Metabolomics Approach Reveals Integrated Metabolic Network Associated with Serotonin Deficiency.

PubMed

Weng, Rui; Shen, Sensen; Tian, Yonglu; Burton, Casey; Xu, Xinyuan; Liu, Yi; Chang, Cuilan; Bai, Yu; Liu, Huwei

2015-07-08

Serotonin is an important neurotransmitter that broadly participates in various biological processes. While serotonin deficiency has been associated with multiple pathological conditions such as depression, schizophrenia, Alzheimer's disease and Parkinson's disease, the serotonin-dependent mechanisms remain poorly understood. This study therefore aimed to identify novel biomarkers and metabolic pathways perturbed by serotonin deficiency using metabolomics approach in order to gain new metabolic insights into the serotonin deficiency-related molecular mechanisms. Serotonin deficiency was achieved through pharmacological inhibition of tryptophan hydroxylase (Tph) using p-chlorophenylalanine (pCPA) or genetic knockout of the neuronal specific Tph2 isoform. This dual approach improved specificity for the serotonin deficiency-associated biomarkers while minimizing nonspecific effects of pCPA treatment or Tph2 knockout (Tph2-/-). Non-targeted metabolic profiling and a targeted pCPA dose-response study identified 21 biomarkers in the pCPA-treated mice while 17 metabolites in the Tph2-/- mice were found to be significantly altered compared with the control mice. These newly identified biomarkers were associated with amino acid, energy, purine, lipid and gut microflora metabolisms. Oxidative stress was also found to be significantly increased in the serotonin deficient mice. These new biomarkers and the overall metabolic pathways may provide new understanding for the serotonin deficiency-associated mechanisms under multiple pathological states.

Metabolomics Approach Reveals Integrated Metabolic Network Associated with Serotonin Deficiency

NASA Astrophysics Data System (ADS)

Weng, Rui; Shen, Sensen; Tian, Yonglu; Burton, Casey; Xu, Xinyuan; Liu, Yi; Chang, Cuilan; Bai, Yu; Liu, Huwei

2015-07-01

Serotonin is an important neurotransmitter that broadly participates in various biological processes. While serotonin deficiency has been associated with multiple pathological conditions such as depression, schizophrenia, Alzheimer’s disease and Parkinson’s disease, the serotonin-dependent mechanisms remain poorly understood. This study therefore aimed to identify novel biomarkers and metabolic pathways perturbed by serotonin deficiency using metabolomics approach in order to gain new metabolic insights into the serotonin deficiency-related molecular mechanisms. Serotonin deficiency was achieved through pharmacological inhibition of tryptophan hydroxylase (Tph) using p-chlorophenylalanine (pCPA) or genetic knockout of the neuronal specific Tph2 isoform. This dual approach improved specificity for the serotonin deficiency-associated biomarkers while minimizing nonspecific effects of pCPA treatment or Tph2 knockout (Tph2-/-). Non-targeted metabolic profiling and a targeted pCPA dose-response study identified 21 biomarkers in the pCPA-treated mice while 17 metabolites in the Tph2-/- mice were found to be significantly altered compared with the control mice. These newly identified biomarkers were associated with amino acid, energy, purine, lipid and gut microflora metabolisms. Oxidative stress was also found to be significantly increased in the serotonin deficient mice. These new biomarkers and the overall metabolic pathways may provide new understanding for the serotonin deficiency-associated mechanisms under multiple pathological states.

Study of the role of anaerobic metabolism in succinate production by Enterobacter aerogenes.

PubMed

Tajima, Yoshinori; Kaida, Kenichi; Hayakawa, Atsushi; Fukui, Keita; Nishio, Yousuke; Hashiguchi, Kenichi; Fudou, Ryosuke; Matsui, Kazuhiko; Usuda, Yoshihiro; Sode, Koji

2014-09-01

Succinate is a core biochemical building block; optimizing succinate production from biomass by microbial fermentation is a focus of basic and applied biotechnology research. Lowering pH in anaerobic succinate fermentation culture is a cost-effective and environmentally friendly approach to reducing the use of sub-raw materials such as alkali, which are needed for neutralization. To evaluate the potential of bacteria-based succinate fermentation under weak acidic (pH <6.2) and anaerobic conditions, we characterized the anaerobic metabolism of Enterobacter aerogenes AJ110637, which rapidly assimilates glucose at pH 5.0. Based on the profile of anaerobic products, we constructed single-gene knockout mutants to eliminate the main anaerobic metabolic pathways involved in NADH re-oxidation. These single-gene knockout studies showed that the ethanol synthesis pathway serves as the dominant NADH re-oxidation pathway in this organism. To generate a metabolically engineered strain for succinate production, we eliminated ethanol formation and introduced a heterogeneous carboxylation enzyme, yielding E. aerogenes strain ΔadhE/PCK. The strain produced succinate from glucose with a 60.5% yield (grams of succinate produced per gram of glucose consumed) at pH <6.2 and anaerobic conditions. Thus, we showed the potential of bacteria-based succinate fermentation under weak acidic conditions.

ROLE OF ESTROGEN RECEPTOR-α ON FOOD DEMAND ELASTICITY

PubMed Central

Minervini, Vanessa; Rowland, Neil E.; Robertson, Kimberly L.; Foster, Thomas C.

2016-01-01

Estrogens have been shown to have an inhibitory effect on food intake under free-feeding conditions, yet the effects of estrogens on food-maintained operant responding have been studied to a much lesser extent and, thus, are not well understood. Therefore, the purpose of the present experiment was to use a behavioral economics paradigm to assess differences in demand elasticity between mice with knockout of the estrogen receptor subtype α, knockout of subtype β, and their wild type controls. The mice responded in a closed economy, and the price of food was increased by increasing the fixed-ratio response requirement every four sessions. Overall, we found that mice with the knockout of receptor subtype α had the most elastic demand functions. Therefore, under these conditions, estrogens increased food seeking via activation of the receptor subtype α. The results were inconsistent with those reported by previous studies that employed free-feeding conditions. PMID:25869426

Impaired eye-blink conditioning in waggler, a mutant mouse with cerebellar BDNF deficiency.

PubMed

Bao, S; Chen, L; Qiao, X; Knusel, B; Thompson, R F

1998-01-01

In addition to their trophic functions, neurotrophins are also implicated in synaptic modulation and learning and memory. Although gene knockout techniques have been used widely in studying the roles of neurotrophins at molecular and cellular levels, behavioral studies using neurotrophin knockouts are limited by the early-onset lethality and various sensory deficits associated with the gene knockout mice. In the present study, we found that in a spontaneous mutant mouse, waggler, the expression of brain-derived neurotrophic factor (BDNF) was selectively absent in the cerebellar granule cells. The cytoarchitecture of the waggler cerebellum appeared to be normal at the light microscope level. The mutant mice exhibited no sensory deficits to auditory stimuli or heat-induced pain. However, they were massively impaired in classic eye-blink conditioning. These results suggest that BDNF may have a role in normal cerebellar neuronal function, which, in turn, is essential for classic eye-blink conditioning.

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.

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.

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.

Mild deficits in mice lacking pituitary adenylate cyclase-activating polypeptide receptor type 1 (PAC1) performing on memory tasks.

PubMed

Sauvage, M; Brabet, P; Holsboer, F; Bockaert, J; Steckler, T

2000-12-08

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor subtype 1 (PAC1) have been suggested to play a role in the modulation of learning and memory. However, behavioral evidence for altered mnemonic function due to altered PAC1 activity is missing. Therefore, the role of PAC1 in learning and memory was studied in mouse mutants lacking this receptor (PAC1 knock-out mice), tested in water maze two-choice spatial discrimination, one-trial contextual and cued fear conditioning, and multiple-session contextual discrimination. Water maze spatial discrimination was unaffected in PAC1 mutants, while a mild deficit was observed in multiple session contextual discrimination in PAC1 knock-out mice. Furthermore, PAC1 knock-out mice were able to learn the association between context and shock in one-trial contextual conditioning, but showed faster return to baseline than wild-type mice. Thus, the effects of PAC1 knock-out on modulating performance in these tasks were subtle and suggest that PAC1 only plays a limited role in learning and memory.

Conditional transgenic mouse models: from the basics to genome-wide sets of knockouts and current studies of tissue regeneration.

PubMed

Bockamp, Ernesto; Sprengel, Rolf; Eshkind, Leonid; Lehmann, Thomas; Braun, Jan M; Emmrich, Frank; Hengstler, Jan G

2008-03-01

Many mouse models are currently available, providing avenues to elucidate gene function and to recapitulate specific pathological conditions. To a large extent, successful translation of clinical evidence or analytical data into appropriate mouse models is possible through progress in transgenic or gene-targeting technology. Beginning with a review of standard mouse transgenics and conventional gene targeting, this article will move on to discussing the basics of conditional gene expression: the tetracycline (tet)-off and tet-on systems based on the transactivators tet-controlled transactivator (Tta) and reverse tet-on transactivator (rtTA) that allow downregulation or induction of gene expression; Cre or Flp recombinase-mediated modifications, including excision, inversion, insertion and interchromosomal translocation; combination of the tet and Cre systems, permitting inducible knockout, reporter gene activation or activation of point mutations; the avian retroviral system based on delivery of rtTA specifically into cells expressing the avian retroviral receptor, which enables cell type-specific, inducible gene expression; the tamoxifen system, one of the most frequently applied steroid receptor-based systems, allows rapid activation of a fusion protein between the gene of interest and a mutant domain of the estrogen receptor, whereby activation does not depend on transcription; and techniques for cell type-specific ablation. The diphtheria toxin receptor system offers the advantage that it can be combined with the 'zoo' of Cre recombinase driver mice. Having described the basics we move on to the cutting edge: generation of genome-wide sets of conditional knockout mice. To this end, large ongoing projects apply two strategies: gene trapping based on random integration of trapping vectors into introns leading to truncation of the transcript, and gene targeting, representing the directed approach using homologous recombination. It can be expected that in the near future genome-wide sets of such mice will be available. Finally, the possibilities of conditional expression systems for investigating gene function in tissue regeneration will be illustrated by examples for neurodegenerative disease, liver regeneration and wound healing of the skin.

Suppression of Autophagy in Osteocytes Mimics Skeletal Aging*

PubMed Central

Onal, Melda; Piemontese, Marilina; Xiong, Jinhu; Wang, Yiying; Han, Li; Ye, Shiqiao; Komatsu, Masaaki; Selig, Martin; Weinstein, Robert S.; Zhao, Haibo; Jilka, Robert L.; Almeida, Maria; Manolagas, Stavros C.; O'Brien, Charles A.

2013-01-01

Bone mass declines with age but the mechanisms responsible remain unclear. Here we demonstrate that deletion of a conditional allele for Atg7, a gene essential for autophagy, from osteocytes caused low bone mass in 6-month-old male and female mice. Cancellous bone volume and cortical thickness were decreased, and cortical porosity increased, in conditional knock-out mice compared with control littermates. These changes were associated with low osteoclast number, osteoblast number, bone formation rate, and wall width in the cancellous bone of conditional knock-out mice. In addition, oxidative stress was higher in the bones of conditional knock-out mice as measured by reactive oxygen species levels in the bone marrow and by p66shc phosphorylation in L6 vertebra. Each of these changes has been previously demonstrated in the bones of old versus young adult mice. Thus, these results demonstrate that suppression of autophagy in osteocytes mimics, in many aspects, the impact of aging on the skeleton and suggest that a decline in autophagy with age may contribute to the low bone mass associated with aging. PMID:23645674

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.

Role of estrogen receptor-α on food demand elasticity.

PubMed

Minervini, Vanessa; Rowland, Neil E; Robertson, Kimberly L; Foster, Thomas C

2015-05-01

Estrogens have been shown to have an inhibitory effect on food intake under free-feeding conditions, yet the effects of estrogens on food-maintained operant responding have been studied to a much lesser extent and, thus, are not well understood. Therefore, the purpose of the present experiment was to use a behavioral economics paradigm to assess differences in demand elasticity between mice with knockout of the estrogen receptor subtype α, knockout of subtype β, and their wild type controls. The mice responded in a closed economy, and the price of food was increased by increasing the fixed-ratio response requirement every four sessions. Overall, we found that mice with the knockout of receptor subtype α had the most elastic demand functions. Therefore, under these conditions, estrogens increased food seeking via activation of the receptor subtype α. The results were inconsistent with those reported by previous studies that employed free-feeding conditions. © Society for the Experimental Analysis of Behavior.

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

Knock-out of a mitochondrial sirtuin protects neurons from degeneration in Caenorhabditis elegans.

PubMed

Sangaletti, Rachele; D'Amico, Massimo; Grant, Jeff; Della-Morte, David; Bianchi, Laura

2017-08-01

Sirtuins are NAD⁺-dependent deacetylases, lipoamidases, and ADP-ribosyltransferases that link cellular metabolism to multiple intracellular pathways that influence processes as diverse as cell survival, longevity, and cancer growth. Sirtuins influence the extent of neuronal death in stroke. However, different sirtuins appear to have opposite roles in neuronal protection. In Caenorhabditis elegans, we found that knock-out of mitochondrial sirtuin sir-2.3, homologous to mammalian SIRT4, is protective in both chemical ischemia and hyperactive channel induced necrosis. Furthermore, the protective effect of sir-2.3 knock-out is enhanced by block of glycolysis and eliminated by a null mutation in daf-16/FOXO transcription factor, supporting the involvement of the insulin/IGF pathway. However, data in Caenorhabditis elegans cell culture suggest that the effects of sir-2.3 knock-out act downstream of the DAF-2/IGF-1 receptor. Analysis of ROS in sir-2.3 knock-out reveals that ROS become elevated in this mutant under ischemic conditions in dietary deprivation (DD), but to a lesser extent than in wild type, suggesting more robust activation of a ROS scavenging system in this mutant in the absence of food. This work suggests a deleterious role of SIRT4 during ischemic processes in mammals that must be further investigated and reveals a novel pathway that can be targeted for the design of therapies aimed at protecting neurons from death in ischemic conditions.

Enhanced hexose fermentation by Saccharomyces cerevisiae through integration of stoichiometric modeling and genetic screening.

PubMed

Quarterman, Josh; Kim, Soo Rin; Kim, Pan-Jun; Jin, Yong-Su

2015-01-20

In order to determine beneficial gene deletions for ethanol production by the yeast Saccharomyces cerevisiae, we performed an in silico gene deletion experiment based on a genome-scale metabolic model. Genes coding for two oxidative phosphorylation reactions (cytochrome c oxidase and ubiquinol cytochrome c reductase) were identified by the model-based simulation as potential deletion targets for enhancing ethanol production and maintaining acceptable overall growth rate in oxygen-limited conditions. Since the two target enzymes are composed of multiple subunits, we conducted a genetic screening study to evaluate the in silico results and compare the effect of deleting various portions of the respiratory enzyme complexes. Over two-thirds of the knockout mutants identified by the in silico study did exhibit experimental behavior in qualitative agreement with model predictions, but the exceptions illustrate the limitation of using a purely stoichiometric model-based approach. Furthermore, there was a substantial quantitative variation in phenotype among the various respiration-deficient mutants that were screened in this study, and three genes encoding respiratory enzyme subunits were identified as the best knockout targets for improving hexose fermentation in microaerobic conditions. Specifically, deletion of either COX9 or QCR9 resulted in higher ethanol production rates than the parental strain by 37% and 27%, respectively, with slight growth disadvantages. Also, deletion of QCR6 led to improved ethanol production rate by 24% with no growth disadvantage. The beneficial effects of these gene deletions were consistently demonstrated in different strain backgrounds and with four common hexoses. The combination of stoichiometric modeling and genetic screening using a systematic knockout collection was useful for narrowing a large set of gene targets and identifying targets of interest. Copyright © 2014 Elsevier B.V. All rights reserved.

Peripheral surgical wounding may induce cognitive impairment through interlukin-6-dependent mechanisms in aged mice.

PubMed

Dong, Yuanlin; Xu, Zhipeng; Huang, Lining; Zhang, Yiying; Xie, Zhongcong

2016-01-01

Post-operative cognitive dysfunction (POCD) is associated with morbidity, mortality and increased cost of medical care. However, the neuropathogenesis and targeted interventions of POCD remain largely to be determined. We have found that the peripheral surgical wounding induces an age-dependent Aβ accumulation, neuroinflammation and cognitive impairment in aged mice. Pro-inflammatory cytokine interlukin-6 (IL-6) has been reported to be associated with cognitive impairment in rodents and humans. However, the role of IL-6 in the neuropathogenesis of POCD is unknown. We therefore employed pharmacological (IL-6 antibody) and genetic (knockout of IL-6) approach to investigate whether IL-6 contributed to the peripheral surgical wounding-induced cognitive impairment in aged mice. Abdominal surgery under local anesthesia (peripheral surgical wounding) was established in 18-month-old wild-type and IL-6 knockout mice ( n = 6 to 10 in each group). Brain level of IL-6 and cognitive function in the mice were determined by western blot, ELISA at the end of procedure, and Fear Conditioning System at 7 days after the procedure. The peripheral surgical wounding increased the level of IL-6 in the hippocampus of aged wild-type, but not IL-6 knockout mice. IL-6 antibody ameliorated the peripheral surgical wounding-induced cognitive impairment in the aged wild-type mice. Finally, the peripheral surgical wounding did not induce cognitive impairment in the aged IL-6 knockout mice. These data suggested that IL-6 would be a required pro-inflammatory cytokine for the peripheral surgical wounding-induced cognitive impairment. Given this, further studies are warranted to investigate the role of IL-6 in the neuropathogenesis and targeted interventions of POCD.

Peripheral surgical wounding may induce cognitive impairment through interlukin-6-dependent mechanisms in aged mice

PubMed Central

Dong, Yuanlin; Xu, Zhipeng; Huang, Lining; Zhang, Yiying; Xie, Zhongcong

2016-01-01

Post-operative cognitive dysfunction (POCD) is associated with morbidity, mortality and increased cost of medical care. However, the neuropathogenesis and targeted interventions of POCD remain largely to be determined. We have found that the peripheral surgical wounding induces an age-dependent Aβ accumulation, neuroinflammation and cognitive impairment in aged mice. Pro-inflammatory cytokine interlukin-6 (IL-6) has been reported to be associated with cognitive impairment in rodents and humans. However, the role of IL-6 in the neuropathogenesis of POCD is unknown. We therefore employed pharmacological (IL-6 antibody) and genetic (knockout of IL-6) approach to investigate whether IL-6 contributed to the peripheral surgical wounding-induced cognitive impairment in aged mice. Abdominal surgery under local anesthesia (peripheral surgical wounding) was established in 18-month-old wild-type and IL-6 knockout mice (n = 6 to 10 in each group). Brain level of IL-6 and cognitive function in the mice were determined by western blot, ELISA at the end of procedure, and Fear Conditioning System at 7 days after the procedure. The peripheral surgical wounding increased the level of IL-6 in the hippocampus of aged wild-type, but not IL-6 knockout mice. IL-6 antibody ameliorated the peripheral surgical wounding-induced cognitive impairment in the aged wild-type mice. Finally, the peripheral surgical wounding did not induce cognitive impairment in the aged IL-6 knockout mice. These data suggested that IL-6 would be a required pro-inflammatory cytokine for the peripheral surgical wounding-induced cognitive impairment. Given this, further studies are warranted to investigate the role of IL-6 in the neuropathogenesis and targeted interventions of POCD. PMID:28217289

Importance of GluA1 Subunit-Containing AMPA Glutamate Receptors for Morphine State-Dependency

PubMed Central

Aitta-aho, Teemu; Möykkynen, Tommi P.; Panhelainen, Anne E.; Vekovischeva, Olga Yu.; Bäckström, Pia; Korpi, Esa R.

2012-01-01

In state-dependency, information retrieval is most efficient when the animal is in the same state as it was during the information acquisition. State-dependency has been implicated in a variety of learning and memory processes, but its mechanisms remain to be resolved. Here, mice deficient in AMPA-type glutamate receptor GluA1 subunits were first conditioned to morphine (10 or 20 mg/kg s.c. during eight sessions over four days) using an unbiased procedure, followed by testing for conditioned place preference at morphine states that were the same as or different from the one the mice were conditioned to. In GluA1 wildtype littermate mice the same-state morphine dose produced the greatest expression of place preference, while in the knockout mice no place preference was then detected. Both wildtype and knockout mice expressed moderate morphine-induced place preference when not at the morphine state (saline treatment at the test); in this case, place preference was weaker than that in the same-state test in wildtype mice. No correlation between place preference scores and locomotor activity during testing was found. Additionally, as compared to the controls, the knockout mice showed unchanged sensitization to morphine, morphine drug discrimination and brain regional μ-opioid receptor signal transduction at the G-protein level. However, the knockout mice failed to show increased AMPA/NMDA receptor current ratios in the ventral tegmental area dopamine neurons of midbrain slices after a single injection of morphine (10 mg/kg, s.c., sliced prepared 24 h afterwards), in contrast to the wildtype mice. The results indicate impaired drug-induced state-dependency in GluA1 knockout mice, correlating with impaired opioid-induced glutamate receptor neuroplasticity. PMID:22675452

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.

Development of a Markerless Knockout Method for Actinobacillus succinogenes

PubMed Central

Joshi, Rajasi V.; Schindler, Bryan D.; McPherson, Nikolas R.; Tiwari, Kanupriya

2014-01-01

Actinobacillus succinogenes is one of the best natural succinate-producing organisms, but it still needs engineering to further increase succinate yield and productivity. In this study, we developed a markerless knockout method for A. succinogenes using natural transformation or electroporation. The Escherichia coli isocitrate dehydrogenase gene with flanking flippase recognition target sites was used as the positive selection marker, making use of A. succinogenes's auxotrophy for glutamate to select for growth on isocitrate. The Saccharomyces cerevisiae flippase recombinase (Flp) was used to remove the selection marker, allowing its reuse. Finally, the plasmid expressing flp was cured using acridine orange. We demonstrate that at least two consecutive deletions can be introduced into the same strain using this approach, that no more than a total of 1 kb of DNA is needed on each side of the selection cassette to protect from exonuclease activity during transformation, and that no more than 200 bp of homologous DNA is needed on each side for efficient recombination. We also demonstrate that electroporation can be used as an alternative transformation method to obtain knockout mutants and that an enriched defined medium can be used for direct selection of knockout mutants on agar plates with high efficiency. Single-knockout mutants of the fumarate reductase and of the pyruvate formate lyase-encoding genes were obtained using this knockout strategy. Double-knockout mutants were also obtained by deleting the citrate lyase-, β-galactosidase-, and aconitase-encoding genes in the pyruvate formate lyase knockout mutant strain. PMID:24610845

Development of a markerless knockout method for Actinobacillus succinogenes.

PubMed

Joshi, Rajasi V; Schindler, Bryan D; McPherson, Nikolas R; Tiwari, Kanupriya; Vieille, Claire

2014-05-01

Actinobacillus succinogenes is one of the best natural succinate-producing organisms, but it still needs engineering to further increase succinate yield and productivity. In this study, we developed a markerless knockout method for A. succinogenes using natural transformation or electroporation. The Escherichia coli isocitrate dehydrogenase gene with flanking flippase recognition target sites was used as the positive selection marker, making use of A. succinogenes's auxotrophy for glutamate to select for growth on isocitrate. The Saccharomyces cerevisiae flippase recombinase (Flp) was used to remove the selection marker, allowing its reuse. Finally, the plasmid expressing flp was cured using acridine orange. We demonstrate that at least two consecutive deletions can be introduced into the same strain using this approach, that no more than a total of 1 kb of DNA is needed on each side of the selection cassette to protect from exonuclease activity during transformation, and that no more than 200 bp of homologous DNA is needed on each side for efficient recombination. We also demonstrate that electroporation can be used as an alternative transformation method to obtain knockout mutants and that an enriched defined medium can be used for direct selection of knockout mutants on agar plates with high efficiency. Single-knockout mutants of the fumarate reductase and of the pyruvate formate lyase-encoding genes were obtained using this knockout strategy. Double-knockout mutants were also obtained by deleting the citrate lyase-, β-galactosidase-, and aconitase-encoding genes in the pyruvate formate lyase knockout mutant strain.

Efficiency of primary saliva secretion: an analysis of parameter dependence in dynamic single-cell and acinus models, with application to aquaporin knockout studies

PubMed Central

Maclaren, Oliver J.; Sneyd, James; Crampin, Edmund J.

2012-01-01

Secretion from the salivary glands is driven by osmosis following the establishment of osmotic gradients between the lumen, the cell and the interstitium by active ion transport. We consider a dynamic model of osmotically-driven primary saliva secretion, and use singular perturbation approaches and scaling assumptions to reduce the model. Our analysis shows that isosmotic secretion is the most efficient secretion regime, and that this holds for single isolated cells and for multiple cells assembled into an acinus. For typical parameter variations, we rule out any significant synergistic effect on total water secretion of an acinar arrangement of cells about a single shared lumen. Conditions for the attainment of isosmotic secretion are considered, and we derive an expression for how the concentration gradient between the interstitium and the lumen scales with water and chloride transport parameters. Aquaporin knockout studies are interpreted in the context of our analysis and further investigated using simulations of transport efficiency with different membrane water permeabilities. We conclude that recent claims that aquaporin knockout studies can be interpreted as evidence against a simple osmotic mechanism are not supported by our work. Many of the results that we obtain are independent of specific transporter details, and our analysis can be easily extended to apply to models that use other proposed ionic mechanisms of saliva secretion. PMID:22258315

Effects of HAb18G/CD147 knockout on hepatocellular carcinoma cells in vitro using a novel zinc-finger nuclease-targeted gene knockout approach.

PubMed

Li, Hong-Wei; Yang, Xiang-Min; Tang, Juan; Wang, Shi-Jie; Chen, Zhi-Nan; Jiang, Jian-Li

2015-03-01

HAb18G/CD147 belongs to the immunoglobulin superfamily and predominantly functions as an inducer of matrix metalloproteinase secretion for tumor invasion and metastasis. This study was designed to investigate the effects of HAb18G/CD147 knockout on hepatocellular carcinoma cells using zinc-finger nuclease (ZFNs)-targeted gene knockout approach. The HCC cell line SMMC-7721 was used for ZFNs-targeted cleavage of the HAb18G/CD147 gene. RT-PCR and Western blot assays were used to detect HAb18G/CD147 expression. HAb18G phenotypic changes following HAb18G/CD147 knockout in SMMC-K7721 cells were assessed using tumor cell adhesion, invasion, migration and colony formation and flow cytometric assays. These data demonstrated that tumor cell adhesion, invasion, migration, and colony formation capabilities of SMMC-K7721 were significantly reduced compared to parental cells or SMMC-7721 with re-expression of HAb18G/CD147 protein transfected with HAb18G/CD147 cDNA. Moreover, knockout of HAb18G/CD147 expression also induced SMMC-K7721 cells to undergo apoptosis compared to SMMC-7721 and SMMC-R7721 (P < 0.01). Molecularly, protein expression of p53 was induced in these cells, but re-expression of HAb18G/CD147 reduced p53 levels in SMMC-R7721 cells, possibly through inhibition of the PI3K-Akt-MDM2 signaling pathway. The findings provide a novel insight into the mechanisms underlying HAb18G/CD147-induced progression of HCC cells.

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.

Knock-out of a mitochondrial sirtuin protects neurons from degeneration in Caenorhabditis elegans

PubMed Central

Sangaletti, Rachele; Grant, Jeff; Della-Morte, David

2017-01-01

Sirtuins are NAD⁺-dependent deacetylases, lipoamidases, and ADP-ribosyltransferases that link cellular metabolism to multiple intracellular pathways that influence processes as diverse as cell survival, longevity, and cancer growth. Sirtuins influence the extent of neuronal death in stroke. However, different sirtuins appear to have opposite roles in neuronal protection. In Caenorhabditis elegans, we found that knock-out of mitochondrial sirtuin sir-2.3, homologous to mammalian SIRT4, is protective in both chemical ischemia and hyperactive channel induced necrosis. Furthermore, the protective effect of sir-2.3 knock-out is enhanced by block of glycolysis and eliminated by a null mutation in daf-16/FOXO transcription factor, supporting the involvement of the insulin/IGF pathway. However, data in Caenorhabditis elegans cell culture suggest that the effects of sir-2.3 knock-out act downstream of the DAF-2/IGF-1 receptor. Analysis of ROS in sir-2.3 knock-out reveals that ROS become elevated in this mutant under ischemic conditions in dietary deprivation (DD), but to a lesser extent than in wild type, suggesting more robust activation of a ROS scavenging system in this mutant in the absence of food. This work suggests a deleterious role of SIRT4 during ischemic processes in mammals that must be further investigated and reveals a novel pathway that can be targeted for the design of therapies aimed at protecting neurons from death in ischemic conditions. PMID:28820880

Merging first principle structure studies and few-body reaction formalism

NASA Astrophysics Data System (ADS)

Crespo, R.; Cravo, E.; Arriaga, A.; Wiringa, R.; Deltuva, A.; Diego, R.

2018-02-01

Calculations for nucleon knockout from a 7Li beam due to the collision with a proton target at 400 MeV/u are shown based on ab initio Quantum Monte Carlo (QMC) and conventional shell-model nuclear structure approaches to describe the relative motion between the knockout particle and the heavy fragment of the projectile. Structure effects on the total cross section are shown.

A Review of Gene Knockout Strategies for Microbial Cells.

PubMed

Tang, Phooi Wah; Chua, Pooi San; Chong, Shiue Kee; Mohamad, Mohd Saberi; Choon, Yee Wen; Deris, Safaai; Omatu, Sigeru; Corchado, Juan Manuel; Chan, Weng Howe; Rahim, Raha Abdul

2015-01-01

Predicting the effects of genetic modification is difficult due to the complexity of metabolic net- works. Various gene knockout strategies have been utilised to deactivate specific genes in order to determine the effects of these genes on the function of microbes. Deactivation of genes can lead to deletion of certain proteins and functions. Through these strategies, the associated function of a deleted gene can be identified from the metabolic networks. The main aim of this paper is to review the available techniques in gene knockout strategies for microbial cells. The review is done in terms of their methodology, recent applications in microbial cells. In addition, the advantages and disadvantages of the techniques are compared and discuss and the related patents are also listed as well. Traditionally, gene knockout is done through wet lab (in vivo) techniques, which were conducted through laboratory experiments. However, these techniques are costly and time consuming. Hence, various dry lab (in silico) techniques, where are conducted using computational approaches, have been developed to surmount these problem. The development of numerous techniques for gene knockout in microbial cells has brought many advancements in the study of gene functions. Based on the literatures, we found that the gene knockout strategies currently used are sensibly implemented with regard to their benefits.

Mapping pathological phenotypes in a mouse model of CDKL5 disorder.

PubMed

Amendola, Elena; Zhan, Yang; Mattucci, Camilla; Castroflorio, Enrico; Calcagno, Eleonora; Fuchs, Claudia; Lonetti, Giuseppina; Silingardi, Davide; Vyssotski, Alexei L; Farley, Dominika; Ciani, Elisabetta; Pizzorusso, Tommaso; Giustetto, Maurizio; Gross, Cornelius T

2014-01-01

Mutations in cyclin-dependent kinase-like 5 (CDKL5) cause early-onset epileptic encephalopathy, a neurodevelopmental disorder with similarities to Rett Syndrome. Here we describe the physiological, molecular, and behavioral phenotyping of a Cdkl5 conditional knockout mouse model of CDKL5 disorder. Behavioral analysis of constitutive Cdkl5 knockout mice revealed key features of the human disorder, including limb clasping, hypoactivity, and abnormal eye tracking. Anatomical, physiological, and molecular analysis of the knockout uncovered potential pathological substrates of the disorder, including reduced dendritic arborization of cortical neurons, abnormal electroencephalograph (EEG) responses to convulsant treatment, decreased visual evoked responses (VEPs), and alterations in the Akt/rpS6 signaling pathway. Selective knockout of Cdkl5 in excitatory and inhibitory forebrain neurons allowed us to map the behavioral features of the disorder to separable cell-types. These findings identify physiological and molecular deficits in specific forebrain neuron populations as possible pathological substrates in CDKL5 disorder.

Proteomic and metabolomic characterization of streptozotocin-induced diabetic nephropathy in TIMP3-deficient mice.

PubMed

Rossi, Claudia; Marzano, Valeria; Consalvo, Ada; Zucchelli, Mirco; Levi Mortera, Stefano; Casagrande, Viviana; Mavilio, Maria; Sacchetta, Paolo; Federici, Massimo; Menghini, Rossella; Urbani, Andrea; Ciavardelli, Domenico

2018-02-01

The tissue inhibitor of metalloproteinase TIMP3 is a stromal protein that restrains the activity of both protease and receptor in the extracellular matrix and has been found to be down-regulated in diabetic nephropathy (DN), the leading cause of end-stage renal disease in developed countries. In order to gain deeper insights on the association of loss of TIMP3 and DN, we performed differential proteomic analysis of kidney and blood metabolic profiling of wild-type and Timp3-knockout mice before and after streptozotocin (STZ) treatment, widely used to induce insulin deficiency and hyperglycemia. Kidney proteomic data and blood metabolic profiles suggest significant alterations of peroxisomal and mitochondrial fatty acids β-oxidation in Timp3-knockout mice compared to wild-type mice under basal condition. These alterations were exacerbated in response to STZ treatment. Proteomic and metabolomic approaches showed that loss of TIMP3 alone or in combination with STZ treatment results in significant alterations of kidney lipid metabolism and peripheral acylcarnitine levels, supporting the idea that loss of TIMP3 may generate a phenotype more prone to DN.

Lymphocyte signaling: beyond knockouts.

