second filial generation rats; second filial generation rats were allocated to the same diet as their mothers. After feeding for 90, 180, and 270 days, rats were sacrificed. One-way analysis of variance was used to analyze body weight, food intake, and food utilization rate data collected during the time of feeding and blood iodine hormone level, which was determined after sacrifice. Results: The LI and LII groups generally demonstrated decreased activity, slow reaction, and growth retardation compared with the NI group. After 270 days, the urine iodine levels of the LI and LII groups were 1.7 and 0.2 μg/L, respectively, which were significantly lower than the NI group (255.2 μg/L) ( P< 0.001). Additionally, the weight of female rats in the LI and LII groups were (288.1±10.5) and (275.7±2.7) g, respectively, which was significantly lower than that of the NI group ((311.0±2.3) g) ( P< 0.001). The weight of male rats were (446.0±4.6) and (451.8±19.1) g, respectively, which were significantly lower than that of the NI group ((517.2±7.8) g) ( P< 0.001). In the LI and LII groups, food intake of female and male rats after 270 days were (465.0±27.7), (658.4±28.6) and (423.0±13.2), (548.0±18.8) g, respectively, which were significantly lower than that of the NI group ((499.5±21.8), (760.8±33.0) g) ( P< 0.001). Moreover, the food utilization rate of female rats in the LI and LII groups was (8.7±0.4)% and (6.0±0.58)%, which was lower than that of the NI group ((11.7±3.5)%) ( P< 0.001); similarly, male rats showed rates of (8.9±1.5)% and (6.9±1.31)%, respectively, which were lower than that of the NI group ((13.7±3.0)%) ( P< 0.001). After 270 days, the level of T3 in the LI and LII groups were (0.45±0.10) and (0.34±0.15) ng/ml, respectively, which was significantly lower than that of the NI group ((0.91±0.49) ng/ml) ( P< 0.01). Moreover, the level of T4 were (69.02±27.87) , (53.18±13.53) ng/ml in LI and LII groups, respectively, which was lower than that of the NI group ((76.69±29.42) ng/ml) ( P< 0.05). Conclusion: This study indicated that iodine deficiency induced by a long-term low-iodine diet can cause changes in weight, food intake, and food utilization rate among second filial iodine deficiency rats. More importantly, the iodine content in low-iodine food impacts these parameters.

The Role of Lymphangiogenesis in Orthotopic Prostatic Tumor-Environment on Regional and Systemic Metastasis

DTIC Science & Technology

2008-01-01

expressing Renilla luciferase and VEGF-C shRNA or irrelevant firefly luciferase shRNA (Ctrl) were implanted orthotopically as before. To determine the...on metastasis in Pten knockout model (Month 10-24): a) Generate Ubc/sVEGFR-3 and Ubc/ Renilla luciferase (RL) lentiviral vectors by subcloning...progression (month 10-12) c) Monitor efficiency of Ubc/lentiviral transduction and expression in Pten (-/-) prostate using optical imaging of Renilla

Metabolic engineering of Synechocystis sp. PCC 6803 for enhanced ethanol production based on flux balance analysis.

PubMed

Yoshikawa, Katsunori; Toya, Yoshihiro; Shimizu, Hiroshi

2017-05-01

Synechocystis sp. PCC 6803 is an attractive host for bio-ethanol production due to its ability to directly convert atmospheric carbon dioxide into ethanol using photosystems. To enhance ethanol production in Synechocystis sp. PCC 6803, metabolic engineering was performed based on in silico simulations, using the genome-scale metabolic model. Comprehensive reaction knockout simulations by flux balance analysis predicted that the knockout of NAD(P)H dehydrogenase enhanced ethanol production under photoautotrophic conditions, where ammonium is the nitrogen source. This deletion inhibits the re-oxidation of NAD(P)H, which is generated by ferredoxin-NADP + reductase and imposes re-oxidation in the ethanol synthesis pathway. The effect of deleting the ndhF1 gene, which encodes NADH dehydrogenase subunit 5, on ethanol production was experimentally evaluated using ethanol-producing strains of Synechocystis sp. PCC 6803. The ethanol titer of the ethanol-producing ∆ndhF1 strain increased by 145%, compared with that of the control strain.

Rho GTPases and their downstream effectors in megakaryocyte biology.

PubMed

Pleines, Irina; Cherpokova, Deya; Bender, Markus

2018-06-18

Megakaryocytes differentiate from hematopoietic stem cells in the bone marrow. The transition of megakaryocytes to platelets is a complex process. Thereby, megakaryocytes extend proplatelets into sinusoidal blood vessels, where the proplatelets undergo fission to release platelets. Defects in platelet production can lead to a low platelet count (thrombocytopenia) with increased bleeding risk. Rho GTPases comprise a family of small signaling G proteins that have been shown to be master regulators of the cytoskeleton controlling many aspects of intracellular processes. The generation of Pf4-Cre transgenic mice was a major breakthrough that enabled studies in megakaryocyte-/platelet-specific knockout mouse lines and provided new insights into the central regulatory role of Rho GTPases in megakaryocyte maturation and platelet production. In this review, we will summarize major findings on the role of Rho GTPases in megakaryocyte biology with a focus on mouse lines in which knockout strategies have been applied to study the function of the best-characterized members Rac1, Cdc42 and RhoA and their downstream effector proteins.

Disease Model Discovery from 3,328 Gene Knockouts by The International Mouse Phenotyping Consortium

PubMed Central

Meehan, Terrence F.; Conte, Nathalie; West, David B.; Jacobsen, Julius O.; Mason, Jeremy; Warren, Jonathan; Chen, Chao-Kung; Tudose, Ilinca; Relac, Mike; Matthews, Peter; Karp, Natasha; Santos, Luis; Fiegel, Tanja; Ring, Natalie; Westerberg, Henrik; Greenaway, Simon; Sneddon, Duncan; Morgan, Hugh; Codner, Gemma F; Stewart, Michelle E; Brown, James; Horner, Neil; Haendel, Melissa; Washington, Nicole; Mungall, Christopher J.; Reynolds, Corey L; Gallegos, Juan; Gailus-Durner, Valerie; Sorg, Tania; Pavlovic, Guillaume; Bower, Lynette R; Moore, Mark; Morse, Iva; Gao, Xiang; Tocchini-Valentini, Glauco P; Obata, Yuichi; Cho, Soo Young; Seong, Je Kyung; Seavitt, John; Beaudet, Arthur L.; Dickinson, Mary E.; Herault, Yann; Wurst, Wolfgang; de Angelis, Martin Hrabe; Lloyd, K.C. Kent; Flenniken, Ann M; Nutter, Lauryl MJ; Newbigging, Susan; McKerlie, Colin; Justice, Monica J.; Murray, Stephen A.; Svenson, Karen L.; Braun, Robert E.; White, Jacqueline K.; Bradley, Allan; Flicek, Paul; Wells, Sara; Skarnes, William C.; Adams, David J.; Parkinson, Helen; Mallon, Ann-Marie; Brown, Steve D.M.; Smedley, Damian

2017-01-01

Although next generation sequencing has revolutionised the ability to associate variants with human diseases, diagnostic rates and development of new therapies are still limited by our lack of knowledge of function and pathobiological mechanism for most genes. To address this challenge, the International Mouse Phenotyping Consortium (IMPC) is creating a genome- and phenome-wide catalogue of gene function by characterizing new knockout mouse strains across diverse biological systems through a broad set of standardised phenotyping tests, with all mice made readily available to the biomedical community. Analysing the first 3328 genes reveals models for 360 diseases including the first for type C Bernard-Soulier, Bardet-Biedl-5 and Gordon Holmes syndromes. 90% of our phenotype annotations are novel, providing the first functional evidence for 1092 genes and candidates in unsolved diseases such as Arrhythmogenic Right Ventricular Dysplasia 3. Finally, we describe our role in variant functional validation with the 100,000 Genomes and other projects. PMID:28650483

CBX7 gene expression plays a negative role in adipocyte cell growth and differentiation

PubMed Central

Forzati, Floriana; Federico, Antonella; Pallante, Pierlorenzo; Colamaio, Marianna; Esposito, Francesco; Sepe, Romina; Gargiulo, Sara; Luciano, Antonio; Arra, Claudio; Palma, Giuseppe; Bon, Giulia; Bucher, Stefania; Falcioni, Rita; Brunetti, Arturo; Battista, Sabrina; Fedele, Monica; Fusco, Alfredo

2014-01-01

ABSTRACT We have recently generated knockout mice for the Cbx7 gene, coding for a polycomb group protein that is downregulated in human malignant neoplasias. These mice develop liver and lung adenomas and carcinomas, which confirms a tumour suppressor role for CBX7. The CBX7 ability to downregulate CCNE1 expression likely accounts for the phenotype of the Cbx7-null mice. Unexpectedly, Cbx7-knockout mice had a higher fat tissue mass than wild-type, suggesting a role of CBX7 in adipogenesis. Consistently, we demonstrate that Cbx7-null mouse embryonic fibroblasts go towards adipocyte differentiation more efficiently than their wild-type counterparts, and this effect is Cbx7 dose-dependent. Similar results were obtained when Cbx7-null embryonic stem cells were induced to differentiate into adipocytes. Conversely, mouse embryonic fibroblasts and human adipose-derived stem cells overexpressing CBX7 show an opposite behaviour. These findings support a negative role of CBX7 in the control of adipocyte cell growth and differentiation. PMID:25190058

Analysis of mammalian gene function through broad-based phenotypic screens across a consortium of mouse clinics.