PubMed

Saveliev, Alexander; Tybulewicz, Victor L J

2009-04-01

The analysis of lymphocyte signaling was greatly enhanced by the advent of gene targeting, which allows the selective inactivation of a single gene. Although this gene 'knockout' approach is often informative, in many cases, the phenotype resulting from gene ablation might not provide a complete picture of the function of the corresponding protein. If a protein has multiple functions within a single or several signaling pathways, or stabilizes other proteins in a complex, the phenotypic consequences of a gene knockout may manifest as a combination of several different perturbations. In these cases, gene targeting to 'knock in' subtle point mutations might provide more accurate insight into protein function. However, to be informative, such mutations must be carefully based on structural and biophysical data.

Characterization of Arabidopsis sterol glycosyltransferase TTG15/UGT80B1 role during freeze and heat stress

PubMed Central

Mishra, Manoj K; Singh, Gaurav; Tiwari, Shalini; Singh, Ruchi; Kumari, Nishi; Misra, Pratibha

2015-01-01

Sterol glycosyltransferases regulate the properties of sterols by catalyzing the transfer of carbohydrate molecules to the sterol moiety for the synthesis of steryl glycosides and acyl steryl glycosides. We have analyzed the functional role of TTG15/UGT80B1 gene of Arabidopsis thaliana in freeze/thaw and heat shock stress using T-DNA insertional sgt knockout mutants. Quantitative study of spatial as well as temporal gene expression showed tissue-specific and dynamic expression patterns throughout the growth stages. Comparative responses of Col-0, TTG15/UGT80B1 knockout mutant and p35S:TTG15/UGT80B1 restored lines were analyzed under heat and freeze stress conditions. Heat tolerance was determined by survival of plants at 42°C for 3 h, MDA analysis and chlorophyll fluorescence image (CFI) analysis. Freezing tolerance was determined by survival of the plants at -1°C temperature in non-acclimatized (NA) and cold acclimatized (CA) conditions and also by CFI analysis, which revealed that, p35S:TTG15/UGT80B1 restored plants were more adapted to freeze stress than TTG15/UGT80B1 knockout mutant under CA condition. HPLC analysis of the plants showed reduced sterol glycoside in mutant seedlings as compared to other genotypes. Following CA condition, both β-sitosterol and sitosterol glycoside quantity was more in Col-0 and p35S:TTG15/UGT80B1 restored lines, whereas it was significantly less in TTG15/UGT80B1 knockout mutants. From these results, it may be concluded that due to low content of free sterols and sterol glycosides, the physiology of mutant plants was more affected during both, the chilling and heat stress. PMID:26382564

Dlgap1 knockout mice exhibit alterations of the postsynaptic density and selective reductions in sociability.

PubMed

Coba, M P; Ramaker, M J; Ho, E V; Thompson, S L; Komiyama, N H; Grant, S G N; Knowles, J A; Dulawa, S C

2018-02-02

The scaffold protein DLGAP1 is localized at the post-synaptic density (PSD) of glutamatergic neurons and is a component of supramolecular protein complexes organized by PSD95. Gain-of-function variants of DLGAP1 have been associated with obsessive-compulsive disorder (OCD), while haploinsufficient variants have been linked to autism spectrum disorder (ASD) and schizophrenia in human genetic studies. We tested male and female Dlgap1 wild type (WT), heterozygous (HT), and knockout (KO) mice in a battery of behavioral tests: open field, dig, splash, prepulse inhibition, forced swim, nest building, social approach, and sucrose preference. We also used biochemical approaches to examine the role of DLGAP1 in the organization of PSD protein complexes. Dlgap1 KO mice were most notable for disruption of protein interactions in the PSD, and deficits in sociability. Other behavioral measures were largely unaffected. Our data suggest that Dlgap1 knockout leads to PSD disruption and reduced sociability, consistent with reports of DLGAP1 haploinsufficient variants in schizophrenia and ASD.

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

A Convenient Cas9-based Conditional Knockout Strategy for Simultaneously Targeting Multiple Genes in Mouse.

PubMed

Chen, Jiang; Du, Yinan; He, Xueyan; Huang, Xingxu; Shi, Yun S

2017-03-31

The most powerful way to probe protein function is to characterize the consequence of its deletion. Compared to conventional gene knockout (KO), conditional knockout (cKO) provides an advanced gene targeting strategy with which gene deletion can be performed in a spatially and temporally restricted manner. However, for most species that are amphiploid, the widely used Cre-flox conditional KO (cKO) system would need targeting loci in both alleles to be loxP flanked, which in practice, requires time and labor consuming breeding. This is considerably significant when one is dealing with multiple genes. CRISPR/Cas9 genome modulation system is advantaged in its capability in targeting multiple sites simultaneously. Here we propose a strategy that could achieve conditional KO of multiple genes in mouse with Cre recombinase dependent Cas9 expression. By transgenic construction of loxP-stop-loxP (LSL) controlled Cas9 (LSL-Cas9) together with sgRNAs targeting EGFP, we showed that the fluorescence molecule could be eliminated in a Cre-dependent manner. We further verified the efficacy of this novel strategy to target multiple sites by deleting c-Maf and MafB simultaneously in macrophages specifically. Compared to the traditional Cre-flox cKO strategy, this sgRNAs-LSL-Cas9 cKO system is simpler and faster, and would make conditional manipulation of multiple genes feasible.

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.

Changes in blood carnitine and acylcarnitine profiles of very long-chain acyl-CoA dehydrogenase-deficient mice subjected to stress.

PubMed

Spiekerkoetter, U; Tokunaga, C; Wendel, U; Mayatepek, E; Exil, V; Duran, M; Wijburg, F A; Wanders, R J A; Strauss, A W

2004-03-01

In humans with deficiency of the very long-chain acyl-CoA dehydrogenase (VLCAD), C14-C18 acylcarnitines accumulate. In this paper we have used the VLCAD knockout mouse as a model to study changes in blood carnitine and acylcarnitine profiles under stress. VLCAD knockout mice exhibit stress-induced hypoglycaemia and skeletal myopathy; symptoms resembling human VLCADD. To study the extent of biochemical derangement in response to different stressors, we determined blood carnitine and acylcarnitine profiles after exercise on a treadmill, fasting, or exposure to cold. Even in a nonstressed, well-fed state, knockout mice presented twofold higher C14-C18 acylcarnitines and a lower free carnitine of 72% as compared to wild-type littermates. After 1 h of intense exercise, the C14-C18 acylcarnitines in blood significantly increased, but free carnitine remained unchanged. After 8 h of fasting at 4 degrees C, the long-chain acylcarnitines were elevated 5-fold in knockout mice in comparison with concentrations in unstressed wild-type mice (P < 0.05), and four out of 12 knockout mice died. Free carnitine decreased to 44% as compared with unstressed wild-type mice. An increase in C14-C18 acylcarnitines and a decrease of free carnitine were also observed in fasted heterozygous and wild-type mice. Long-chain acylcarnitines in blood increase in knockout mice in response to different stressors and concentrations correlate with the clinical condition. A decrease in blood free carnitine in response to severe stress is observed in knockout mice but also in wild-type littermates. Monitoring blood acylcarnitine profiles in response to different stressors may allow systematic analysis of therapeutic interventions in VLCAD knockout mice.

Differential sensitivity of Pak5, Pak6, and Pak5/Pak6 double-knockout mice to the stimulant effects of amphetamine and exercise-induced alterations in body weight.

PubMed

Furnari, Melody A; Jobes, Michelle L; Nekrasova, Tanya; Minden, Audrey; Wagner, George C

2014-04-01

PAK5 and PAK6 are protein kinases highly expressed in the brain. Previously, we observed that Pak6 knockout mice gained significantly more weight during development than Pak5 knockout mice as well as wild-type controls and double-knockout mice lacking both Pak5 and Pak6. In this study, we assessed the effects of exercise on food intake and weight gain of these mice as well as their sensitivity to the stimulant effects of amphetamine. Mice of each genotype were placed in cages with free access to run wheel exercise or in cages without run wheels for a total of 74 days. Food and fluid intake as well as body weight of each mouse were measured on a weekly basis. Finally, mice were given a high dose of amphetamine and activity levels were observed immediately thereafter for 90 minutes. Brains and testes of mice were assayed for protein levels of the estrogen alpha and progesterone receptors. While run wheel mice consumed significantly more food, they weighed less than non-run wheel mice. In addition, although Pak6 knockout mice consumed the same amount of food as wild-type mice, they were significantly heavier regardless of run wheel condition. Pak5 knockout mice were found to be more active than other genotypes after amphetamine treatment. Finally, protein levels of the progesterone and estrogen alpha receptors were altered in brain and testes of the Pak6 knockout mice. Collectively, these data suggest that PAK6 play a role in weight gain unrelated to exercise and caloric intake and that Pak5 knockout mice are more sensitive to the stimulant effects of amphetamine.

Distinct Roles of Opioid and Dopamine Systems in Lateral Hypothalamic Intracranial Self-Stimulation.

PubMed

Ide, Soichiro; Takahashi, Takehiro; Takamatsu, Yukio; Uhl, George R; Niki, Hiroaki; Sora, Ichiro; Ikeda, Kazutaka

2017-05-01

Opioid and dopamine systems play crucial roles in reward. Similarities and differences in the neural mechanisms of reward that are mediated by these 2 systems have remained largely unknown. Thus, in the present study, we investigated the differences in reward function in both µ-opioid receptor knockout mice and dopamine transporter knockout mice, important molecules in the opioid and dopamine systems. Mice were implanted with electrodes into the right lateral hypothalamus (l hour). Mice were then trained to put their muzzle into the hole in the head-dipping chamber for intracranial electrical stimulation, and the influences of gene knockout were assessed. Significant differences are observed between opioid and dopamine systems in reward function. µ-Opioid receptor knockout mice exhibited enhanced intracranial electrical stimulation, which induced dopamine release. They also exhibited greater motility under conditions of "despair" in both the tail suspension test and water wheel test. In contrast, dopamine transporter knockout mice maintained intracranial electrical stimulation responding even when more active efforts were required to obtain the reward. The absence of µ-opioid receptor or dopamine transporter did not lead to the absence of intracranial electrical stimulation responsiveness but rather differentially altered it. The present results in µ-opioid receptor knockout mice are consistent with the suppressive involvement of µ-opioid receptors in both positive incentive motivation associated with intracranial electrical stimulation and negative incentive motivation associated with depressive states. In contrast, the results in dopamine transporter knockout mice are consistent with the involvement of dopamine transporters in positive incentive motivation, especially its persistence. Differences in intracranial electrical stimulation in µ-opioid receptor and dopamine transporter knockout mice underscore the multidimensional nature of reward. © The Author 2016. Published by Oxford University Press on behalf of CINP.

Petri Nets with Fuzzy Logic (PNFL): Reverse Engineering and Parametrization

PubMed Central

Küffner, Robert; Petri, Tobias; Windhager, Lukas; Zimmer, Ralf

2010-01-01

Background The recent DREAM4 blind assessment provided a particularly realistic and challenging setting for network reverse engineering methods. The in silico part of DREAM4 solicited the inference of cycle-rich gene regulatory networks from heterogeneous, noisy expression data including time courses as well as knockout, knockdown and multifactorial perturbations. Methodology and Principal Findings We inferred and parametrized simulation models based on Petri Nets with Fuzzy Logic (PNFL). This completely automated approach correctly reconstructed networks with cycles as well as oscillating network motifs. PNFL was evaluated as the best performer on DREAM4 in silico networks of size 10 with an area under the precision-recall curve (AUPR) of 81%. Besides topology, we inferred a range of additional mechanistic details with good reliability, e.g. distinguishing activation from inhibition as well as dependent from independent regulation. Our models also performed well on new experimental conditions such as double knockout mutations that were not included in the provided datasets. Conclusions The inference of biological networks substantially benefits from methods that are expressive enough to deal with diverse datasets in a unified way. At the same time, overly complex approaches could generate multiple different models that explain the data equally well. PNFL appears to strike the balance between expressive power and complexity. This also applies to the intuitive representation of PNFL models combining a straightforward graphical notation with colloquial fuzzy parameters. PMID:20862218

Motor Deficits and Decreased Striatal Dopamine Receptor 2 Binding Activity in the Striatum-Specific Dyt1 Conditional Knockout Mice

PubMed Central

Yokoi, Fumiaki; Dang, Mai Tu; Li, Jianyong; Standaert, David G.; Li, Yuqing

2011-01-01

DYT1 early-onset generalized dystonia is a hyperkinetic movement disorder caused by mutations in DYT1 (TOR1A), which codes for torsinA. Recently, significant progress has been made in studying pathophysiology of DYT1 dystonia using targeted mouse models. Dyt1 ΔGAG heterozygous knock-in (KI) and Dyt1 knock-down (KD) mice exhibit motor deficits and alterations of striatal dopamine metabolisms, while Dyt1 knockout (KO) and Dyt1 ΔGAG homozygous KI mice show abnormal nuclear envelopes and neonatal lethality. However, it has not been clear whether motor deficits and striatal abnormality are caused by Dyt1 mutation in the striatum itself or the end results of abnormal signals from other brain regions. To identify the brain region that contributes to these phenotypes, we made a striatum-specific Dyt1 conditional knockout (Dyt1 sKO) mouse. Dyt1 sKO mice exhibited motor deficits and reduced striatal dopamine receptor 2 (D2R) binding activity, whereas they did not exhibit significant alteration of striatal monoamine contents. Furthermore, we also found normal nuclear envelope structure in striatal medium spiny neurons (MSNs) of an adult Dyt1 sKO mouse and cerebral cortical neurons in cerebral cortex-specific Dyt1 conditional knockout (Dyt1 cKO) mice. The results suggest that the loss of striatal torsinA alone is sufficient to produce motor deficits, and that this effect may be mediated, at least in part, through changes in D2R function in the basal ganglia circuit. PMID:21931745

Evaluation and Design of Genome-Wide CRISPR/SpCas9 Knockout Screens

PubMed Central

Hart, Traver; Tong, Amy Hin Yan; Chan, Katie; Van Leeuwen, Jolanda; Seetharaman, Ashwin; Aregger, Michael; Chandrashekhar, Megha; Hustedt, Nicole; Seth, Sahil; Noonan, Avery; Habsid, Andrea; Sizova, Olga; Nedyalkova, Lyudmila; Climie, Ryan; Tworzyanski, Leanne; Lawson, Keith; Sartori, Maria Augusta; Alibeh, Sabriyeh; Tieu, David; Masud, Sanna; Mero, Patricia; Weiss, Alexander; Brown, Kevin R.; Usaj, Matej; Billmann, Maximilian; Rahman, Mahfuzur; Costanzo, Michael; Myers, Chad L.; Andrews, Brenda J.; Boone, Charles; Durocher, Daniel; Moffat, Jason

2017-01-01

The adaptation of CRISPR/SpCas9 technology to mammalian cell lines is transforming the study of human functional genomics. Pooled libraries of CRISPR guide RNAs (gRNAs) targeting human protein-coding genes and encoded in viral vectors have been used to systematically create gene knockouts in a variety of human cancer and immortalized cell lines, in an effort to identify whether these knockouts cause cellular fitness defects. Previous work has shown that CRISPR screens are more sensitive and specific than pooled-library shRNA screens in similar assays, but currently there exists significant variability across CRISPR library designs and experimental protocols. In this study, we reanalyze 17 genome-scale knockout screens in human cell lines from three research groups, using three different genome-scale gRNA libraries. Using the Bayesian Analysis of Gene Essentiality algorithm to identify essential genes, we refine and expand our previously defined set of human core essential genes from 360 to 684 genes. We use this expanded set of reference core essential genes, CEG2, plus empirical data from six CRISPR knockout screens to guide the design of a sequence-optimized gRNA library, the Toronto KnockOut version 3.0 (TKOv3) library. We then demonstrate the high effectiveness of the library relative to reference sets of essential and nonessential genes, as well as other screens using similar approaches. The optimized TKOv3 library, combined with the CEG2 reference set, provide an efficient, highly optimized platform for performing and assessing gene knockout screens in human cell lines. PMID:28655737

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.

Sequence determinants of improved CRISPR sgRNA design.

PubMed

Xu, Han; Xiao, Tengfei; Chen, Chen-Hao; Li, Wei; Meyer, Clifford A; Wu, Qiu; Wu, Di; Cong, Le; Zhang, Feng; Liu, Jun S; Brown, Myles; Liu, X Shirley

2015-08-01

The CRISPR/Cas9 system has revolutionized mammalian somatic cell genetics. Genome-wide functional screens using CRISPR/Cas9-mediated knockout or dCas9 fusion-mediated inhibition/activation (CRISPRi/a) are powerful techniques for discovering phenotype-associated gene function. We systematically assessed the DNA sequence features that contribute to single guide RNA (sgRNA) efficiency in CRISPR-based screens. Leveraging the information from multiple designs, we derived a new sequence model for predicting sgRNA efficiency in CRISPR/Cas9 knockout experiments. Our model confirmed known features and suggested new features including a preference for cytosine at the cleavage site. The model was experimentally validated for sgRNA-mediated mutation rate and protein knockout efficiency. Tested on independent data sets, the model achieved significant results in both positive and negative selection conditions and outperformed existing models. We also found that the sequence preference for CRISPRi/a is substantially different from that for CRISPR/Cas9 knockout and propose a new model for predicting sgRNA efficiency in CRISPRi/a experiments. These results facilitate the genome-wide design of improved sgRNA for both knockout and CRISPRi/a studies. © 2015 Xu et al.; Published by Cold Spring Harbor Laboratory Press.

Targeted deletion of miR-132/-212 impairs memory and alters the hippocampal transcriptome.

PubMed

Hansen, Katelin F; Sakamoto, Kensuke; Aten, Sydney; Snider, Kaitlin H; Loeser, Jacob; Hesse, Andrea M; Page, Chloe E; Pelz, Carl; Arthur, J Simon C; Impey, Soren; Obrietan, Karl

2016-02-01

miR-132 and miR-212 are structurally related microRNAs that have been found to exert powerful modulatory effects within the central nervous system (CNS). Notably, these microRNAs are tandomly processed from the same noncoding transcript, and share a common seed sequence: thus it has been difficult to assess the distinct contribution of each microRNA to gene expression within the CNS. Here, we employed a combination of conditional knockout and transgenic mouse models to examine the contribution of the miR-132/-212 gene locus to learning and memory, and then to assess the distinct effects that each microRNA has on hippocampal gene expression. Using a conditional deletion approach, we show that miR-132/-212 double-knockout mice exhibit significant cognitive deficits in spatial memory, recognition memory, and in tests of novel object recognition. Next, we utilized transgenic miR-132 and miR-212 overexpression mouse lines and the miR-132/-212 double-knockout line to explore the distinct effects of these two miRNAs on the transcriptional profile of the hippocampus. Illumina sequencing revealed that miR-132/-212 deletion increased the expression of 1138 genes; Venn analysis showed that 96 of these genes were also downregulated in mice overexpressing miR-132. Of the 58 genes that were decreased in animals overexpressing miR-212, only four of them were also increased in the knockout line. Functional gene ontology analysis of downregulated genes revealed significant enrichment of genes related to synaptic transmission, neuronal proliferation, and morphogenesis, processes known for their roles in learning, and memory formation. These data, coupled with previous studies, firmly establish a role for the miR-132/-212 gene locus as a key regulator of cognitive capacity. Further, although miR-132 and miR-212 share a seed sequence, these data indicate that these miRNAs do not exhibit strongly overlapping mRNA targeting profiles, thus indicating that these two genes may function in a complex, nonredundant manner to shape the transcriptional profile of the CNS. The dysregulation of miR-132/-212 expression could contribute to signaling mechanisms that are involved in an array of cognitive disorders. © 2016 Hansen et al.; Published by Cold Spring Harbor Laboratory Press.

Targeted deletion of miR-132/-212 impairs memory and alters the hippocampal transcriptome

PubMed Central

Hansen, Katelin F.; Sakamoto, Kensuke; Aten, Sydney; Snider, Kaitlin H.; Loeser, Jacob; Hesse, Andrea M.; Page, Chloe E.; Pelz, Carl; Arthur, J. Simon C.; Impey, Soren

2016-01-01

miR-132 and miR-212 are structurally related microRNAs that have been found to exert powerful modulatory effects within the central nervous system (CNS). Notably, these microRNAs are tandomly processed from the same noncoding transcript, and share a common seed sequence: thus it has been difficult to assess the distinct contribution of each microRNA to gene expression within the CNS. Here, we employed a combination of conditional knockout and transgenic mouse models to examine the contribution of the miR-132/-212 gene locus to learning and memory, and then to assess the distinct effects that each microRNA has on hippocampal gene expression. Using a conditional deletion approach, we show that miR-132/-212 double-knockout mice exhibit significant cognitive deficits in spatial memory, recognition memory, and in tests of novel object recognition. Next, we utilized transgenic miR-132 and miR-212 overexpression mouse lines and the miR-132/-212 double-knockout line to explore the distinct effects of these two miRNAs on the transcriptional profile of the hippocampus. Illumina sequencing revealed that miR-132/-212 deletion increased the expression of 1138 genes; Venn analysis showed that 96 of these genes were also downregulated in mice overexpressing miR-132. Of the 58 genes that were decreased in animals overexpressing miR-212, only four of them were also increased in the knockout line. Functional gene ontology analysis of downregulated genes revealed significant enrichment of genes related to synaptic transmission, neuronal proliferation, and morphogenesis, processes known for their roles in learning, and memory formation. These data, coupled with previous studies, firmly establish a role for the miR-132/-212 gene locus as a key regulator of cognitive capacity. Further, although miR-132 and miR-212 share a seed sequence, these data indicate that these miRNAs do not exhibit strongly overlapping mRNA targeting profiles, thus indicating that these two genes may function in a complex, nonredundant manner to shape the transcriptional profile of the CNS. The dysregulation of miR-132/-212 expression could contribute to signaling mechanisms that are involved in an array of cognitive disorders. PMID:26773099

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.

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

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.

Mapping Pathological Phenotypes in a Mouse Model of CDKL5 Disorder

PubMed Central

Amendola, Elena; Zhan, Yang; Mattucci, Camilla; Castroflorio, Enrico; Calcagno, Eleonora; Fuchs, Claudia; Lonetti, Giuseppina; Silingardi, Davide; Vyssotski, Alexei L.; Farley, Dominika; Ciani, Elisabetta; Pizzorusso, Tommaso; Giustetto, Maurizio; Gross, Cornelius T.

2014-01-01

Mutations in cyclin-dependent kinase-like 5 (CDKL5) cause early-onset epileptic encephalopathy, a neurodevelopmental disorder with similarities to Rett Syndrome. Here we describe the physiological, molecular, and behavioral phenotyping of a Cdkl5 conditional knockout mouse model of CDKL5 disorder. Behavioral analysis of constitutive Cdkl5 knockout mice revealed key features of the human disorder, including limb clasping, hypoactivity, and abnormal eye tracking. Anatomical, physiological, and molecular analysis of the knockout uncovered potential pathological substrates of the disorder, including reduced dendritic arborization of cortical neurons, abnormal electroencephalograph (EEG) responses to convulsant treatment, decreased visual evoked responses (VEPs), and alterations in the Akt/rpS6 signaling pathway. Selective knockout of Cdkl5 in excitatory and inhibitory forebrain neurons allowed us to map the behavioral features of the disorder to separable cell-types. These findings identify physiological and molecular deficits in specific forebrain neuron populations as possible pathological substrates in CDKL5 disorder. PMID:24838000

Deficiency of iNOS-derived NO accelerates lipid accumulation-independent liver fibrosis in non-alcoholic steatohepatitis mouse model.

PubMed

Nozaki, Yuichi; Fujita, Koji; Wada, Koichiro; Yoneda, Masato; Kessoku, Takaomi; Shinohara, Yoshiyasu; Imajo, Kento; Ogawa, Yuji; Nakamuta, Makoto; Saito, Satoru; Masaki, Naohiko; Nagashima, Yoji; Terauchi, Yasuo; Nakajima, Atsushi

2015-04-01

Although many of the factors and molecules closely associated with non-alcoholic steatohepatitis (NASH) have been reported, the role of inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO) on the progression of NASH remains unclear. We therefore investigated the role of iNOS-derived NO in NASH pathogenesis with a long-term follow-up study using systemic iNOS-knockout mice under high-fat diet (HFD) conditions. iNOS-knockout and wild-type mice were fed a basal or HFD for 10 or 48 weeks. Lipid accumulation, fibrosis, and inflammation were evaluated, and various factors and molecules closely associated with NASH were analyzed. Marked fibrosis and inflammation (indicators of NASH) were observed in the livers of iNOS-knockout mice compared to wild-type mice after 48 weeks of a HFD; however, lipid accumulation in iNOS-knockout mice livers was less than in the wild-type. Increased expressions of various cytokines that are transcriptionally controlled by NF-kB in iNOS-deficient mice livers were observed during HFD conditions. iNOS-derived NO may play a protective role against the progression to NASH during an HFD by preventing fibrosis and inflammation, which are mediated by NF-kB activation in Kupffer cells. A lack of iNOS-derived NO accelerates progression to NASH without excessive lipid accumulation.

Neutral endopeptidase (EC 3.4.24.11) terminates colitis by degrading substance P.

PubMed

Sturiale, S; Barbara, G; Qiu, B; Figini, M; Geppetti, P; Gerard, N; Gerard, C; Grady, E F; Bunnett, N W; Collins, S M

1999-09-28

Neurogenic inflammation is regulated by sensory nerves and characterized by extravasation of plasma proteins and infiltration of neutrophils from post-capillary venules and arteriolar vasodilatation. Although it is well established that substance P (SP) interacts with the neurokinin 1 receptor (NK1R) to initiate neurogenic inflammation, the mechanisms that terminate inflammation are unknown. We examined whether neutral endopeptidase (NEP), a cell-surface enzyme that degrades SP in the extracellular fluid, terminates neurogenic inflammation in the colon. In NEP knockout mice, the SP concentration in the colon was approximately 2.5-fold higher than in wild-type mice, suggesting increased bioavailability of SP. The extravasation of Evans blue-labeled plasma proteins in the colon of knockout mice under basal conditions was approximately 4-fold higher than in wild-type mice. This elevated plasma leak was attenuated by recombinant NEP or the NK1R antagonist SR140333, and is thus caused by diminished degradation of SP. To determine whether deletion of NEP predisposes mice to uncontrolled inflammation, we compared dinitrobenzene sulfonic acid-induced colitis in wild-type and knockout mice. The severity of colitis, determined by macroscopic and histologic scoring and by myeloperoxidase activity, was markedly worse in knockout than wild-type mice after 3 and 7 days. The exacerbated inflammation in knockout mice was prevented by recombinant NEP and SR140333. Thus, NEP maintains low levels of SP in the extracellular fluid under basal conditions and terminates its proinflammatory effects. Because we have previously shown that intestinal inflammation results in down-regulation of NEP and diminished degradation of SP, our present results suggest that defects in NEP expression contribute to uncontrolled inflammation.

Neutral endopeptidase (EC 3.4.24.11) terminates colitis by degrading substance P

PubMed Central

Sturiale, S.; Barbara, G.; Qiu, B.; Figini, M.; Geppetti, P.; Gerard, N.; Gerard, C.; Grady, E. F.; Bunnett, N. W.; Collins, S. M.

1999-01-01

Neurogenic inflammation is regulated by sensory nerves and characterized by extravasation of plasma proteins and infiltration of neutrophils from post-capillary venules and arteriolar vasodilatation. Although it is well established that substance P (SP) interacts with the neurokinin 1 receptor (NK1R) to initiate neurogenic inflammation, the mechanisms that terminate inflammation are unknown. We examined whether neutral endopeptidase (NEP), a cell-surface enzyme that degrades SP in the extracellular fluid, terminates neurogenic inflammation in the colon. In NEP knockout mice, the SP concentration in the colon was ≈2.5-fold higher than in wild-type mice, suggesting increased bioavailability of SP. The extravasation of Evans blue-labeled plasma proteins in the colon of knockout mice under basal conditions was ≈4-fold higher than in wild-type mice. This elevated plasma leak was attenuated by recombinant NEP or the NK1R antagonist SR140333, and is thus caused by diminished degradation of SP. To determine whether deletion of NEP predisposes mice to uncontrolled inflammation, we compared dinitrobenzene sulfonic acid-induced colitis in wild-type and knockout mice. The severity of colitis, determined by macroscopic and histologic scoring and by myeloperoxidase activity, was markedly worse in knockout than wild-type mice after 3 and 7 days. The exacerbated inflammation in knockout mice was prevented by recombinant NEP and SR140333. Thus, NEP maintains low levels of SP in the extracellular fluid under basal conditions and terminates its proinflammatory effects. Because we have previously shown that intestinal inflammation results in down-regulation of NEP and diminished degradation of SP, our present results suggest that defects in NEP expression contribute to uncontrolled inflammation. PMID:10500232

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

Using corticosteroids to reshape the gut microbiome: implications for inflammatory bowel diseases.

PubMed

Huang, Edmond Y; Inoue, Takuya; Leone, Vanessa A; Dalal, Sushila; Touw, Ketrija; Wang, Yunwei; Musch, Mark W; Theriault, Betty; Higuchi, Kazuhide; Donovan, Sharon; Gilbert, Jack; Chang, Eugene B

2015-05-01

Commensal gut microbiota play an important role in regulating metabolic and inflammatory conditions. Reshaping intestinal microbiota through pharmacologic means may be a viable treatment option. We sought to delineate the functional characteristics of glucocorticoid-mediated alterations on gut microbiota and their subsequent repercussions on host mucin regulation and colonic inflammation. Adult male C57Bl/6 mice, germ-free, Muc2-heterozygote (±), or Muc2-knockout (-/-) were injected with dexamethasone, a synthetic glucocorticoid, for 4 weeks. Fecal samples were collected for gut microbiota analysis through 16S rRNA terminal restriction fragment length polymorphism and amplicon sequencing. Intestinal mucosa was collected for mucin gene expression studies. Germ-free mice were conventionalized with gut microbes from treated and nontreated groups to determine their functional capacities in recipient hosts. Exposure to dexamethasone in wild-type mice led to substantial shifts in gut microbiota over a 4-week period. Furthermore, a significant downregulation of colonic Muc2 gene expression was observed after treatment. Muc2-knockout mice harbored a proinflammatory environment of gut microbes, characterized by the increase or decrease in prevalence of specific microbiota populations such as Clostridiales and Lactobacillaceae, respectively. This colitogenic phenotype was transmissible to IL10-knockout mice, a genetically susceptible model of colonic inflammatory disorders. Microbiota from donors pretreated with dexamethasone, however, ameliorated symptoms of inflammation. Commensal gut bacteria may be a key mediator of the anti-inflammatory effects observed in the large intestine after glucocorticoid exposure. These findings underscore the notion that intestinal microbes comprise a "microbial organ" essential for host physiology that can be targeted by therapeutic approaches to restore intestinal homeostasis.

Murine GRPR and Stathmin Control in Opposite Directions both Cued Fear Extinction and Neural Activities of the Amygdala and Prefrontal Cortex

PubMed Central

Martel, Guillaume; Hevi, Charles; Wong, Alexandra; Zushida, Ko; Uchida, Shusaku; Shumyatsky, Gleb P.

2012-01-01

Extinction is an integral part of normal healthy fear responses, while it is compromised in several fear-related mental conditions in humans, such as post-traumatic stress disorder (PTSD). Although much research has recently been focused on fear extinction, its molecular and cellular underpinnings are still unclear. The development of animal models for extinction will greatly enhance our approaches to studying its neural circuits and the mechanisms involved. Here, we describe two gene-knockout mouse lines, one with impaired and another with enhanced extinction of learned fear. These mutant mice are based on fear memory-related genes, stathmin and gastrin-releasing peptide receptor (GRPR). Remarkably, both mutant lines showed changes in fear extinction to the cue but not to the context. We performed indirect imaging of neuronal activity on the second day of cued extinction, using immediate-early gene c-Fos. GRPR knockout mice extinguished slower (impaired extinction) than wildtype mice, which was accompanied by an increase in c-Fos activity in the basolateral amygdala and a decrease in the prefrontal cortex. By contrast, stathmin knockout mice extinguished faster (enhanced extinction) and showed a decrease in c-Fos activity in the basolateral amygdala and an increase in the prefrontal cortex. At the same time, c-Fos activity in the dentate gyrus was increased in both mutant lines. These experiments provide genetic evidence that the balance between neuronal activities of the amygdala and prefrontal cortex defines an impairment or facilitation of extinction to the cue while the hippocampus is involved in the context-specificity of extinction. PMID:22312434

NODAL in the Uterus Is Necessary for Proper Placental Development and Maintenance of Pregnancy1

PubMed Central

Park, Craig B.; DeMayo, Francesco J.; Lydon, John P.; Dufort, Daniel

2012-01-01

Preterm birth is the single leading cause of perinatal mortality in developed countries, affecting approximately 12% of pregnancies and accounting for 75% of neonatal loss in the United States. Despite the prevalence and severity of premature delivery, the causes and mechanisms that underlie spontaneous and idiopathic preterm birth remain unknown. Our inability to elucidate these fundamental causes has been attributed to a poor understanding of the signaling pathways associated with the premature induction of parturition and a lack of suitable animal models available for preterm birth research. In this study, we describe the generation and analysis of a novel conditional knockout of the transforming growth factor beta (TGFB) superfamily member, Nodal, from the maternal reproductive tract of mice. Strikingly, uterine Nodal knockout females exhibited a severe malformation of the maternal decidua basalis during placentation, leading to significant intrauterine growth restriction, and ultimately preterm birth and fetal loss on Day 17.5 of gestation. Using several approaches, we characterized aberrant placental development and demonstrated that reduced proliferation combined with increased apoptosis resulted in a diminished decidua basalis and compromised maternal-fetal interface. Last, we evaluated various components of the established parturition cascade and determined that preterm birth derived from the maternal Nodal knockout occurs prior to PTGS2 (COX-2) upregulation at the placental interface. Taken together, the results presented in this study highlight an in vivo role for maternal NODAL during placentation, present an interesting link between disrupted decidua basalis formation and premature parturition, and describe a potentially valuable model toward elucidating the complex processes that underlie preterm birth. PMID:22378764

Murine GRPR and stathmin control in opposite directions both cued fear extinction and neural activities of the amygdala and prefrontal cortex.

PubMed

Martel, Guillaume; Hevi, Charles; Wong, Alexandra; Zushida, Ko; Uchida, Shusaku; Shumyatsky, Gleb P

2012-01-01

Extinction is an integral part of normal healthy fear responses, while it is compromised in several fear-related mental conditions in humans, such as post-traumatic stress disorder (PTSD). Although much research has recently been focused on fear extinction, its molecular and cellular underpinnings are still unclear. The development of animal models for extinction will greatly enhance our approaches to studying its neural circuits and the mechanisms involved. Here, we describe two gene-knockout mouse lines, one with impaired and another with enhanced extinction of learned fear. These mutant mice are based on fear memory-related genes, stathmin and gastrin-releasing peptide receptor (GRPR). Remarkably, both mutant lines showed changes in fear extinction to the cue but not to the context. We performed indirect imaging of neuronal activity on the second day of cued extinction, using immediate-early gene c-Fos. GRPR knockout mice extinguished slower (impaired extinction) than wildtype mice, which was accompanied by an increase in c-Fos activity in the basolateral amygdala and a decrease in the prefrontal cortex. By contrast, stathmin knockout mice extinguished faster (enhanced extinction) and showed a decrease in c-Fos activity in the basolateral amygdala and an increase in the prefrontal cortex. At the same time, c-Fos activity in the dentate gyrus was increased in both mutant lines. These experiments provide genetic evidence that the balance between neuronal activities of the amygdala and prefrontal cortex defines an impairment or facilitation of extinction to the cue while the hippocampus is involved in the context-specificity of extinction.

CRISPR/Cas9 knockout of HAS2 in rat chondrosarcoma chondrocytes demonstrates the requirement of hyaluronan for aggrecan retention

PubMed Central

Huang, Yi; Askew, Emily B.; Knudson, Cheryl B.; Knudson, Warren

2016-01-01

Hyaluronan (HA) plays an essential role in cartilage where it functions to retain aggrecan. Previous studies have suggested that aggrecan is anchored indirectly to the plasma membrane of chondrocytes via its binding to cell-associated HA. However, reagents used to test these observations such as hyaluronidase and HA oligosaccharides are short term and may have side activities that complicate interpretation. Using the CRISPR/Cas9 gene editing approach, a model system was developed by generating HA-deficient chondrocyte cell lines. HA synthase-2 (Has2)-specific single guide RNA was introduced into two different variant lines of rat chondrosarcoma chondrocytes; knockout clones were isolated and characterized. Two other members of the HA synthase gene family were expressed at very low relative copy number but showed no compensatory response in the Has2 knockouts. Wild type chondrocytes of both variants exhibited large pericellular matrices or coats extending from the plasma membrane. Addition of purified aggrecan monomer expanded the size of these coats as the proteoglycan became retained within the pericellular matrix. Has2 knockout chondrocytes lost all capacity to assemble a particle-excluding pericellular matrix and more importantly, no matrices formed around the knockout cells following the addition of purified aggrecan. When grown as pellet cultures so as to generate a bioengineered neocartilage tissue, the Has2 knockout chondrocytes assumed a tightly-compacted morphology as compared to the wild type cells. When knockout chondrocytes were transduced with Adeno-ZsGreen1-mycHas2, the cell-associated pericellular matrices were restored including the capacity to bind and incorporate additional exogenous aggrecan into the matrix. These results suggest that HA is essential for aggrecan retention and maintaining cell separation during tissue formation. PMID:27094859

The heat shock protein 90 of Toxoplasma gondii is essential for invasion of host cells and tachyzoite growth

PubMed Central

Sun, Hongchao; Zhuo, Xunhui; Zhao, Xianfeng; Yang, Yi; Chen, Xueqiu; Yao, Chaoqun; Du, Aifang

2017-01-01

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that infects almost all warm-blooded vertebrates. Heat shock proteins (HSP) regulate key signal transduction events in many organisms, and heat shock protein 90 (Hsp90) plays an important role in growth, development, and virulence in several parasitic protozoa. Here, we discovered increased transcription of the Hsp90 gene under conditions for bradyzoite differentiation, i.e. alkaline and heat shock conditions in vitro, suggesting that Hsp90 may be connected with bradyzoite development in T. gondii. A knockout of the TgHsp90 strain (ΔHsp90) and a complementation strain were constructed. The TgHsp90 knockout cells were found to be defective in host-cell invasion, were not able to proliferate in vitro in Vero cells, and did not show long-time survival in mice in vivo. These inabilities of the knockout parasites were restored upon complementation of TgHsp90. These data unequivocally show that TgHsp90 contributes to bradyzoite development, and to invasion and replication of T. gondii in host cells. PMID:28627357

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.

[Upregulation of P2X3 receptors in dorsal root ganglion of TRPV1 knockout female mice].

PubMed

Fang, Xiao; Shi, Xiao-Han; Huang, Li-Bin; Rong, Wei-Fang; Ma, Bei

2014-08-25

The study was aimed to investigate the changes in mechanical pain threshold in the condition of chronic inflammatory pain after transient receptor potential vanilloid 1 (TRPV1) gene was knockout. Hind-paw intraplantar injection of complete freund's adjuvant (CFA, 20 μL) produced peripheral inflammation in wild-type and TRPV1 knockout female mice. The mechanical pain thresholds were measured during the 8 days after injection and pre-injection by using Von-Frey hair. Nine days after injection, mice were killed and the differences of expression of c-Fos and P2X3 receptor in the dorsal root ganglia (DRG) and spinal cord dorsal horn were examined by Western blotting between the two groups. Compared with that in wild-type mice, the mechanical pain threshold was increased significantly in TRPV1 knockout mice (P < 0.05); 3 days after CFA injection, the baseline mechanical pain threshold in the TRPV1 knockout mice group was significantly higher than that in the wild-type mice group (P < 0.05); The result of Western blotting showed that the expression of c-Fos protein both in DRG and spinal cord dorsal horn of TRPV1 knockout mice group was decreased significantly compared with that in wild-type mice group (P < 0.01, P < 0.05), while the expression of P2X3 receptor in DRG of TRPV1 knockout mice group was increased significantly compared with that in wild-type mice group (P < 0.05). Our findings indicate that TRPV1 may influence the peripheral mechanical pain threshold by mediating the expression of c-Fos protein both in DRG and spinal cord dorsal horn and changing the expression of P2X3 receptor in DRG.

Behavioral characterization of mice deficient in the phosphodiesterase-10A (PDE10A) enzyme on a C57/Bl6N congenic background.

PubMed

Siuciak, Judith A; McCarthy, Sheryl A; Chapin, Douglas S; Martin, Ashley N; Harms, John F; Schmidt, Christopher J

2008-02-01

The phenotype of genetically modified animals is strongly influenced by both the genetic background of the animal as well as environmental factors. We have previously reported the behavioral and neurochemical characterization of PDE10A knockout mice maintained on a DBA1LacJ (PDE10A(DBA)) genetic background. The aim of the present studies was to assess the behavioral and neurochemical phenotype of PDE10A knockout mice on an alternative congenic C57BL/6N (PDE10A(C57)) genetic background. Consistent with our previous results, PDE10A(C57) knockout mice showed a decrease in exploratory locomotor activity and a delay in the acquisition of conditioned avoidance responding. Also consistent with previous studies, the elimination of PDE10A did not alter basal levels of striatal cGMP or cAMP or affect behavior in several other well-characterized behavioral assays. PDE10A(C57) knockout mice showed a blunted response to MK-801, although to a lesser degree than previously observed in the PDE10A(DBA) knockout mice, and no differences were observed following a PCP challenge. PDE10A(C57) knockout mice showed a significant change in striatal dopamine turnover, which was accompanied by an enhanced locomotor response to AMPH, These studies demonstrate that while many of the behavioral effects of the PDE10A gene deletion appear to be independent of genetic background, the impact of the deletion on behavior can vary in magnitude. Furthermore, the effects on the dopaminergic system appear to be background-dependent, with significant effects observed only in knockout mice on the C57BL6N genetic background.

[Quantitative changes of main components of erythrocyte membranes which define architectonics of cells under pttg gene knockout].

PubMed

Kaniuka, O P; Filiak, Ie Z; Kulachkovs'kyĭ, O R; Osyp, Iu L; Sybirna, N O

2014-01-01

A pttg gene knockout affects the functional state of erythron in mice which could be associated with structural changes in the structure of erythrocyte membranes. The pttg gene knockout causes a significant modification of fatty acids composition of erythrocyte membrane lipids by reducing the content of palmitic acid and increasing of polyunsaturated fatty acids amount by 18%. Analyzing the erythrocyte surface architectonics of mice under pttg gene knockout, it was found that on the background of reduction of the functionally complete biconcave discs population one could observe an increase of the number of transformed cells at different degeneration stages. Researches have shown that in mice with a pttg gene knockout compared with a control group of animals cytoskeletal protein--beta-spectrin was reduced by 17.03%. However, there is a reduction of membrane protein band 3 by 33.04%, simultaneously the content of anion transport protein band 4.5 increases by 35.2% and protein band 4.2 by 32.1%. The lectin blot analysis has helped to reveal changes in the structure of the carbohydrate determinants of erythrocyte membrane glycoproteins under conditions of directed pttg gene inactivation, accompanied by changes in the type of communication, which joins the terminal residue in carbohydrate determinant of glycoproteins. Thus, a significant redistribution of protein and fatty acids contents in erythrocyte membranes that manifested in the increase of the deformed shape of red blood cells is observed underpttg gene knockout.

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

Quasi-free Proton Knockout Reactions on the Oxygen Isotopic Chain

NASA Astrophysics Data System (ADS)

Atar, Leyla; Aumann, Thomas; Bertulani, Carlos; Paschalis, Stefanos; R3B Collaboration

2017-09-01

It is well known from electron-induced knockout data that the single-particle (SP) strength is reduced to about 60-70% for stable nuclei in comparison to the independent particle model due to the presence of short- and long-range correlations. This finding has been confirmed by nuclear knockout reactions using stable and exotic beams, however, with a strong dependency on the proton-neutron asymmetry. The observed strong reduction of SP cross sections for the deeply bound valence nucleons in asymmetric nuclei is theoretically not understood. To understand this dependency quantitatively a complementary approach, quasi-free (QF) knockout reactions in inverse kinematics, is introduced. We have performed a systematic study of spectroscopic strength of oxygen isotopes using QF (p,2p) knockout reactions in complete kinematics at the R3B/LAND setup at GSI with secondary beams containing 13-24O. The oxygen isotopic chain covers a large variation of separ ation energies, which allow a systematic study of SF with respect to isospin asymmetry. We will present results on the (p,2p) cross sections for the entire oxygen isotopic chain obtained from a single experiment. By comparison with the Eikonal reaction theory the SF and reduction factors will be presented. The work is supported by GSI-TU Darmstadt cooperation and BMBF project 05P15RDFN1.

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.

Inhibition of the cluster of differentiation 14 innate immunity pathway with IAXO-101 improves chronic microelectrode performance

NASA Astrophysics Data System (ADS)

Hermann, John K.; Ravikumar, Madhumitha; Shoffstall, Andrew J.; Ereifej, Evon S.; Kovach, Kyle M.; Chang, Jeremy; Soffer, Arielle; Wong, Chun; Srivastava, Vishnupriya; Smith, Patrick; Protasiewicz, Grace; Jiang, Jingle; Selkirk, Stephen M.; Miller, Robert H.; Sidik, Steven; Ziats, Nicholas P.; Taylor, Dawn M.; Capadona, Jeffrey R.

2018-04-01

Objective. Neuroinflammatory mechanisms are hypothesized to contribute to intracortical microelectrode failures. The cluster of differentiation 14 (CD14) molecule is an innate immunity receptor involved in the recognition of pathogens and tissue damage to promote inflammation. The goal of the study was to investigate the effect of CD14 inhibition on intracortical microelectrode recording performance and tissue integration. Approach. Mice implanted with intracortical microelectrodes in the motor cortex underwent electrophysiological characterization for 16 weeks, followed by endpoint histology. Three conditions were examined: (1) wildtype control mice, (2) knockout mice lacking CD14, and (3) wildtype control mice administered a small molecule inhibitor to CD14 called IAXO-101. Main results. The CD14 knockout mice exhibited acute but not chronic improvements in intracortical microelectrode performance without significant differences in endpoint histology. Mice receiving IAXO-101 exhibited significant improvements in recording performance over the entire 16 week duration without significant differences in endpoint histology. Significance. Full removal of CD14 is beneficial at acute time ranges, but limited CD14 signaling is beneficial at chronic time ranges. Innate immunity receptor inhibition strategies have the potential to improve long-term intracortical microelectrode performance.

Discrimination between cocaine-associated context and cue in a modified conditioned place preference paradigm: role of the nNOS gene in cue conditioning.

PubMed

Itzhak, Yossef; Roger-Sánchez, Concepción; Kelley, Jonathan B; Anderson, Karen L

2010-03-01

The conditioned place preference (CPP) paradigm entails appetitive learning and is utilized to investigate the motivational effects of drug and natural reward in rodents. However, a typical CPP design does not allow dissociation between cue- and context-dependent appetitive learning. In humans, context and cues that had been associated with drug reward can elicit conditioned response and drug craving. Therefore, we investigated (a) methods by which to discriminate between cue- and context-dependent appetitive learning, and (b) the role of the neuronal nitric oxide synthase (nNOS) gene in appetitive learning. Wild-type (WT) and nNOS knockout (KO) mice were trained by cocaine (20 mg/kg) in a discrete context paired with a light cue (a compound context-cue stimulus). In test 1, approach behaviour to either the training context or to the cue in a novel context was determined. WT mice showed robust preference for both cocaine-associated context and cue. nNOS KO mice acquired approach behaviour for the cocaine-associated context but not cue. This finding suggests that the nNOS gene is required for cue-dependent appetitive learning. On the following day (test 2), mice were tested for approach behaviour to the compound context-cue stimulus. Context but not cue exposure in test 1 reduced approach behaviour to the compound context-cue stimulus in test 2, suggesting that repeated context but not cue exposures diminished the conditioned response. Hence, this modified CPP paradigm is useful for the investigation of approach behaviour for both drug-associated context and cue, and allows further investigation of mechanisms underlying cue- and context-dependent appetitive learning.

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.

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

Differentially expressed genes during the imbibition of dormant and after-ripened seeds - a reverse genetics approach.

PubMed

Yazdanpanah, Farzaneh; Hanson, Johannes; Hilhorst, Henk W M; Bentsink, Leónie

2017-09-11

Seed dormancy, defined as the incapability of a viable seed to germinate under favourable conditions, is an important trait in nature and agriculture. Despite extensive research on dormancy and germination, many questions about the molecular mechanisms controlling these traits remain unanswered, likely due to its genetic complexity and the large environmental effects which are characteristic of these quantitative traits. To boost research towards revealing mechanisms in the control of seed dormancy and germination we depend on the identification of genes controlling those traits. We used transcriptome analysis combined with a reverse genetics approach to identify genes that are prominent for dormancy maintenance and germination in imbibed seeds of Arabidopsis thaliana. Comparative transcriptomics analysis was employed on freshly harvested (dormant) and after-ripened (AR; non-dormant) 24-h imbibed seeds of four different DELAY OF GERMINATION near isogenic lines (DOGNILs) and the Landsberg erecta (Ler) wild type with varying levels of primary dormancy. T-DNA knock-out lines of the identified genes were phenotypically investigated for their effect on dormancy and AR. We identified conserved sets of 46 and 25 genes which displayed higher expression in seeds of all dormant and all after-ripened DOGNILs and Ler, respectively. Knock-out mutants in these genes showed dormancy and germination related phenotypes. Most of the identified genes had not been implicated in seed dormancy or germination. This research will be useful to further decipher the molecular mechanisms by which these important ecological and commercial traits are regulated.

Altered sleep and affect in the neurotensin receptor 1 knockout mouse.

PubMed

Fitzpatrick, Karrie; Winrow, Christopher J; Gotter, Anthony L; Millstein, Joshua; Arbuzova, Janna; Brunner, Joseph; Kasarskis, Andrew; Vitaterna, Martha H; Renger, John J; Turek, Fred W

2012-07-01

Sleep and mood disorders have long been understood to have strong genetic components, and there is considerable comorbidity of sleep abnormalities and mood disorders, suggesting the involvement of common genetic pathways. Here, we examine a candidate gene implicated in the regulation of both sleep and affective behavior using a knockout mouse model. Previously, we identified a quantitative trait locus (QTL) for REM sleep amount, REM sleep bout number, and wake amount in a genetically segregating population of mice. Here, we show that traits mapping to this QTL correlated with an expression QTL for neurotensin receptor 1 (Ntsr1), a receptor for neurotensin, a ligand known to be involved in several psychiatric disorders. We examined sleep as well as behaviors indicative of anxiety and depression in the NTSR1 knockout mouse. NTSR1 knockouts had a lower percentage of sleep time spent in REM sleep in the dark phase and a larger diurnal variation in REM sleep duration than wild types under baseline conditions. Following sleep deprivation, NTSR1 knockouts exhibited more wake and less NREM rebound sleep. NTSR1 knockouts also showed increased anxious and despair behaviors. Here we illustrate a link between expression of the Ntsr1 gene and sleep traits previously associated with a particular QTL. We also demonstrate a relationship between Ntsr1 and anxiety and despair behaviors. Given the considerable evidence that anxiety and depression are closely linked with abnormalities in sleep, the data presented here provide further evidence that neurotensin and Ntsr1 may be a component of a pathway involved in both sleep and mood disorders.

Mu opioid receptors on primary afferent nav1.8 neurons contribute to opiate-induced analgesia: insight from conditional knockout mice.

PubMed

Weibel, Raphaël; Reiss, David; Karchewski, Laurie; Gardon, Olivier; Matifas, Audrey; Filliol, Dominique; Becker, Jérôme A J; Wood, John N; Kieffer, Brigitte L; Gaveriaux-Ruff, Claire

2013-01-01

Opiates are powerful drugs to treat severe pain, and act via mu opioid receptors distributed throughout the nervous system. Their clinical use is hampered by centrally-mediated adverse effects, including nausea or respiratory depression. Here we used a genetic approach to investigate the potential of peripheral mu opioid receptors as targets for pain treatment. We generated conditional knockout (cKO) mice in which mu opioid receptors are deleted specifically in primary afferent Nav1.8-positive neurons. Mutant animals were compared to controls for acute nociception, inflammatory pain, opiate-induced analgesia and constipation. There was a 76% decrease of mu receptor-positive neurons and a 60% reduction of mu-receptor mRNA in dorsal root ganglia of cKO mice. Mutant mice showed normal responses to heat, mechanical, visceral and chemical stimuli, as well as unchanged morphine antinociception and tolerance to antinociception in models of acute pain. Inflammatory pain developed similarly in cKO and controls mice after Complete Freund's Adjuvant. In the inflammation model, however, opiate-induced (morphine, fentanyl and loperamide) analgesia was reduced in mutant mice as compared to controls, and abolished at low doses. Morphine-induced constipation remained intact in cKO mice. We therefore genetically demonstrate for the first time that mu opioid receptors partly mediate opiate analgesia at the level of Nav1.8-positive sensory neurons. In our study, this mechanism operates under conditions of inflammatory pain, but not nociception. Previous pharmacology suggests that peripheral opiates may be clinically useful, and our data further demonstrate that Nav1.8 neuron-associated mu opioid receptors are feasible targets to alleviate some forms of persistent pain.

Mu Opioid Receptors on Primary Afferent Nav1.8 Neurons Contribute to Opiate-Induced Analgesia: Insight from Conditional Knockout Mice

PubMed Central

Karchewski, Laurie; Gardon, Olivier; Matifas, Audrey; Filliol, Dominique; Becker, Jérôme A. J.; Wood, John N.; Kieffer, Brigitte L.; Gaveriaux-Ruff, Claire

2013-01-01

Opiates are powerful drugs to treat severe pain, and act via mu opioid receptors distributed throughout the nervous system. Their clinical use is hampered by centrally-mediated adverse effects, including nausea or respiratory depression. Here we used a genetic approach to investigate the potential of peripheral mu opioid receptors as targets for pain treatment. We generated conditional knockout (cKO) mice in which mu opioid receptors are deleted specifically in primary afferent Nav1.8-positive neurons. Mutant animals were compared to controls for acute nociception, inflammatory pain, opiate-induced analgesia and constipation. There was a 76% decrease of mu receptor-positive neurons and a 60% reduction of mu-receptor mRNA in dorsal root ganglia of cKO mice. Mutant mice showed normal responses to heat, mechanical, visceral and chemical stimuli, as well as unchanged morphine antinociception and tolerance to antinociception in models of acute pain. Inflammatory pain developed similarly in cKO and controls mice after Complete Freund’s Adjuvant. In the inflammation model, however, opiate-induced (morphine, fentanyl and loperamide) analgesia was reduced in mutant mice as compared to controls, and abolished at low doses. Morphine-induced constipation remained intact in cKO mice. We therefore genetically demonstrate for the first time that mu opioid receptors partly mediate opiate analgesia at the level of Nav1.8-positive sensory neurons. In our study, this mechanism operates under conditions of inflammatory pain, but not nociception. Previous pharmacology suggests that peripheral opiates may be clinically useful, and our data further demonstrate that Nav1.8 neuron-associated mu opioid receptors are feasible targets to alleviate some forms of persistent pain. PMID:24069332

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.

Transcriptional and phenotypic comparisons of Ppara knockout and siRNA knockdown mice

PubMed Central

De Souza, Angus T.; Dai, Xudong; Spencer, Andrew G.; Reppen, Tom; Menzie, Ann; Roesch, Paula L.; He, Yudong; Caguyong, Michelle J.; Bloomer, Sherri; Herweijer, Hans; Wolff, Jon A.; Hagstrom, James E.; Lewis, David L.; Linsley, Peter S.; Ulrich, Roger G.

2006-01-01

RNA interference (RNAi) has great potential as a tool for studying gene function in mammals. However, the specificity and magnitude of the in vivo response to RNAi remains to be fully characterized. A molecular and phenotypic comparison of a genetic knockout mouse and the corresponding knockdown version would help clarify the utility of the RNAi approach. Here, we used hydrodynamic delivery of small interfering RNA (siRNA) to knockdown peroxisome proliferator activated receptor alpha (Ppara), a gene that is central to the regulation of fatty acid metabolism. We found that Ppara knockdown in the liver results in a transcript profile and metabolic phenotype that is comparable to those of Ppara−/− mice. Combining the profiles from mice treated with the PPARα agonist fenofibrate, we confirmed the specificity of the RNAi response and identified candidate genes proximal to PPARα regulation. Ppara knockdown animals developed hypoglycemia and hypertriglyceridemia, phenotypes observed in Ppara−/− mice. In contrast to Ppara−/− mice, fasting was not required to uncover these phenotypes. Together, these data validate the utility of the RNAi approach and suggest that siRNA can be used as a complement to classical knockout technology in gene function studies. PMID:16945951

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.

Proteome analysis of a hepatocyte-specific BIRC5 (survivin)-knockout mouse model during liver regeneration.

PubMed

Bracht, Thilo; Hagemann, Sascha; Loscha, Marius; Megger, Dominik A; Padden, Juliet; Eisenacher, Martin; Kuhlmann, Katja; Meyer, Helmut E; Baba, Hideo A; Sitek, Barbara

2014-06-06

The Baculoviral IAP repeat-containing protein 5 (BIRC5), also known as inhibitor of apoptosis protein survivin, is a member of the chromosomal passenger complex and a key player in mitosis. To investigate the function of BIRC5 in liver regeneration, we analyzed a hepatocyte-specific BIRC5-knockout mouse model using a quantitative label-free proteomics approach. Here, we present the analyses of the proteome changes in hepatocyte-specific BIRC5-knockout mice compared to wildtype mice, as well as proteome changes during liver regeneration induced by partial hepatectomy in wildtype mice and mice lacking hepatic BIRC5, respectively. The BIRC5-knockout mice showed an extensive overexpression of proteins related to cellular maintenance, organization and protein synthesis. Key regulators of cell growth, transcription and translation MTOR and STAT1/STAT2 were found to be overexpressed. During liver regeneration proteome changes representing a response to the mitotic stimulus were detected in wildtype mice. Mainly proteins corresponding to proliferation, cell cycle and cytokinesis were up-regulated. The hepatocyte-specific BIRC5-knockout mice showed impaired liver regeneration, which had severe consequences on the proteome level. However, several proteins with function in mitosis were found to be up-regulated upon the proliferative stimulus. Our results show that the E3 ubiquitin-protein ligase UHRF1 is strongly up-regulated during liver regeneration independently of BIRC5.

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.

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.

Macrophage Migration Inhibitory Factor (MIF) Deficiency Exacerbates Aging-Induced Cardiac Remodeling and Dysfunction Despite Improved Inflammation: Role of Autophagy Regulation.

PubMed

Xu, Xihui; Pang, Jiaojiao; Chen, Yuguo; Bucala, Richard; Zhang, Yingmei; Ren, Jun

2016-03-04

Aging leads to unfavorable geometric and functional sequelae in the heart. The proinflammatory cytokine macrophage migration inhibitory factor (MIF) plays a role in the maintenance of cardiac homeostasis under stress conditions although its impact in cardiac aging remains elusive. This study was designed to evaluate the role of MIF in aging-induced cardiac anomalies and the underlying mechanism involved. Cardiac geometry, contractile and intracellular Ca(2+) properties were examined in young (3-4 mo) or old (24 mo) wild type and MIF knockout (MIF(-/-)) mice. Our data revealed that MIF knockout exacerbated aging-induced unfavorable structural and functional changes in the heart. The detrimental effect of MIF knockout was associated with accentuated loss in cardiac autophagy with aging. Aging promoted cardiac inflammation, the effect was attenuated by MIF knockout. Intriguingly, aging-induced unfavorable responses were reversed by treatment with the autophagy inducer rapamycin, with improved myocardial ATP availability in aged WT and MIF(-/-) mice. Using an in vitro model of senescence, MIF knockdown exacerbated doxorubicin-induced premature senescence in H9C2 myoblasts, the effect was ablated by MIF replenishment. Our data indicated that MIF knockout exacerbates aging-induced cardiac remodeling and functional anomalies despite improved inflammation, probably through attenuating loss of autophagy and ATP availability in the heart.

Using Corticosteroids to Reshape the Gut Microbiome: Implications for Inflammatory Bowel Diseases

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

Huang, Edmond Y.; Inoue, Takuya; Leone, Vanessa A.

Introduction—Commensal gut microbiota play an important role in regulating metabolic and inflammatory conditions. Reshaping intestinal microbiota through pharmacologic means may be a viable treatment option. Here we sought to delineate the functional characteristics of glucocorticoid-mediated alterations on gut microbiota and their subsequent repercussions on host mucin regulation and colonic inflammation. Methods—Adult male C57Bl/6 mice, germ-free (GF), Muc2-heterozygote (±), or Muc2-knockout (-/-) were injected with dexamethasone, a synthetic glucocorticoid, for four weeks. Fecal samples were collected for gut microbiota analysis via 16S rRNA T-RFLP and amplicon sequencing. Intestinal mucosa was collected for mucin gene expression studies. GF mice were conventionalized withmore » gut microbes from treated- and non-treated groups to determine their functional capacities in recipient hosts. Results—Exposure to DEX in WT mice led to substantial shifts in gut microbiota over a four-week period. Furthermore, a significant down-regulation of colonic Muc2 gene expression was observed after treatment. Muc2-knockout mice harbored a pro-inflammatory environment of gut microbes, characterized by the increase or decrease in prevalence of specific microbiota populations such as Clostridiales and Lactobacillaceae, respectively. This colitogenic phenotype was transmissible to IL10-knockout (IL10-KO) mice, a genetically susceptible model of colonic inflammatory disorders. Microbiota from donors pre-treated with DEX, however, ameliorated symptoms of inflammation. We conclude that commensal gut bacteria may be a key mediator of the anti-inflammatory effects observed in the large intestine after GC exposure. These findings underscore the notion that intestinal microbes comprise a “microbial organ” essential for host physiology that can be targeted by therapeutic approaches to restore intestinal homeostasis.« less

Using Corticosteroids to Reshape the Gut Microbiome: Implications for Inflammatory Bowel Diseases

DOE PAGES

Huang, Edmond Y.; Inoue, Takuya; Leone, Vanessa A.; ...

2015-05-01

Introduction—Commensal gut microbiota play an important role in regulating metabolic and inflammatory conditions. Reshaping intestinal microbiota through pharmacologic means may be a viable treatment option. Here we sought to delineate the functional characteristics of glucocorticoid-mediated alterations on gut microbiota and their subsequent repercussions on host mucin regulation and colonic inflammation. Methods—Adult male C57Bl/6 mice, germ-free (GF), Muc2-heterozygote (±), or Muc2-knockout (-/-) were injected with dexamethasone, a synthetic glucocorticoid, for four weeks. Fecal samples were collected for gut microbiota analysis via 16S rRNA T-RFLP and amplicon sequencing. Intestinal mucosa was collected for mucin gene expression studies. GF mice were conventionalized withmore » gut microbes from treated- and non-treated groups to determine their functional capacities in recipient hosts. Results—Exposure to DEX in WT mice led to substantial shifts in gut microbiota over a four-week period. Furthermore, a significant down-regulation of colonic Muc2 gene expression was observed after treatment. Muc2-knockout mice harbored a pro-inflammatory environment of gut microbes, characterized by the increase or decrease in prevalence of specific microbiota populations such as Clostridiales and Lactobacillaceae, respectively. This colitogenic phenotype was transmissible to IL10-knockout (IL10-KO) mice, a genetically susceptible model of colonic inflammatory disorders. Microbiota from donors pre-treated with DEX, however, ameliorated symptoms of inflammation. We conclude that commensal gut bacteria may be a key mediator of the anti-inflammatory effects observed in the large intestine after GC exposure. These findings underscore the notion that intestinal microbes comprise a “microbial organ” essential for host physiology that can be targeted by therapeutic approaches to restore intestinal homeostasis.« less

Whirlin Replacement Restores the Formation of the USH2 Protein Complex in Whirlin Knockout Photoreceptors

PubMed Central

Zou, Junhuang; Luo, Ling; Shen, Zuolian; Chiodo, Vince A.; Ambati, Balamurali K.; Hauswirth, William W.

2011-01-01

Purpose. Whirlin is the causative gene for Usher syndrome type IID (USH2D), a condition manifested as both retinitis pigmentosa and congenital deafness. Mutations in this gene cause disruption of the USH2 protein complex composed of USH2A and VLGR1 at the periciliary membrane complex (PMC) in photoreceptors. In this study, the adeno-associated virus (AAV)-mediated whirlin replacement was evaluated as a treatment option. Methods. Murine whirlin cDNA driven by the human rhodopsin kinase promoter (hRK) was packaged as an AAV2/5 vector and delivered into the whirlin knockout retina through subretinal injection. The efficiency, efficacy, and safety of this treatment were examined using immunofluorescent staining, confocal imaging, immunoelectron microscopy, Western blot analysis, histologic analysis, and electroretinogram. Results. The AAV-mediated whirlin expression started at two weeks, reached its maximum level at 10 weeks, and lasted up to six months post injection. The transgenic whirlin product had a molecular size and an expression level comparable to the wild-type. It was distributed at the PMC in both rod and cone photoreceptors from the central to peripheral retina. Importantly, the transgenic whirlin restored the cellular localization and expression level of both USH2A and VLGR1 and did not cause defects in the retinal histology and function in the whirlin knockout mouse. Conclusions. Whirlin transgene recruits USH2A and VLGR1 to the PMC and is sufficient for the formation of the USH2 protein complex in photoreceptors. The combined hRK and AAV gene delivery system could be an effective gene therapy approach to treat retinal degeneration in USH2D patients. PMID:21212183

Selection on Network Dynamics Drives Differential Rates of Protein Domain Evolution

PubMed Central

Mannakee, Brian K.; Gutenkunst, Ryan N.

2016-01-01

The long-held principle that functionally important proteins evolve slowly has recently been challenged by studies in mice and yeast showing that the severity of a protein knockout only weakly predicts that protein’s rate of evolution. However, the relevance of these studies to evolutionary changes within proteins is unknown, because amino acid substitutions, unlike knockouts, often only slightly perturb protein activity. To quantify the phenotypic effect of small biochemical perturbations, we developed an approach to use computational systems biology models to measure the influence of individual reaction rate constants on network dynamics. We show that this dynamical influence is predictive of protein domain evolutionary rate within networks in vertebrates and yeast, even after controlling for expression level and breadth, network topology, and knockout effect. Thus, our results not only demonstrate the importance of protein domain function in determining evolutionary rate, but also the power of systems biology modeling to uncover unanticipated evolutionary forces. PMID:27380265

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

Altered Sleep and Affect in the Neurotensin Receptor 1 Knockout Mouse

PubMed Central

Fitzpatrick, Karrie; Winrow, Christopher J.; Gotter, Anthony L.; Millstein, Joshua; Arbuzova, Janna; Brunner, Joseph; Kasarskis, Andrew; Vitaterna, Martha H.; Renger, John J.; Turek, Fred W.