PubMed

de Angelis, Martin Hrabě; Nicholson, George; Selloum, Mohammed; White, Jacqui; Morgan, Hugh; Ramirez-Solis, Ramiro; Sorg, Tania; Wells, Sara; Fuchs, Helmut; Fray, Martin; Adams, David J; Adams, Niels C; Adler, Thure; Aguilar-Pimentel, Antonio; Ali-Hadji, Dalila; Amann, Gregory; André, Philippe; Atkins, Sarah; Auburtin, Aurelie; Ayadi, Abdel; Becker, Julien; Becker, Lore; Bedu, Elodie; Bekeredjian, Raffi; Birling, Marie-Christine; Blake, Andrew; Bottomley, Joanna; Bowl, Mike; Brault, Véronique; Busch, Dirk H; Bussell, James N; Calzada-Wack, Julia; Cater, Heather; Champy, Marie-France; Charles, Philippe; Chevalier, Claire; Chiani, Francesco; Codner, Gemma F; Combe, Roy; Cox, Roger; Dalloneau, Emilie; Dierich, André; Di Fenza, Armida; Doe, Brendan; Duchon, Arnaud; Eickelberg, Oliver; Esapa, Chris T; El Fertak, Lahcen; Feigel, Tanja; Emelyanova, Irina; Estabel, Jeanne; Favor, Jack; Flenniken, Ann; Gambadoro, Alessia; Garrett, Lilian; Gates, Hilary; Gerdin, Anna-Karin; Gkoutos, George; Greenaway, Simon; Glasl, Lisa; Goetz, Patrice; Da Cruz, Isabelle Goncalves; Götz, Alexander; Graw, Jochen; Guimond, Alain; Hans, Wolfgang; Hicks, Geoff; Hölter, Sabine M; Höfler, Heinz; Hancock, John M; Hoehndorf, Robert; Hough, Tertius; Houghton, Richard; Hurt, Anja; Ivandic, Boris; Jacobs, Hughes; Jacquot, Sylvie; Jones, Nora; Karp, Natasha A; Katus, Hugo A; Kitchen, Sharon; Klein-Rodewald, Tanja; Klingenspor, Martin; Klopstock, Thomas; Lalanne, Valerie; Leblanc, Sophie; Lengger, Christoph; le Marchand, Elise; Ludwig, Tonia; Lux, Aline; McKerlie, Colin; Maier, Holger; Mandel, Jean-Louis; Marschall, Susan; Mark, Manuel; Melvin, David G; Meziane, Hamid; Micklich, Kateryna; Mittelhauser, Christophe; Monassier, Laurent; Moulaert, David; Muller, Stéphanie; Naton, Beatrix; Neff, Frauke; Nolan, Patrick M; Nutter, Lauryl Mj; Ollert, Markus; Pavlovic, Guillaume; Pellegata, Natalia S; Peter, Emilie; Petit-Demoulière, Benoit; Pickard, Amanda; Podrini, Christine; Potter, Paul; Pouilly, Laurent; Puk, Oliver; Richardson, David; Rousseau, Stephane; Quintanilla-Fend, Leticia; Quwailid, Mohamed M; Racz, Ildiko; Rathkolb, Birgit; Riet, Fabrice; Rossant, Janet; Roux, Michel; Rozman, Jan; Ryder, Ed; Salisbury, Jennifer; Santos, Luis; Schäble, Karl-Heinz; Schiller, Evelyn; Schrewe, Anja; Schulz, Holger; Steinkamp, Ralf; Simon, Michelle; Stewart, Michelle; Stöger, Claudia; Stöger, Tobias; Sun, Minxuan; Sunter, David; Teboul, Lydia; Tilly, Isabelle; Tocchini-Valentini, Glauco P; Tost, Monica; Treise, Irina; Vasseur, Laurent; Velot, Emilie; Vogt-Weisenhorn, Daniela; Wagner, Christelle; Walling, Alison; Weber, Bruno; Wendling, Olivia; Westerberg, Henrik; Willershäuser, Monja; Wolf, Eckhard; Wolter, Anne; Wood, Joe; Wurst, Wolfgang; Yildirim, Ali Önder; Zeh, Ramona; Zimmer, Andreas; Zimprich, Annemarie; Holmes, Chris; Steel, Karen P; Herault, Yann; Gailus-Durner, Valérie; Mallon, Ann-Marie; Brown, Steve Dm

2015-09-01

The function of the majority of genes in the mouse and human genomes remains unknown. The mouse embryonic stem cell knockout resource provides a basis for the characterization of relationships between genes and phenotypes. The EUMODIC consortium developed and validated robust methodologies for the broad-based phenotyping of knockouts through a pipeline comprising 20 disease-oriented platforms. We developed new statistical methods for pipeline design and data analysis aimed at detecting reproducible phenotypes with high power. We acquired phenotype data from 449 mutant alleles, representing 320 unique genes, of which half had no previous functional annotation. We captured data from over 27,000 mice, finding that 83% of the mutant lines are phenodeviant, with 65% demonstrating pleiotropy. Surprisingly, we found significant differences in phenotype annotation according to zygosity. New phenotypes were uncovered for many genes with previously unknown function, providing a powerful basis for hypothesis generation and further investigation in diverse systems.

Bm65 is essential for the propagation of Bombyx mori nucleopolyhedrovirus.

PubMed

Tang, Qi; Li, Guohui; Yao, Qin; Chen, Liang; Feng, Fan; Yuan, Yi; Chen, Keping

2013-01-01

Orf65 (Bm65) of Bombyx mori nucleopolyhedrovirus (BmNPV) is a highly conserved gene that encodes an unknown 104-amino acid protein. In the present study, we have shown the role of Bm65 in the baculovirus life cycle. 5'-RACE analysis showed that the transcription start site of Bm65 was 14 nucleotides upstream of the start codon ATG. The transcription profile of Bm65 was detected from 6 to 72 h postinfection (p. i.) by RT-PCR. A Bm65-knockout bacmid was constructed by homologous recombination to characterize the role of Bm65 in viral life cycle. Fluorescence microscopy showed that Bm65-knockout virus was unable to generate infectious budded virus in BmN cells. Furthermore, quantitative real-time PCR analysis demonstrated that Bm65 deletion did not affect the viral DNA replication. To conclude, Bm65 is essential for the propagation of BmNPV, but is unnecessary for the replication of viral DNA.

Generating mouse lines for lineage tracing and knockout studies.

PubMed

Kraus, Petra; Sivakamasundari, V; Xing, Xing; Lufkin, Thomas

2014-01-01

In 2007 Capecchi, Evans, and Smithies received the Nobel Prize in recognition for discovering the principles for introducing specific gene modifications in mice via embryonic stem cells, a technology, which has revolutionized the field of biomedical science allowing for the generation of genetically engineered animals. Here we describe detailed protocols based on and developed from these ground-breaking discoveries, allowing for the modification of genes not only to create mutations to study gene function but additionally to modify genes with fluorescent markers, thus permitting the isolation of specific rare wild-type and mutant cell types for further detailed analysis at the biochemical, pathological, and genomic levels.

The long pursued Holy Grail of the true "alcoholic" rat.

PubMed

Gessa, Gian Luigi

2016-08-15

An anthology of microdialysis and electrophysiological studies on ethanol effect on mesolimbic dopaminergic neurons is presented. The usefulness of rats with innate preference for ethanol, such as the Sardinian alcohol-preferring (sP), in studying ethanol rewarding and reinforcing effects is signaled. The generation of the long sought "alcoholics rat" from sP rats is announced. Rats of the sP line avoid the shortcomings of using rats non selected for ethanol preference. Copyright © 2016. Published by Elsevier B.V.

Prior fear conditioning does not impede enhanced active avoidance in serotonin transporter knockout rats.

PubMed

Schipper, Pieter; Henckens, Marloes J A G; Borghans, Bart; Hiemstra, Marlies; Kozicz, Tamas; Homberg, Judith R

2017-05-30

Stressors can be actively or passively coped with, and adequate adaption of the coping response to environmental conditions can reduce their potential deleterious effects. One major factor influencing stress coping behaviour is serotonin transporter (5-HTT) availability. Abolishment of 5-HTT is known to impair fear extinction but facilitates acquisition of signalled active avoidance (AA), a behavioural task in which an animal learns to avoid an aversive stimulus that is predicted by a cue. Flexibility in adapting coping behaviour to the nature of the stressor shapes resilience to stress-related disorders. Therefore, we investigated the relation between 5-HTT expression and ability to adapt a learned coping response to changing environmental conditions. To this end, we first established and consolidated a cue-conditioned passive fear response in 5-HTT -/- and wildtype rats. Next, we used the conditioned stimulus (CS) to signal oncoming shocks during signalled AA training in 5-HTT -/- and wildtype rats to study their capability to acquire an active coping response to the CS following fear conditioning. Finally, we investigated the behavioural response to the CS in a novel environment and measured freezing, exploration and self-grooming, behaviours reflective of stress coping strategy. We found that fear conditioned and sham conditioned 5-HTT -/- animals acquired the signalled AA response faster than wildtypes, while prior conditioning briefly delayed AA learning similarly in both genotypes. Subsequent exposure to the CS in the novel context reduced freezing and increased locomotion in 5-HTT -/- compared to wildtype rats. This indicates that improved AA performance in 5-HTT -/- rats resulted in a weaker residual passive fear response to the CS in a novel context. Fear conditioning prior to AA training did not affect freezing upon re-encountering the CS, although it did reduce locomotion in 5-HTT -/- rats. We conclude that independent of 5-HTT signalling, prior fear conditioning does not greatly impair the acquisition of subsequent active coping behaviour when the situation allows for it. Abolishment of 5-HTT results in a more active coping style in case of novelty-induced fear and upon CS encounter in a novel context after AA learning. Copyright © 2017 Elsevier B.V. All rights reserved.

Generation of immunodeficient rats with Rag1 and Il2rg gene deletions and human tissue grafting models.

PubMed

Ménoret, Séverine; Ouisse, Laure-Hélène; Tesson, Laurent; Delbos, Frédéric; Garnier, Delphine; Remy, Séverine; Usal, Claire; Concordet, Jean-Paul; Giovannangeli, Carine; Chenouard, Vanessa; Brusselle, Lucas; Merieau, Emmanuel; Nerrière-Daguin, Véronique; Duteille, Franck; Bellier-Waast, Frédérique; Fraichard, Alexandre; Nguyen, Tuan H; Anegon, Ignacio

2018-04-24

Immunodeficient mice are invaluable tools to analyze the long-term effects of potentially immunogenic molecules in the absence of adaptive immune responses. Nevertheless, there are models and experimental situations that would beneficiate of larger immunodeficient recipients. Rats are ideally suited to perform experiments in which larger size is needed and are still a small animal model suitable for rodent facilities. Additionally, rats reproduce certain human diseases better than mice, such as ankylosing spondylitis and Duchenne disease and these disease models would greatly benefit of immunodeficient rats to test different immunogenic treatments. We describe the generation of Il2rg-deficient rats and their crossing with previously described Rag1-deficient rats to generate double-mutant RRG animals. As compared to Rag1-deficient rats, Il2rg-deficient rats were more immunodeficient since partially lacked not only T and B cells but also NK cells. RRG animals showed a more profound immunossuppressed phenotype since they displayed undetectable levels of T, B and NK cells. Similarly, all immunoglobulin isotypes in sera were decreased in Rag1 or Il2rg-deficient rats and undetectable in RRG animals. Rag1 or Il2rg-deficient rats rejected allogeneic skin transplants and human tumors whereas RRG animals not only accepted allogeneic rat skin but also xenogeneic human tumors, skin and hepatocytes. Immune humanization of RRG animals was unsuccessful. Thus, immunodeficient RRG animals are useful recipients for long term studies in which immune responses could be an obstacle, including tissue humanization of different tissues.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.

Generation of murine tumor cell lines deficient in MHC molecule surface expression using the CRISPR/Cas9 system.

PubMed

Das, Krishna; Eisel, David; Lenkl, Clarissa; Goyal, Ashish; Diederichs, Sven; Dickes, Elke; Osen, Wolfram; Eichmüller, Stefan B

2017-01-01

In this study, the CRISPR/Cas9 technology was used to establish murine tumor cell lines, devoid of MHC I or MHC II surface expression, respectively. The melanoma cell line B16F10 and the murine breast cancer cell line EO-771, the latter stably expressing the tumor antigen NY-BR-1 (EO-NY), were transfected with an expression plasmid encoding a β2m-specific single guide (sg)RNA and Cas9. The resulting MHC I negative cells were sorted by flow cytometry to obtain single cell clones, and loss of susceptibility of peptide pulsed MHC I negative clones to peptide-specific CTL recognition was determined by IFNγ ELISpot assay. The β2m knockout (KO) clones did not give rise to tumors in syngeneic mice (C57BL/6N), unless NK cells were depleted, suggesting that outgrowth of the β2m KO cell lines was controlled by NK cells. Using sgRNAs targeting the β-chain encoding locus of the IAb molecule we also generated several B16F10 MHC II KO clones. Peptide loaded B16F10 MHC II KO cells were insusceptible to recognition by OT-II cells and tumor growth was unaltered compared to parental B16F10 cells. Thus, in our hands the CRISPR/Cas9 system has proven to be an efficient straight forward strategy for the generation of MHC knockout cell lines. Such cell lines could serve as parental cells for co-transfection of compatible HLA alleles together with human tumor antigens of interest, thereby facilitating the generation of HLA matched transplantable tumor models, e.g. in HLAtg mouse strains of the newer generation, lacking cell surface expression of endogenous H2 molecules. In addition, our tumor cell lines established might offer a useful tool to investigate tumor reactive T cell responses that function independently from MHC molecule surface expression by the tumor.

Diversity at the Locus Associated with Transcription of a Variable Surface Antigen of Pneumocystis carinii as an Index of Population Structure and Dynamics in Infected Rats

PubMed Central

Keely, Scott P.; Cushion, Melanie T.; Stringer, James R.

2003-01-01

Pneumocystis carinii expresses a surface glycoprotein called MSG. Different isoforms of MSG are encoded by a gene family spread over at least 15 telomeric sites. Only one locus, called UCS, supports the production of MSG mRNA. Previous studies showed that P. carinii populations from individual rats exhibited high degrees of diversity with respect to the MSG genes attached to the UCS locus. This diversity could have been generated primarily in the rats studied. Alternatively, the rats may have been infected by P. carinii organisms that were already different at the UCS locus. To investigate this issue, we examined the UCS locus in P. carinii from rats that had been exposed to few of the microbes at a specified time, which produced a bottleneck in the microbial population. Some of the rats with bottlenecks produced P. carinii populations in which a single MSG sequence resided at the UCS locus in 80 to 90% of the organisms, showing that P. carinii can proliferate within a rat without generating the very high levels of UCS diversity previously seen. From the degree of diversity observed in the bottlenecked populations, the maximum rate of switching appeared to be 0.01 event per generation. These data also suggest that the infectious dose is as low as one organism, that rats that share a cage readily infect each other, and that the doubling time of P. carinii in vivo is ∼3 days. In addition, we found that inoculation with 107 P. carinii organisms from a population highly heterogeneous at the UCS locus reproduced this heterogeneity. By contrast, shifts in population structure occurred in rats given 104 P. carinii organisms, suggesting that a small fraction of these proliferated. PMID:12496148

Deletion of PDZD7 disrupts the Usher syndrome type 2 protein complex in cochlear hair cells and causes hearing loss in mice.

PubMed

Zou, Junhuang; Zheng, Tihua; Ren, Chongyu; Askew, Charles; Liu, Xiao-Ping; Pan, Bifeng; Holt, Jeffrey R; Wang, Yong; Yang, Jun

2014-05-01

Usher syndrome type 2 (USH2) is the predominant form of USH, a leading genetic cause of combined deafness and blindness. PDZD7, a paralog of two USH causative genes, USH1C and USH2D (WHRN), was recently reported to be implicated in USH2 and non-syndromic deafness. It encodes a protein with multiple PDZ domains. To understand the biological function of PDZD7 and the pathogenic mechanism caused by PDZD7 mutations, we generated and thoroughly characterized a Pdzd7 knockout mouse model. The Pdzd7 knockout mice exhibit congenital profound deafness, as assessed by auditory brainstem response, distortion product otoacoustic emission and cochlear microphonics tests, and normal vestibular function, as assessed by their behaviors. Lack of PDZD7 leads to the disorganization of stereocilia bundles and a reduction in mechanotransduction currents and sensitivity in cochlear outer hair cells. At the molecular level, PDZD7 determines the localization of the USH2 protein complex, composed of USH2A, GPR98 and WHRN, to ankle links in developing cochlear hair cells, likely through its direct interactions with these three proteins. The localization of PDZD7 to the ankle links of cochlear hair bundles also relies on USH2 proteins. In photoreceptors of Pdzd7 knockout mice, the three USH2 proteins largely remain unchanged at the periciliary membrane complex. The electroretinogram responses of both rod and cone photoreceptors are normal in knockout mice at 1 month of age. Therefore, although the organization of the USH2 complex appears different in photoreceptors, it is clear that PDZD7 plays an essential role in organizing the USH2 complex at ankle links in developing cochlear hair cells. GenBank accession numbers: KF041446, KF041447, KF041448, KF041449, KF041450, KF041451.

Sm2, a paralog of the Trichoderma cerato-platanin elicitor Sm1, is also highly important for plant protection conferred by the fungal-root interaction of Trichoderma with maize.

PubMed

Gaderer, Romana; Lamdan, Netta L; Frischmann, Alexa; Sulyok, Michael; Krska, Rudolf; Horwitz, Benjamin A; Seidl-Seiboth, Verena

2015-01-16

The proteins Sm1 and Sm2 from the biocontrol fungus Trichoderma virens belong to the cerato-platanin protein family. Members of this family are small, secreted proteins that are abundantly produced by filamentous fungi with all types of life-styles. Some species of the fungal genus Trichoderma are considered as biocontrol fungi because they are mycoparasites and are also able to directly interact with plants, thereby stimulating plant defense responses. It was previously shown that the cerato-platanin protein Sm1 from T. virens - and to a lesser extent its homologue Epl1 from Trichoderma atroviride - induce plant defense responses. The plant protection potential of other members of the cerato-platanin protein family in Trichoderma, however, has not yet been investigated. In order to analyze the function of the cerato-platanin protein Sm2, sm1 and sm2 knockout strains were generated and characterized. The effect of the lack of Sm1 and Sm2 in T. virens on inducing systemic resistance in maize seedlings, challenged with the plant pathogen Cochliobolus heterostrophus, was tested. These plant experiments were also performed with T. atroviride epl1 and epl2 knockout strains. In our plant-pathogen system T. virens was a more effective plant protectant than T. atroviride and the results with both Trichoderma species showed concordantly that the level of plant protection was more strongly reduced in plants treated with the sm2/epl2 knockout strains than with sm1/epl1 knockout strains. Although the cerato-platanin genes sm1/epl1 are more abundantly expressed than sm2/epl2 during fungal growth, Sm2/Epl2 are, interestingly, more important than Sm1/Epl1 for the promotion of plant protection conferred by Trichoderma in the maize-C. heterostrophus pathosystem.

Hypervitaminosis D mediates compensatory Ca2+ hyperabsorption in TRPV5 knockout mice.

PubMed

Renkema, Kirsten Y; Nijenhuis, Tom; van der Eerden, Bram C J; van der Kemp, Annemiete W C M; Weinans, Harrie; van Leeuwen, Johannes P T M; Bindels, René J M; Hoenderop, Joost G J

2005-11-01

Vitamin D plays an important role in Ca(2+) homeostasis by controlling Ca(2+) (re)absorption in intestine, kidney, and bone. The epithelial Ca(2+) channel TRPV5 mediates the Ca(2+) entry step in active Ca(2+) reabsorption. TRPV5 knockout (TRPV5(-/-)) mice show impaired Ca(2+) reabsorption, hypercalciuria, hypervitaminosis D, and intestinal hyperabsorption of Ca(2+). Moreover, these mice demonstrate upregulation of intestinal TRPV6 and calbindin-D(9K) expression compared with wild-type mice. For addressing the role of the observed hypervitaminosis D in the maintenance of Ca(2+) homeostasis and the regulation of expression levels of the Ca(2+) transport proteins in kidney and intestine, TRPV5/25-hydroxyvitamin-D(3)-1alpha-hydroxylase double knockout (TRPV5(-/-)/1alpha-OHase(-/-)) mice, which show undetectable serum 1,25(OH)(2)D(3) levels, were generated. TRPV5(-/-)/1alpha-OHase(-/-) mice displayed a significant hypocalcemia compared with wild-type mice (1.10 +/- 0.02 and 2.54 +/- 0.01 mM, respectively; P < 0.05). mRNA levels of renal calbindin-D(28K) (7 +/- 2%), calbindin-D(9K) (32 +/- 4%), Na(+)/Ca(2+) exchanger (12 +/- 2%), and intestinal TRPV6 (40 +/- 8%) and calbindin-D(9K) (26 +/- 4%) expression levels were decreased compared with wild-type mice. Hyperparathyroidism and rickets were present in TRPV5(-/-)/1alpha-OHase(-/-) mice, more pronounced than observed in single TRPV5 or 1alpha-OHase knockout mice. It is interesting that a renal Ca(2+) leak, as demonstrated in TRPV5(-/-) mice, persisted in TRPV5(-/-)/1alpha-OHase(-/-) mice, but a compensatory upregulation of intestinal Ca(2+) transporters was abolished. In conclusion, the elevation of serum 1,25(OH)(2)D(3) levels in TRPV5(-/-) mice is responsible for the upregulation of intestinal Ca(2+) transporters and Ca(2+) hyperabsorption. Hypervitaminosis D, therefore, is of crucial importance to maintain normocalcemia in impaired Ca(2+) reabsorption in TRPV5(-/-) mice.