2012-01-01

Study Objective: Sleep and mood disorders have long been understood to have strong genetic components, and there is considerable comorbidity of sleep abnormalities and mood disorders, suggesting the involvement of common genetic pathways. Here, we examine a candidate gene implicated in the regulation of both sleep and affective behavior using a knockout mouse model. Design: Previously, we identified a quantitative trait locus (QTL) for REM sleep amount, REM sleep bout number, and wake amount in a genetically segregating population of mice. Here, we show that traits mapping to this QTL correlated with an expression QTL for neurotensin receptor 1 (Ntsr1), a receptor for neurotensin, a ligand known to be involved in several psychiatric disorders. We examined sleep as well as behaviors indicative of anxiety and depression in the NTSR1 knockout mouse. Measurements and Results: NTSR1 knockouts had a lower percentage of sleep time spent in REM sleep in the dark phase and a larger diurnal variation in REM sleep duration than wild types under baseline conditions. Following sleep deprivation, NTSR1 knockouts exhibited more wake and less NREM rebound sleep. NTSR1 knockouts also showed increased anxious and despair behaviors. Conclusions: Here we illustrate a link between expression of the Ntsr1 gene and sleep traits previously associated with a particular QTL. We also demonstrate a relationship between Ntsr1 and anxiety and despair behaviors. Given the considerable evidence that anxiety and depression are closely linked with abnormalities in sleep, the data presented here provide further evidence that neurotensin and Ntsr1 may be a component of a pathway involved in both sleep and mood disorders. Citation: Fitzpatrick K; Winrow CJ; Gotter AL; Millstein J; Arbuzova J; Brunner J; Kasarskis A; Vitaterna MH; Renger JJ; Turek FW. Altered sleep and affect in the neurotensin receptor 1 knockout mouse. SLEEP 2012;35(7):949-956. PMID:22754041

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

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.

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.

The Unexpected Effects of Beneficial and Adverse Social Experiences during Adolescence on Anxiety and Aggression and Their Modulation by Genotype

PubMed Central

Meyer, Neele; Richter, S. Helene; Schreiber, Rebecca S.; Kloke, Vanessa; Kaiser, Sylvia; Lesch, Klaus-Peter; Sachser, Norbert

2016-01-01

Anxiety and aggression are part of the behavioral repertoire of humans and animals. However, in their exaggerated form both can become maladaptive and result in psychiatric disorders. On the one hand, genetic predisposition has been shown to play a crucial modulatory role in anxiety and aggression. On the other hand, social experiences have been implicated in the modulation of these traits. However, so far, mainly experiences in early life phases have been considered crucial for shaping anxiety-like and aggressive behavior, while the phase of adolescence has largely been neglected. Therefore, the aim of the present study was to elucidate how levels of anxiety-like and aggressive behavior are shaped by social experiences during adolescence and serotonin transporter (5-HTT) genotype. For this purpose, male mice of a 5-HTT knockout mouse model including all three genotypes (wildtype, heterozygous and homozygous 5-HTT knockout mice) were either exposed to an adverse social situation or a beneficial social environment during adolescence. This was accomplished in a custom-made cage system where mice experiencing the adverse environment were repeatedly introduced to the territory of a dominant opponent but had the possibility to escape to a refuge cage. Mice encountering beneficial social conditions had free access to a female mating partner. Afterwards, anxiety-like and aggressive behavior was assessed in a battery of tests. Surprisingly, unfavorable conditions during adolescence led to a decrease in anxiety-like behavior and an increase in exploratory locomotion. Additionally, aggressive behavior was augmented in animals that experienced social adversity. Concerning genotype, homozygous 5-HTT knockout mice were more anxious and less aggressive than heterozygous 5-HTT knockout and wildtype mice. In summary, adolescence is clearly an important phase in which anxiety-like and aggressive behavior can be shaped. Furthermore, it seems that having to cope with challenge during adolescence instead of experiencing throughout beneficial social conditions leads to reduced levels of anxiety-like behavior. PMID:27303275

Translation Initiation Factor AteIF(iso)4E Is Involved in Selective mRNA Translation in Arabidopsis Thaliana Seedlings

PubMed Central

Martínez-Silva, Ana Valeria; Aguirre-Martínez, César; Flores-Tinoco, Carlos E.; Alejandri-Ramírez, Naholi D.; Dinkova, Tzvetanka D.

2012-01-01

One of the most regulated steps of translation initiation is the recruitment of mRNA by the translation machinery. In eukaryotes, this step is mediated by the 5′end cap-binding factor eIF4E bound to the bridge protein eIF4G and forming the eIF4F complex. In plants, different isoforms of eIF4E and eIF4G form the antigenically distinct eIF4F and eIF(iso)4F complexes proposed to mediate selective translation. Using a microarray analysis of polyribosome- and non-polyribosome-purified mRNAs from 15 day-old Arabidopsis thaliana wild type [WT] and eIF(iso)4E knockout mutant [(iso)4E-1] seedlings we found 79 transcripts shifted from polyribosomes toward non-polyribosomes, and 47 mRNAs with the opposite behavior in the knockout mutant. The translationally decreased mRNAs were overrepresented in root-preferentially expressed genes and proteins from the endomembrane system, including several transporters such as the phosphate transporter PHOSPHATE1 (PHO1), Sucrose transporter 3 (SUC3), ABC transporter-like with ATPase activity (MRP11) and five electron transporters, as well as signal transduction-, protein modification- and transcription-related proteins. Under normal growth conditions, eIF(iso)4E expression under the constitutive promoter 35 S enhanced the polyribosomal recruitment of PHO1 supporting its translational preference for eIF(iso)4E. Furthermore, under phosphate deficiency, the PHO1 protein increased in the eIF(iso)4E overexpressing plants and decreased in the knockout mutant as compared to wild type. In addition, the knockout mutant had larger root, whereas the 35 S directed expression of eIF(iso)4E caused shorter root under normal growth conditions, but not under phosphate deficiency. These results indicate that selective translation mediated by eIF(iso)4E is relevant for Arabidopsis root development under normal growth conditions. PMID:22363683

Phosphoproteomic analysis of the striatum from pleiotrophin knockout and midkine knockout mice treated with cocaine reveals regulation of oxidative stress-related proteins potentially underlying cocaine-induced neurotoxicity and neurodegeneration.

PubMed

Vicente-Rodríguez, Marta; Gramage, Esther; Herradón, Gonzalo; Pérez-García, Carmen

2013-12-06

The neurotrophic factors pleiotrophin (PTN) and midkine (MK) are highly upregulated in different brain areas relevant to drug addiction after administrations of different drugs of abuse, including psychostimulants. We have previously demonstrated that PTN and MK modulate amphetamine-induced neurotoxicity and that PTN prevents cocaine-induced cytotoxicity in NG108-15 and PC12 cells. In an effort to dissect the different mechanisms of action triggered by PTN and MK to exert their protective roles against psychostimulant neurotoxicity, we have now used a proteomic approach to study protein phosphorylation, in which we combined phosphoprotein enrichment, by immobilized metal affinity chromatography (IMAC), with two-dimensional gel electrophoresis and mass spectrometry, in order to identify the phosphoproteins regulated in the striatum of PTN knockout, MK knockout and wild type mice treated with a single dose of cocaine (15mg/kg, i.p.). We identified 7 differentially expressed phosphoproteins: 5'(3')-deoxyribonucleotidase, endoplasmic reticulum resident protein 60 (ERP60), peroxiredoxin-6 (PRDX6), glutamate dehydrogenase 1 (GLUD1), aconitase and two subunits of hemoglobin. Most of these proteins are related to neurodegeneration processes and oxidative stress and their variations specially affect the PTN knockout mice, suggesting a protective role of endogenous PTN against cocaine-induced neural alterations. Further studies are needed to validate these proteins as possible targets against neural alterations induced by cocaine. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Analysis of the proteolysis of bioactive peptides using a peptidomics approach

PubMed Central

Kim, Yun-Gon; Lone, Anna Mari; Saghatelian, Alan

2014-01-01

Identifying the peptidases that inactivate bioactive peptides (e.g. peptide hormones and neuropeptides) in mammals is an important unmet challenge. This protocol describes a recent approach that combines liquid chromatography-mass spectrometry peptidomics to identify endogenous cleavage sites of a bioactive peptide, the subsequent biochemical purification of a candidate peptidase based on these cleavage sites, and validation of the candidate peptidase’s role in the physiological regulation of the bioactive peptide by examining a peptidase knockout mouse. We highlight successful application of this protocol to discover that insulin-degrading enzyme (IDE) regulates physiological calcitonin gene-related peptide (CGRP) levels and detail the key stages and steps in this approach. This protocol requires 7 days of work; however, the total time for this protocol is highly variable because of its dependence on the availability of biological reagents, namely purified enzymes and knockout mice. The protocol is valuable because it expedites the characterization of mammalian peptidases, such as IDE, which in certain instances can be used to develop novel therapeutics. PMID:23949379

[Influence of tissue-specific superoxide dismutase genes expression in brain cells on Drosophila melanogaster sensitivity to oxidative stress and viability].

PubMed

Vitushynska, M V; Matiytsiv, N P; Chernyk, Y

2015-01-01

The study has shown that both functional gene knockout Sodl and Sod2 and their overexpression in neurons and glial tissue increase the sensitivity of Drosophila melanogaster to oxidative stress (OS) conditions. The lowest survival rate was only 20.5% in insects with Sod2 knockout in neurons. Comparative analysis of the survival curves showed that adults with altered tissue-specific expression of the studied genes had reduced average and maximum life span. Under OS conditions induced by 5% hydrogen peroxide the life spans of wild type Oregon R and transgenic insects were significantly reduced. Altered Sod gene expression in glial tissue leads to degenerative changes in Drosophila brain at the young age. During the aging of insects and the action of pro-oxidants increasing of neurodegenerative phenotype is observed.

Independent degeneration of photoreceptors and retinal pigment epithelium in conditional knockout mouse models of choroideremia

PubMed Central

Tolmachova, Tanya; Anders, Ross; Abrink, Magnus; Bugeon, Laurence; Dallman, Margaret J.; Futter, Clare E.; Ramalho, José S.; Tonagel, Felix; Tanimoto, Naoyuki; Seeliger, Mathias W.; Huxley, Clare; Seabra, Miguel C.

2006-01-01

Choroideremia (CHM) is an X-linked degeneration of the retinal pigment epithelium (RPE), photoreceptors, and choroid, caused by loss of function of the CHM/REP1 gene. REP1 is involved in lipid modification (prenylation) of Rab GTPases, key regulators of intracellular vesicular transport and organelle dynamics. To study the pathogenesis of CHM and to develop a model for assessing gene therapy, we have created a conditional mouse knockout of the Chm gene. Heterozygous-null females exhibit characteristic hallmarks of CHM: progressive degeneration of the photoreceptors, patchy depigmentation of the RPE, and Rab prenylation defects. Using tamoxifen-inducible and tissue-specific Cre expression in combination with floxed Chm alleles, we show that CHM pathogenesis involves independently triggered degeneration of photoreceptors and the RPE, associated with different subsets of defective Rabs. PMID:16410831

Dysregulation of synaptic plasticity precedes appearance of morphological defects in a Pten conditional knockout mouse model of autism.

PubMed

Takeuchi, Koichi; Gertner, Michael J; Zhou, Jing; Parada, Luis F; Bennett, Michael V L; Zukin, R Suzanne

2013-03-19

The phosphoinositide signaling system is a crucial regulator of neural development, cell survival, and plasticity. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) negatively regulates phosphatidylinositol 3-kinase signaling and downstream targets. Nse-Cre Pten conditional knockout mice, in which Pten is ablated in granule cells of the dentate gyrus and pyramidal neurons of the hippocampal CA3, but not CA1, recapitulate many of the symptoms of humans with inactivating PTEN mutations, including progressive hypertrophy of the dentate gyrus and deficits in hippocampus-based social and cognitive behaviors. However, the impact of Pten loss on activity-dependent synaptic plasticity in this clinically relevant mouse model of Pten inactivation remains unclear. Here, we show that two phosphatidylinositol 3-kinase- and protein synthesis-dependent forms of synaptic plasticity, theta burst-induced long-term potentiation and metabotropic glutamate receptor (mGluR)-dependent long-term depression, are dysregulated at medial perforant path-to-dentate gyrus synapses of young Nse-Cre Pten conditional knockout mice before the onset of visible morphological abnormalities. In contrast, long-term potentiation and mGluR-dependent long-term depression are normal at CA3-CA1 pyramidal cell synapses at this age. Our results reveal that deletion of Pten in dentate granule cells dysregulates synaptic plasticity, a defect that may underlie abnormal social and cognitive behaviors observed in humans with Pten inactivating mutations and potentially other autism spectrum disorders.

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.

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

Shorter duration of non-rapid eye movement sleep slow waves in EphA4 knockout mice.

PubMed

Freyburger, Marlène; Poirier, Gaétan; Carrier, Julie; Mongrain, Valérie

2017-10-01

Slow waves occurring during non-rapid eye movement sleep have been associated with neurobehavioural performance and memory. In addition, the duration of previous wakefulness and sleep impacts characteristics of these slow waves. However, molecular mechanisms regulating the dynamics of slow-wave characteristics remain poorly understood. The EphA4 receptor regulates glutamatergic transmission and synaptic plasticity, which have both been linked to sleep slow waves. To investigate if EphA4 regulates slow-wave characteristics during non-rapid eye movement sleep, we compared individual parameters of slow waves between EphA4 knockout mice and wild-type littermates under baseline conditions and after a 6-h sleep deprivation. We observed that, compared with wild-type mice, knockout mice display a shorter duration of positive and negative phases of slow waves under baseline conditions and after sleep deprivation. However, the mutation did not change slow-wave density, amplitude and slope, and did not affect the sleep deprivation-dependent changes in slow-wave characteristics, suggesting that EphA4 is not involved in the response to elevated sleep pressure. Our present findings suggest a role for EphA4 in shaping cortical oscillations during sleep that is independent from sleep need. © 2017 European Sleep Research Society.

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

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.

Quasifree (p, 2p) Reactions on Oxygen Isotopes: Observation of Isospin Independence of the Reduced Single-Particle Strength.

PubMed

Atar, L; Paschalis, S; Barbieri, C; Bertulani, C A; Díaz Fernández, P; Holl, M; Najafi, M A; Panin, V; Alvarez-Pol, H; Aumann, T; Avdeichikov, V; Beceiro-Novo, S; Bemmerer, D; Benlliure, J; Boillos, J M; Boretzky, K; Borge, M J G; Caamaño, M; Caesar, C; Casarejos, E; Catford, W; Cederkall, J; Chartier, M; Chulkov, L; Cortina-Gil, D; Cravo, E; Crespo, R; Dillmann, I; Elekes, Z; Enders, J; Ershova, O; Estrade, A; Farinon, F; Fraile, L M; Freer, M; Galaviz Redondo, D; Geissel, H; Gernhäuser, R; Golubev, P; Göbel, K; Hagdahl, J; Heftrich, T; Heil, M; Heine, M; Heinz, A; Henriques, A; Hufnagel, A; Ignatov, A; Johansson, H T; Jonson, B; Kahlbow, J; Kalantar-Nayestanaki, N; Kanungo, R; Kelic-Heil, A; Knyazev, A; Kröll, T; Kurz, N; Labiche, M; Langer, C; Le Bleis, T; Lemmon, R; Lindberg, S; Machado, J; Marganiec-Gałązka, J; Movsesyan, A; Nacher, E; Nikolskii, E Y; Nilsson, T; Nociforo, C; Perea, A; Petri, M; Pietri, S; Plag, R; Reifarth, R; Ribeiro, G; Rigollet, C; Rossi, D M; Röder, M; Savran, D; Scheit, H; Simon, H; Sorlin, O; Syndikus, I; Taylor, J T; Tengblad, O; Thies, R; Togano, Y; Vandebrouck, M; Velho, P; Volkov, V; Wagner, A; Wamers, F; Weick, H; Wheldon, C; Wilson, G L; Winfield, J S; Woods, P; Yakorev, D; Zhukov, M; Zilges, A; Zuber, K

2018-02-02

Quasifree one-proton knockout reactions have been employed in inverse kinematics for a systematic study of the structure of stable and exotic oxygen isotopes at the R^{3}B/LAND setup with incident beam energies in the range of 300-450  MeV/u. The oxygen isotopic chain offers a large variation of separation energies that allows for a quantitative understanding of single-particle strength with changing isospin asymmetry. Quasifree knockout reactions provide a complementary approach to intermediate-energy one-nucleon removal reactions. Inclusive cross sections for quasifree knockout reactions of the type ^{A}O(p,2p)^{A-1}N have been determined and compared to calculations based on the eikonal reaction theory. The reduction factors for the single-particle strength with respect to the independent-particle model were obtained and compared to state-of-the-art ab initio predictions. The results do not show any significant dependence on proton-neutron asymmetry.

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.

The contribution of TWIK-1 channels to astrocyte K+ current is limited by retention in intracellular compartments

PubMed Central

Wang, Wei; Putra, Adhytia; Schools, Gary P.; Ma, Baofeng; Chen, Haijun; Kaczmarek, Leonard K.; Barhanin, Jacques; Lesage, Florian; Zhou, Min

2013-01-01

TWIK-1 two-pore domain K+ channels are expressed abundantly in astrocytes. In the present study, we examined the extent to which TWIK-1 contributes to the linear current-voltage (I–V) relationship (passive) K+ membrane conductance, a dominant electrophysiological feature of mature hippocampal astrocytes. Astrocytes from TWIK-1 knockout mice have a more negative resting potential than those from wild type animals and a reduction in both inward rectification and Cs+ permeability. Nevertheless, the overall whole-cell passive conductance is not altered significantly in TWIK-1 knockout astrocytes. The expression of Kir4.1 and TREK-1, two other major astrocytic K+ channels, or of other two-pore K+ channels is not altered in TWIK-1 knockout mice, suggesting that the mild effect of TWIK-1 knockout does not result from compensation by these channels. Fractionation experiments showed that TWIK-1 is primarily localized in intracellular cytoplasmic fractions (55%) and mildly hydrophobic internal compartment fractions (41%), with only 5% in fractions containing plasma membranes. Our study revealed that TWIK-1 proteins are mainly located in the intracellular compartments of hippocampal astrocyte under physiological condition, therefore a minimal contribution of TWIK-1 channels to whole-cell currents is likely attributable to a relatively low level presence of channels in the plasma membrane. PMID:24368895

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.

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.

Role of Medial Prefrontal Cortex Narp in the Extinction of Morphine Conditioned Place Preference

ERIC Educational Resources Information Center

Blouin, Ashley M.; Han, Sungho; Pearce, Anne M.; Cheng, KaiLun; Lee, JongAh J.; Johnson, Alexander W.; Wang, Chuansong; During, Matthew J.; Holland, Peter C.; Shaham, Yavin; Baraban, Jay M.; Reti, Irving M.

2013-01-01

Narp knockout (KO) mice demonstrate an impaired extinction of morphine conditioned place preference (CPP). Because the medial prefrontal cortex (mPFC) has been implicated in extinction learning, we tested whether Narp cells in this region play a role in the extinction of morphine CPP. We found that intracranial injections of adenoassociated virus…

Dysfunction of intraflagellar transport-A causes hyperphagia-induced obesity and metabolic syndrome

PubMed Central

Jacobs, Damon T.; Silva, Luciane M.; Allard, Bailey A.; Schonfeld, Michael P.; Chatterjee, Anindita; Talbott, George C.

2016-01-01

ABSTRACT Primary cilia extend from the plasma membrane of most vertebrate cells and mediate signaling pathways. Ciliary dysfunction underlies ciliopathies, which are genetic syndromes that manifest multiple clinical features, including renal cystic disease and obesity. THM1 (also termed TTC21B or IFT139) encodes a component of the intraflagellar transport-A complex and mutations in THM1 have been identified in 5% of individuals with ciliopathies. Consistent with this, deletion of murine Thm1 during late embryonic development results in cystic kidney disease. Here, we report that deletion of murine Thm1 during adulthood results in obesity, diabetes, hypertension and fatty liver disease, with gender differences in susceptibility to weight gain and metabolic dysfunction. Pair-feeding of Thm1 conditional knock-out mice relative to control littermates prevented the obesity and related disorders, indicating that hyperphagia caused the obese phenotype. Thm1 ablation resulted in increased localization of adenylyl cyclase III in primary cilia that were shortened, with bulbous distal tips on neurons of the hypothalamic arcuate nucleus, an integrative center for signals that regulate feeding and activity. In pre-obese Thm1 conditional knock-out mice, expression of anorexogenic pro-opiomelanocortin (Pomc) was decreased by 50% in the arcuate nucleus, which likely caused the hyperphagia. Fasting of Thm1 conditional knock-out mice did not alter Pomc nor orexogenic agouti-related neuropeptide (Agrp) expression, suggesting impaired sensing of changes in peripheral signals. Together, these data indicate that the Thm1-mutant ciliary defect diminishes sensitivity to feeding signals, which alters appetite regulation and leads to hyperphagia, obesity and metabolic disease. PMID:27482817

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

Takemura, Takayo; Yoshida, Yuichi; Kiso, Shinichi, E-mail: kiso@gh.med.osaka-u.ac.jp

Highlights: •HB-EGF expression was increased during the development of liver fibrosis. •Conditional HB-EGF knockout mouse showed enhanced experimental liver fibrosis. •HB-EGF antagonized TGF-β-induced activation of hepatic stellate cells. •We report a possible protective role of HB-EGF in cholestatic liver fibrosis. -- Abstract: Our aims were to evaluate the involvement of heparin-binding EGF-like growth factor (HB-EGF) in liver fibrogenesis of humans and mice and to elucidate the effect of HB-EGF deficiency on cholestatic liver fibrosis using conditional HB-EGF knockout (KO) mice. We first demonstrated that gene expression of HB-EGF had a positive significant correlation with that of collagen in human fibroticmore » livers, and was increased in bile duct ligation (BDL)-induced fibrotic livers in mouse. We then generated conditional HB-EGF knockout (KO) mice using the interferon inducible Mx-1 promoter driven Cre recombinase transgene and wild type (WT) and KO mice were subjected to BDL. After BDL, KO mice exhibited enhanced liver fibrosis with increased expression of collagen, compared with WT mice. Finally, we used mouse hepatic stellate cells (HSCs) to examine the role of HB-EGF in the activation of these cells and showed that HB-EGF antagonized TGF-β-induced gene expression of collagen in mouse primary HSCs. Interestingly, HB-EGF did not prevent the TGF-β-induced nuclear accumulation of Smad3, but did lead to stabilization of the Smad transcriptional co-repressor TG-interacting factor. In conclusion, our data suggest a possible protective role of HB-EGF in cholestatic liver fibrosis.« less

GPR39 (zinc receptor) knockout mice exhibit depression-like behavior and CREB/BDNF down-regulation in the hippocampus.

PubMed

Młyniec, Katarzyna; Budziszewska, Bogusława; Holst, Birgitte; Ostachowicz, Beata; Nowak, Gabriel

2014-10-31

Zinc may act as a neurotransmitter in the central nervous system by activation of the GPR39 metabotropic receptors. In the present study, we investigated whether GPR39 knockout would cause depressive-like and/or anxiety-like behavior, as measured by the forced swim test, tail suspension test, and light/dark test. We also investigated whether lack of GPR39 would change levels of cAMP response element-binding protein (CREB),brain-derived neurotrophic factor (BDNF) and tropomyosin related kinase B (TrkB) protein in the hippocampus and frontal cortex of GPR39 knockout mice subjected to the forced swim test, as measured by Western-blot analysis. In this study, GPR39 knockout mice showed an increased immobility time in both the forced swim test and tail suspension test, indicating depressive-like behavior and displayed anxiety-like phenotype. GPR39 knockout mice had lower CREB and BDNF levels in the hippocampus, but not in the frontal cortex, which indicates region specificity for the impaired CREB/BDNF pathway (which is important in antidepressant response) in the absence of GPR39. There were no changes in TrkB protein in either structure. In the present study, we also investigated activity in the hypothalamus-pituitary-adrenal axis under both zinc- and GPR39-deficient conditions. Zinc-deficient mice had higher serum corticosterone levels and lower glucocorticoid receptor levels in the hippocampus and frontal cortex. There were no changes in the GPR39 knockout mice in comparison with the wild-type control mice, which does not support a role of GPR39 in hypothalamus-pituitary-adrenal axis regulation. The results of this study indicate the involvement of the GPR39 Zn(2+)-sensing receptor in the pathophysiology of depression with component of anxiety. © The Author 2015. Published by Oxford University Press on behalf of CINP.

Rabbit models for biomedical research revisited via genome editing approaches

PubMed Central

HONDA, Arata; OGURA, Atsuo

2017-01-01

Although the laboratory rabbit has long contributed to many paradigmatic studies in biology and medicine, it is often considered to be a “classical animal model” because in the last 30 years, the laboratory mouse has been more often used, thanks to the availability of embryonic stem cells that have allowed the generation of gene knockout (KO) animals. However, recent genome-editing strategies have changed this unrivaled condition; so far, more than 10 mammalian species have been added to the list of KO animals. Among them, the rabbit has distinct advantages for application of genome-editing systems, such as easy application of superovulation, consistency with fertile natural mating, well-optimized embryo manipulation techniques, and the short gestation period. The rabbit has now returned to the stage of advanced biomedical research. PMID:28579598

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

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.

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.

Feasibility study for clinical application of caspase-3 inhibitors in Pemphigus vulgaris.

PubMed

Hariton, William V J; Galichet, Arnaud; Vanden Berghe, Tom; Overmiller, Andrew M; Mahoney, My G; Declercq, Wim; Müller, Eliane J

2017-12-01

The potentially severe side effects of systemic corticosteroids and immunosuppressants used in Pemphigus vulgaris (PV) call for novel therapeutic approaches. In this context, pharmacological inhibition of major pathogenic signalling effectors represents a promising alternative. However, we have also shown that overinhibition of effectors required for epidermal homeostasis can exacerbate PV pathophysiology implicating transepidermal keratinocyte fragility. A feedforward target validation therefore preferentially includes studies on knockout mouse models. We previously reported on successful amelioration of PV blisters following inhibition of non-apoptotic, low-level caspase-3. Here, we use conditional, keratinocyte-specific caspase-3-deficient mice (casp3 EKO ) to demonstrate (i) absence of keratinocyte fragility upon injection of the potent Dsg3-specific antibody AK23 and (ii) amelioration of blistering on the background of known signalling effectors. Our results provide the experimental proof of concept justifying translation of the caspase-3 inhibitor approach into PV clinical trials. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Both mineralocorticoid and glucocorticoid receptors regulate emotional memory in mice.

PubMed

Zhou, Ming; Bakker, Eveline H M; Velzing, Els H; Berger, Stefan; Oitzl, Melly; Joëls, Marian; Krugers, Harm J

2010-11-01

Corticosteroid hormones are thought to promote optimal behavioral adaptation under fearful conditions, primarily via glucocorticoid receptors (GRs). Here, we examined - using pharmacological and genetic approaches in mice - if mineralocorticoid receptors (MRs) also play a role in fearful memory formation. As expected, administration of the GR-antagonist RU38486 prior to training in a fear conditioning paradigm impaired contextual memory when tested 24 (but not when tested 3) h after training. Tone-cue memory was enhanced by RU38486 when tested at 4 (but not 25) h after training. Interestingly, pre (but not post)-training administration of MR antagonist spironolactone impaired contextual memory, both at 3 and 24h after training. Similar effects were also found in forebrain-specific MR knockout mice. Spironolactone also impaired tone-cue memory, but only at 4h after training. These results reveal that - in addition to GRs - MRs also play a critical role in establishing fear memories, particularly in the early phase of memory formation. Copyright © 2010 Elsevier Inc. All rights reserved.

Mouse phenotyping.

PubMed

Fuchs, Helmut; Gailus-Durner, Valérie; Adler, Thure; Aguilar-Pimentel, Juan Antonio; Becker, Lore; Calzada-Wack, Julia; Da Silva-Buttkus, Patricia; Neff, Frauke; Götz, Alexander; Hans, Wolfgang; Hölter, Sabine M; Horsch, Marion; Kastenmüller, Gabi; Kemter, Elisabeth; Lengger, Christoph; Maier, Holger; Matloka, Mikolaj; Möller, Gabriele; Naton, Beatrix; Prehn, Cornelia; Puk, Oliver; Rácz, Ildikó; Rathkolb, Birgit; Römisch-Margl, Werner; Rozman, Jan; Wang-Sattler, Rui; Schrewe, Anja; Stöger, Claudia; Tost, Monica; Adamski, Jerzy; Aigner, Bernhard; Beckers, Johannes; Behrendt, Heidrun; Busch, Dirk H; Esposito, Irene; Graw, Jochen; Illig, Thomas; Ivandic, Boris; Klingenspor, Martin; Klopstock, Thomas; Kremmer, Elisabeth; Mempel, Martin; Neschen, Susanne; Ollert, Markus; Schulz, Holger; Suhre, Karsten; Wolf, Eckhard; Wurst, Wolfgang; Zimmer, Andreas; Hrabě de Angelis, Martin

2011-02-01

Model organisms like the mouse are important tools to learn more about gene function in man. Within the last 20 years many mutant mouse lines have been generated by different methods such as ENU mutagenesis, constitutive and conditional knock-out approaches, knock-down, introduction of human genes, and knock-in techniques, thus creating models which mimic human conditions. Due to pleiotropic effects, one gene may have different functions in different organ systems or time points during development. Therefore mutant mouse lines have to be phenotyped comprehensively in a highly standardized manner to enable the detection of phenotypes which might otherwise remain hidden. The German Mouse Clinic (GMC) has been established at the Helmholtz Zentrum München as a phenotyping platform with open access to the scientific community (www.mousclinic.de; [1]). The GMC is a member of the EUMODIC consortium which created the European standard workflow EMPReSSslim for the systemic phenotyping of mouse models (http://www.eumodic.org/[2]). Copyright © 2010 Elsevier Inc. All rights reserved.

The role of ventral striatal cAMP signaling in stress-induced behaviors

PubMed Central

Plattner, Florian; Hayashi, Kanehiro; Hernandez, Adan; Benavides, David R.; Tassin, Tara C.; Tan, Chunfeng; Day, Jonathan; Fina, Maggy W.; Yuen, Eunice Y.; Yan, Zhen; Goldberg, Matthew S.; Nairn, Angus C.; Greengard, Paul; Nestler, Eric J.; Taussig, Ronald; Nishi, Akinori; Houslay, Miles D.; Bibb, James A.

2015-01-01

The cAMP/PKA signaling cascade is a ubiquitous pathway acting downstream of multiple neuromodulators. We found that the phosphorylation of phosphodiesterase-4 (PDE4) by cyclin-dependent protein kinase 5 (Cdk5) facilitates cAMP degradation and homeostasis of cAMP/PKA signaling. In mice, loss of Cdk5 throughout the forebrain elevated cAMP levels and increased PKA activity in striatal neurons, and altered behavioral responses to acute or chronic stressors. Ventral striatum- or D1 dopamine receptor-specific conditional knockout of Cdk5, or ventral striatum infusion of a small interfering peptide that selectively targets the regulation of PDE4 by Cdk5, all produced analogical effects on stress-induced behavioral responses. Together, our results demonstrate that altering cAMP signaling in medium spiny neurons of the ventral striatum can effectively modulate stress-induced behavioral states. We propose that targeting the Cdk5 regulation of PDE4 could be a new therapeutic approach for clinical conditions associated with stress, such as depression. PMID:26192746

Deletion of the δ opioid receptor gene impairs place conditioning but preserves morphine reinforcement.

PubMed

Le Merrer, Julie; Plaza-Zabala, Ainhoa; Del Boca, Carolina; Matifas, Audrey; Maldonado, Rafael; Kieffer, Brigitte L

2011-04-01

Converging experimental data indicate that δ opioid receptors contribute to mediate drug reinforcement processes. Whether their contribution reflects a role in the modulation of drug reward or an implication in conditioned learning, however, has not been explored. In the present study, we investigated the impact of δ receptor gene knockout on reinforced conditioned learning under several experimental paradigms. We assessed the ability of δ receptor knockout mice to form drug-context associations with either morphine (appetitive)- or lithium (aversive)-induced Pavlovian place conditioning. We also examined the efficiency of morphine to serve as a positive reinforcer in these mice and their motivation to gain drug injections, with operant intravenous self-administration under fixed and progressive ratio schedules and at two different doses. Mutant mice showed impaired place conditioning in both appetitive and aversive conditions, indicating disrupted context-drug association. In contrast, mutant animals displayed intact acquisition of morphine self-administration and reached breaking-points comparable to control subjects. Thus, reinforcing effects of morphine and motivation to obtain the drug were maintained. Collectively, the data suggest that δ receptor activity is not involved in morphine reinforcement but facilitates place conditioning. This study reveals a novel aspect of δ opioid receptor function in addiction-related behaviors. Copyright © 2011 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Inference of gene regulatory networks from genome-wide knockout fitness data

PubMed Central

Wang, Liming; Wang, Xiaodong; Arkin, Adam P.; Samoilov, Michael S.

2013-01-01

Motivation: Genome-wide fitness is an emerging type of high-throughput biological data generated for individual organisms by creating libraries of knockouts, subjecting them to broad ranges of environmental conditions, and measuring the resulting clone-specific fitnesses. Since fitness is an organism-scale measure of gene regulatory network behaviour, it may offer certain advantages when insights into such phenotypical and functional features are of primary interest over individual gene expression. Previous works have shown that genome-wide fitness data can be used to uncover novel gene regulatory interactions, when compared with results of more conventional gene expression analysis. Yet, to date, few algorithms have been proposed for systematically using genome-wide mutant fitness data for gene regulatory network inference. Results: In this article, we describe a model and propose an inference algorithm for using fitness data from knockout libraries to identify underlying gene regulatory networks. Unlike most prior methods, the presented approach captures not only structural, but also dynamical and non-linear nature of biomolecular systems involved. A state–space model with non-linear basis is used for dynamically describing gene regulatory networks. Network structure is then elucidated by estimating unknown model parameters. Unscented Kalman filter is used to cope with the non-linearities introduced in the model, which also enables the algorithm to run in on-line mode for practical use. Here, we demonstrate that the algorithm provides satisfying results for both synthetic data as well as empirical measurements of GAL network in yeast Saccharomyces cerevisiae and TyrR–LiuR network in bacteria Shewanella oneidensis. Availability: MATLAB code and datasets are available to download at http://www.duke.edu/∼lw174/Fitness.zip and http://genomics.lbl.gov/supplemental/fitness-bioinf/ Contact: wangx@ee.columbia.edu or mssamoilov@lbl.gov Supplementary information: Supplementary data are available at Bioinformatics online PMID:23271269

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.