A novel CBL-Bflox/flox mouse model allows tissue-selective fully conditional CBL/CBL-B double-knockout: CD4-Cre mediated CBL/CBL-B deletion occurs in both T-cells and hematopoietic stem cells

PubMed Central

Goetz, Benjamin; An, Wei; Mohapatra, Bhopal; Zutshi, Neha; Iseka, Fany; Storck, Matthew D.; Meza, Jane; Sheinin, Yuri; Band, Vimla; Band, Hamid

2016-01-01

CBL-family ubiquitin ligases are critical negative regulators of tyrosine kinase signaling, with a clear redundancy between CBL and CBL-B evident in the immune cell and hematopoietic stem cell studies. Since CBL and CBL-B are negative regulators of immune cell activation, elimination of their function to boost immune cell activities could be beneficial in tumor immunotherapy. However, mutations of CBL are associated with human leukemias, pointing to tumor suppressor roles of CBL proteins; hence, it is critical to assess the tumor-intrinsic roles of CBL and CBL-B in cancers. This has not been possible since the only available whole-body CBL-B knockout mice exhibit constitutive tumor rejection. We engineered a new CBL-Bflox/flox mouse, combined this with an existing CBLflox/flox mouse to generate CBLflox/flox; CBL-Bflox/flox mice, and tested the tissue-specific concurrent deletion of CBL and CBL-B using the widely-used CD4-Cre transgenic allele to produce a T-cell-specific double knockout. Altered T-cell development, constitutive peripheral T-cell activation, and a lethal multi-organ immune infiltration phenotype largely resembling the previous Lck-Cre driven floxed-CBL deletion on a CBL-B knockout background establish the usefulness of the new model for tissue-specific CBL/CBL-B deletion. Unexpectedly, CD4-Cre-induced deletion in a small fraction of hematopoietic stem cells led to expansion of certain non-T-cell lineages, suggesting caution in the use of CD4-Cre for T-cell-restricted gene deletion. The establishment of a new model of concurrent tissue-selective CBL/CBL-B deletion should allow a clear assessment of the tumor-intrinsic roles of CBL/CBL-B in non-myeloid malignancies and help test the potential for CBL/CBL-B inactivation in immunotherapy of tumors. PMID:27276677

Random phenotypic variation of yeast (Saccharomyces cerevisiae) single-gene knockouts fits a double pareto-lognormal distribution.

PubMed

Graham, John H; Robb, Daniel T; Poe, Amy R

2012-01-01

Distributed robustness is thought to influence the buffering of random phenotypic variation through the scale-free topology of gene regulatory, metabolic, and protein-protein interaction networks. If this hypothesis is true, then the phenotypic response to the perturbation of particular nodes in such a network should be proportional to the number of links those nodes make with neighboring nodes. This suggests a probability distribution approximating an inverse power-law of random phenotypic variation. Zero phenotypic variation, however, is impossible, because random molecular and cellular processes are essential to normal development. Consequently, a more realistic distribution should have a y-intercept close to zero in the lower tail, a mode greater than zero, and a long (fat) upper tail. The double Pareto-lognormal (DPLN) distribution is an ideal candidate distribution. It consists of a mixture of a lognormal body and upper and lower power-law tails. If our assumptions are true, the DPLN distribution should provide a better fit to random phenotypic variation in a large series of single-gene knockout lines than other skewed or symmetrical distributions. We fit a large published data set of single-gene knockout lines in Saccharomyces cerevisiae to seven different probability distributions: DPLN, right Pareto-lognormal (RPLN), left Pareto-lognormal (LPLN), normal, lognormal, exponential, and Pareto. The best model was judged by the Akaike Information Criterion (AIC). Phenotypic variation among gene knockouts in S. cerevisiae fits a double Pareto-lognormal (DPLN) distribution better than any of the alternative distributions, including the right Pareto-lognormal and lognormal distributions. A DPLN distribution is consistent with the hypothesis that developmental stability is mediated, in part, by distributed robustness, the resilience of gene regulatory, metabolic, and protein-protein interaction networks. Alternatively, multiplicative cell growth, and the mixing of lognormal distributions having different variances, may generate a DPLN distribution.

Distinct roles of the DmNav and DSC1 channels in the action of DDT and pyrethroids.

PubMed

Rinkevich, Frank D; Du, Yuzhe; Tolinski, Josh; Ueda, Atsushi; Wu, Chun-Fang; Zhorov, Boris S; Dong, Ke

2015-03-01

Voltage-gated sodium channels (Nav channels) are critical for electrical signaling in the nervous system and are the primary targets of the insecticides DDT and pyrethroids. In Drosophila melanogaster, besides the canonical Nav channel, Para (also called DmNav), there is a sodium channel-like cation channel called DSC1 (Drosophila sodium channel 1). Temperature-sensitive paralytic mutations in DmNav (para(ts)) confer resistance to DDT and pyrethroids, whereas DSC1 knockout flies exhibit enhanced sensitivity to pyrethroids. To further define the roles and interaction of DmNav and DSC1 channels in DDT and pyrethroid neurotoxicology, we generated a DmNav/DSC1 double mutant line by introducing a para(ts1) allele (carrying the I265N mutation) into a DSC1 knockout line. We confirmed that the I265N mutation reduced the sensitivity to two pyrethroids, permethrin and deltamethrin of a DmNav variant expressed in Xenopus oocytes. Computer modeling predicts that the I265N mutation confers pyrethroid resistance by allosterically altering the second pyrethroid receptor site on the DmNav channel. Furthermore, we found that I265N-mediated pyrethroid resistance in para(ts1) mutant flies was almost completely abolished in para(ts1);DSC1(-/-) double mutant flies. Unexpectedly, however, the DSC1 knockout flies were less sensitive to DDT, compared to the control flies (w(1118A)), and the para(ts1);DSC1(-/-) double mutant flies were even more resistant to DDT compared to the DSC1 knockout or para(ts1) mutant. Our findings revealed distinct roles of the DmNav and DSC1 channels in the neurotoxicology of DDT vs. pyrethroids and implicate the exciting possibility of using DSC1 channel blockers or modifiers in the management of pyrethroid resistance. Copyright © 2015 Elsevier Inc. All rights reserved.

Independent effects of apolipoprotein AV and apolipoprotein CIII on plasma triglyceride concentrations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baroukh, Nadine N.; Bauge, Eric; Akiyama, Jennifer

2003-08-15

Both the apolipoprotein A5 and C3 genes have repeatedly been shown to play an important role in determining plasma triglyceride concentrations in humans and mice. In mice, transgenic and knockout experiments indicate that plasma triglyceride levels are negatively and positively correlated with APOA5 and APOC3 expression, respectively. In humans, common polymorphisms in both genes have also been associated with plasma triglyceride concentrations. The evolutionary relationship among these two apolipoprotein genes and their close proximity on human chromosome 11q23 have largely precluded the determination of their relative contribution to altered Both the apolipoprotein A5 and C3 genes have repeatedly been shownmore » to play an important role in determining plasma triglyceride concentrations in humans and mice. In mice, transgenic and knockout experiments indicate that plasma triglyceride levels are negatively and positively correlated with APOA5 and APOC3 expression, respectively. In humans, common polymorphisms in both genes have also been associated with plasma triglyceride concentrations. The evolutionary relationship among these two apolipoprotein genes and their close proximity on human chromosome 11q23 have largely precluded the determination of their relative contribution to altered triglycerides. To overcome these confounding factors and address their relationship, we generated independent lines of mice that either over-expressed (''double transgenic'') or completely lacked (''double knockout'') both apolipoprotein genes. We report that both ''double transgenic'' and ''double knockout'' mice display intermedia tetriglyceride concentrations compared to over-expression or deletion of either gene alone. Furthermore, we find that human ApoAV plasma protein levels in the ''double transgenic'' mice are approximately 500-fold lower than human ApoCIII levels, supporting ApoAV is a potent triglyceride modulator despite its low concentration. Together, these data indicate that APOA5 and APOC3 independently influence plasma triglyceride concentrations but in an opposing manner.« less

PrP{sup C} displays an essential protective role from oxidative stress in an astrocyte cell line derived from PrP{sup C} knockout mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bertuchi, Fernanda R.; Bourgeon, Dominique M.G.; Landemberger, Michele C.