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.

Connexin-deficiency affects expression levels of glial glutamate transporters within the cerebrum.

PubMed

Unger, Tina; Bette, Stefanie; Zhang, Jiong; Theis, Martin; Engele, Jürgen

2012-01-06

The glial glutamate transporter subtypes, GLT-1/EAAT-2 and GLAST/EAAT-1 clear the bulk of extracellular glutamate and are severely dysregulated in various acute and chronic brain diseases. Despite the previous identification of several extracellular factors modulating glial glutamate transporter expression, our knowledge of the regulatory network controlling glial glutamate transport in health and disease still remains incomplete. In studies with cultured cortical astrocytes, we previously obtained evidence that glial glutamate transporter expression is also affected by gap junctions/connexins. To assess whether gap junctions would likewise control the in vivo expression of glial glutamate transporters, we have now assessed their expression levels in brains of conditional Cx43 knockout mice, total Cx30 knockouts, as well as Cx43/Cx30 double knockouts. We found that either knocking out Cx30, Cx43, or both increases GLT-1/EAAT-2 protein levels in the cerebral cortex to a similar extent. By contrast, GLAST/EAAT-1 protein levels maximally increased in cerebral cortices of Cx30/Cx43 double knockouts, implying that gap junctions differentially affect the expression of GLT-1/EAAT-2 and GLAST/EAAT-1. Quantitative PCR analysis further revealed that increases in glial glutamate transporter expression are brought about by transcriptional and translational/posttranslational processes. Moreover, GLT-1/EAAT-2- and GLAST/EAAT-1 protein levels remained unchanged in the hippocampi of Cx43/Cx30 double knockouts when compared to Cx43fl/fl controls, indicating brain region-specific effects of gap junctions on glial glutamate transport. Since astrocytic gap junction coupling is affected in various forms of brain injuries, our findings point to gap junctions/connexins as important regulators of glial glutamate turnover in the diseased cerebral cortex. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

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

Robust and Sensitive Analysis of Mouse Knockout Phenotypes

PubMed Central

Karp, Natasha A.; Melvin, David; Mott, Richard F.

2012-01-01

A significant challenge of in-vivo studies is the identification of phenotypes with a method that is robust and reliable. The challenge arises from practical issues that lead to experimental designs which are not ideal. Breeding issues, particularly in the presence of fertility or fecundity problems, frequently lead to data being collected in multiple batches. This problem is acute in high throughput phenotyping programs. In addition, in a high throughput environment operational issues lead to controls not being measured on the same day as knockouts. We highlight how application of traditional methods, such as a Student’s t-Test or a 2-way ANOVA, in these situations give flawed results and should not be used. We explore the use of mixed models using worked examples from Sanger Mouse Genome Project focusing on Dual-Energy X-Ray Absorptiometry data for the analysis of mouse knockout data and compare to a reference range approach. We show that mixed model analysis is more sensitive and less prone to artefacts allowing the discovery of subtle quantitative phenotypes essential for correlating a gene’s function to human disease. We demonstrate how a mixed model approach has the additional advantage of being able to include covariates, such as body weight, to separate effect of genotype from these covariates. This is a particular issue in knockout studies, where body weight is a common phenotype and will enhance the precision of assigning phenotypes and the subsequent selection of lines for secondary phenotyping. The use of mixed models with in-vivo studies has value not only in improving the quality and sensitivity of the data analysis but also ethically as a method suitable for small batches which reduces the breeding burden of a colony. This will reduce the use of animals, increase throughput, and decrease cost whilst improving the quality and depth of knowledge gained. PMID:23300663

The Role of Beta-Adrenergic Receptors in the Regulation of Circadian Intraocular Pressure Rhythm in Mice.

PubMed

Tsuchiya, Shunsuke; Higashide, Tomomi; Toida, Kazunori; Sugiyama, Kazuhisa

2017-07-01

To investigate whether the elimination of β1- and β2-adrenergic receptors alters the diurnal intraocular pressure (IOP) rhythm in mice. β1-/β2-adrenergic receptor double-knockout and C57BL/6J mice were anesthetized intraperitoneally, with their IOPs measured via microneedle method. After entrainment to a 12-h light-dark (LD) cycle (light phase 6:00-18:00), IOPs were measured every 3 h from 9:00 to 24:00 (group 1, β1-/β2-adrenergic receptor double-knockout mice, n = 11; C57BL/6J, n = 15). The IOP measurements at 15:00 and 24:00 under a 12-h LD cycle and in the constant darkness (1 day and 8 days after exposure to darkness, respectively) were performed in another group of β1-/β2-adrenergic receptor double-knockout mice (group 2, n = 12). IOP variance throughout the day and mean IOP differences among time points were evaluated using a linear mixed model. β1-/β2-adrenergic receptor double-knockout and C57BL/6J mice showed biphasic IOP curves, low during the light phase and high during the dark phase; the fluctuation was significant (P < 0.001). The peak IOP (18.7 ± 1.4 mmHg) occurred at 24:00 and the trough IOP (13.5 ± 1.5 mmHg) occurred at 15:00 in β1-/β2-adrenergic receptor double-knockout mice group. IOP curves of β1-/β2-adrenergic receptor double-knockout and C57BL/6J were nearly parallel, and the IOPs of β1-/β2-adrenergic receptor double-knockout mice were significantly higher than those of C57BL/6J mice (P < 0.001). Under constant dark (DD) conditions, IOP at 24:00 (18.1 ± 1.5 mmHg) was significantly higher than that at 15:00 (13.3 ± 1.2 mmHg) (P < 0.001). The transition from the LD cycle to DD environment produced no significant change in IOP (P = 0.728). Elimination of both β1- and β2-adrenergic receptors did not disturb the biphasic diurnal IOP rhythm in mice.

In silico experiment system for testing hypothesis on gene functions using three condition specific biological networks.

PubMed

Lee, Chai-Jin; Kang, Dongwon; Lee, Sangseon; Lee, Sunwon; Kang, Jaewoo; Kim, Sun

2018-05-25

Determining functions of a gene requires time consuming, expensive biological experiments. Scientists can speed up this experimental process if the literature information and biological networks can be adequately provided. In this paper, we present a web-based information system that can perform in silico experiments of computationally testing hypothesis on the function of a gene. A hypothesis that is specified in English by the user is converted to genes using a literature and knowledge mining system called BEST. Condition-specific TF, miRNA and PPI (protein-protein interaction) networks are automatically generated by projecting gene and miRNA expression data to template networks. Then, an in silico experiment is to test how well the target genes are connected from the knockout gene through the condition-specific networks. The test result visualizes path from the knockout gene to the target genes in the three networks. Statistical and information-theoretic scores are provided on the resulting web page to help scientists either accept or reject the hypothesis being tested. Our web-based system was extensively tested using three data sets, such as E2f1, Lrrk2, and Dicer1 knockout data sets. We were able to re-produce gene functions reported in the original research papers. In addition, we comprehensively tested with all disease names in MalaCards as hypothesis to show the effectiveness of our system. Our in silico experiment system can be very useful in suggesting biological mechanisms which can be further tested in vivo or in vitro. http://biohealth.snu.ac.kr/software/insilico/. Copyright © 2018 Elsevier Inc. All rights reserved.

Embryonic ablation of neuronal VGF increases energy expenditure and reduces body weight

PubMed Central

Jiang, Cheng; Lin, Wei-Jye; Sadahiro, Masato; Shin, Andrew C.; Buettner, Christoph; Salton, Stephen R.

2016-01-01

Germline ablation of VGF, a secreted neuronal, neuroendocrine, and endocrine peptide precursor, results in lean, hypermetabolic, and infertile adult mice that are resistant to diet-, lesion-, and genetically-induced obesity and diabetes (Hahm et al., 1999, 2002). To assess whether this phenotype is predominantly driven by reduced VGF expression in developing and/or adult neurons, or in peripheral endocrine and neuroendocrine tissues, we generated and analyzed conditional VGF knockout mice, obtained by mating loxP-flanked (floxed) Vgf mice with either pan-neuronal Synapsin-Cre- or forebrain alpha-CaMKII-Cre-recombinase-expressing transgenic mice. Adult male and female mice, with conditional ablation of the Vgf gene in embryonic neurons had significantly reduced body weight, increased energy expenditure, and were resistant to diet-induced obesity. Conditional forebrain postnatal ablation of VGF in male mice, primarily in adult excitatory neurons, had no measurable effect on body weight nor on energy expenditure, but led to a modest increase in adiposity, partially overlapping the effect of AAV-Cre-mediated targeted ablation of VGF in the adult ventromedial hypothalamus and arcuate nucleus of floxed Vgf mice (Foglesong et al., 2016), and also consistent with results of icv delivery of the VGF-derived peptide TLQP-21 to adult mice, which resulted in increased energy expenditure and reduced adiposity (Bartolomucci et al., 2006). Because the lean, hypermetabolic phenotype of germline VGF knockout mice is to a great extent recapitulated in Syn-Cre+/−,Vgfflpflox/flpflox mice, we conclude that the metabolic profile of germline VGF knockout mice is largely the result of VGF ablation in embryonic CNS neurons, rather than peripheral endocrine and/or neuroendocrine cells, and that in forebrain structures such as hypothalamus, VGF and/or VGF-derived peptides play uniquely different roles in the developing and adult nervous system. PMID:28024880

Dysfunction of intraflagellar transport-A causes hyperphagia-induced obesity and metabolic syndrome.

PubMed

Jacobs, Damon T; Silva, Luciane M; Allard, Bailey A; Schonfeld, Michael P; Chatterjee, Anindita; Talbott, George C; Beier, David R; Tran, Pamela V

2016-07-01

Primary cilia extend from the plasma membrane of most vertebrate cells and mediate signaling pathways. Ciliary dysfunction underlies ciliopathies, which are genetic syndromes that manifest multiple clinical features, including renal cystic disease and obesity. THM1 (also termed TTC21B or IFT139) encodes a component of the intraflagellar transport-A complex and mutations in THM1 have been identified in 5% of individuals with ciliopathies. Consistent with this, deletion of murine Thm1 during late embryonic development results in cystic kidney disease. Here, we report that deletion of murine Thm1 during adulthood results in obesity, diabetes, hypertension and fatty liver disease, with gender differences in susceptibility to weight gain and metabolic dysfunction. Pair-feeding of Thm1 conditional knock-out mice relative to control littermates prevented the obesity and related disorders, indicating that hyperphagia caused the obese phenotype. Thm1 ablation resulted in increased localization of adenylyl cyclase III in primary cilia that were shortened, with bulbous distal tips on neurons of the hypothalamic arcuate nucleus, an integrative center for signals that regulate feeding and activity. In pre-obese Thm1 conditional knock-out mice, expression of anorexogenic pro-opiomelanocortin (Pomc) was decreased by 50% in the arcuate nucleus, which likely caused the hyperphagia. Fasting of Thm1 conditional knock-out mice did not alter Pomc nor orexogenic agouti-related neuropeptide (Agrp) expression, suggesting impaired sensing of changes in peripheral signals. Together, these data indicate that the Thm1-mutant ciliary defect diminishes sensitivity to feeding signals, which alters appetite regulation and leads to hyperphagia, obesity and metabolic disease. © 2016. Published by The Company of Biologists Ltd.

PAR-2 mediates increased inflammatory cell adhesion and neointima formation following vascular injury in the mouse.

PubMed

Tennant, Gail M; Wadsworth, Roger M; Kennedy, Simon

2008-05-01

Activation of PAR-2 in the vasculature affects vascular tone and adhesion of leukocytes to the endothelium. Since adhesion of leukocytes is increased following vascular injury and is important in determining the extent of neointima formation, we hypothesised that mice lacking PAR-2 may have reduced neointima formation following vascular injury. PAR-2 activating peptides and trypsin induced endothelium-dependent relaxation of mouse carotid artery which was absent in the knockout mouse. Lack of a PAR-2 receptor did not affect lymphocyte adhesion under basal conditions, but reduced the contractile response produced by lymphocytes. Twenty-eight days after denuding injury, vessel contraction to lymphocytes was reduced in both strains while lymphocyte adhesion was significantly greater in PAR-2(+/+) mice compared to the PAR-2 knockout mice. Neointimal area was markedly reduced in the PAR-2 knockout mouse. Our data show that PAR-2 modulates inflammatory cell adhesion when stimulated and in mice lacking the PAR-2 receptor, adhesion to injured vessels is reduced with a consequent reduction in neointima formation.

Requirement of Smad4 from Ocular Surface Ectoderm for Retinal Development.

PubMed

Li, Jing; Wang, Shusheng; Anderson, Chastain; Zhao, Fangkun; Qin, Yu; Wu, Di; Wu, Xinwei; Liu, Jia; He, Xuefei; Zhao, Jiangyue; Zhang, Jinsong

2016-01-01

Microphthalmia is characterized by abnormally small eyes and usually retinal dysplasia, accounting for up to 11% of the blindness in children. Right now there is no effective treatment for the disease, and the underlying mechanisms, especially how retinal dysplasia develops from microphthalmia and whether it depends on the signals from lens ectoderm are still unclear. Mutations in genes of the TGF-β superfamily have been noted in patients with microphthalmia. Using conditional knockout mice, here we address the question that whether ocular surface ectoderm-derived Smad4 modulates retinal development. We found that loss of Smad4 specifically on surface lens ectoderm leads to microphthalmia and dysplasia of retina. Retinal dysplasia in the knockout mice is caused by the delayed or failed differentiation and apoptosis of retinal cells. Microarray analyses revealed that members of Hedgehog and Wnt signaling pathways are affected in the knockout retinas, suggesting that ocular surface ectoderm-derived Smad4 can regulate Hedgehog and Wnt signaling in the retina. Our studies suggest that defective of ocular surface ectoderm may affect retinal development.

Requirement of Smad4 from Ocular Surface Ectoderm for Retinal Development

PubMed Central

Li, Jing; Wang, Shusheng; Anderson, Chastain; Zhao, Fangkun; Qin, Yu; Wu, Di; Wu, Xinwei; Liu, Jia; He, Xuefei; Zhao, Jiangyue; Zhang, Jinsong

2016-01-01

Microphthalmia is characterized by abnormally small eyes and usually retinal dysplasia, accounting for up to 11% of the blindness in children. Right now there is no effective treatment for the disease, and the underlying mechanisms, especially how retinal dysplasia develops from microphthalmia and whether it depends on the signals from lens ectoderm are still unclear. Mutations in genes of the TGF-β superfamily have been noted in patients with microphthalmia. Using conditional knockout mice, here we address the question that whether ocular surface ectoderm-derived Smad4 modulates retinal development. We found that loss of Smad4 specifically on surface lens ectoderm leads to microphthalmia and dysplasia of retina. Retinal dysplasia in the knockout mice is caused by the delayed or failed differentiation and apoptosis of retinal cells. Microarray analyses revealed that members of Hedgehog and Wnt signaling pathways are affected in the knockout retinas, suggesting that ocular surface ectoderm-derived Smad4 can regulate Hedgehog and Wnt signaling in the retina. Our studies suggest that defective of ocular surface ectoderm may affect retinal development. PMID:27494603

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

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.

Calorie restriction ameliorates neurodegenerative phenotypes in forebrain-specific presenilin-1 and presenilin-2 double knockout mice.

PubMed

Wu, Pu; Shen, Qian; Dong, Suzhen; Xu, Zhiliang; Tsien, Joe Z; Hu, Yinghe

2008-10-01

Conditional double knockout of presenilin-1 and presenilin-2 (cDKO) in forebrain of mice led to brain atrophy, tau hyperphosphorylation, synaptic dysfunction and cognitive deficit. These brain changes recapitulated most of the neurodegenerative phenotypes of Alzheimer's disease (AD). In this report, we have investigated the effects of 4-month calorie restriction (CR) regimen on different phenotypes in cDKO mice. We found that CR improved novel object recognition and contextual fear conditioning memory in the cDKO mice. Histological and biochemical analysis showed that CR attenuated ventricle enlargement, caspase-3 activation and astrogliosis. In addition, the induction of tau hyperphosphorylation in the cDKO mice was reduced by CR, possibly through reduction of p25 accumulation and aberrant CDK5 activation. Finally, DNA microarray analysis demonstrated that CR could increase the expression of neurogenesis related genes and decrease the expression of inflammation related genes in the hippocampus of cDKO mice. The possible molecular mechanisms of the CR effects on alleviating AD pathogenesis have been discussed.

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.

Lymphocyte signaling : beyond knockouts

PubMed Central

Saveliev, Alexander; Tybulewicz, Victor L. J.

2016-01-01

The analysis of lymphocyte signaling was greatly enhanced by the advent of gene targeting, which allows the selective inactivation of a single gene. Whereas this gene ‘knockout’ approach is often informative, in many cases the phenotype resulting from gene ablation might not provide a complete picture of the function of the corresponding protein. If a protein has multiple functions within a single or several signaling pathways, or stabilizes other proteins in a complex, the phenotypic consequences of a gene knockout may manifest as a combination of several different perturbations. In these cases, gene targeting to ‘knockin’ subtle point mutations might provide more accurate insight into protein function. However, to be informative, such mutations must be carefully designed based on structural and biophysical data. PMID:19295633

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

Primary Coenzyme Q Deficiency in Pdss2 Mutant Mice Causes Isolated Renal Disease

PubMed Central

Haase, Volker H.; King, Rhonda; Polyak, Erzsebet; Selak, Mary; Yudkoff, Marc; Hancock, Wayne W.; Meade, Ray; Saiki, Ryoichi; Lunceford, Adam L.; Clarke, Catherine F.; Gasser, David L.

2008-01-01

Coenzyme Q (CoQ) is an essential electron carrier in the respiratory chain whose deficiency has been implicated in a wide variety of human mitochondrial disease manifestations. Its multi-step biosynthesis involves production of polyisoprenoid diphosphate in a reaction that requires the enzymes be encoded by PDSS1 and PDSS2. Homozygous mutations in either of these genes, in humans, lead to severe neuromuscular disease, with nephrotic syndrome seen in PDSS2 deficiency. We now show that a presumed autoimmune kidney disease in mice with the missense Pdss2kd/kd genotype can be attributed to a mitochondrial CoQ biosynthetic defect. Levels of CoQ9 and CoQ10 in kidney homogenates from B6.Pdss2kd/kd mutants were significantly lower than those in B6 control mice. Disease manifestations originate specifically in glomerular podocytes, as renal disease is seen in Podocin/cre,Pdss2loxP/loxP knockout mice but not in conditional knockouts targeted to renal tubular epithelium, monocytes, or hepatocytes. Liver-conditional B6.Alb/cre,Pdss2loxP/loxP knockout mice have no overt disease despite demonstration that their livers have undetectable CoQ9 levels, impaired respiratory capacity, and significantly altered intermediary metabolism as evidenced by transcriptional profiling and amino acid quantitation. These data suggest that disease manifestations of CoQ deficiency relate to tissue-specific respiratory capacity thresholds, with glomerular podocytes displaying the greatest sensitivity to Pdss2 impairment. PMID:18437205

Problem-Solving Test: Conditional Gene Targeting Using the Cre/loxP Recombination System

ERIC Educational Resources Information Center

Szeberényi, József

2013-01-01

Terms to be familiar with before you start to solve the test: gene targeting, knock-out mutation, bacteriophage, complementary base-pairing, homologous recombination, deletion, transgenic organisms, promoter, polyadenylation element, transgene, DNA replication, RNA polymerase, Shine-Dalgarno sequence, restriction endonuclease, polymerase chain…

Contribution of chloride channel permease to fluoride resistance in Streptococcus mutans.

PubMed

Murata, Takatoshi; Hanada, Nobuhiro

2016-06-01

Genes encoding fluoride transporters have been identified in bacterial and archaeal species. The genome sequence of the cariogenic Streptococcus mutans bacteria suggests the presence of a putative fluoride transporter, which is referred to as a chloride channel permease. Two homologues of this gene (GenBank locus tags SMU_1290c and SMU_1289c) reside in tandem in the genome of S. mutans The aim of this study was to determine whether the chloride channel permeases contribute to fluoride resistance. We constructed SMU_1290c- and SMU_1289c-knockout S. mutans UA159 strains. We also constructed a double-knockout strain lacking both genes. SMU_1290c or SMU_1289c was transformed into a fluoride transporter- disrupted Escherichia coli strain. All bacterial strains were cultured under appropriate conditions with or without sodium fluoride, and fluoride resistance was evaluated. All three gene-knockout S. mutans strains showed lower resistance to sodium fluoride than did the wild-type strain. No significant changes in resistance to other sodium halides were recognized between the wild-type and double-knockout strains. Both SMU_1290c and SMU_1289c transformation rescued fluoride transporter-disrupted E. coli cell from fluoride toxicity. We conclude that the chloride channel permeases contribute to fluoride resistance in S. mutans. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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.

The receptor-like pseudokinase MRH1 interacts with the voltage-gated potassium channel AKT2.

PubMed

Sklodowski, Kamil; Riedelsberger, Janin; Raddatz, Natalia; Riadi, Gonzalo; Caballero, Julio; Chérel, Isabelle; Schulze, Waltraud; Graf, Alexander; Dreyer, Ingo

2017-03-16

The potassium channel AKT2 plays important roles in phloem loading and unloading. It can operate as inward-rectifying channel that allows H + -ATPase-energized K + uptake. Moreover, through reversible post-translational modifications it can also function as an open, K + -selective channel, which taps a 'potassium battery', providing additional energy for transmembrane transport processes. Knowledge about proteins involved in the regulation of the operational mode of AKT2 is very limited. Here, we employed a large-scale yeast two-hybrid screen in combination with fluorescence tagging and null-allele mutant phenotype analysis and identified the plasma membrane localized receptor-like kinase MRH1/MDIS2 (AT4G18640) as interaction partner of AKT2. The phenotype of the mrh1-1 knockout plant mirrors that of akt2 knockout plants in energy limiting conditions. Electrophysiological analyses showed that MRH1/MDIS2 failed to exert any functional regulation on AKT2. Using structural protein modeling approaches, we instead gathered evidence that the putative kinase domain of MRH1/MDIS2 lacks essential sites that are indispensable for a functional kinase suggesting that MRH1/MDIS2 is a pseudokinase. We propose that MRH1/MDIS2 and AKT2 are likely parts of a bigger protein complex. MRH1 might help to recruit other, so far unknown partners, which post-translationally regulate AKT2. Additionally, MRH1 might be involved in the recognition of chemical signals.

Female preproenkephalin-knockout mice display altered emotional responses

PubMed Central

Ragnauth, A.; Schuller, A.; Morgan, M.; Chan, J.; Ogawa, S.; Pintar, J.; Bodnar, R. J.; Pfaff, D. W.

2001-01-01

The endogenous opioid system has been implicated in sexual behavior, palatable intake, fear, and anxiety. The present study examined whether ovariectomized female transgenic preproenkephalin-knockout (PPEKO) mice and their wild-type and heterozygous controls displayed alterations in fear and anxiety paradigms, sucrose intake, and lordotic behavior. To examine stability of responding, three squads of the genotypes were tested across seasons over a 20-month period. In a fear-conditioning paradigm, PPEKO mice significantly increased freezing to both fear and fear + shock stimuli relative to controls. In the open field, PPEKO mice spent significantly less time and traversed significantly less distance in the center of an open field than wild-type controls. Further, PPEKO mice spent significantly less time and tended to be less active on the light side of a dark–light chamber than controls, indicating that deletion of the enkephalin gene resulted in exaggerated responses to fear or anxiety-provoking environments. These selective deficits were observed consistently across testing squads spanning 20 months and different seasons. In contrast, PPEKO mice failed to differ from corresponding controls in sucrose, chow, or water intake across a range (0.0001–20%) of sucrose concentrations and failed to differ in either lordotic or female approach to male behaviors when primed with estradiol and progesterone, thereby arguing strongly for the selectivity of a fear and anxiety deficit which was not caused by generalized and nonspecific debilitation. These transgenic data strongly suggest that opioids, and particularly enkephalin gene products, are acting naturally to inhibit fear and anxiety. PMID:11172058

The receptor-like pseudokinase MRH1 interacts with the voltage-gated potassium channel AKT2

NASA Astrophysics Data System (ADS)

Sklodowski, Kamil; Riedelsberger, Janin; Raddatz, Natalia; Riadi, Gonzalo; Caballero, Julio; Chérel, Isabelle; Schulze, Waltraud; Graf, Alexander; Dreyer, Ingo

2017-03-01

The potassium channel AKT2 plays important roles in phloem loading and unloading. It can operate as inward-rectifying channel that allows H+-ATPase-energized K+ uptake. Moreover, through reversible post-translational modifications it can also function as an open, K+-selective channel, which taps a ‘potassium battery’, providing additional energy for transmembrane transport processes. Knowledge about proteins involved in the regulation of the operational mode of AKT2 is very limited. Here, we employed a large-scale yeast two-hybrid screen in combination with fluorescence tagging and null-allele mutant phenotype analysis and identified the plasma membrane localized receptor-like kinase MRH1/MDIS2 (AT4G18640) as interaction partner of AKT2. The phenotype of the mrh1-1 knockout plant mirrors that of akt2 knockout plants in energy limiting conditions. Electrophysiological analyses showed that MRH1/MDIS2 failed to exert any functional regulation on AKT2. Using structural protein modeling approaches, we instead gathered evidence that the putative kinase domain of MRH1/MDIS2 lacks essential sites that are indispensable for a functional kinase suggesting that MRH1/MDIS2 is a pseudokinase. We propose that MRH1/MDIS2 and AKT2 are likely parts of a bigger protein complex. MRH1 might help to recruit other, so far unknown partners, which post-translationally regulate AKT2. Additionally, MRH1 might be involved in the recognition of chemical signals.

Double knockout of pendrin and Na-Cl cotransporter (NCC) causes severe salt wasting, volume depletion, and renal failure.

PubMed

Soleimani, Manoocher; Barone, Sharon; Xu, Jie; Shull, Gary E; Siddiqui, Faraz; Zahedi, Kamyar; Amlal, Hassane

2012-08-14

The Na-Cl cotransporter (NCC), which is the target of inhibition by thiazides, is located in close proximity to the chloride-absorbing transporter pendrin in the kidney distal nephron. Single deletion of pendrin or NCC does not cause salt wasting or excessive diuresis under basal conditions, raising the possibility that these transporters are predominantly active during salt depletion or in response to excess aldosterone. We hypothesized that pendrin and NCC compensate for loss of function of the other under basal conditions, thereby masking the role that each plays in salt absorption. To test our hypothesis, we generated pendrin/NCC double knockout (KO) mice by crossing pendrin KO mice with NCC KO mice. Pendrin/NCC double KO mice displayed severe salt wasting and sharp increase in urine output under basal conditions. As a result, animals developed profound volume depletion, renal failure, and metabolic alkalosis without hypokalemia, which were all corrected with salt replacement. We propose that the combined inhibition of pendrin and NCC can provide a strong diuretic regimen without causing hypokalemia for patients with fluid overload, including patients with congestive heart failure, nephrotic syndrome, diuretic resistance, or generalized edema.

Double knockout of pendrin and Na-Cl cotransporter (NCC) causes severe salt wasting, volume depletion, and renal failure

PubMed Central

Soleimani, Manoocher; Barone, Sharon; Xu, Jie; Shull, Gary E.; Siddiqui, Faraz; Zahedi, Kamyar; Amlal, Hassane

2012-01-01

The Na-Cl cotransporter (NCC), which is the target of inhibition by thiazides, is located in close proximity to the chloride-absorbing transporter pendrin in the kidney distal nephron. Single deletion of pendrin or NCC does not cause salt wasting or excessive diuresis under basal conditions, raising the possibility that these transporters are predominantly active during salt depletion or in response to excess aldosterone. We hypothesized that pendrin and NCC compensate for loss of function of the other under basal conditions, thereby masking the role that each plays in salt absorption. To test our hypothesis, we generated pendrin/NCC double knockout (KO) mice by crossing pendrin KO mice with NCC KO mice. Pendrin/NCC double KO mice displayed severe salt wasting and sharp increase in urine output under basal conditions. As a result, animals developed profound volume depletion, renal failure, and metabolic alkalosis without hypokalemia, which were all corrected with salt replacement. We propose that the combined inhibition of pendrin and NCC can provide a strong diuretic regimen without causing hypokalemia for patients with fluid overload, including patients with congestive heart failure, nephrotic syndrome, diuretic resistance, or generalized edema. PMID:22847418

Linking Yeast Gcn5p Catalytic Function and Gene Regulation Using a Quantitative, Graded Dominant Mutant Approach

PubMed Central

Lanza, Amanda M.; Blazeck, John J.; Crook, Nathan C.; Alper, Hal S.

2012-01-01

Establishing causative links between protein functional domains and global gene regulation is critical for advancements in genetics, biotechnology, disease treatment, and systems biology. This task is challenging for multifunctional proteins when relying on traditional approaches such as gene deletions since they remove all domains simultaneously. Here, we describe a novel approach to extract quantitative, causative links by modulating the expression of a dominant mutant allele to create a function-specific competitive inhibition. Using the yeast histone acetyltransferase Gcn5p as a case study, we demonstrate the utility of this approach and (1) find evidence that Gcn5p is more involved in cell-wide gene repression, instead of the accepted gene activation associated with HATs, (2) identify previously unknown gene targets and interactions for Gcn5p-based acetylation, (3) quantify the strength of some Gcn5p-DNA associations, (4) demonstrate that this approach can be used to correctly identify canonical chromatin modifications, (5) establish the role of acetyltransferase activity on synthetic lethal interactions, and (6) identify new functional classes of genes regulated by Gcn5p acetyltransferase activity—all six of these major conclusions were unattainable by using standard gene knockout studies alone. We recommend that a graded dominant mutant approach be utilized in conjunction with a traditional knockout to study multifunctional proteins and generate higher-resolution data that more accurately probes protein domain function and influence. PMID:22558379

Characterization of Breast Cancer Cell Death Induced by Interferons and Retinoids

DTIC Science & Technology

1999-07-01

treated cells. Cells were treated for 48 hr, before RNA extraction . Figure 4: Expression of GRIM-I in different mouse tissues. A multiple tissue...knockout approach (12). In this teria were scraped from the plates, and plasmid DNA was extracted and purified approach specific cell death-associated genes...ml), and Hirt DNA extracts intracellular redox regulatory enzyme (16). We show that cel- were prepared (22). DNA was digested with DpnI and

40 CFR 60.5430 - What definitions apply to this subpart?

Code of Federal Regulations, 2014 CFR

2014-07-01

... control vessels, bottoms receivers or knockout vessels. (3) Pressure vessels designed to operate in excess... supply natural gas to the process control device (e.g., level control, temperature control, pressure control) where the supply gas pressure is modulated by the process condition, and then flows to the valve...

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.

Characterization of a Bacillus subtilis surfactin synthetase knockout and antimicrobial activity analysis.

PubMed

Liu, Hongxia; Qu, Xiaoxu; Gao, Ling; Zhao, Shengming; Lu, Zhaoxin; Zhang, Chong; Bie, Xiaomei

2016-11-10

Gene knockout is an important approach to improve the production of antimicrobial compounds. B. subtilis PB2-LS10, derived from B. subtilis PB2-L by a surfactin synthetase (srf) genes knockout, exhibits stronger inhibitory action than its parental strain against all tested pathogenic bacteria and fungi. The antimicrobial extracts produced by B. subtilis PB2-L and B. subtilis PB2-LS10 respectively were characterized by the high-resolution LC-ESI-MS. To provide further insight into the distinct antimicrobial activities, we investigated the impact of the srf genes deletion on the growth and gene transcriptional profile of the strains. The mutant strain grew quickly and reached stationary phase 2h earlier than the wild-type. Prominent expression changes in the modified strain involved genes that were essential to metabolic pathways and processes. Genes related to amino acid transport, ATP-binding cassette (ABC) transporters and protein export were up-regulated in strain PB2-LS10. However, amino acid metabolism, carbohydrate metabolism and fatty acid metabolism were repressed. Because of its excellent antimicrobial activity, strain PB2-LS10 has potential for use in food preservation. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

The effect of JAK2 knockout on inhibition of liver tumor growth by inducing apoptosis, autophagy and anti-proliferation via STATs and PI3K/AKT signaling pathways.

PubMed

Xu, Ying; Lv, Sheng-Xiang

2016-12-01

Liver cancer is a leading cause of cancer death, making it as the second most common cause for death from cancer globally. Though many studies before have explored a lot for liver cancer prevention and treatment, there are still a lot far from to know based on the molecular mechanisms. Janus kinase 2 (JAK2) has been reported to play an essential role in the progression of apoptosis, autophagy and proliferation for cells. Therefore, we were aimed to investigate the underlying mechanisms by which JAK2 performed its role in ameliorating liver cancer. JAK2 knockout liver cancer cell lines were involved for our experiments in vitro and in vivo. Western blotting, quantitative RT-PCR (qRT-PCR), ELISA, Immunohistochemistry, and flow-cytometric analysis were used to determine the key signaling pathway regulated by JAK2 for liver cancer progression. Data here indicated that JAK2, indeed, expressed highly in cancer cell lines compared to the normal liver cells. And apoptosis and autophagy were found in JAK2 knockout liver cancer cells through activating Caspase-3, Cyclin-D1 and mTOR regulated by STAT3/5 and PI3K/AKT signaling pathway. And also, the liver cancer cells proliferation was inhibited. In addition, tumor size and weight were reduced by knockout of JAK2 in vivo experiments. These findings demonstrated that JAK2 and its down-streaming signaling pathways play a direct role in the progression of liver cancer possibly. To our knowledge, it was the first time to evaluate the role of JAK2 knockout in improving liver cancer from apoptosis, autophagy and proliferation, which could be a potential target for future therapeutic approach clinically. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

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

Neuronal BDNF Signaling Is Necessary for the Effects of Treadmill Exercise on Synaptic Stripping of Axotomized Motoneurons

PubMed Central

Krakowiak, Joey; Liu, Caiyue; Papudesu, Chandana; Ward, P. Jillian; Wilhelm, Jennifer C.; English, Arthur W.