2012-02-03

Highlights: Black-Right-Pointing-Pointer PrP{sup C} in solution acts as a radical scavenger. Black-Right-Pointing-Pointer PrP{sup C} reduces hydrogen peroxide toxicity in astrocytes. Black-Right-Pointing-Pointer Increase in ROS disrupted the cell cycle in the PrP{sup C}-knockout astrocytes. Black-Right-Pointing-Pointer PrP{sup C} prevents the cell death independently of an SOD-like activity. -- Abstract: The PrP{sup C} protein, which is especially present in the cellular membrane of nervous system cells, has been extensively studied for its controversial antioxidant activity. In this study, we elucidated the free radical scavenger activity of purified murine PrP{sup C} in solution and its participation as a cell protector in astrocytes that weremore » subjected to treatment with an oxidant. In vitro and using an EPR spin-trapping technique, we observed that PrP{sup C} decreased the oxidation of the DMPO trap in a Fenton reaction system (Cu{sup 2+}/ascorbate/H{sub 2}O{sub 2}), which was demonstrated by approximately 70% less DMPO/OH{sup {center_dot}}. In cultured PrP{sup C}-knockout astrocytes from mice, the absence of PrP{sup C} caused an increase in intracellular ROS (reactive oxygen species) generation during the first 3 h of H{sub 2}O{sub 2} treatment. This rapid increase in ROS disrupted the cell cycle in the PrP{sup C}-knockout astrocytes, which increased the population of cells in the sub-G1 phase when compared with cultured wild-type astrocytes. We conclude that PrP{sup C} in solution acts as a radical scavenger, and in astrocytes, it is essential for protection from oxidative stress caused by an external chemical agent, which is a likely condition in human neurodegenerative CNS disorders and pathological conditions such as ischemia.« less

New insight into the role of the β3 subunit of the GABAA-R in development, behavior, body weight regulation, and anesthesia revealed by conditional gene knockout

PubMed Central

Ferguson, Carolyn; Hardy, Steven L; Werner, David F; Hileman, Stanley M; DeLorey, Timothy M; Homanics, Gregg E

2007-01-01

Background The β3 subunit of the γ-aminobutyric acid type A receptor (GABAA-R) has been reported to be important for palate formation, anesthetic action, and normal nervous system function. This subunit has also been implicated in the pathogenesis of Angelman syndrome and autism spectrum disorder. To further investigate involvement of this subunit, we previously produced mice with a global knockout of β3. However, developmental abnormalities, compensation, reduced viability, and numerous behavioral abnormalities limited the usefulness of that murine model. To overcome many of these limitations, a mouse line with a conditionally inactivated β3 gene was engineered. Results Gene targeting and embryonic stem cell technologies were used to create mice in which exon 3 of the β3 subunit was flanked by loxP sites (i.e., floxed). Crossing the floxed β3 mice to a cre general deleter mouse line reproduced the phenotype of the previously described global knockout. Pan-neuronal knockout of β3 was achieved by crossing floxed β3 mice to Synapsin I-cre transgenic mice. Palate development was normal in pan-neuronal β3 knockouts but ~61% died as neonates. Survivors were overtly normal, fertile, and were less sensitive to etomidate. Forebrain selective knockout of β3 was achieved using α CamKII-cre transgenic mice. Palate development was normal in forebrain selective β3 knockout mice. These knockouts survived the neonatal period, but ~30% died between 15–25 days of age. Survivors had reduced reproductive fitness, reduced sensitivity to etomidate, were hyperactive, and some became obese. Conclusion Conditional inactivation of the β3 gene revealed novel insight into the function of this GABAA-R subunit. The floxed β3 knockout mice described here will be very useful for conditional knockout studies to further investigate the role of the β3 subunit in development, ethanol and anesthetic action, normal physiology, and pathophysiologic processes. PMID:17927825

LncRNA uc.48+ is involved in diabetic neuropathic pain mediated by the P2X3 receptor in the dorsal root ganglia.

PubMed

Wang, Shouyu; Xu, Hong; Zou, Lifang; Xie, Jinyang; Wu, Hong; Wu, Bing; Yi, Zhihua; Lv, Qiulan; Zhang, Xi; Ying, Mofeng; Liu, Shuangmei; Li, Guilin; Gao, Yun; Xu, Changshui; Zhang, Chunping; Xue, Yun; Liang, Shangdong

2016-03-01

Some long non-coding RNAs (lncRNAs) participate in physiological processes that maintain cellular and tissue homeostasis, and thus, the dysregulated expression of lncRNAs is involved in the onset and progression of many pathological conditions. Research has indicated that the genetic knockout of some lncRNAs in mice resulted in peri- or postnatal lethality or developmental defects. Diabetes mellitus (DM) is a major cause of peripheral neuropathy. Our studies showed that the expression levels of lncRNA uc.48+ in the diabetic rat dorsal root ganglia (DRG) and the DM patients' serum samples were increased. It suggested that lncRNA uc.48+ was involved in the pathophysiological process of DM. The aim of this study was to investigate the effects of lncRNA uc.48+ small interfering RNA (siRNA) on diabetic neuropathic pain (DNP) mediated by the P2X3 receptor in the DRG. The values of the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured by the von Frey test and Hargreaves' test, respectively. The levels of P2X3 protein and messenger RNA (mRNA) in the DRG were detected by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and western blotting. The experiments showed that the MWT and TWL values in DM rats were lower than those in the control rats. The MWT and TWL values in DM rats treated with lncRNA uc.48+ siRNA were increased compared to those in DM rats, but there was no significant difference between the DM rat group and the DM + scramble siRNA group. The levels of P2X3 protein and mRNA in the DM DRG were higher than those in the control, while the levels of P2X3 protein and mRNA in the DG of DM rats treated with uc.48+ siRNA were significantly decreased compared to those in DM rats. The expression levels of TNF-α in the DRG of DM rats treated with uc.48+ siRNA were significantly decreased compared to those in the DM group. The phosphorylation and activation of ERK1/2 in the DM DRG were decreased by uc.48+ siRNA treatment. Therefore, uc.48+ siRNA treatment may alleviate the DNP by inhibiting the excitatory transmission mediated by the P2X3 receptor in DRG.

High-resolution imaging of selenium in kidneys: a localized selenium pool associated with glutathione peroxidase 3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Malinouski, M.; Kehr, S.; Finney, L.

2012-04-17

Recent advances in quantitative methods and sensitive imaging techniques of trace elements provide opportunities to uncover and explain their biological roles. In particular, the distribution of selenium in tissues and cells under both physiological and pathological conditions remains unknown. In this work, we applied high-resolution synchrotron X-ray fluorescence microscopy (XFM) to map selenium distribution in mouse liver and kidney. Liver showed a uniform selenium distribution that was dependent on selenocysteine tRNA{sup [Ser]Sec} and dietary selenium. In contrast, kidney selenium had both uniformly distributed and highly localized components, the latter visualized as thin circular structures surrounding proximal tubules. Other parts ofmore » the kidney, such as glomeruli and distal tubules, only manifested the uniformly distributed selenium pattern that co-localized with sulfur. We found that proximal tubule selenium localized to the basement membrane. It was preserved in Selenoprotein P knockout mice, but was completely eliminated in glutathione peroxidase 3 (GPx3) knockout mice, indicating that this selenium represented GPx3. We further imaged kidneys of another model organism, the naked mole rat, which showed a diminished uniformly distributed selenium pool, but preserved the circular proximal tubule signal. We applied XFM to image selenium in mammalian tissues and identified a highly localized pool of this trace element at the basement membrane of kidneys that was associated with GPx3. XFM allowed us to define and explain the tissue topography of selenium in mammalian kidneys at submicron resolution.« less

Effects of endogenous nitric oxide and of DETA NONOate in arteriogenesis.

PubMed

Troidl, Kerstin; Tribulova, Silvia; Cai, Wei-Jun; Rüding, Inka; Apfelbeck, Hanna; Schierling, Wilma; Troidl, Christian; Schmitz-Rixen, Thomas; Schaper, Wolfgang

2010-02-01

Previous studies showed that targeted endothelial nitric oxide synthase (eNOS) disruption in mice with femoral artery occlusion does not impede and transgenic eNOS overexpression does not stimulate collateral artery growth after femoral artery occlusion, suggesting that nitric oxide from eNOS does not play a role in arteriogenesis. However, pharmacologic nitric oxide synthase inhibition with L-NAME markedly blocks arteriogenesis, suggestive of an important role of nitric oxide. To solve the paradox, we studied targeted deletion of eNOS and of inducible nitric oxide synthase (iNOS) in mice and found that only iNOS knockout could partially inhibit arteriogenesis. However, the combination of eNOS knockout and treatment with the iNOS inhibitor L-NIL completely abolished arteriogenesis. mRNA transcription studies (reverse transcriptase-polymerase chain reaction) performed on collateral arteries of rats showed that eNOS and especially iNOS (but not neural nitric oxide synthase) become upregulated in shear stress-stimulated collateral vessels, which supports the hypothesis that nitric oxide is necessary for arteriogenesis but that iNOS plays an important part. This was strengthened by the observation that the nitric oxide donor DETA NONOate strongly stimulated collateral artery growth, activated perivascular monocytes, and increased proliferation markers. Shear stress-induced nitric oxide may activate the innate immune system and activate iNOS. In conclusion, arteriogenesis is completely dependent on the presence of nitric oxide, a large part of it coming from mononuclear cells.

Liquiritigenin and liquiritin alleviated MCT-induced HSOS by activating Nrf2 antioxidative defense system.

PubMed

Huang, Zhenlin; Sheng, Yuchen; Chen, Minwei; Hao, Zhanxia; Hu, Feifei; Ji, Lili

2018-06-14

Hepatic sinusoidal obstruction syndrome (HSOS) is a serious and life-threatening liver disease. Liquiritigenin (LG) and liquiritin (LQ) are natural flavonoids distributed in Glycyrrhizae Radix et Rhizoma (Gan-cao). This study aims to investigate the protective effect and mechanism of LG and LQ against monocrotaline (MCT)-induced HSOS. Results of serum alanine/aspartate aminotransferases (ALT/AST) activities, liver histological evaluation and scanning electron microscope observation, and hepatic metalloproteinase-9 (MMP-9) expression demonstrated that LG and LQ both alleviated HSOS induced by MCT in rats. Results of hepatic reactive oxygen species (ROS), malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), oxidized glutathione (GSSG) and reduced glutathione (GSH) contents, glutathione reductase (GR) and superoxide dismutase (SOD) activities showed that LG and LQ attenuated MCT-induced liver oxidative stress injury. Furthermore, LG and LQ were found to promote Nrf2 nuclear translocation and lead to the increased expression of Nrf2 downstream antioxidative genes. Molecule docking analysis indicated the potential interaction of LG and LQ with Nrf2 binding site in the kelch-like ECH-associated protein-1 (Keap1) protein. Finally, Nrf2 knock-out mice were used. The results showed that LG and LQ both alleviated MCT-induced HSOS in wild-type mice, but such protection was totally diminished in Nrf2 knock-out mice. In conclusion, our study revealed that LG and LQ alleviated MCT-induced HSOS by inducing the activation of hepatic Nrf2 antioxidative defense system. Copyright © 2018. Published by Elsevier Inc.