2015-01-01

The withdrawal of synaptic inputs from the somata and proximal dendrites of spinal motoneurons following peripheral nerve injury could contribute to poor functional recovery. Decreased availability of neurotrophins to afferent terminals on axotomized motoneurons has been implicated as one cause of the withdrawal. No reduction in contacts made by synaptic inputs immunoreactive to the vesicular glutamate transporter 1 and glutamic acid decarboxylase 67 is noted on axotomized motoneurons if modest treadmill exercise, which stimulates the production of neurotrophins by spinal motoneurons, is applied after nerve injury. In conditional, neuron-specific brain-derived neurotrophic factor (BDNF) knockout mice, a reduction in synaptic contacts onto motoneurons was noted in intact animals which was similar in magnitude to that observed after nerve transection in wild-type controls. No further reduction in coverage was found if nerves were cut in knockout mice. Two weeks of moderate daily treadmill exercise following nerve injury in these BDNF knockout mice did not affect synaptic inputs onto motoneurons. Treadmill exercise has a profound effect on synaptic inputs to motoneurons after peripheral nerve injury which requires BDNF production by those postsynaptic cells. PMID:25918648

Rines E3 ubiquitin ligase regulates MAO-A levels and emotional responses.

PubMed

Kabayama, Miyuki; Sakoori, Kazuto; Yamada, Kazuyuki; Ornthanalai, Veravej G; Ota, Maya; Morimura, Naoko; Katayama, Kei-ichi; Murphy, Niall P; Aruga, Jun

2013-08-07

Monoamine oxidase A (MAO-A), the catabolic enzyme of norepinephrine and serotonin, plays a critical role in emotional and social behavior. However, the control and impact of endogenous MAO-A levels in the brain remains unknown. Here we show that the RING finger-type E3 ubiquitin ligase Rines/RNF180 regulates brain MAO-A subset, monoamine levels, and emotional behavior. Rines interacted with MAO-A and promoted its ubiquitination and degradation. Rines knock-out mice displayed impaired stress responses, enhanced anxiety, and affiliative behavior. Norepinephrine and serotonin levels were altered in the locus ceruleus, prefrontal cortex, and amygdala in either stressed or resting conditions, and MAO-A enzymatic activity was enhanced in the locus ceruleus in Rines knock-out mice. Treatment of Rines knock-out mice with MAO inhibitors showed genotype-specific effects on some of the abnormal affective behaviors. These results indicated that the control of emotional behavior by Rines is partly due to the regulation of MAO-A levels. These findings verify that Rines is a critical regulator of the monoaminergic system and emotional behavior and identify a promising candidate drug target for treating diseases associated with emotion.

Impairments in the Initiation of Maternal Behavior in Oxytocin Receptor Knockout Mice

PubMed Central

Rich, Megan E.; deCárdenas, Emily J.; Lee, Heon-Jin; Caldwell, Heather K.

2014-01-01

Oxytocin (Oxt) acting through its single receptor subtype, the Oxtr, is important for the coordination of physiology and behavior associated with parturition and maternal care. Knockout mouse models have been helpful in exploring the contributions of Oxt to maternal behavior, including total body Oxt knockout (Oxt −/−) mice, forebrain conditional Oxtr knockout (Oxtr FB/FB) mice, and total body Oxtr knockout (Oxtr −/−) mice. Since Oxtr −/− mice are unable to lactate, maternal behavior has only been examined in virgin females, or in dams within a few hours of parturition, and there have been no studies that have examined their anxiety-like and depression-like behavior following parturition. To improve our understanding of how the absence of Oxt signaling affects maternal behavior, mood and anxiety, we designed a study using Oxtr −/− mice that separated nursing behavior from other aspects of maternal care, such as licking and grooming by thelectomizing (i.e. removing the nipples) of Oxtr +/+ mice and sham-thelectomizing Oxtr −/− mice, and pairing both genotypes with a wet nurse. We then measured pup abandonment, maternal behavior, and postpartum anxiety-like and depression-like behaviors. We hypothesized that genetic disruption of the Oxtr would impact maternal care, mood and anxiety. Specifically, we predicted that Oxtr −/− dams would have impaired maternal care and increased anxiety-like and depression-like behaviors in the postpartum period. We found that Oxtr −/− dams had significantly higher levels of pup abandonment compared to controls, which is consistent with previous work in Oxtr FB/FB mice. Interestingly, Oxtr −/− dams that initiated maternal care did not differ from wildtype controls in measures of maternal behavior. We also did not find any evidence of altered anxiety-like or depressive-like behavior in the postpartum period of Oxtr −/− dams. Thus, our data suggest that Oxt lowers the threshold for the initiation of maternal behavior. PMID:24892749

Injury risk in professional boxing.

PubMed

Bledsoe, Gregory H; Li, Guohu; Levy, Fred

2005-10-01

Although a popular endeavor, boxing has fallen under increased scrutiny because of its association with traumatic brain injury. However, few studies have investigated the overall epidemiology of boxing injuries from representative samples, and no study has ever documented the incidence of injuries in female boxers. This study is a review of professional boxing data from the state of Nevada from September 2001 through March 2003. Medical and outcome data for all professional boxing matches occurring in Nevada between September 2001 and March 2003 (n = 524 matches) were analyzed on the basis of a pair-matched, case-control design. Cases were boxers who received an injury during the boxing matches. Boxers who were not injured served as control subjects. Both conditional and unconditional logistic regression models were used to assess risk factors for injury. The overall incidence rate of injury was 17.1 per 100 boxer-matches, or 3.4 per 100 boxer-rounds. Facial laceration accounted for 51% of all injuries, followed by hand injury (17%), eye injury (14%), and nose injury (5%). Male boxers were significantly more likely than female boxers to receive injuries (3.6 versus 1.2 per 100 boxer-rounds, P = 0.01). Male boxing matches also ended in knockouts and technical knockouts more often than did female matches (P < 0.001). The risk of injury for those who lost the matches was nearly twice the risk for the winners. Those who lost by knockout had double the risk of injury compared with those who lost by other means. Neither age nor weight was significantly associated with the risk of injury. The injury rate in professional boxing matches is high, particularly among male boxers. Superficial facial lacerations are the most common injury reported. Male boxers have a higher rate of knockout and technical knockouts than female boxers. Further research is necessary to determine the outcomes of injury, particularly the long-term neurologic outcome differences between sexes.

Conditional genomic rearrangement by designed meiotic recombination using VDE (PI-SceI) in yeast.

PubMed

Fukuda, Tomoyuki; Ohya, Yoshikazu; Ohta, Kunihiro

2007-10-01

Meiotic recombination plays critical roles in the acquisition of genetic diversity and has been utilized for conventional breeding of livestock and crops. The frequency of meiotic recombination is normally low, and is extremely low in regions called "recombination cold domains". Here, we describe a new and highly efficient method to modulate yeast meiotic gene rearrangements using VDE (PI-SceI), an intein-encoded endonuclease that causes an efficient unidirectional meiotic gene conversion at its recognition sequence (VRS). We designed universal targeting vectors, by use of which the strain that inserts the VRS at a desired site is acquired. Meiotic induction of the strains provided unidirectional gene conversions and frequent genetic rearrangements of flanking genes with little impact on cell viability. This system thus opens the way for the designed modulation of meiotic gene rearrangements, regardless of recombinational activity of chromosomal domains. Finally, the VDE-VRS system enabled us to conduct meiosis-specific conditional knockout of genes where VDE-initiated gene conversion disrupts the target gene during meiosis, serving as a novel approach to examine the functions of genes during germination of resultant spores.

Role of fibroblast growth factor receptor signaling in kidney development

PubMed Central

2011-01-01

Fibroblast growth factor receptors (Fgfrs) consist of four signaling family members and one nonsignaling “decoy” receptor, Fgfr-like 1 (Fgfrl1), all of which are expressed in the developing kidney. Several studies have shown that exogenous fibroblast growth factors (Fgfs) affect growth and maturation of the metanephric mesenchyme (MM) and ureteric bud (UB) in cultured tissues. Transgenic and conditional knockout approaches in whole animals have shown that Fgfr1 and Fgfr2 (predominantly the IIIc isoform) in kidney mesenchyme are critical for early MM and UB formation. Conditional deletion of the ligand, Fgf8, in nephron precursors or global deletion of Fgfrl1 interrupts nephron formation. Fgfr2 (likely the IIIb isoform signaling downstream of Fgf7 and Fgf10) is critical for ureteric morphogenesis. Moreover, Fgfr2 appears to act independently of Frs2α (the major signaling adapter for Fgfrs) in regulating UB branching. Loss of Fgfr2 in the MM leads to many kidney and urinary tract anomalies, including vesicoureteral reflux. Thus Fgfr signaling is critical for patterning of virtually all renal lineages at early and later stages of development. PMID:21613421

Role of fibroblast growth factor receptor signaling in kidney development.

PubMed

Bates, Carlton M

2011-08-01

Fibroblast growth factor receptors (Fgfrs) consist of four signaling family members and one nonsignaling "decoy" receptor, Fgfr-like 1 (Fgfrl1), all of which are expressed in the developing kidney. Several studies have shown that exogenous fibroblast growth factors (Fgfs) affect growth and maturation of the metanephric mesenchyme (MM) and ureteric bud (UB) in cultured tissues. Transgenic and conditional knockout approaches in whole animals have shown that Fgfr1 and Fgfr2 (predominantly the IIIc isoform) in kidney mesenchyme are critical for early MM and UB formation. Conditional deletion of the ligand, Fgf8, in nephron precursors or global deletion of Fgfrl1 interrupts nephron formation. Fgfr2 (likely the IIIb isoform signaling downstream of Fgf7 and Fgf10) is critical for ureteric morphogenesis. Moreover, Fgfr2 appears to act independently of Frs2α (the major signaling adapter for Fgfrs) in regulating UB branching. Loss of Fgfr2 in the MM leads to many kidney and urinary tract anomalies, including vesicoureteral reflux. Thus Fgfr signaling is critical for patterning of virtually all renal lineages at early and later stages of development.

Manipulation of arginase expression modulates abiotic stress tolerance in Arabidopsis: effect on arginine metabolism and ROS accumulation

PubMed Central

Chan, Zhulong

2013-01-01

Arginine is an important medium for the transport and storage of nitrogen, and arginase (also known as arginine amidohydrolase, ARGAH) is responsible for catalyse of arginine into ornithine and urea in plants. In this study, the impact of AtARGAHs on abiotic stress response was investigated by manipulating AtARGAHs expression. In the knockout mutants of AtARGAHs, enhanced tolerances were observed to multiple abiotic stresses including water deficit, salt, and freezing stresses, while AtARGAH1- and AtARGAH2-overexpressing lines exhibited reduced abiotic stress tolerances compared to the wild type. Consistently, the enhanced tolerances were confirmed by the changes of physiological parameters including electrolyte leakage, water loss rate, stomatal aperture, and survival rate. Interestingly, the direct downstream products of arginine catabolism including polyamines and nitric oxide (NO) concentrations significantly increased in the AtARGAHs-knockout lines, but decreased in overexpressing lines under control conditions. Additionally, the AtARGAHs-overexpressing and -knockout lines displayed significantly reduced relative arginine (% of total free amino acids) relative to the wild type. Similarly, reactive oxygen species accumulation was remarkably regulated by AtARGAHs under abiotic stress conditions, as shown from hydrogen peroxide (H2O2), superoxide radical () concentrations, and antioxidant enzyme activities. Taken together, this is the first report, as far as is known, to provide evidence that AtARGAHs negatively regulate many abiotic stress tolerances, at least partially, attribute to their roles in modulating arginine metabolism and reactive oxygen species accumulation. Biotechnological strategy based on manipulation of AtARGAHs expression will be valuable for future crop breeding. PMID:23378380

Effect of IAA on in vitro growth and colonization of Nostoc in plant roots

PubMed Central

Hussain, Anwar; Shah, Syed T.; Rahman, Hazir; Irshad, Muhammad; Iqbal, Amjad

2015-01-01

Nostoc is widely known for its ability to fix atmospheric nitrogen and the establishment of symbiotic relationship with a wide range of plants from various taxonomic groups. Several strains of Nostoc produce phytohormones that promote growth of its plant partners. Nostoc OS-1 was therefore selected for study because of the presence of putative ipdC gene that encodes a key enzyme to produce Indole-3-acetic acid (IAA). The results indicated that both cellular and released IAA was found high with increasing incubation time and reached to a peak value (i.e., 21 pmol mg-1ch-a) on the third week as determined by UPLC-ESI-MS/MS. Also the Nostoc OS-1 strain efficiently colonized the roots and promoted the growth of rice as well as wheat under axenic conditions and induced ipdC gene that suggested the possible involvement of IAA in these phenotypes. To confirm the impact of IAA on root colonization efficiency and plant promoting phenotypes of Nostoc OS-1, an ipdC knockout mutant was generated by homologous recombinant method. The amount of releasing IAA, in vitro growth, root colonization, and plant promoting efficiency of the ipdC knockout mutant was observed significantly lower than wild type strain under axenic conditions. Importantly, these phenotypes were restored to wild-type levels when the ipdC knockout mutant was complemented with wild type ipdC gene. These results together suggested that ipdC and/or synthesized IAA of Nostoc OS-1 is required for its efficient root colonization and plant promoting activity. PMID:25699072

Ssb1 and Ssb2 cooperate to regulate mouse hematopoietic stem and progenitor cells by resolving replicative stress.

PubMed

Shi, Wei; Vu, Therese; Boucher, Didier; Biernacka, Anna; Nde, Jules; Pandita, Raj K; Straube, Jasmin; Boyle, Glen M; Al-Ejeh, Fares; Nag, Purba; Jeffery, Jessie; Harris, Janelle L; Bain, Amanda L; Grzelak, Marta; Skrzypczak, Magdalena; Mitra, Abhishek; Dojer, Norbert; Crosetto, Nicola; Cloonan, Nicole; Becherel, Olivier J; Finnie, John; Skaar, Jeffrey R; Walkley, Carl R; Pandita, Tej K; Rowicka, Maga; Ginalski, Krzysztof; Lane, Steven W; Khanna, Kum Kum

2017-05-04

Hematopoietic stem and progenitor cells (HSPCs) are vulnerable to endogenous damage and defects in DNA repair can limit their function. The 2 single-stranded DNA (ssDNA) binding proteins SSB1 and SSB2 are crucial regulators of the DNA damage response; however, their overlapping roles during normal physiology are incompletely understood. We generated mice in which both Ssb1 and Ssb2 were constitutively or conditionally deleted. Constitutive Ssb1/Ssb2 double knockout (DKO) caused early embryonic lethality, whereas conditional Ssb1/Ssb2 double knockout (cDKO) in adult mice resulted in acute lethality due to bone marrow failure and intestinal atrophy featuring stem and progenitor cell depletion, a phenotype unexpected from the previously reported single knockout models of Ssb1 or Ssb2 Mechanistically, cDKO HSPCs showed altered replication fork dynamics, massive accumulation of DNA damage, genome-wide double-strand breaks enriched at Ssb-binding regions and CpG islands, together with the accumulation of R -loops and cytosolic ssDNA. Transcriptional profiling of cDKO HSPCs revealed the activation of p53 and interferon (IFN) pathways, which enforced cell cycling in quiescent HSPCs, resulting in their apoptotic death. The rapid cell death phenotype was reproducible in in vitro cultured cDKO-hematopoietic stem cells, which were significantly rescued by nucleotide supplementation or after depletion of p53. Collectively, Ssb1 and Ssb2 control crucial aspects of HSPC function, including proliferation and survival in vivo by resolving replicative stress to maintain genomic stability. © 2017 by The American Society of Hematology.

Ssb1 and Ssb2 cooperate to regulate mouse hematopoietic stem and progenitor cells by resolving replicative stress

PubMed Central

Vu, Therese; Boucher, Didier; Biernacka, Anna; Nde, Jules; Pandita, Raj K.; Straube, Jasmin; Boyle, Glen M.; Al-Ejeh, Fares; Jeffery, Jessie; Harris, Janelle L.; Bain, Amanda L.; Grzelak, Marta; Skrzypczak, Magdalena; Mitra, Abhishek; Dojer, Norbert; Crosetto, Nicola; Cloonan, Nicole; Becherel, Olivier J.; Finnie, John; Skaar, Jeffrey R.; Walkley, Carl R.; Pandita, Tej K.; Rowicka, Maga; Ginalski, Krzysztof

2017-01-01

Hematopoietic stem and progenitor cells (HSPCs) are vulnerable to endogenous damage and defects in DNA repair can limit their function. The 2 single-stranded DNA (ssDNA) binding proteins SSB1 and SSB2 are crucial regulators of the DNA damage response; however, their overlapping roles during normal physiology are incompletely understood. We generated mice in which both Ssb1 and Ssb2 were constitutively or conditionally deleted. Constitutive Ssb1/Ssb2 double knockout (DKO) caused early embryonic lethality, whereas conditional Ssb1/Ssb2 double knockout (cDKO) in adult mice resulted in acute lethality due to bone marrow failure and intestinal atrophy featuring stem and progenitor cell depletion, a phenotype unexpected from the previously reported single knockout models of Ssb1 or Ssb2. Mechanistically, cDKO HSPCs showed altered replication fork dynamics, massive accumulation of DNA damage, genome-wide double-strand breaks enriched at Ssb-binding regions and CpG islands, together with the accumulation of R-loops and cytosolic ssDNA. Transcriptional profiling of cDKO HSPCs revealed the activation of p53 and interferon (IFN) pathways, which enforced cell cycling in quiescent HSPCs, resulting in their apoptotic death. The rapid cell death phenotype was reproducible in in vitro cultured cDKO-hematopoietic stem cells, which were significantly rescued by nucleotide supplementation or after depletion of p53. Collectively, Ssb1 and Ssb2 control crucial aspects of HSPC function, including proliferation and survival in vivo by resolving replicative stress to maintain genomic stability. PMID:28270450

Conditional loss of progranulin in neurons is not sufficient to cause neuronal ceroid lipofuscinosis-like neuropathology in mice.

PubMed

Petkau, Terri L; Blanco, Jake; Leavitt, Blair R

2017-10-01

Progranulin deficiency due to heterozygous null mutations in the GRN gene is a common cause of familial frontotemporal lobar degeneration (FTLD), while homozygous loss-of-function GRN mutations cause neuronal ceroid lipofuscinosis (NCL). Aged progranulin-knockout mice display highly exaggerated lipofuscinosis, microgliosis, and astrogliosis, as well as mild cell loss in specific brain regions. Progranulin is a secreted glycoprotein expressed in both neurons and microglia, but not astrocytes, in the brain. We generated conditional progranulin-knockout mice that lack progranulin in nestin-expressing cells (Nes-cKO mice), which include most neurons as well as astrocytes. We confirmed near complete knockout of progranulin in neurons in Nes-cKO mice, while microglial progranulin levels remained similar to that of wild-type animals. Overall brain progranulin levels were reduced by about 50% in Nes-cKO, and no Grn was detected in primary Nes-cKO neurons. Nes-cKO mice aged to 12months did not display any increase in lipofuscin deposition, microgliosis, or astrogliosis in the four brain regions examined, though increases were observed for most of these measures in Grn-null animals. We conclude that neuron-specific loss of progranulin is not sufficient to cause similar neuropathological changes to those seen in constitutive Grn-null animals. Our results suggest that increased lipofuscinosis and gliosis in Grn-null animals are not caused by intrinsic progranulin deficiency in neurons, and that microglia-derived progranulin may be sufficient to maintain neuronal health and homeostasis in the brain. Copyright © 2017 Elsevier Inc. All rights reserved.

A conditioned aversion study of sucrose and SC45647 taste in TRPM5 knockout mice.

PubMed

Eddy, Meghan C; Eschle, Benjamin K; Peterson, Darlene; Lauras, Nathan; Margolskee, Robert F; Delay, Eugene R

2012-06-01

Previously, published studies have reported mixed results regarding the role of the TRPM5 cation channel in signaling sweet taste by taste sensory cells. Some studies have reported a complete loss of sweet taste preference in TRPM5 knockout (KO) mice, whereas others have reported only a partial loss of sweet taste preference. This study reports the results of conditioned aversion studies designed to motivate wild-type (WT) and KO mice to respond to sweet substances. In conditioned taste aversion experiments, WT mice showed nearly complete LiCl-induced response suppression to sucrose and SC45647. In contrast, TRPM5 KO mice showed a much smaller conditioned aversion to either sweet substance, suggesting a compromised, but not absent, ability to detect sweet taste. A subsequent conditioned flavor aversion experiment was conducted to determine if TRPM5 KO mice were impaired in their ability to learn a conditioned aversion. In this experiment, KO and WT mice were conditioned to a mixture of SC45647 and amyl acetate (an odor cue). Although WT mice avoided both components of the stimulus mixture, they avoided SC45647 more than the odor cue. The KO mice also avoided both stimuli, but they avoided the odor component more than SC45647, suggesting that while the KO mice are capable of learning an aversion, to them the odor cue was more salient than the taste cue. Collectively, these findings suggest the TRPM5 KO mice have some residual ability to detect SC45647 and sucrose, and, like bitter, there may be a TRPM5-independent transduction pathway for detecting these substances.

Problem-Solving Test: Targeted Gene Disruption

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2008-01-01

Mutational inactivation of a specific gene is the most powerful technique to analyze the biological function of the gene. This approach has been used for a long time in viruses, bacteria, yeast, and fruit fly, but looked quite hopeless in more complex organisms. Targeted inactivation of specific genes (also known as knock-out mutation) in mice is…

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.

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.

Dnmt1 regulates the myogenic lineage specification of muscle stem cells.

PubMed

Liu, Renjing; Kim, Kun-Yong; Jung, Yong-Wook; Park, In-Hyun

2016-10-18

DNA methylation is an important epigenetic mark that regulates gene expression. Dnmt1 plays an important role in maintaining DNA methylation patterns on daughter DNA strands. Studies have shed light into the functional role of Dnmt1 regulation in the hematopoietic and epidermal systems. Here we show that Dnmt1 is required for myogenesis. Loss of Dnmt1 results in reduced expression of myogenic genes and defects in myogenic differentiation. We have utilized a conditional knockout mouse approach to examine the functional consequences of Dnmt1 depletion specifically in the developing muscle. These mice were born runted, with smaller body weights, and reduced ability to form myotubes in vitro. We show that expression of Id-1, a negative regulator of myogenesis, is enhanced in Dnmt1-deficient cultures, leading to enhanced transdifferentiation of myoblasts toward the osteogenic lineage. Thus, these studies demonstrate that Dnmt1 influences cellular identity and determines lineage fidelity.

Dnmt1 regulates the myogenic lineage specification of muscle stem cells

PubMed Central

Liu, Renjing; Kim, Kun-Yong; Jung, Yong-Wook; Park, In-Hyun

2016-01-01

DNA methylation is an important epigenetic mark that regulates gene expression. Dnmt1 plays an important role in maintaining DNA methylation patterns on daughter DNA strands. Studies have shed light into the functional role of Dnmt1 regulation in the hematopoietic and epidermal systems. Here we show that Dnmt1 is required for myogenesis. Loss of Dnmt1 results in reduced expression of myogenic genes and defects in myogenic differentiation. We have utilized a conditional knockout mouse approach to examine the functional consequences of Dnmt1 depletion specifically in the developing muscle. These mice were born runted, with smaller body weights, and reduced ability to form myotubes in vitro. We show that expression of Id-1, a negative regulator of myogenesis, is enhanced in Dnmt1-deficient cultures, leading to enhanced transdifferentiation of myoblasts toward the osteogenic lineage. Thus, these studies demonstrate that Dnmt1 influences cellular identity and determines lineage fidelity. PMID:27752090

Measuring reward with the conditioned place preference (CPP) paradigm: update of the last decade.

PubMed

Tzschentke, Thomas M

2007-09-01

Conditioned place preference (CPP) continues to be one of the most popular models to study the motivational effects of drugs and non-drug treatments in experimental animals. This is obvious from a steady year-to-year increase in the number of publications reporting the use this model. Since the compilation of the preceding review in 1998, more than 1000 new studies using place conditioning have been published, and the aim of the present review is to provide an overview of these recent publications. There are a number of trends and developments that are obvious in the literature of the last decade. First, as more and more knockout and transgenic animals become available, place conditioning is increasingly used to assess the motivational effects of drugs or non-drug rewards in genetically modified animals. Second, there is a still small but growing literature on the use of place conditioning to study the motivational aspects of pain, a field of pre-clinical research that has so far received little attention, because of the lack of appropriate animal models. Third, place conditioning continues to be widely used to study tolerance and sensitization to the rewarding effects of drugs induced by pre-treatment regimens. Fourth, extinction/reinstatement procedures in place conditioning are becoming increasingly popular. This interesting approach is thought to model certain aspects of relapse to addictive behavior and has previously almost exclusively been studied in drug self-administration paradigms. It has now also become established in the place conditioning literature and provides an additional and technically easy approach to this important phenomenon. The enormous number of studies to be covered in this review prevented in-depth discussion of many methodological, pharmacological or neurobiological aspects; to a large extent, the presentation of data had to be limited to a short and condensed summary of the most relevant findings.

Characterization of adult ghrelin and ghrelin receptor knockout mice under positive and negative energy balance

USDA-ARS?s Scientific Manuscript database

Ghrelin and the ghrelin receptor (GH secretagogue receptor, GHS-R) are believed to have important roles in energy homeostasis. We describe results from the first studies to be conducted in congenic (N10) adult ghrelin(-/-) and Ghsr(-/-) mice under conditions of both positive (high-fat diet) and nega...

New animal models of cystic fibrosis: what are they teaching us?

PubMed Central

Keiser, Nicholas W.; Engelhardt, John F.

2013-01-01

Purpose of review Cystic fibrosis is the first human genetic disease to benefit from the directed engineering of three different species of animal models (mice, pigs, and ferrets). Recent studies on the cystic fibrosis pig and ferret models are providing new information about the pathophysiology of cystic fibrosis in various organ systems. Additionally, new conditional cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice are teaching unexpected lessons about CFTR function in surprising cellular locations. Comparisons between these animal models and the human condition are key to dissecting the complexities of disease pathophysiology in cystic fibrosis. Recent findings Cystic fibrosis pigs and ferrets have provided new models to study the spontaneous development of disease in the lung and pancreas, two organs that are largely spared overt spontaneous disease in cystic fibrosis mice. New cystic fibrosis mouse models are now interrogating CFTR functions involved in growth and inflammation at an organ-based level using conditional knockout technology. Together, these models are providing new insights on the human condition. Summary Basic and clinical cystic fibrosis research will benefit greatly from the comparative pathophysiology of cystic fibrosis mice, pigs, and ferrets. Both similarities and differences between these three cystic fibrosis models will inform pathophysiologically important mechanisms of CFTR function in humans and aid in the development of both organ-specific and general therapies for cystic fibrosis. PMID:21857224

Impaired long-term memory retention: common denominator for acutely or genetically reduced hippocampal neurogenesis in adult mice.

PubMed

Ben Abdallah, Nada M-B; Filipkowski, Robert K; Pruschy, Martin; Jaholkowski, Piotr; Winkler, Juergen; Kaczmarek, Leszek; Lipp, Hans-Peter

2013-09-01

In adult rodents, decreasing hippocampal neurogenesis experimentally using different approaches often impairs performance in hippocampus-dependent processes. Nonetheless, functional relevance of adult neurogenesis is far from being unraveled, and deficits so far described in animal models often lack reproducibility. One hypothesis is that such differences might be the consequence of the extent of the methodological specificity used to alter neurogenesis rather than the extent to which adult neurogenesis is altered. To address this, we focused on cranial irradiation, the most widely used technique to impair hippocampal neurogenesis and consequentially induce hippocampus-dependent behavioral deficits. To investigate the specificity of the technique, we thus exposed 4-5 months old female cyclin D2 knockout mice, a model lacking physiological levels of olfactory and hippocampal neurogenesis, to an X-ray dose of 10 Gy, reported to specifically affect transiently amplifying precursors. After a recovery period of 1.5 months, behavioral tests were performed and probed for locomotor activity, habituation, anxiety, and spatial learning and memory. Spatial learning in the Morris water maze was intact in all experimental groups. Although spatial memory retention assessed 24h following acquisition was also intact in all mice, irradiated wild type and cyclin D2 knockout mice displayed memory deficits one week after acquisition. In addition, we observed significant differences in tests addressing anxiety and locomotor activity dependent on the technique used to alter neurogenesis. Whereas irradiated mice were hyperactive regardless of their genotype, cyclin D2 knockout mice were hypoactive in most of the tests and displayed altered habituation. The present study emphasizes that different approaches aimed at decreasing adult hippocampal neurogenesis may result in distinct behavioral impairments related to locomotion and anxiety. In contrast, spatial long-term memory retention is consistently altered after both approaches suggesting a plausible implication of hippocampal neurogenesis in this cognitive process. Copyright © 2013 Elsevier B.V. All rights reserved.

15 years of genetic approaches in vivo for addiction research: Opioid receptor and peptide gene knockout in mouse models of drug abuse.

PubMed

Charbogne, Pauline; Kieffer, Brigitte L; Befort, Katia

2014-01-01

The endogenous opioid system is expressed throughout the brain reinforcement circuitry, and plays a major role in reward processing, mood control and the development of addiction. This neuromodulator system is composed of three receptors, mu, delta and kappa, interacting with a family of opioid peptides derived from POMC (β-endorphin), preproenkephalin (pEnk) and preprodynorphin (pDyn) precursors. Knockout mice targeting each gene of the opioid system have been created almost two decades ago. Extending classical pharmacology, these mutant mice represent unique tools to tease apart the specific role of each opioid receptor and peptide in vivo, and a powerful approach to understand how the opioid system modulates behavioral effects of drugs of abuse. The present review summarizes these studies, with a focus on major drugs of abuse including morphine/heroin, cannabinoids, psychostimulants, nicotine or alcohol. Genetic data, altogether, set the mu receptor as the primary target for morphine and heroin. In addition, this receptor is essential to mediate rewarding properties of non-opioid drugs of abuse, with a demonstrated implication of β-endorphin for cocaine and nicotine. Delta receptor activity reduces levels of anxiety and depressive-like behaviors, and facilitates morphine-context association. pEnk is involved in these processes and delta/pEnk signaling likely regulates alcohol intake. The kappa receptor mainly interacts with pDyn peptides to limit drug reward, and mediate dysphoric effects of cannabinoids and nicotine. Kappa/dynorphin activity also increases sensitivity to cocaine reward under stressful conditions. The opioid system remains a prime candidate to develop successful therapies in addicted individuals, and understanding opioid-mediated processes at systems level, through emerging genetic and imaging technologies, represents the next challenging goal and a promising avenue in addiction research. This article is part of a Special Issue entitled 'NIDA 40th Anniversary Issue'. Copyright © 2013 Elsevier Ltd. All rights reserved.

Requirement for the eIF4E binding proteins for the synergistic down-regulation of protein synthesis by hypertonic conditions and mTOR inhibition.

PubMed

Clemens, Michael J; Elia, Androulla; Morley, Simon J

2013-01-01

The protein kinase mammalian target of rapamycin (mTOR) regulates the phosphorylation and activity of several proteins that have the potential to control translation, including p70S6 kinase and the eIF4E binding proteins 4E-BP1 and 4E-BP2. In spite of this, in exponentially growing cells overall protein synthesis is often resistant to mTOR inhibitors. We report here that sensitivity of wild-type mouse embryonic fibroblasts (MEFs) to mTOR inhibitors can be greatly increased when the cells are subjected to the physiological stress imposed by hypertonic conditions. In contrast, protein synthesis in MEFs with a double knockout of 4E-BP1 and 4E-BP2 remains resistant to mTOR inhibitors under these conditions. Phosphorylation of p70S6 kinase and protein kinase B (Akt) is blocked by the mTOR inhibitor Ku0063794 equally well in both wild-type and 4E-BP knockout cells, under both normal and hypertonic conditions. The response of protein synthesis to hypertonic stress itself does not require the 4E-BPs. These data suggest that under certain stress conditions: (i) translation has a greater requirement for mTOR activity and (ii) there is an absolute requirement for the 4E-BPs for regulation by mTOR. Importantly, dephosphorylation of p70S6 kinase and Akt is not sufficient to affect protein synthesis acutely.

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

Embryonic ablation of neuronal VGF increases energy expenditure and reduces body weight.

PubMed

Jiang, Cheng; Lin, Wei-Jye; Sadahiro, Masato; Shin, Andrew C; Buettner, Christoph; Salton, Stephen R

2017-08-01

Germline ablation of VGF, a secreted neuronal, neuroendocrine, and endocrine peptide precursor, results in lean, hypermetabolic, and infertile adult mice that are resistant to diet-, lesion-, and genetically-induced obesity and diabetes (Hahm et al., 1999, 2002). To assess whether this phenotype is predominantly driven by reduced VGF expression in developing and/or adult neurons, or in peripheral endocrine and neuroendocrine tissues, we generated and analyzed conditional VGF knockout mice, obtained by mating loxP-flanked (floxed) Vgf mice with either pan-neuronal Synapsin-Cre- or forebrain alpha-CaMKII-Cre-recombinase-expressing transgenic mice. Adult male and female mice, with conditional ablation of the Vgf gene in embryonic neurons had significantly reduced body weight, increased energy expenditure, and were resistant to diet-induced obesity. Conditional forebrain postnatal ablation of VGF in male mice, primarily in adult excitatory neurons, had no measurable effect on body weight nor on energy expenditure, but led to a modest increase in adiposity, partially overlapping the effect of AAV-Cre-mediated targeted ablation of VGF in the adult ventromedial hypothalamus and arcuate nucleus of floxed Vgf mice (Foglesong et al., 2016), and also consistent with results of icv delivery of the VGF-derived peptide TLQP-21 to adult mice, which resulted in increased energy expenditure and reduced adiposity (Bartolomucci et al., 2006). Because the lean, hypermetabolic phenotype of germline VGF knockout mice is to a great extent recapitulated in Syn-Cre +/- ,Vgf flpflox/flpflox mice, we conclude that the metabolic profile of germline VGF knockout mice is largely the result of VGF ablation in embryonic CNS neurons, rather than peripheral endocrine and/or neuroendocrine cells, and that in forebrain structures such as hypothalamus, VGF and/or VGF-derived peptides play uniquely different roles in the developing and adult nervous system. Copyright © 2016 Elsevier Ltd. All rights reserved.

The gene for a lectin-like protein is transcriptionally activated during sexual development, but is not essential for fruiting body formation in the filamentous fungus Sordaria macrospora.

PubMed

Nowrousian, Minou; Cebula, Patricia

2005-11-03

The filamentous fungus Sordaria macrospora forms complex three-dimensional fruiting bodies called perithecia that protect the developing ascospores and ensure their proper discharge. In previous microarray analyses, several genes have been identified that are downregulated in sterile mutants compared to the wild type. Among these genes was tap1 (transcript associated with perithecial development), a gene encoding a putative lectin homolog. Analysis of tap1 transcript levels in the wild type under conditions allowing only vegetative growth compared to conditions that lead to fruiting body development showed that tap1 is not only downregulated in developmental mutants but is also upregulated in the wild type during fruiting body development. We have cloned and sequenced a 3.2 kb fragment of genomic DNA containing the tap1 open reading frame and adjoining sequences. The genomic region comprising tap1 is syntenic to its homologous region in the closely related filamentous fungus Neurospora crassa. To determine whether tap1 is involved in fruiting body development in S. macrospora, a knockout construct was generated in which the tap1 open reading frame was replaced by the hygromycin B resistance gene hph under the control of fungal regulatory regions. Transformation of the S. macrospora wild type with this construct resulted in a tap1 deletion strain where tap1 had been replaced by the hph cassette. The knockout strain displayed no phenotypic differences under conditions of vegetative growth and sexual development when compared to the wild type. Double mutants carrying the Deltatap1 allele in several developmental mutant backgrounds were phenotypically similar to the corresponding developmental mutant strains. The tap1 transcript is strongly upregulated during sexual development in S. macrospora; however, analysis of a tap1 knockout strain shows that tap1 is not essential for fruiting body formation in S. macrospora.

The gene for a lectin-like protein is transcriptionally activated during sexual development, but is not essential for fruiting body formation in the filamentous fungus Sordaria macrospora

PubMed Central

Nowrousian, Minou; Cebula, Patricia

2005-01-01

Background The filamentous fungus Sordaria macrospora forms complex three-dimensional fruiting bodies called perithecia that protect the developing ascospores and ensure their proper discharge. In previous microarray analyses, several genes have been identified that are downregulated in sterile mutants compared to the wild type. Among these genes was tap1 (transcript associated with perithecial development), a gene encoding a putative lectin homolog. Results Analysis of tap1 transcript levels in the wild type under conditions allowing only vegetative growth compared to conditions that lead to fruiting body development showed that tap1 is not only downregulated in developmental mutants but is also upregulated in the wild type during fruiting body development. We have cloned and sequenced a 3.2 kb fragment of genomic DNA containing the tap1 open reading frame and adjoining sequences. The genomic region comprising tap1 is syntenic to its homologous region in the closely related filamentous fungus Neurospora crassa. To determine whether tap1 is involved in fruiting body development in S. macrospora, a knockout construct was generated in which the tap1 open reading frame was replaced by the hygromycin B resistance gene hph under the control of fungal regulatory regions. Transformation of the S. macrospora wild type with this construct resulted in a tap1 deletion strain where tap1 had been replaced by the hph cassette. The knockout strain displayed no phenotypic differences under conditions of vegetative growth and sexual development when compared to the wild type. Double mutants carrying the Δtap1 allele in several developmental mutant backgrounds were phenotypically similar to the corresponding developmental mutant strains. Conclusion The tap1 transcript is strongly upregulated during sexual development in S. macrospora; however, analysis of a tap1 knockout strain shows that tap1 is not essential for fruiting body formation in S. macrospora. PMID:16266439

Photosynthetic Response of Poplars (Populus) to Climatic Stressors: Investigating Isoprene's Role in Increasing Tolerance to Temperature and Atmospheric Water Stress in Arizona

NASA Astrophysics Data System (ADS)

Pfeiffer, A. W.; Minor, R. L.; Heard, M. M.; Barron-Gafford, G.

2014-12-01

The southwestern United States is expected to become warmer and drier under future climate projections. The way in which plant and ecosystems respond to these changes is valuable for predicting carbon and water cycling, ecosystem resilience, phenology, and future agriculture, including biofuel production. We examined the interacting effects of dominant climate stressors--vapor pressure deficit (VPD) and temperature--on photosynthesis. Specifically, we tested whether or not plant production of the terpene isoprene imparts heat and water-stress tolerance. Within an experimental common garden of poplars (Populus) at University of Arizona's Biosphere 2 we measured four separate genetic lines - two that retained isoprene production capacity and two that had this gene "knocked out". VPD was altered at temperatures of 30, 35, and 40C to present both heat and aridity stresses. Maximum photosynthetic capacity (Amax), the VPD at which Amax occurred (VPDopt), and the VPD range between Amax and ninety percent of Amax (Ω90) were calculated to quantify how VPD differentially affected the lines. Amax was significantly lower in knockout lines than in control lines. Moreover, the difference in Amax between lines increased from 19.3% at 30C to 28.4% at 35C to 39.0% at 40C, indicating that trees without isoprene production are less equipped to handle hot and dry conditions. Ω90 and VPDopt response were not the same, though. Isoprene knockouts had significantly higher VPD optimums (1.9749 kPA vs. 1.6451 kPa) compared to isoprene-producing lines. Although maximum photosynthesis is diminished without isoprene production under water and heat stress, isoprene knockout lines were still fairly active at a high VPD and under a wide range of VPD conditions. Beyond advancing our basic understanding of plant ecophysiology, these results will inform the potential use of poplars as a source of biofuel production across a range of current and projected climate conditions.

Heparan Sulfate Biosynthesis Enzyme, Ext1, Contributes to Outflow Tract Development of Mouse Heart via Modulation of FGF Signaling.

PubMed

Zhang, Rui; Cao, Peijuan; Yang, Zhongzhou; Wang, Zhenzhen; Wu, Jiu-Lin; Chen, Yan; Pan, Yi

2015-01-01

Glycosaminoglycans are important regulators of multiple signaling pathways. As a major constituent of the heart extracellular matrix, glycosaminoglycans are implicated in cardiac morphogenesis through interactions with different signaling morphogens. Ext1 is a glycosyltransferase responsible for heparan sulfate synthesis. Here, we evaluate the function of Ext1 in heart development by analyzing Ext1 hypomorphic mutant and conditional knockout mice. Outflow tract alignment is sensitive to the dosage of Ext1. Deletion of Ext1 in the mesoderm induces a cardiac phenotype similar to that of a mutant with conditional deletion of UDP-glucose dehydrogenase, a key enzyme responsible for synthesis of all glycosaminoglycans. The outflow tract defect in conditional Ext1 knockout(Ext1f/f:Mesp1Cre) mice is attributable to the reduced contribution of second heart field and neural crest cells. Ext1 deletion leads to downregulation of FGF signaling in the pharyngeal mesoderm. Exogenous FGF8 ameliorates the defects in the outflow tract and pharyngeal explants. In addition, Ext1 expression in second heart field and neural crest cells is required for outflow tract remodeling. Our results collectively indicate that Ext1 is crucial for outflow tract formation in distinct progenitor cells, and heparan sulfate modulates FGF signaling during early heart development.

Histone Deacetylase 3 Is Necessary for Proper Brain Development*

PubMed Central

Norwood, Jordan; Franklin, Jade M.; Sharma, Dharmendra; D'Mello, Santosh R.

2014-01-01

The functional role of histone deacetylase 3 (HDAC3) in the developing brain has yet to be elucidated. We show that mice lacking HDAC3 in neurons and glia of the central nervous system, Nes-Cre/HDAC3 conditional KO mice, show major abnormalities in the cytoarchitecture of the neocortex and cerebellum and die within 24 h of birth. Later-born neurons do not localize properly in the cortex. A similar mislocalization is observed with cerebellar Purkinje neurons. Although the proportion of astrocytes is higher than normal, the numbers of oligodendrocytes are reduced. In contrast, conditional knockout of HDAC3 in neurons of the forebrain and certain other brain regions, using Thy1-Cre and calcium/calmodulin dependent protein kinase II α-Cre for ablation, produces no overt abnormalities in the organization of cells within the cortex or of cerebellar Purkinje neurons at birth. However, both lines of conditional knockout mice suffer from progressive hind limb paralysis and ataxia and die around 6 weeks after birth. The mice display an increase in overall numbers of cells, higher numbers of astrocytes, and Purkinje neuron degeneration. Taken together, our results demonstrate that HDAC3 plays an essential role in regulating brain development, with effects on both neurons and glia in different brain regions. PMID:25339172

Remote Memory and Cortical Synaptic Plasticity Require Neuronal CCCTC-Binding Factor (CTCF).

PubMed

Kim, Somi; Yu, Nam-Kyung; Shim, Kyu-Won; Kim, Ji-Il; Kim, Hyopil; Han, Dae Hee; Choi, Ja Eun; Lee, Seung-Woo; Choi, Dong Il; Kim, Myung Won; Lee, Dong-Sung; Lee, Kyungmin; Galjart, Niels; Lee, Yong-Seok; Lee, Jae-Hyung; Kaang, Bong-Kiun

2018-05-30

The molecular mechanism of long-term memory has been extensively studied in the context of the hippocampus-dependent recent memory examined within several days. However, months-old remote memory maintained in the cortex for long-term has not been investigated much at the molecular level yet. Various epigenetic mechanisms are known to be important for long-term memory, but how the 3D chromatin architecture and its regulator molecules contribute to neuronal plasticity and systems consolidation is still largely unknown. CCCTC-binding factor (CTCF) is an 11-zinc finger protein well known for its role as a genome architecture molecule. Male conditional knock-out mice in which CTCF is lost in excitatory neurons during adulthood showed normal recent memory in the contextual fear conditioning and spatial water maze tasks. However, they showed remarkable impairments in remote memory in both tasks. Underlying the remote memory-specific phenotypes, we observed that female CTCF conditional knock-out mice exhibit disrupted cortical LTP, but not hippocampal LTP. Similarly, we observed that CTCF deletion in inhibitory neurons caused partial impairment of remote memory. Through RNA sequencing, we observed that CTCF knockdown in cortical neuron culture caused altered expression of genes that are highly involved in cell adhesion, synaptic plasticity, and memory. These results suggest that remote memory storage in the cortex requires CTCF-mediated gene regulation in neurons, whereas recent memory formation in the hippocampus does not. SIGNIFICANCE STATEMENT CCCTC-binding factor (CTCF) is a well-known 3D genome architectural protein that regulates gene expression. Here, we use two different CTCF conditional knock-out mouse lines and reveal, for the first time, that CTCF is critically involved in the regulation of remote memory. We also show that CTCF is necessary for appropriate expression of genes, many of which we found to be involved in the learning- and memory-related processes. Our study provides behavioral and physiological evidence for the involvement of CTCF-mediated gene regulation in the remote long-term memory and elucidates our understanding of systems consolidation mechanisms. Copyright © 2018 the authors 0270-6474/18/385042-11$15.00/0.

Astrocyte- and endothelial-targeted CCL2 conditional knockout mice: critical tools for studying the pathogenesis of neuroinflammation.

PubMed

Ge, Shujun; Murugesan, Nivetha; Pachter, Joel S

2009-09-01

While the expression of the C-C chemokine ligand 2 (CCL2) in the central nervous system (CNS) is associated with numerous neuroinflammatory conditions, the critical cellular sources of this chemokine, which is responsible for disease processes-as well as associated pathogenic mechanisms, remain unresolved. As the potential for anti-CCL2 therapeutics in treating neuroinflammatory disease is likely to be contingent upon effective drug delivery to the source(s) and/or target(s) of CCL2 action in the CNS, tools to highlight the course of CCL2 action during neuroinflammation are imperative. In response to this need, we used the Cre/loxP and FLP-FRT recombination system to develop the first two, cell-conditional CCL2 knockout mice-separately targeting CCL2 gene elimination to astrocytes and endothelial cells, both of which have been considered to play crucial though undefined roles in neuroinflammatory disease. Specifically, mice containing a floxed CCL2 allele were intercrossed with GFAP-Cre or Tie2-Cre transgenic mice to generate mice with CCL2-deficient astrocytes (astrocyte KO) or endothelial cells (endothelial KO), respectively. Polymerase chain reaction, reverse transcription polymerase chain reaction/quantitative reverse transcriptase polymerase chain reaction, and enzyme-linked immunosorbent assay of CCL2 gene, RNA, and protein, respectively, from cultured astrocytes and brain microvascular endothelial cells (BMEC) established the efficiency and specificity of the CCL2 gene deletions and a CCL2 null phenotype in these CNS cells. Effective cell-conditional knockout of CCL2 was also confirmed in an in vivo setting, wherein astrocytes and BMEC were retrieved by immune-guided laser capture microdissection from their in situ positions in the brains of mice experiencing acute, lipopolysaccharide-mediated endotoxemia to induce CCL2 gene expression. In vivo analysis further revealed apparent cross-talk between BMEC and astrocytes regarding the regulation of astrocyte CCL2 expression. Use of astrocyte KO and endothelial KO mice should prove critical in elaborating the pathogenic mechanisms of and optimizing the treatments for neuroinflammatory disease.

Elimination of Kalrn Expression in POMC Cells Reduces Anxiety-Like Behavior and Contextual Fear Learning

PubMed Central

Mandela, Prashant; Yan, Yan; LaRese, Taylor; Eipper, Betty A.; Mains, Richard E.

2014-01-01

Kalirin, a Rho GDP/GTP exchange factor for Rac1 and RhoG, is known to play an essential role in the formation and maintenance of excitatory synapses and in the secretion of neuropeptides. Mice unable to express any of the isoforms of Kalrn in cells that produce POMC at any time during development (POMC cells) exhibited reduced anxiety-like behavior and reduced acquisition of passive avoidance behavior, along with sex-specific alteration in the corticosterone response to restraint stress. Strikingly, lack of Kalrn expression in POMC cells closely mimicked the effects of global Kalrn knockout on anxiety-like behavior and passive avoidance conditioning without causing the other deficits noted in Kalrn knockout mice. Our data suggest that deficits in excitatory inputs onto POMC neurons are responsible for the behavioral phenotypes observed. PMID:25014196

Elongation factor P is dispensable in Escherichia coli and Pseudomonas aeruginosa.

PubMed

Balibar, Carl J; Iwanowicz, Dorothy; Dean, Charles R

2013-09-01

Elongation factor P (EF-P) is a highly conserved ribosomal initiation factor responsible for stimulating formation of the first peptide bond. Its essentiality has been debated and may differ depending on the organism. Here, we demonstrate that EF-P is dispensable in Escherichia coli and Pseudomonas aeruginosa under laboratory growth conditions. Although knockouts are viable, growth rates are diminished compared with wild-type strains. Despite this cost in fitness, these mutants are not more susceptible to a wide range of antibiotics; including ribosome targeting antibiotics, such as lincomycin, chloramphenicol, and streptomycin, which have been shown previously to disrupt EF-P function in vitro. In Pseudomonas, knockout of efp leads to an upregulation of mexX, a phenotype previously observed with other genetic lesions affecting ribosome function and that can be induced by the treatment with antibiotics affecting protein synthesis.

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

Meta-analysis identifies gene-by-environment interactions as demonstrated in a study of 4,965 mice.

PubMed

Kang, Eun Yong; Han, Buhm; Furlotte, Nicholas; Joo, Jong Wha J; Shih, Diana; Davis, Richard C; Lusis, Aldons J; Eskin, Eleazar

2014-01-01

Identifying environmentally-specific genetic effects is a key challenge in understanding the structure of complex traits. Model organisms play a crucial role in the identification of such gene-by-environment interactions, as a result of the unique ability to observe genetically similar individuals across multiple distinct environments. Many model organism studies examine the same traits but under varying environmental conditions. For example, knock-out or diet-controlled studies are often used to examine cholesterol in mice. These studies, when examined in aggregate, provide an opportunity to identify genomic loci exhibiting environmentally-dependent effects. However, the straightforward application of traditional methodologies to aggregate separate studies suffers from several problems. First, environmental conditions are often variable and do not fit the standard univariate model for interactions. Additionally, applying a multivariate model results in increased degrees of freedom and low statistical power. In this paper, we jointly analyze multiple studies with varying environmental conditions using a meta-analytic approach based on a random effects model to identify loci involved in gene-by-environment interactions. Our approach is motivated by the observation that methods for discovering gene-by-environment interactions are closely related to random effects models for meta-analysis. We show that interactions can be interpreted as heterogeneity and can be detected without utilizing the traditional uni- or multi-variate approaches for discovery of gene-by-environment interactions. We apply our new method to combine 17 mouse studies containing in aggregate 4,965 distinct animals. We identify 26 significant loci involved in High-density lipoprotein (HDL) cholesterol, many of which are consistent with previous findings. Several of these loci show significant evidence of involvement in gene-by-environment interactions. An additional advantage of our meta-analysis approach is that our combined study has significantly higher power and improved resolution compared to any single study thus explaining the large number of loci discovered in the combined study.

Meta-Analysis Identifies Gene-by-Environment Interactions as Demonstrated in a Study of 4,965 Mice

PubMed Central

Joo, Jong Wha J.; Shih, Diana; Davis, Richard C.; Lusis, Aldons J.; Eskin, Eleazar

2014-01-01

Identifying environmentally-specific genetic effects is a key challenge in understanding the structure of complex traits. Model organisms play a crucial role in the identification of such gene-by-environment interactions, as a result of the unique ability to observe genetically similar individuals across multiple distinct environments. Many model organism studies examine the same traits but under varying environmental conditions. For example, knock-out or diet-controlled studies are often used to examine cholesterol in mice. These studies, when examined in aggregate, provide an opportunity to identify genomic loci exhibiting environmentally-dependent effects. However, the straightforward application of traditional methodologies to aggregate separate studies suffers from several problems. First, environmental conditions are often variable and do not fit the standard univariate model for interactions. Additionally, applying a multivariate model results in increased degrees of freedom and low statistical power. In this paper, we jointly analyze multiple studies with varying environmental conditions using a meta-analytic approach based on a random effects model to identify loci involved in gene-by-environment interactions. Our approach is motivated by the observation that methods for discovering gene-by-environment interactions are closely related to random effects models for meta-analysis. We show that interactions can be interpreted as heterogeneity and can be detected without utilizing the traditional uni- or multi-variate approaches for discovery of gene-by-environment interactions. We apply our new method to combine 17 mouse studies containing in aggregate 4,965 distinct animals. We identify 26 significant loci involved in High-density lipoprotein (HDL) cholesterol, many of which are consistent with previous findings. Several of these loci show significant evidence of involvement in gene-by-environment interactions. An additional advantage of our meta-analysis approach is that our combined study has significantly higher power and improved resolution compared to any single study thus explaining the large number of loci discovered in the combined study. PMID:24415945

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.

Functional Analysis of Nuclear Estrogen Receptors in Zebrafish Reproduction by Genome Editing Approach.

PubMed

Lu, Huijie; Cui, Yong; Jiang, Liwen; Ge, Wei

2017-07-01

Estrogens signal through both nuclear and membrane receptors with most reported effects being mediated via the nuclear estrogen receptors (nERs). Although much work has been reported on nERs in the zebrafish, there is a lack of direct genetic evidence for their functional roles and importance in reproduction. To address this issue, we undertook this study to disrupt all three nERs in the zebrafish, namely esr1 (ERα), esr2a (ERβII), and esr2b (ERβI), by the genome-editing technology clustered regularly interspaced short palindromic repeats and its associated nuclease (CRISPR/Cas9). Using this loss-of-function genetic approach, we successfully created three mutant zebrafish lines with each nER knocked out. In addition, we also generated all possible double and triple knockouts of the three nERs. The phenotypes of these mutants in reproduction were analyzed in all single, double, and triple nER knockouts in both females and males. Surprisingly, all three single nER mutant fish lines display normal reproductive development and function in both females and males, suggesting functional redundancy among these nERs. Further analysis of double and triple knockouts showed that nERs, especially Esr2a and Esr2b, were essential for female reproduction, and loss of these two nERs led to an arrest of folliculogenesis at previtellogenic stage II followed by sex reversal from female to male. In addition, the current study also revealed a unique role for Esr2a in follicle cell proliferation and transdifferentiation, follicle growth, and chorion formation. Taken together, this study provides the most comprehensive genetic analysis for differential functions of esr1, esr2a, and esr2b in fish reproduction. Copyright © 2017 Endocrine Society.

Sensitive single-stage PCR using custom-synthesized internal controls.

PubMed

Zimmermann, K; Rieger, M; Gross, P; Turecek, P L; Schwarz, H P

2000-04-01

A new approach for an internally controlled PCR was developed using a custom-synthesized oligonucleotide as the internal control. Three different PCR setups demonstrated the usefulness of this approach: (i) the addition of the respective internal control to samples containing ssDNA virus Parvo B19; (ii) the co-extraction of plasma samples and the respective internal control for the detection of the ssDNA virus TTV; and (iii) the addition of the respective internal control to a crude lysate of tail pieces for the genotyping of FVIII knockout mice, demonstrating that this approach is also applicable for dsDNA.

Alternative polyadenylation drives genome-to-phenome information detours in the AMPKα1 and AMPKα2 knockout mice.

PubMed

Zhang, Shuwen; Zhang, Yangzi; Zhou, Xiang; Fu, Xing; Michal, Jennifer J; Ji, Guoli; Du, Min; Davis, Jon F; Jiang, Zhihua

2018-04-24

Currently available mouse knockout (KO) lines remain largely uncharacterized for genome-to-phenome (G2P) information flows. Here we test our hypothesis that altered myogenesis seen in AMPKα1- and AMPKα2-KO mice is caused by use of alternative polyadenylation sites (APSs). AMPKα1 and AMPKα2 are two α subunits of adenosine monophosphate-activated protein kinase (AMPK), which serves as a cellular sensor in regulation of many biological events. A total of 56,483 APSs were derived from gastrocnemius muscles. The differentially expressed APSs (DE-APSs) that were down-regulated tended to be distal. The DE-APSs that were related to reduced and increased muscle mass were down-regulated in AMPKα1-KO mice, but up-regulated in AMPKα2-KO mice, respectively. Five genes: Car3 (carbonic anhydrase 3), Mylk4 (myosin light chain kinase family, member 4), Neb (nebulin), Obscn (obscurin) and Pfkm (phosphofructokinase, muscle) utilized different APSs with potentially antagonistic effects on muscle function. Overall, gene knockout triggers genome plasticity via use of APSs, completing the G2P processes. However, gene-based analysis failed to reach such a resolution. Therefore, we propose that alternative transcripts are minimal functional units in genomes and the traditional central dogma concept should be now examined under a systems biology approach.

Subregion-Specific p300 Conditional Knock-Out Mice Exhibit Long-Term Memory Impairments

ERIC Educational Resources Information Center

Oliveira, Ana M. M.; Estevez, Marcel A.; Hawk, Joshua D.; Grimes, Shannon; Brindle, Paul K.; Abel, Ted

2011-01-01

Histone acetylation plays a critical role during long-term memory formation. Several studies have demonstrated that the histone acetyltransferase (HAT) CBP is required during long-term memory formation, but the involvement of other HAT proteins has not been extensively investigated. The HATs CBP and p300 have at least 400 described interacting…

Notch Signaling Regulates Ovarian Follicle Formation and Coordinates Follicular Growth

PubMed Central

Vanorny, Dallas A.; Prasasya, Rexxi D.; Chalpe, Abha J.; Kilen, Signe M.

2014-01-01

Ovarian follicles form through a process in which somatic pregranulosa cells encapsulate individual germ cells from germ cell syncytia. Complementary expression of the Notch ligand, Jagged1, in germ cells and the Notch receptor, Notch2, in pregranulosa cells suggests a role for Notch signaling in mediating cellular interactions during follicle assembly. Using a Notch reporter mouse, we demonstrate that Notch signaling is active within somatic cells of the embryonic ovary, and these cells undergo dramatic reorganization during follicle histogenesis. This coincides with a significant increase in the expression of the ligands, Jagged1 and Jagged2; the receptor, Notch2; and the target genes, Hes1 and Hey2. Histological examination of ovaries from mice with conditional deletion of Jagged1 within germ cells (J1 knockout [J1KO]) or Notch2 within granulosa cells (N2 knockout [N2KO]) reveals changes in follicle dynamics, including perturbations in the primordial follicle pool and antral follicle development. J1KO and N2KO ovaries also contain multi-oocytic follicles, which represent a failure to resolve germ cell syncytia, and follicles with enlarged oocytes but lacking somatic cell growth, signifying a potential role of Notch signaling in follicle activation and the coordination of follicle development. We also observed decreased cell proliferation and increased apoptosis in the somatic cells of both conditional knockout lines. As a consequence of these defects, J1KO female mice are subfertile; however, N2KO female mice remain fertile. This study demonstrates important functions for Jagged1 and Notch2 in the resolution of germ cell syncytia and the coordination of somatic and germ cell growth within follicles of the mouse ovary. PMID:24552588

Neuronal SIRT1 (Silent Information Regulator 2 Homologue 1) Regulates Glycolysis and Mediates Resveratrol-Induced Ischemic Tolerance.

PubMed

Koronowski, Kevin B; Khoury, Nathalie; Saul, Isabel; Loris, Zachary B; Cohan, Charles H; Stradecki-Cohan, Holly M; Dave, Kunjan R; Young, Juan I; Perez-Pinzon, Miguel A

2017-11-01

Resveratrol, at least in part via SIRT1 (silent information regulator 2 homologue 1) activation, protects against cerebral ischemia when administered 2 days before injury. However, it remains unclear if SIRT1 activation must occur, and in which brain cell types, for the induction of neuroprotection. We hypothesized that neuronal SIRT1 is essential for resveratrol-induced ischemic tolerance and sought to characterize the metabolic pathways regulated by neuronal Sirt1 at the cellular level in the brain. We assessed infarct size and functional outcome after transient 60 minute middle cerebral artery occlusion in control and inducible, neuronal-specific SIRT1 knockout mice. Nontargeted primary metabolomics analysis identified putative SIRT1-regulated pathways in brain. Glycolytic function was evaluated in acute brain slices from adult mice and primary neuronal-enriched cultures under ischemic penumbra-like conditions. Resveratrol-induced neuroprotection from stroke was lost in neuronal Sirt1 knockout mice. Metabolomics analysis revealed alterations in glucose metabolism on deletion of neuronal Sirt1 , accompanied by transcriptional changes in glucose metabolism machinery. Furthermore, glycolytic ATP production was impaired in acute brain slices from neuronal Sirt1 knockout mice. Conversely, resveratrol increased glycolytic rate in a SIRT1-dependent manner and under ischemic penumbra-like conditions in vitro. Our data demonstrate that resveratrol requires neuronal SIRT1 to elicit ischemic tolerance and identify a novel role for SIRT1 in the regulation of glycolytic function in brain. Identification of robust neuroprotective mechanisms that underlie ischemia tolerance and the metabolic adaptations mediated by SIRT1 in brain are crucial for the translation of therapies in cerebral ischemia and other neurological disorders. © 2017 American Heart Association, Inc.

Conditional N-WASP knockout in mouse brain implicates actin cytoskeleton regulation in hydrocephalus pathology.

PubMed

Jain, Neeraj; Lim, Lee Wei; Tan, Wei Ting; George, Bhawana; Makeyev, Eugene; Thanabalu, Thirumaran

2014-04-01

Cerebrospinal fluid (CSF) is produced by the choroid plexus and moved by multi-ciliated ependymal cells through the ventricular system of the vertebrate brain. Defects in the ependymal layer functionality are a common cause of hydrocephalus. N-WASP (Neural-Wiskott Aldrich Syndrome Protein) is a brain-enriched regulator of actin cytoskeleton and N-WASP knockout caused embryonic lethality in mice with neural tube and cardiac abnormalities. To shed light on the role of N-WASP in mouse brain development, we generated N-WASP conditional knockout mouse model N-WASP(fl/fl); Nestin-Cre (NKO-Nes). NKO-Nes mice were born with Mendelian ratios but exhibited reduced growth characteristics compared to their littermates containing functional N-WASP alleles. Importantly, all NKO-Nes mice developed cranial deformities due to excessive CSF accumulation and did not survive past weaning. Coronal brain sections of these animals revealed dilated lateral ventricles, defects in ciliogenesis, loss of ependymal layer integrity, reduced thickness of cerebral cortex and aqueductal stenosis. Immunostaining for N-cadherin suggests that ependymal integrity in NKO-Nes mice is lost as compared to normal morphology in the wild-type controls. Moreover, scanning electron microscopy and immunofluorescence analyses of coronal brain sections with anti-acetylated tubulin antibodies revealed the absence of cilia in ventricular walls of NKO-Nes mice indicative of ciliogenesis defects. N-WASP deficiency does not lead to altered expression of N-WASP regulatory proteins, Fyn and Cdc42, which have been previously implicated in hydrocephalus pathology. Taken together, our results suggest that N-WASP plays a critical role in normal brain development and implicate actin cytoskeleton regulation as a vulnerable axis frequently deregulated in hydrocephalus. Copyright © 2014 Elsevier Inc. All rights reserved.

Improving the production of acetyl-CoA-derived chemicals in Escherichia coli BL21(DE3) through iclR and arcA deletion.

PubMed

Liu, Min; Ding, Yamei; Chen, Hailin; Zhao, Zhe; Liu, Huizhou; Xian, Mo; Zhao, Guang

2017-01-07

Acetyl-CoA-derived chemicals are suitable for multiple applications in many industries. The bio-production of these chemicals has become imperative owing to the economic and environmental problems. However, acetate overflow is the major drawback for acetyl-CoA-derived chemicals production. Approaches for overcoming acetate overflow may be beneficial for the production of acetyl-CoA-derived chemicals. In this study, a transcriptional regulator iclR was knocked out in E.coli BL21(DE3) to overcome acetate overflow and improve the chemicals production. Two important acetyl-CoA-derived chemicals, phloroglucinol (PG) and 3-hydroxypropionate (3HP) were used to evaluate it. It is revealed that knockout of iclR significantly increased expressions of aceBAK operon. The cell yields and glucose utilization efficiencies were higher than those of control strains. The acetate concentrations were decreased by more than 50% and the productions of PG and 3HP were increased more than twice in iclR mutants. The effects of iclR knockout on cell physiology, cell metabolism and production of acetyl-CoA-derived chemicals were similar to those of arcA knockout in our previous study. However, the arcA-iclR double mutants couldn't gain higher productions of PG and 3HP. The mechanisms are unclear and needed to be resolved in future. Knockout of iclR significantly increased gene expression of aceBAK operon and concomitantly activated glyoxylate pathway. This genetic modification may be a good way to overcome acetate overflow, and improve the production of a wide range of acetyl-CoA-derived chemicals.

Haloperidol inhibits the development of atherosclerotic lesions in LDL receptor knockout mice

PubMed Central

van der Sluis, Ronald J; Nahon, Joya E; Reuwer, Anne Q; Van Eck, Miranda; Hoekstra, Menno

2015-01-01

Background and Purpose Antipsychotic drugs have been shown to modulate the expression of ATP-binding cassette transporter A1 (ABCA1), a key factor in the anti-atherogenic reverse cholesterol transport process, in vitro. Here we evaluated the potential of the typical antipsychotic drug haloperidol to modulate the cholesterol efflux function of macrophages in vitro and their susceptibility to atherosclerosis in vivo. Experimental Approach Thioglycollate-elicited peritoneal macrophages were used for in vitro studies. Hyperlipidaemic low-density lipoprotein (LDL) receptor knockout mice were implanted with a haloperidol-containing pellet and subsequently fed a Western-type diet for 5 weeks to induce the development of atherosclerotic lesions in vivo. Key Results Haloperidol induced a 54% decrease in the mRNA expression of ABCA1 in peritoneal macrophages. This coincided with a 30% decrease in the capacity of macrophages to efflux cholesterol to apolipoprotein A1. Haloperidol treatment stimulated the expression of ABCA1 (+51%) and other genes involved in reverse cholesterol transport, that is, CYP7A1 (+98%) in livers of LDL receptor knockout mice. No change in splenic ABCA1 expression was noted. However, the average size of the atherosclerotic size was significantly smaller (−31%) in the context of a mildly more atherogenic metabolic phenotype upon haloperidol treatment. More importantly, haloperidol markedly lowered MCP-1 expression (−70%) and secretion (−28%) by peritoneal macrophages. Conclusions and Implications Haloperidol treatment lowered the susceptibility of hyperlipidaemic LDL receptor knockout mice to develop atherosclerotic lesions. Our findings suggest that the beneficial effect of haloperidol on atherosclerosis susceptibility can be attributed to its ability to inhibit macrophage chemotaxis. PMID:25572138

Role of Galectin-3 in Obesity and Impaired Glucose Homeostasis

PubMed Central

Menini, Stefano; Iacobini, Carla; Blasetti Fantauzzi, Claudia; Pesce, Carlo M.; Pugliese, Giuseppe

2016-01-01

Galectin-3 is an important modulator of several biological functions. It has been implicated in numerous disease conditions, particularly in the long-term complications of diabetes because of its ability to bind the advanced glycation/lipoxidation end products that accumulate in target organs and exert their toxic effects by triggering proinflammatory and prooxidant pathways. Recent evidence suggests that galectin-3 may also participate in the development of obesity and type 2 diabetes. It has been shown that galectin-3 levels are higher in obese and diabetic individuals and parallel deterioration of glucose homeostasis. Two studies in galectin-3 knockout mice fed a high-fat diet (HFD) have shown increased adiposity and adipose tissue and systemic inflammation associated with altered glucose homeostasis, suggesting that galectin-3 negatively modulates the responsiveness of innate and adaptive immunity to overnutrition. However, these studies have also shown that impaired glucose homeostasis occurs in galectin-3 knockout animals independently of obesity. Moreover, another study reported decreased weight and fat mass in HFD-fed galectin-3 knockout mice. In vitro, galectin-3 was found to stimulate differentiation of preadipocytes into mature adipocytes. Altogether, these data indicate that galectin-3 deserves further attention in order to clarify its role as a potential player and therapeutic target in obesity and type 2 diabetes. PMID:26770660

β2-Adrenergic Receptor Knockout Mice Exhibit A Diabetic Retinopathy Phenotype

PubMed Central

Jiang, Youde; Zhang, Qiuhua; Liu, Li; Tang, Jie; Kern, Timothy S.; Steinle, Jena J.