The Role of Dopamine Receptors in the Neurobehavioral Syndrome Provoked by Activation of L-Type Calcium Channels in Rodents

PubMed Central

Kasim, Suhail; Blake, Bonita L.; Fan, Xueliang; Chartoff, Elena; Egami, Kiyoshi; Breese, George R.; Hess, Ellen J.; Jinnah, H.A.

2010-01-01

In rodents, activation of L-type calcium channels with ± BayK 8644 causes an unusual behavioral syndrome that includes dystonia and self-biting. Prior studies have linked both of these behaviors to dysfunction of dopaminergic transmission in the striatum. The current studies were designed to further elucidate the relationship between ± BayK 8644 and dopaminergic transmission in the expression of the behavioral syndrome. The drug does not appear to release presynaptic dopamine stores, since microdialysis of the striatum revealed dopamine release was unaltered by ± BayK 8644. In addition, the behaviors were preserved or even exaggerated in mice or rats with virtually complete dopamine depletion. On the other hand, pretreatment of mice with D3 or D1/5 dopamine receptor antagonists attenuated the behavioral effects of ± BayK 8644, while pretreatment with D2 or D4 antagonists had no effect. In D3 receptor knockout mice, ± BayK 8644 elicited both dystonia and self-biting, but these behaviors were less severe than in matched controls. In D1 receptor knockout mice, behavioral responses to ± BayK 8644 appeared exaggerated. These results argue that the behavioral effects of ± BayK 8644 are not mediated by a presynaptic influence. Instead, the behaviors appear to result from a postsynaptic activation of the drug, which does not require but can be modified by D3 or D1/5 receptors. PMID:17028428

Cholecystokinin Plays a Novel Protective Role in Diabetic Kidney Through Anti-inflammatory Actions on Macrophage

PubMed Central

Miyamoto, Satoshi; Shikata, Kenichi; Miyasaka, Kyoko; Okada, Shinichi; Sasaki, Motofumi; Kodera, Ryo; Hirota, Daisho; Kajitani, Nobuo; Takatsuka, Tetsuharu; Kataoka, Hitomi Usui; Nishishita, Shingo; Sato, Chikage; Funakoshi, Akihiro; Nishimori, Hisakazu; Uchida, Haruhito Adam; Ogawa, Daisuke; Makino, Hirofumi

2012-01-01

Inflammatory process is involved in the pathogenesis of diabetic nephropathy. In this article, we show that cholecystokinin (CCK) is expressed in the kidney and exerts renoprotective effects through its anti-inflammatory actions. DNA microarray showed that CCK was upregulated in the kidney of diabetic wild-type (WT) mice but not in diabetic intracellular adhesion molecule-1 knockout mice. We induced diabetes in CCK-1 receptor (CCK-1R) and CCK-2R double-knockout (CCK-1R−/−,-2R−/−) mice, and furthermore, we performed a bone marrow transplantation study using CCK-1R−/− mice to determine the role of CCK-1R on macrophages in the diabetic kidney. Diabetic CCK-1R−/−,-2R−/− mice revealed enhanced albuminuria and inflammation in the kidney compared with diabetic WT mice. In addition, diabetic WT mice with CCK-1R−/− bone marrow–derived cells developed more albuminuria than diabetic CCK-1R−/− mice with WT bone marrow–derived cells. Administration of sulfated cholecystokinin octapeptide (CCK-8S) ameliorated albuminuria, podocyte loss, expression of proinflammatory genes, and infiltration of macrophages in the kidneys of diabetic rats. Furthermore, CCK-8S inhibited both expression of tumor necrosis factor-α and chemotaxis in cultured THP-1 cells. These results suggest that CCK suppresses the activation of macrophage and expression of proinflammatory genes in diabetic kidney. Our findings may provide a novel strategy of therapy for the early stage of diabetic nephropathy. PMID:22357963

Lactobacillus acidophilus ATCC 4356 prevents atherosclerosis via inhibition of intestinal cholesterol absorption in apolipoprotein E-knockout mice.

PubMed

Huang, Ying; Wang, Jinfeng; Quan, Guihua; Wang, Xiaojun; Yang, Longfei; Zhong, Lili

2014-12-01

The objective of this study was to investigate the effect of Lactobacillus acidophilus ATCC 4356 on the development of atherosclerosis in apolipoprotein E-knockout (ApoE(-/-)) mice. Eight-week-old ApoE(-/-) mice were fed a Western diet with or without L. acidophilus ATCC 4356 daily for 16 weeks. L. acidophilus ATCC 4356 protected ApoE(-/-) mice from atherosclerosis by reducing their plasma cholesterol levels from 923 ± 44 to 581 ± 18 mg/dl, likely via a marked decrease in cholesterol absorption caused by modulation of Niemann-Pick C1-like 1 (NPC1L1). In addition, suppression of cholesterol absorption induced reverse cholesterol transport (RCT) in macrophages through the peroxisome proliferator-activated receptor/liver X receptor (PPAR/LXR) pathway. Fecal lactobacillus and bifidobacterium counts were significantly (P < 0.05) higher in the L. acidophilus ATCC 4356 treatment groups than in the control groups. Furthermore, L. acidophilus ATCC 4356 was detected in the rat small intestine, colon, and feces during the feeding trial. The bacterial levels remained high even after the administration of lactic acid bacteria had been stopped for 2 weeks. These results suggest that administration of L. acidophilus ATCC 4356 can protect against atherosclerosis through the inhibition of intestinal cholesterol absorption. Therefore, L. acidophilus ATCC 4356 may be a potential therapeutic material for preventing the progression of atherosclerosis. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Sensorimotor development in neonatal progesterone receptor knockout mice.

PubMed

Willing, Jari; Wagner, Christine K

2014-01-01

Early exposure to steroid hormones can permanently and dramatically alter neural development. This is best understood in the organizational effects of hormones during development of brain regions involved in reproductive behaviors or neuroendocrine function. However, recent evidence strongly suggests that steroid hormones play a vital role in shaping brain regions involved in cognitive behavior such as the cerebral cortex. The most abundantly expressed steroid hormone receptor in the developing rodent cortex is the progesterone receptor (PR). In the rat, PR is initially expressed in the developmentally-critical subplate at E18, and subsequently in laminas V and II/III through the first three postnatal weeks (Quadros et al. [2007] J Comp Neurol 504:42-56; Lopez & Wagner [2009]: J Comp Neurol 512:124-139), coinciding with significant periods of dendritic maturation, the arrival of afferents and synaptogenesis. In the present study, we investigated PR expression in the neonatal mouse somatosensory cortex. Additionally, to investigate the potential role of PR in developing cortex, we examined sensorimotor function in the first two postnatal weeks in PR knockout mice and their wildtype (WT) and heterozygous (HZ) counterparts. While the three genotypes were similar in most regards, PRKO and HZ mice lost the rooting reflex 2-3 days earlier than WT mice. These studies represent the first developmental behavioral assessment of PRKO mice and suggest PR expression may play an important role in the maturation of cortical connectivity and sensorimotor integration. Copyright © 2013 Wiley Periodicals, Inc.

Structural and Functional Analysis of HIV-1 Coreceptors: Roles of Charged Residues and Posttranslational Modifications on Coreceptor Activity

DTIC Science & Technology

2000-01-01

to sites of inflammation. They may have additional functions. For example analysis of CXCR4 knockout mice show that CXCR4, which is chemotactic for... mice had similar phenotypes (195). Homozygous knockout of CXCR4 or SDF-1 results in embyonic lethality. Though CCR5 appears to be dispensable, other...chemokine receptors have vital functions. CXCR5 knockout mice have B-cell homing defects (118), and CXCR2 knockout mice overproduce B-cells and

[Preliminary exploration on knockout drops (Meng Han Agents)].

PubMed

Zhang, Z

1996-05-01

This author points out, based on relevant materials, that knockout drops were vertigo powder. Due to homophonic reasons in Chinese language, the term "mingxuan" was transliterated into the former Chinese term (menghan). Knockout drops for medicinal use were merely made up of compound recipes containing stramonium flowers. The knockout drops in old fictions and opera books were powder of stramonium flower. The ingredients and application of such recipes are discussed here, the anti-remedies for such recipes are also mentioned.

Neisseria gonorrhoeae PIII has a role on NG1873 outer membrane localization and is involved in bacterial adhesion to human cervical and urethral epithelial cells.

PubMed

Leuzzi, Rosanna; Nesta, Barbara; Monaci, Elisabetta; Cartocci, Elena; Serino, Laura; Soriani, Marco; Rappuoli, Rino; Pizza, Mariagrazia

2013-11-09

Protein PIII is one of the major outer membrane proteins of Neisseria gonorrhoeae, 95% identical to RmpM (reduction modifiable protein M) or class 4 protein of Neisseria meningitidis. RmpM is known to be a membrane protein associated by non-covalent bonds to the peptidoglycan layer and interacting with PorA/PorB porin complexes resulting in the stabilization of the bacterial membrane. The C-terminal domain of PIII (and RmpM) is highly homologous to members of the OmpA family, known to have a role in adhesion/invasion in many bacterial species. The contribution of PIII in the membrane architecture and its role in the interaction with epithelial cells has never been investigated. We generated a ΔpIII knock-out mutant strain and evaluated the effects of the loss of PIII expression on bacterial morphology and on outer membrane composition. Deletion of the pIII gene does not cause any alteration in bacterial morphology or sensitivity to detergents. Moreover, the expression profile of the main membrane proteins remains the same for the wild-type and knock-out strains, with the exception of the NG1873 which is not exported to the outer membrane and accumulates in the inner membrane in the ΔpIII knock-out mutant strain.We also show that purified PIII protein is able to bind human cervical and urethral cells and that the ΔpIII knock-out mutant strain has a lower ability to adhere to human cervical and urethral cells. Here we demonstrated that the PIII protein does not play a key structural role in the membrane organization of gonococcus and does not induce major effects on the expression of the main outer membrane proteins. However, in the PIII knock-out strain, the NG1873 protein is not localized in the outer membrane as it is in the wild-type strain suggesting a possible interaction of PIII with NG1873. The evidence that PIII binds to human epithelial cells derived from the female and male genital tract highlights a possible role of PIII in the virulence of gonococcus and suggests that the structural homology to OmpA is conserved also at functional level.

Generation of a Tph2 Conditional Knockout Mouse Line for Time- and Tissue-Specific Depletion of Brain Serotonin

PubMed Central

Migliarini, Sara; Pacini, Giulia; Pasqualetti, Massimo

2015-01-01

Serotonin has been gaining increasing attention during the last two decades due to the dual function of this monoamine as key regulator during critical developmental events and as neurotransmitter. Importantly, unbalanced serotonergic levels during critical temporal phases might contribute to the onset of neuropsychiatric disorders, such as schizophrenia and autism. Despite increasing evidences from both animal models and human genetic studies have underpinned the importance of serotonin homeostasis maintenance during central nervous system development and adulthood, the precise role of this molecule in time-specific activities is only beginning to be elucidated. Serotonin synthesis is a 2-step process, the first step of which is mediated by the rate-limiting activity of Tph enzymes, belonging to the family of aromatic amino acid hydroxylases and existing in two isoforms, Tph1 and Tph2, responsible for the production of peripheral and brain serotonin, respectively. In the present study, we generated and validated a conditional knockout mouse line, Tph2 flox/flox, in which brain serotonin can be effectively ablated with time specificity. We demonstrated that the Cre-mediated excision of the third exon of Tph2 gene results in the production of a Tph2 null allele in which we observed the near-complete loss of brain serotonin, as well as the growth defects and perinatal lethality observed in serotonin conventional knockouts. We also revealed that in mice harbouring the Tph2 null allele, but not in wild-types, two distinct Tph2 mRNA isoforms are present, namely Tph2Δ3 and Tph2Δ3Δ4, with the latter showing an in-frame deletion of amino acids 84–178 and coding a protein that could potentially retain non-negligible enzymatic activity. As we could not detect Tph1 expression in the raphe, we made the hypothesis that the Tph2Δ3Δ4 isoform can be at the origin of the residual, sub-threshold amount of serotonin detected in the brain of Tph2 null/null mice. Finally, we set up a tamoxifen administration protocol that allows an efficient, time-specific inactivation of brain serotonin synthesis. On the whole, we generated a suitable genetic tool to investigate how serotonin depletion impacts on time-specific events during central nervous system development and adulthood life. PMID:26291320

Behavioral and neurochemical characterization of mice deficient in the phosphodiesterase-1B (PDE1B) enzyme.

PubMed

Siuciak, J A; McCarthy, S A; Chapin, D S; Reed, T M; Vorhees, C V; Repaske, D R

2007-07-01

PDE1B is a calcium-dependent cyclic nucleotide phosphodiesterase that is highly expressed in the striatum. In order to investigate the physiological role of PDE1B in the central nervous system, PDE1B knockout mice (C57BL/6N background) were assessed in behavioral tests and their brains were assayed for monoamine content. In a variety of well-characterized behavioral tasks, including the elevated plus maze (anxiety-like behavior), forced swim test (depression-like behavior), hot plate (nociception) and two cognition models (passive avoidance and acquisition of conditioned avoidance responding), PDE1B knockout mice performed similarly to wild-type mice. PDE1B knockout mice showed increased baseline exploratory activity when compared to wild-type mice. When challenged with amphetamine (AMPH) and methamphetamine (METH), male and female PDE1B knockout mice showed an exaggerated locomotor response. Male PDE1B knockout mice also showed increased locomotor responses to higher doses of phencyclidine (PCP) and MK-801; however, this effect was not consistently observed in female knockout mice. In the striatum, increased dopamine turnover (DOPAC/DA and HVA/DA ratios) was found in both male and female PDE1B knockout mice. Striatal serotonin (5-HT) levels were also decreased in PDE1B knockout mice, although levels of the metabolite, 5HIAA, were unchanged. The present studies demonstrate increased striatal dopamine turnover in PDE1B knockout mice associated with increased baseline motor activity and an exaggerated locomotor response to dopaminergic stimulants such as methamphetamine and amphetamine. These data further support a role for PDE1B in striatal function.

The influence of electromagnetic radiation generated by a mobile phone on the skeletal system of rats.

PubMed

Sieroń-Stołtny, Karolina; Teister, Łukasz; Cieślar, Grzegorz; Sieroń, Dominik; Śliwinski, Zbigniew; Kucharzewski, Marek; Sieroń, Aleksander

2015-01-01

The study was focused on the influence of electromagnetic field generated by mobile phone on the skeletal system of rats, assessed by measuring the macrometric parameters of bones, mechanical properties of long bones, calcium and phosphorus content in bones, and the concentration of osteogenesis (osteocalcin) and bone resorption (NTX, pyridinoline) markers in blood serum. The study was carried out on male rats divided into two groups: experimental group subjected to 28-day cycle of exposures in electromagnetic field of 900 MHz frequency generated by mobile phone and a control, sham-exposed one. The mobile phone-generated electromagnetic field did not influence the macrometric parameters of long bones and L4 vertebra, it altered mechanical properties of bones (stress and energy at maximum bending force, stress at fracture), it decreased the content of calcium in long bones and L4 vertebra, and it altered the concentration of osteogenesis and bone resorption markers in rats. On the basis of obtained results, it was concluded that electromagnetic field generated by 900 MHz mobile phone does not have a direct impact on macrometric parameters of bones; however, it alters the processes of bone mineralization and the intensity of bone turnover processes and thus influences the mechanical strength of bones.

The Influence of Electromagnetic Radiation Generated by a Mobile Phone on the Skeletal System of Rats

PubMed Central

Sieroń-Stołtny, Karolina; Teister, Łukasz; Cieślar, Grzegorz; Sieroń, Dominik; Śliwinski, Zbigniew; Sieroń, Aleksander

2015-01-01

The study was focused on the influence of electromagnetic field generated by mobile phone on the skeletal system of rats, assessed by measuring the macrometric parameters of bones, mechanical properties of long bones, calcium and phosphorus content in bones, and the concentration of osteogenesis (osteocalcin) and bone resorption (NTX, pyridinoline) markers in blood serum. The study was carried out on male rats divided into two groups: experimental group subjected to 28-day cycle of exposures in electromagnetic field of 900 MHz frequency generated by mobile phone and a control, sham-exposed one. The mobile phone-generated electromagnetic field did not influence the macrometric parameters of long bones and L4 vertebra, it altered mechanical properties of bones (stress and energy at maximum bending force, stress at fracture), it decreased the content of calcium in long bones and L4 vertebra, and it altered the concentration of osteogenesis and bone resorption markers in rats. On the basis of obtained results, it was concluded that electromagnetic field generated by 900 MHz mobile phone does not have a direct impact on macrometric parameters of bones; however, it alters the processes of bone mineralization and the intensity of bone turnover processes and thus influences the mechanical strength of bones. PMID:25705697

Swimming in Light: A Large-Scale Computational Analysis of the Metabolism of Dinoroseobacter shibae

PubMed Central

Rex, Rene; Bill, Nelli; Schmidt-Hohagen, Kerstin; Schomburg, Dietmar

2013-01-01

The Roseobacter clade is a ubiquitous group of marine α-proteobacteria. To gain insight into the versatile metabolism of this clade, we took a constraint-based approach and created a genome-scale metabolic model (iDsh827) of Dinoroseobacter shibae DFL12T. Our model is the first accounting for the energy demand of motility, the light-driven ATP generation and experimentally determined specific biomass composition. To cover a large variety of environmental conditions, as well as plasmid and single gene knock-out mutants, we simulated 391,560 different physiological states using flux balance analysis. We analyzed our results with regard to energy metabolism, validated them experimentally, and revealed a pronounced metabolic response to the availability of light. Furthermore, we introduced the energy demand of motility as an important parameter in genome-scale metabolic models. The results of our simulations also gave insight into the changing usage of the two degradation routes for dimethylsulfoniopropionate, an abundant compound in the ocean. A side product of dimethylsulfoniopropionate degradation is dimethyl sulfide, which seeds cloud formation and thus enhances the reflection of sunlight. By our exhaustive simulations, we were able to identify single-gene knock-out mutants, which show an increased production of dimethyl sulfide. In addition to the single-gene knock-out simulations we studied the effect of plasmid loss on the metabolism. Moreover, we explored the possible use of a functioning phosphofructokinase for D. shibae. PMID:24098096

Severely altered guanidino compound levels, disturbed body weight homeostasis and impaired fertility in a mouse model of guanidinoacetate N-methyltransferase (GAMT) deficiency.

PubMed

Schmidt, Andreas; Marescau, Bart; Boehm, Ernest A; Renema, W Klaas Jan; Peco, Ruben; Das, Anib; Steinfeld, Robert; Chan, Sharon; Wallis, Julie; Davidoff, Michail; Ullrich, Kurt; Waldschütz, Ralph; Heerschap, Arend; De Deyn, Peter P; Neubauer, Stefan; Isbrandt, Dirk

2004-05-01

We generated a knockout mouse model for guanidinoacetate N-methyltransferase (GAMT) deficiency (MIM 601240), the first discovered human creatine deficiency syndrome, by gene targeting in embryonic stem cells. Disruption of the open reading frame of the murine GAMT gene in the first exon resulted in the elimination of 210 of the 237 amino acids present in mGAMT. The creation of an mGAMT null allele was verified at the genetic, RNA and protein levels. GAMT knockout mice have markedly increased guanidinoacetate (GAA) and reduced creatine and creatinine levels in brain, serum and urine, which are key findings in human GAMT patients. In vivo (31)P magnetic resonance spectroscopy showed high levels of PGAA and reduced levels of creatine phosphate in heart, skeletal muscle and brain. These biochemical alterations were comparable to those found in human GAMT patients and can be attributed to the very similar GAMT expression patterns found by us in human and mouse tissues. We provide evidence that GAMT deficiency in mice causes biochemical adaptations in brain and skeletal muscle. It is associated with increased neonatal mortality, muscular hypotonia, decreased male fertility and a non-leptin-mediated life-long reduction in body weight due to reduced body fat mass. Therefore, GAMT knockout mice are a valuable creatine deficiency model for studying the effects of high-energy phosphate depletion in brain, heart, skeletal muscle and other organs.

Romk1 Knockout Mice Do Not Produce Bartter Phenotype but Exhibit Impaired K Excretion*

PubMed Central

Dong, Ke; Yan, Qingshang; Lu, Ming; Wan, Laxiang; Hu, Haiyan; Guo, Junhua; Boulpaep, Emile; Wang, WenHui; Giebisch, Gerhard; Hebert, Steven C.; Wang, Tong

2016-01-01

Romk knock-out mice show a similar phenotype to Bartter syndrome of salt wasting and dehydration due to reduced Na-K-2Cl-cotransporter activity. At least three ROMK isoforms have been identified in the kidney; however, unique functions of any of the isoforms in nephron segments are still poorly understood. We have generated a mouse deficient only in Romk1 by selective deletion of the Romk1-specific first exon using an ES cell Cre-LoxP strategy and examined the renal phenotypes, ion transporter expression, ROMK channel activity, and localization under normal and high K intake. Unlike Romk−/− mice, there was no Bartter phenotype with reduced NKCC2 activity and increased NCC expression in Romk1−/− mice. The small conductance K channel (SK) activity showed no difference of channel properties or gating in the collecting tubule between Romk1+/+ and Romk1−/− mice. High K intake increased SK channel number per patch and increased the ROMK channel intensity in the apical membrane of the collecting tubule in Romk1+/+, but such regulation by high K intake was diminished with significant hyperkalemia in Romk1−/− mice. We conclude that 1) animal knockouts of ROMK1 do not produce Bartter phenotype. 2) There is no functional linking of ROMK1 and NKCC2 in the TAL. 3) ROMK1 is critical in response to high K intake-stimulated K+ secretion in the collecting tubule. PMID:26728465

Romk1 Knockout Mice Do Not Produce Bartter Phenotype but Exhibit Impaired K Excretion.

PubMed

Dong, Ke; Yan, Qingshang; Lu, Ming; Wan, Laxiang; Hu, Haiyan; Guo, Junhua; Boulpaep, Emile; Wang, WenHui; Giebisch, Gerhard; Hebert, Steven C; Wang, Tong

2016-03-04

Romk knock-out mice show a similar phenotype to Bartter syndrome of salt wasting and dehydration due to reduced Na-K-2Cl-cotransporter activity. At least three ROMK isoforms have been identified in the kidney; however, unique functions of any of the isoforms in nephron segments are still poorly understood. We have generated a mouse deficient only in Romk1 by selective deletion of the Romk1-specific first exon using an ES cell Cre-LoxP strategy and examined the renal phenotypes, ion transporter expression, ROMK channel activity, and localization under normal and high K intake. Unlike Romk(-/-) mice, there was no Bartter phenotype with reduced NKCC2 activity and increased NCC expression in Romk1(-/-) mice. The small conductance K channel (SK) activity showed no difference of channel properties or gating in the collecting tubule between Romk1(+/+) and Romk1(-/-) mice. High K intake increased SK channel number per patch and increased the ROMK channel intensity in the apical membrane of the collecting tubule in Romk1(+/+), but such regulation by high K intake was diminished with significant hyperkalemia in Romk1(-/-) mice. We conclude that 1) animal knockouts of ROMK1 do not produce Bartter phenotype. 2) There is no functional linking of ROMK1 and NKCC2 in the TAL. 3) ROMK1 is critical in response to high K intake-stimulated K(+) secretion in the collecting tubule. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Dysregulation of Uterine Signaling Pathways in Progesterone Receptor-Cre Knockout of Dicer

PubMed Central

Andreu-Vieyra, Claudia V.; Kim, Tae Hoon; Jeong, Jae-Wook; Hodgson, Myles C.; Chen, Ruihong; Creighton, Chad J.; Lydon, John P.; Gunaratne, Preethi H.; DeMayo, Francesco J.; Matzuk, Martin M.

2012-01-01

Epithelial-stromal interactions in the uterus are required for normal uterine functions such as pregnancy, and multiple signaling pathways are essential for this process. Although Dicer and microRNA (miRNA) have been implicated in several reproductive processes, the specific roles of Dicer and miRNA in uterine development are not known. To address the roles of miRNA in the regulation of key uterine pathways, we generated a conditional knockout of Dicer in the postnatal uterine epithelium and stroma using progesterone receptor-Cre. These Dicer conditional knockout females are sterile with small uteri, which demonstrate significant defects, including absence of glandular epithelium and enhanced stromal apoptosis, beginning at approximately postnatal d 15, with coincident expression of Cre and deletion of Dicer. Specific miRNA (miR-181c, −200b, −101, let-7d) were down-regulated and corresponding predicted proapoptotic target genes (Bcl2l11, Aldh1a3) were up-regulated, reflecting the apoptotic phenomenon. Although these mice had normal serum hormone levels, critical uterine signaling pathways, including progesterone-responsive genes, Indian hedgehog signaling, and the Wnt/β-catenin canonical pathway, were dysregulated at the mRNA level. Importantly, uterine stromal cell proliferation in response to progesterone was absent, whereas uterine epithelial cell proliferation in response to estradiol was maintained in adult uteri. These data implicate Dicer and appropriate miRNA expression as essential players in the regulation of multiple uterine signaling pathways required for uterine development and appropriate function. PMID:22798293

Knockout silkworms reveal a dispensable role for juvenile hormones in holometabolous life cycle

PubMed Central

Daimon, Takaaki; Uchibori, Miwa; Nakao, Hajime; Sezutsu, Hideki; Shinoda, Tetsuro

2015-01-01

Insect juvenile hormones (JHs) prevent precocious metamorphosis and allow larvae to undergo multiple rounds of status quo molts. However, the roles of JHs during the embryonic and very early larval stages have not been fully understood. We generated and characterized knockout silkworms (Bombyx mori) with null mutations in JH biosynthesis or JH receptor genes using genome-editing tools. We found that embryonic growth and morphogenesis are largely independent of JHs in Bombyx and that, even in the absence of JHs or JH signaling, pupal characters are not formed in first- or second-instar larvae, and precocious metamorphosis is induced after the second instar at the earliest. We also show by mosaic analysis that a pupal specifier gene broad, which is dramatically up-regulated in the late stage of the last larval instar, is essential for pupal commitment in the epidermis. Importantly, the mRNA expression level of broad, which is thought to be repressed by JHs, remained at very low basal levels during the early larval instars of JH-deficient or JH signaling-deficient knockouts. Therefore, our study suggests that the long-accepted paradigm that JHs maintain the juvenile status throughout larval life should be revised because the larval status can be maintained by a JH-independent mechanism in very early larval instars. We propose that the lack of competence for metamorphosis during the early larval stages may result from the absence of an unidentified broad-inducing factor, i.e., a competence factor. PMID:26195792

Central carbon metabolism influences cellulase production in Bacillus licheniformis.

PubMed

Wang, J; Liu, S; Li, Y; Wang, H; Xiao, S; Li, C; Liu, B

2018-01-01

Bacillus licheniformis that can produce cellulase including endo glucanase and glucosidase is an important industrial microbe for cellulose degradation. The purpose of this research was to assess the effect of endo glucanase gene bglC and glucosidase gene bglH on the central metabolic flux in B. licheniformis. bglC and bglH were knocked out using homologous recombination method, respectively, and the corresponding knockout strains were obtained for 13 C metabolic flux analysis. A significant change was observed in metabolic fluxes after 13 C metabolic flux ratio analysis. In both of the knockout strains, the increased fluxes of the pentose phosphate pathway and malic enzyme reaction enabled an elevated supply of NADPH which provided enough reducing power for the in vivo synthesis reactions. The fluxes through tricarboxylic acid cycle and anaplerotic reactions increased fast in the two knockout strains, which meant more energy generated. The changed fluxes in central carbon metabolism provided a holistic view of the physiological status in B. licheniformis and possible targets for further strain engineering. Cellulase is very important in the field of agriculture and bioenergy because of its degrading effect on cellulosic biomass. This study presented the effect of central carbon metabolism on cellulase production in Bacillus licheniformis. The study also provided a holistic view of the physiological status in B. licheniformis. The shifted metabolism provided a quantitative evaluation of the biosynthesis of cellulase and a priority ranked target list for further strain engineering. © 2017 The Society for Applied Microbiology.

Cdk5 modulates cocaine reward, motivation, and striatal neuron excitability.

PubMed

Benavides, David R; Quinn, Jennifer J; Zhong, Ping; Hawasli, Ammar H; DiLeone, Ralph J; Kansy, Janice W; Olausson, Peter; Yan, Zhen; Taylor, Jane R; Bibb, James A

2007-11-21

Cyclin-dependent kinase 5 (Cdk5) regulates dopamine neurotransmission and has been suggested to serve as a homeostatic target of chronic psychostimulant exposure. To study the role of Cdk5 in the modulation of the cellular and behavioral effects of psychoactive drugs of abuse, we developed Cre/loxP conditional knock-out systems that allow temporal and spatial control of Cdk5 expression in the adult brain. Here, we report the generation of Cdk5 conditional knock-out (cKO) mice using the alphaCaMKII promoter-driven Cre transgenic line (CaMKII-Cre). In this model system, loss of Cdk5 in the adult forebrain increased the psychomotor-activating effects of cocaine. Additionally, these CaMKII-Cre Cdk5 cKO mice show enhanced incentive motivation for food as assessed by instrumental responding on a progressive ratio schedule of reinforcement. Behavioral changes were accompanied by increased excitability of medium spiny neurons in the nucleus accumbens (NAc) in Cdk5 cKO mice. To study NAc-specific effects of Cdk5, another model system was used in which recombinant adeno-associated viruses expressing Cre recombinase caused restricted loss of Cdk5 in NAc neurons. Targeted knock-out of Cdk5 in the NAc facilitated cocaine-induced locomotor sensitization and conditioned place preference for cocaine. These results suggest that Cdk5 acts as a negative regulator of neuronal excitability in the NAc and that Cdk5 may govern the behavioral effects of cocaine and motivation for reinforcement.