2013-01-01

There is considerable evidence from our lab and others for a functional link between β-adrenergic receptor and insulin receptor signaling pathways in retina. Furthermore, we hypothesize that this link may contribute to lesions similar to diabetic retinopathy in that the loss of adrenergic input observed in diabetic retinopathy may disrupt normal anti-apoptotic insulin signaling, leading to retinal cell death. Our studies included assessment of neural retina function (ERG), vascular degeneration, and Müller glial cells (which express only β1 and β2-adrenergic receptor subtypes). In the current study, we produced β2-adrenergic receptor knockout mice to examine this deletion on retinal neurons and vasculature, and to identify specific pathways through which β2-adrenergic receptor modulates insulin signaling. As predicted from our hypothesis, β2-adrenergic receptor knockout mice display certain features similar to diabetic retinopathy. In addition, loss of β2-adrenergic input resulted in an increase in TNFα, a key inhibitor of insulin receptor signaling. Increased TNFα may be associated with insulin-dependent production of the anti-apoptotic factor, Akt. Since the effects occurred in vivo under normal glucose conditions, we postulate that aspects of the diabetic retinopathy phenotype might be triggered by loss of β2-adrenergic receptor signaling. PMID:23894672

GABA-B Agonist Baclofen Normalizes Auditory-Evoked Neural Oscillations and Behavioral Deficits in the Fmr1 Knockout Mouse Model of Fragile X Syndrome

PubMed Central

Featherstone, R.; Naschek, M.; Nam, J.; Du, A.; Wright, S.; Weger, R.; Akuzawa, S.

2017-01-01

Abstract Fragile X syndrome is a genetic condition resulting from FMR1 gene mutation that leads to intellectual disability, autism-like symptoms, and sensory hypersensitivity. Arbaclofen, a GABA-B agonist, has shown efficacy in some individuals with FXS but has become unavailable after unsuccessful clinical trials, prompting interest in publicly available, racemic baclofen. The present study investigated whether racemic baclofen can remediate abnormalities of neural circuit function, sensory processing, and behavior in Fmr1 knockout mice, a rodent model of fragile X syndrome. Fmr1 knockout mice showed increased baseline and auditory-evoked high-frequency gamma (30–80 Hz) power relative to C57BL/6 controls, as measured by electroencephalography. These deficits were accompanied by decreased T maze spontaneous alternation, decreased social interactions, and increased open field center time, suggestive of diminished working memory, sociability, and anxiety-like behavior, respectively. Abnormal auditory-evoked gamma oscillations, working memory, and anxiety-related behavior were normalized by treatment with baclofen, but impaired sociability was not. Improvements in working memory were evident predominantly in mice whose auditory-evoked gamma oscillations were dampened by baclofen. These findings suggest that racemic baclofen may be useful for targeting sensory and cognitive disturbances in fragile X syndrome. PMID:28451631

TAM receptors affect adult brain neurogenesis by negative regulation of microglial cell activation.

PubMed

Ji, Rui; Tian, Shifu; Lu, Helen J; Lu, Qingjun; Zheng, Yan; Wang, Xiaomin; Ding, Jixiang; Li, Qiutang; Lu, Qingxian

2013-12-15

TAM tyrosine kinases play multiple functional roles, including regulation of the target genes important in homeostatic regulation of cytokine receptors or TLR-mediated signal transduction pathways. In this study, we show that TAM receptors affect adult hippocampal neurogenesis and loss of TAM receptors impairs hippocampal neurogenesis, largely attributed to exaggerated inflammatory responses by microglia characterized by increased MAPK and NF-κB activation and elevated production of proinflammatory cytokines that are detrimental to neuron stem cell proliferation and neuronal differentiation. Injection of LPS causes even more severe inhibition of BrdU incorporation in the Tyro3(-/-)Axl(-/-)Mertk(-/-) triple-knockout (TKO) brains, consistent with the LPS-elicited enhanced expression of proinflammatory mediators, for example, IL-1β, IL-6, TNF-α, and inducible NO synthase, and this effect is antagonized by coinjection of the anti-inflammatory drug indomethacin in wild-type but not TKO brains. Conditioned medium from TKO microglia cultures inhibits neuron stem cell proliferation and neuronal differentiation. IL-6 knockout in Axl(-/-)Mertk(-/-) double-knockout mice overcomes the inflammatory inhibition of neurogenesis, suggesting that IL-6 is a major downstream neurotoxic mediator under homeostatic regulation by TAM receptors in microglia. Additionally, autonomous trophic function of the TAM receptors on the proliferating neuronal progenitors may also promote progenitor differentiation into immature neurons.

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

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.

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.

Glutathione S-transferase pi mediates MPTP-induced c-Jun N-terminal kinase activation in the nigrostriatal pathway.

PubMed

Castro-Caldas, Margarida; Carvalho, Andreia Neves; Rodrigues, Elsa; Henderson, Colin; Wolf, C Roland; Gama, Maria João

2012-06-01

Parkinson's disease (PD) is a progressive movement disorder resulting from the death of dopaminergic neurons in the substantia nigra. Neurotoxin-based models of PD using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) recapitulate the neurological features of the disease, triggering a cascade of deleterious events through the activation of the c-Jun N-terminal kinase (JNK). The molecular mechanisms underlying the regulation of JNK activity under cellular stress conditions involve the activation of several upstream kinases along with the fine-tuning of different endogenous JNK repressors. Glutathione S-transferase pi (GSTP), a phase II detoxifying enzyme, has been shown to inhibit JNK-activated signaling by protein-protein interactions, preventing c-Jun phosphorylation and the subsequent trigger of the cell death cascade. Here, we use C57BL/6 wild-type and GSTP knockout mice treated with MPTP to evaluate the regulation of JNK signaling by GSTP in both the substantia nigra and the striatum. The results presented herein show that GSTP knockout mice are more susceptible to the neurotoxic effects of MPTP than their wild-type counterparts. Indeed, the administration of MPTP induces a progressive demise of nigral dopaminergic neurons together with the degeneration of striatal fibers at an earlier time-point in the GSTP knockout mice when compared to the wild-type mice. Also, MPTP treatment leads to increased p-JNK levels and JNK catalytic activity in both wild-type and GSTP knockout mice midbrain and striatum. Moreover, our results demonstrate that in vivo GSTP acts as an endogenous regulator of the MPTP-induced cellular stress response by controlling JNK activity through protein-protein interactions.

Glutathione peroxidase mimic ebselen improves glucose-stimulated insulin secretion in murine islets.

PubMed

Wang, Xinhui; Yun, Jun-Won; Lei, Xin Gen

2014-01-10

Glutathione peroxidase (GPX) mimic ebselen and superoxide dismutase (SOD) mimic copper diisopropylsalicylate (CuDIPs) were used to rescue impaired glucose-stimulated insulin secretion (GSIS) in islets of GPX1 and(or) SOD1-knockout mice. Ebselen improved GSIS in islets of all four tested genotypes. The rescue in the GPX1 knockout resulted from a coordinated transcriptional regulation of four key GSIS regulators and was mediated by the peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α)-mediated signaling pathways. In contrast, CuDIPs improved GSIS only in the SOD1 knockout and suppressed gene expression of the PGC-1α pathway. Islets from the GPX1 and(or) SOD1 knockout mice provided metabolically controlled intracellular hydrogen peroxide (H2O2) and superoxide conditions for the present study to avoid confounding effects. Bioinformatics analyses of gene promoters and expression profiles guided the search for upstream signaling pathways to link the ebselen-initiated H2O2 scavenging to downstream key events of GSIS. The RNA interference was applied to prove PGC-1α as the main mediator for that link. Our study revealed a novel metabolic use and clinical potential of ebselen in rescuing GSIS in the GPX1-deficient islets and mice, along with distinct differences between the GPX and SOD mimics in this regard. These findings highlight the necessities and opportunities of discretional applications of various antioxidant enzyme mimics in treating insulin secretion disorders. REBOUND TRACK: This work was rejected during standard peer review and rescued by Rebound Peer Review (Antioxid Redox Signal 16: 293-296, 2012) with the following serving as open reviewers: Regina Brigelius-Flohe, Vadim Gladyshev, Dexing Hou, and Holger Steinbrenner.

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

Effects of sex and COMT genotype on environmentally modulated cognitive control in mice

PubMed Central

Papaleo, Francesco; Erickson, Lucy; Liu, Guangping; Chen, Jingshan; Weinberger, Daniel R.

2012-01-01

Cognitive functioning differs between males and females, likely in part related to genetic dimorphisms. An example of a common genetic variation reported to have sexually dimorphic effects on cognition and temperament in humans is the Val/Met polymorphism in catechol-O-methyltransferase (COMT). We tested male and female wild-type mice (+/+) and their COMT knockout littermates (+/− and −/−) in the five-choice serial reaction time task (5CSRTT) to investigate the effects of sex, COMT genotype, and their interactions with environmental manipulations of cognitive functions such as attention, impulsivity, compulsivity, motivation, and rule-reversal learning. No sex- or COMT-dependent differences were present in the basic acquisition of the five-choice serial reaction time task. In contrast, specific environmental manipulations revealed a variety of sex- and COMT-dependent effects. Following an experimental change to trigger impulsive responding, the sexes showed similar increases in impulsiveness, but males eventually habituated whereas females did not. Moreover, COMT knockout mice were more impulsive compared with wild-type littermates. Manipulations involving mild stress adversely affected cognitive performance in males, and particularly COMT knockout males, but not in females. In contrast, following amphetamine treatment, subtle sex by genotype and sex by treatment interactions emerged primarily limited to compulsive behavior. After repeated testing, female mice showed improved performance, working harder and eventually outperforming males. Finally, removing the food-restriction condition enhanced sex and COMT differences, revealing that overall, females outperform males and COMT knockout males outperform their wild-type littermates. These findings illuminate complex sex- and COMT-related effects and their interactions with environmental factors to influence specific executive cognitive domains. PMID:23169629

ANTXR2 Knock-Out Does Not Result in the Development of Hypertension in Rats.

PubMed

Liu, Xiaoyan; Yuan, Wen; Li, Jing; Yang, Lei; Cai, Jun

2017-02-01

Our recent genetic study as well as robust evidences reported by previous genome-wide association studies (GWASs) have indicated that the single nucleotide polymorphism rs16998073, located near gene anthrax toxin receptor 2 (ANTXR2), was significantly associated with hypertension in Asians and Europeans. The aim of the present study was to determine whether ANTXR2 is the causal gene of hypertension at the 4q21 locus using an ANTXR2 knock-out model. Relative expression of ANTXR2 in Wistar-Kyoto rats (WKYs) and spontaneously hypertensive rats (SHRs) were determined by real-time quantitative polymerase chain reaction and western blot analysis. ANTXR2 knock-out rats were created using CRISPR/Cas9-mediated genome editing and blood pressure values were measured in ANTXR2 -/- and wild type (WT) rats by tail-cuff method and carotid arterial catheterization method. Neither the mRNA nor protein levels of ANTXR2 were significantly different between tissues from SHRs and WKYs. To create ANTXR2 -/- rats, 67 base pairs were deleted in exon 1 of ANTXR2 using CRISPR/Cas9-mediated genome editing. ANTXR2 protein decreased significantly in aortas of ANTXR2 -/- rats, suggesting sufficient efficiency of ANTXR2 knock-out in this model. However, ANTXR2 -/- rats exhibited nearly the same blood pressure as WT rats at baseline conditions as well as during Angiotensin II (400ng/kg/min) infusion or high-salt diet treatment. These findings suggest that ANTXR2 might not be associated with hypertension and thus further functional analysis is warranted to identify the causal gene at this locus. © American Journal of Hypertension, Ltd 2016. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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.

Genetic inactivation of the vesicular glutamate transporter 2 (VGLUT2) in the mouse: what have we learnt about functional glutamatergic neurotransmission?

PubMed

Wallén-Mackenzie, Asa; Wootz, Hanna; Englund, Hillevi

2010-02-01

During the past decade, three proteins that possess the capability of packaging glutamate into presynaptic vesicles have been identified and characterized. These three vesicular glutamate transporters, VGLUT1-3, are encoded by solute carrier genes Slc17a6-8. VGLUT1 (Slc17a7) and VGLUT2 (Slc17a6) are expressed in glutamatergic neurons, while VGLUT3 (Slc17a8) is expressed in neurons classically defined by their use of another transmitter, such as acetylcholine and serotonin. As glutamate is both a ubiquitous amino acid and the most abundant neurotransmitter in the adult central nervous system, the discovery of the VGLUTs made it possible for the first time to identify and specifically target glutamatergic neurons. By molecular cloning techniques, different VGLUT isoforms have been genetically targeted in mice, creating models with alterations in their glutamatergic signalling. Glutamate signalling is essential for life, and its excitatory function is involved in almost every neuronal circuit. The importance of glutamatergic signalling was very obvious when studying full knockout models of both VGLUT1 and VGLUT2, none of which were compatible with normal life. While VGLUT1 full knockout mice die after weaning, VGLUT2 full knockout mice die immediately after birth. Many neurological diseases have been associated with altered glutamatergic signalling in different brain regions, which is why conditional knockout mice with abolished VGLUT-mediated signalling only in specific circuits may prove helpful in understanding molecular mechanisms behind such pathologies. We review the recent studies in which mouse genetics have been used to characterize the functional role of VGLUT2 in the central nervous system.

Co-regulation of intragenic microRNA miR-153 and its host gene Ia-2 β: identification of miR-153 target genes with functions related to IA-2β in pancreas and brain.

PubMed

Mandemakers, W; Abuhatzira, L; Xu, H; Caromile, L A; Hébert, S S; Snellinx, A; Morais, V A; Matta, S; Cai, T; Notkins, A L; De Strooper, B

2013-07-01

We analysed the genomic organisation of miR-153, a microRNA embedded in genes that encode two of the major type 1 diabetes autoantigens, islet-associated protein (IA)-2 and IA-2β. We also identified miR-153 target genes that correlated with IA-2β localisation and function. A bioinformatics approach was used to identify miR-153's genomic organisation. To analyse the co-regulation of miR-153 and IA-2β, quantitative PCR analysis of miR-153 and Ia-2β (also known as Ptprn2) was performed after a glucose stimulation assay in MIN6B cells and isolated murine pancreatic islets, and also in wild-type Ia-2 (also known as Ptprn), Ia-2β single knockout and Ia-2/Ia-2β double knockout mouse brain and pancreatic islets. Bioinformatics identification of miR-153 target genes and validation via luciferase reporter assays, western blotting and quantitative PCR were also carried out. Two copies of miR-153, miR-153-1 and miR-153-2, are localised in intron 19 of Ia-2 and Ia-2β, respectively. In rodents, only miR-153-2 is conserved. We demonstrated that expression of miR-153-2 and Ia-2β in rodents is partially co-regulated as demonstrated by a strong reduction of miR-153 expression levels in Ia-2β knockout and Ia-2/Ia-2β double knockout mice. miR-153 levels were unaffected in Ia-2 knockout mice. In addition, glucose stimulation, which increases Ia-2 and Ia-2β expression, also significantly increased expression of miR-153. Several predicted targets of miR-153 were reduced after glucose stimulation in vitro, correlating with the increase in miR-153 levels. This study suggests the involvement of miR-153, IA-2β and miR-153 target genes in a regulatory network, which is potentially relevant to insulin and neurotransmitter release.

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.

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.

Sortilin 1 knockout alters basal adipose glucose metabolism but not diet-induced obesity in mice.

PubMed

Li, Jibiao; Matye, David J; Wang, Yifeng; Li, Tiangang

2017-04-01

Sortilin 1 (Sort1) is a trafficking receptor that has been implicated in the regulation of plasma cholesterol in humans and mice. Here, we use metabolomics and hyperinsulinemic-euglycemic clamp approaches to obtain further understanding of the in vivo effects of Sort1 deletion on diet-induced obesity as well as on adipose lipid and glucose metabolism. Results show that Sort1 knockout (KO) does not affect Western diet-induced obesity nor adipose fatty acid and ceramide concentrations. Under the basal fasting state, chow-fed Sort1 KO mice have decreased adipose glycolytic metabolites, but Sort1 deletion does not affect insulin-stimulated tissue glucose uptake during the insulin clamp. These results suggest that Sort1 loss-of-function in vivo does not affect obesity development, but differentially modulates adipose glucose metabolism under fasting and insulin-stimulated states. © 2017 Federation of European Biochemical Societies.

Molecular manipulation of the mating-type system and development of a new approach for characterizing pathogen virulence in Pyrenophora tritici-repentis

USDA-ARS?s Scientific Manuscript database

The ascomycete Pyrenophora tritici-repentis (Ptr) is an important fungal pathogen worldwide that causes tan spot of wheat. The fungus is self-fertile because each isolate contains both mating type (MAT) idiomorphs. In this work, we developed knockouts of the MAT genes in Ptr and tested fertility of ...

A DOUBLE KNOCKOUT; A NOVEL APPROACH TO UNDERSTANDING STRESS-INDUCIBLE 70 KDA HEAT SHOCK PROTEINS (HSP70S) ON DEVELOPMENT AND REPRODUCTION

EPA Science Inventory

Heat and chemical toxicants which disrupt spermatogenesis and cause male infertility are thought to induce the expression of Hsp70-1 and 70-3, the major inducible heat shock proteins of the 70kDa family. Previous studies from several laboratories including our own have characteri...

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.

Glutamate Delta-1 Receptor Regulates Metabotropic Glutamate Receptor 5 Signaling in the Hippocampus.

PubMed

Suryavanshi, Pratyush S; Gupta, Subhash C; Yadav, Roopali; Kesherwani, Varun; Liu, Jinxu; Dravid, Shashank M

2016-08-01

The delta family of ionotropic glutamate receptors consists of glutamate delta-1 (GluD1) and glutamate delta-2 receptors. We have previously shown that GluD1 knockout mice exhibit features of developmental delay, including impaired spine pruning and switch in the N-methyl-D-aspartate receptor subunit, which are relevant to autism and other neurodevelopmental disorders. Here, we identified a novel role of GluD1 in regulating metabotropic glutamate receptor 5 (mGlu5) signaling in the hippocampus. Immunohistochemical analysis demonstrated colocalization of mGlu5 with GluD1 punctas in the hippocampus. Additionally, GluD1 protein coimmunoprecipitated with mGlu5 in the hippocampal membrane fraction, as well as when overexpressed in human embryonic kidney 293 cells, demonstrating that GluD1 and mGlu5 may cooperate in a signaling complex. The interaction of mGlu5 with scaffold protein effector Homer, which regulates mechanistic target of rapamycin (mTOR) signaling, was abnormal both under basal conditions and in response to mGlu1/5 agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) in GluD1 knockout mice. The basal levels of phosphorylated mTOR and protein kinase B, the signaling proteins downstream of mGlu5 activation, were higher in GluD1 knockout mice, and no further increase was induced by DHPG. We also observed higher basal protein translation and an absence of DHPG-induced increase in GluD1 knockout mice. In accordance with a role of mGlu5-mediated mTOR signaling in synaptic plasticity, DHPG-induced internalization of surface α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunits was impaired in the GluD1 knockout mice. These results demonstrate that GluD1 interacts with mGlu5, and loss of GluD1 impairs normal mGlu5 signaling potentially by dysregulating coupling to its effector. These studies identify a novel role of the enigmatic GluD1 subunit in hippocampal function. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Elimination of Kalrn expression in POMC cells reduces anxiety-like behavior and contextual fear learning.

PubMed

Mandela, Prashant; Yan, Yan; LaRese, Taylor; Eipper, Betty A; Mains, Richard E

2014-07-01

Kalirin, a Rho GDP/GTP exchange factor for Rac1 and RhoG, is known to play an essential role in the formation and maintenance of excitatory synapses and in the secretion of neuropeptides. Mice unable to express any of the isoforms of Kalrn in cells that produce POMC at any time during development (POMC cells) exhibited reduced anxiety-like behavior and reduced acquisition of passive avoidance behavior, along with sex-specific alteration in the corticosterone response to restraint stress. Strikingly, lack of Kalrn expression in POMC cells closely mimicked the effects of global Kalrn knockout on anxiety-like behavior and passive avoidance conditioning without causing the other deficits noted in Kalrn knockout mice. Our data suggest that deficits in excitatory inputs onto POMC neurons are responsible for the behavioral phenotypes observed. Copyright © 2014 Elsevier Inc. All rights reserved.

APLP2 regulates neuronal stem cell differentiation during cortical development.

PubMed

Shariati, S Ali M; Lau, Pierre; Hassan, Bassem A; Müller, Ulrike; Dotti, Carlos G; De Strooper, Bart; Gärtner, Annette

2013-03-01

Expression of amyloid precursor protein (APP) and its two paralogues, APLP1 and APLP2 during brain development coincides with key cellular events such as neuronal differentiation and migration. However, genetic knockout and shRNA studies have led to contradictory conclusions about their role during embryonic brain development. To address this issue, we analysed in depth the role of APLP2 during neurogenesis by silencing APLP2 in vivo in an APP/APLP1 double knockout mouse background. We find that under these conditions cortical progenitors remain in their undifferentiated state much longer, displaying a higher number of mitotic cells. In addition, we show that neuron-specific APLP2 downregulation does not impact the speed or position of migrating excitatory cortical neurons. In summary, our data reveal that APLP2 is specifically required for proper cell cycle exit of neuronal progenitors, and thus has a distinct role in priming cortical progenitors for neuronal differentiation.

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.

RSK2 Signaling in Brain Habenula Contributes to Place Aversion Learning

ERIC Educational Resources Information Center

Darcq, Emmanuel; Koebel, Pascale; Del Boca, Carolina; Pannetier, Solange; Kirstetter, Anne-Sophie; Garnier, Jean-Marie; Hanauer, Andre; Befort, Katia; Kieffer, Brigitte L.

2011-01-01

RSK2 is a Ser/Thr kinase acting in the Ras/MAPK pathway. "Rsk2" gene deficiency leads to the Coffin-Lowry Syndrome, notably characterized by cognitive deficits. We found that "mrsk2" knockout mice are unable to associate an aversive stimulus with context in a lithium-induced conditioned place aversion task requiring both high-order cognition and…

Impairment of photoreceptor ribbon synapses in a novel Pomt1 conditional knockout mouse model of dystroglycanopathy.

PubMed

Rubio-Fernández, Marcos; Uribe, Mary Luz; Vicente-Tejedor, Javier; Germain, Francisco; Susín-Lara, Cristina; Quereda, Cristina; Montoliu, Lluis; de la Villa, Pedro; Martín-Nieto, José; Cruces, Jesús

2018-06-04

Hypoglycosylation of α-dystroglycan (α-DG) resulting from deficiency of protein O-mannosyltransferase 1 (POMT1) may cause severe neuromuscular dystrophies with brain and eye anomalies, named dystroglycanopathies. The retinal involvement of these disorders motivated us to generate a conditional knockout (cKO) mouse experiencing a Pomt1 intragenic deletion (exons 3-4) during the development of photoreceptors, mediated by the Cre recombinase expressed from the cone-rod homeobox (Crx) gene promoter. In this mouse, retinal α-DG was unglycosylated and incapable of binding laminin. Retinal POMT1 deficiency caused significant impairments in both electroretinographic recordings and optokinetic reflex in Pomt1 cKO mice, and immunohistochemical analyses revealed the absence of β-DG and of the α-DG-interacting protein, pikachurin, in the outer plexiform layer (OPL). At the ultrastructural level, noticeable alterations were observed in the ribbon synapses established between photoreceptors and bipolar cells. Therefore, O-mannosylation of α-DG in the retina carried out by POMT1 is crucial for the establishment of proper synapses at the OPL and transmission of visual information from cones and rods to their postsynaptic neurons.

FGF signaling in the osteoprogenitor lineage non-autonomously regulates postnatal chondrocyte proliferation and skeletal growth

PubMed Central

Karuppaiah, Kannan; Yu, Kai; Lim, Joohyun; Chen, Jianquan; Smith, Craig; Long, Fanxin

2016-01-01

ABSTRACT Fibroblast growth factor (FGF) signaling is important for skeletal development; however, cell-specific functions, redundancy and feedback mechanisms regulating bone growth are poorly understood. FGF receptors 1 and 2 (Fgfr1 and Fgfr2) are both expressed in the osteoprogenitor lineage. Double conditional knockout mice, in which both receptors were inactivated using an osteoprogenitor-specific Cre driver, appeared normal at birth; however, these mice showed severe postnatal growth defects that include an ∼50% reduction in body weight and bone mass, and impaired longitudinal bone growth. Histological analysis showed reduced cortical and trabecular bone, suggesting cell-autonomous functions of FGF signaling during postnatal bone formation. Surprisingly, the double conditional knockout mice also showed growth plate defects and an arrest in chondrocyte proliferation. We provide genetic evidence of a non-cell-autonomous feedback pathway regulating Fgf9, Fgf18 and Pthlh expression, which led to increased expression and signaling of Fgfr3 in growth plate chondrocytes and suppression of chondrocyte proliferation. These observations show that FGF signaling in the osteoprogenitor lineage is obligately coupled to chondrocyte proliferation and the regulation of longitudinal bone growth. PMID:27052727

Role of kinase-independent and -dependent functions of FAK in endothelial cell survival and barrier function during embryonic development.

PubMed

Zhao, Xiaofeng; Peng, Xu; Sun, Shaogang; Park, Ann Y J; Guan, Jun-Lin

2010-06-14

Focal adhesion kinase (FAK) is essential for vascular development as endothelial cell (EC)-specific knockout of FAK (conditional FAK knockout [CFKO] mice) leads to embryonic lethality. In this study, we report the differential kinase-independent and -dependent functions of FAK in vascular development by creating and analyzing an EC-specific FAK kinase-defective (KD) mutant knockin (conditional FAK knockin [CFKI]) mouse model. CFKI embryos showed apparently normal development through embryonic day (E) 13.5, whereas the majority of CFKO embryos died at the same stage. Expression of KD FAK reversed increased EC apoptosis observed with FAK deletion in embryos and in vitro through suppression of up-regulated p21. However, vessel dilation and defective angiogenesis of CFKO embryos were not rescued in CFKI embryos. ECs without FAK or expressing KD FAK showed increased permeability, abnormal distribution of vascular endothelial cadherin (VE-cadherin), and reduced VE-cadherin Y658 phosphorylation. Together, our data suggest that kinase-independent functions of FAK can support EC survival in vascular development through E13.5 but are insufficient for maintaining EC function to allow for completion of embryogenesis.

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.

An optimal serum-free defined condition for in vitro culture of kidney organoids.

PubMed

Nishikawa, Masaki; Kimura, Hiroshi; Yanagawa, Naomi; Hamon, Morgan; Hauser, Peter; Zhao, Lifu; Jo, Oak D; Yanagawa, Norimoto

2018-07-02

Kidney organoid is an emerging topic of importance for research in kidney development and regeneration. Conventional culture systems for kidney organoids reported thus far use culture media containing serum, which may compromise our understanding and the potential clinical applicability of the organoid system. In our present study, we tested two serum-free culture conditions and compared their suitability for the maintenance and growth of kidney organoids in culture. One of the serum-free culture conditions was the combination of keratinocytes serum free medium (KSFM) with knockout serum replacement (KSR) (KSFM + KSR), and the other was the combination of knockout DMEM/F12 (KD/F12) and KSR (KD/F12 + KSR). With cell aggregates derived from E12.5 mouse embryonic kidneys, we found that KD/F12 + KSR was superior to KSFM + KSR in promoting the growth of the aggregate with expansion of Six2 + nephron progenitor cells (NPC) and elaborated ureteric branching morphogenesis. With KD/F12 + KSR, we found that lower concentrations of KSR at 5-10% were superior to a higher concentration (20%) in promoting the growth of aggregates without affecting the expression levels of NPC marker genes. We also found that NPC in aggregates retained their differentiation potential to develop nephron tubules through mesenchyme-to-epithelial transition (MET), after being maintained in culture under these conditions for up to 7 days. In conclusion, we have identified a defined serum-free culture condition suitable for the maintenance and growth of kidney organoids that retain the differentiation potential to develop nephron structures. This defined serum-free culture condition may serve as a useful platform for further investigation of kidney organoids in vitro. Copyright © 2018 Elsevier Inc. All rights reserved.

Cyanobacterial carbon metabolism: Fluxome plasticity and oxygen dependence: Cyanobacterial Carbon Metabolism

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

Wan, Ni; DeLorenzo, Drew M.; He, Lian

Synechocystis sp. strain PCC 6803 has been widely used as a photo-biorefinery chassis. Based on its genome annotation, this species contains a complete TCA cycle, an Embden-Meyerhof-Parnas pathway (EMPP), an oxidative pentose phosphate pathway (OPPP), and an Entner–Doudoroff pathway (EDP). To evaluate how Synechocystis 6803 catabolizes glucose under heterotrophic conditions, we performed 13C metabolic flux analysis, metabolite pool size analysis, gene knockouts, and heterologous expressions. The results revealed a cyclic mode of flux through the OPPP. Small, but non-zero, fluxes were observed through the TCA cycle and the malic shunt. Independent knockouts of 6-phosphogluconate dehydrogenase (gnd) and malic enzyme (me)more » corroborated these results, as neither mutant could grow under dark heterotrophic conditions. Our data also indicate that Synechocystis 6803 metabolism relies upon oxidative phosphorylation to generate ATP from NADPH under dark or insufficient light conditions. The pool sizes of intermediates in the TCA cycle, particularly acetyl-CoA, were found to be several fold lower in Synechocystis 6803 (compared to E. coli metabolite pool sizes), while its sugar phosphate intermediates were several-fold higher. Moreover, negligible flux was detected through the native, or heterologous, EDP in the wild type or Δgnd strains under heterotrophic conditions. Comparing photoautotrophic, photomixotrophic, and heterotrophic conditions, the Calvin cycle, OPPP, and EMPP in Synechocystis 6803 possess the ability to regulate their fluxes under various growth conditions (plastic), whereas its TCA cycle always maintains at low levels (rigid). This work also demonstrates how genetic profiles do not always reflect actual metabolic flux through native or heterologous pathways. Biotechnol. Bioeng. 2017;114: 1593–1602. © 2017 Wiley Periodicals, Inc.« less

RNaseT2 knockout rats exhibit hippocampal neuropathology and deficits in memory.

PubMed

Sinkevicius, Kerstin W; Morrison, Thomas R; Kulkarni, Praveen; Caffrey Cagliostro, Martha K; Iriah, Sade; Malmberg, Samantha; Sabrick, Julia; Honeycutt, Jennifer A; Askew, Kim L; Trivedi, Malav; Ferris, Craig F

2018-06-27

RNASET2 deficiency in humans is associated with infant cystic leukoencephalopathy, which causes psychomotor impairment, spasticity and epilepsy. A zebrafish mutant model suggests that loss of RNASET2 function leads to neurodegeneration due to the accumulation of non-degraded RNA in the lysosomes. The goal of this study was to characterize the first rodent model of RNASET2 deficiency. The brains of 3- and 12-month-old RNaseT2 knockout rats were studied using multiple magnetic resonance imaging modalities and behavioral tests. While T1- and T2-weighted images of RNaseT2 knockout rats exhibited no evidence of cystic lesions, the prefrontal cortex and hippocampal complex were enlarged in knockout animals. Diffusion-weighted imaging showed altered anisotropy and putative gray matter changes in the hippocampal complex of the RNaseT2 knockout rats. Immunohistochemistry for glial fibrillary acidic protein (GFAP) showed the presence of hippocampal neuroinflammation. Decreased levels of lysosome-associated membrane protein 2 (LAMP2) and elevated acid phosphatase and β-N-acetylglucosaminidase (NAG) activities indicated that the RNASET2 knockout rats likely had altered lysosomal function and potential defects in autophagy. Object recognition tests confirmed that RNaseT2 knockout rats exhibited memory deficits. However, the Barnes maze, and balance beam and rotarod tests indicated there were no differences in spatial memory or motor impairments, respectively. Overall, patients with RNASET2 deficiency exhibited a more severe neurodegeneration phenotype than was observed in the RNaseT2 knockout rats. However, the vulnerability of the knockout rat hippocampus as evidenced by neuroinflammation, altered lysosomal function and cognitive defects indicates that this is still a useful in vivo model to study RNASET2 function. © 2018. Published by The Company of Biologists Ltd.

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.

Chronic Desipramine Treatment Rescues Depression-Related, Social and Cognitive Deficits in Engrailed-2 Knockout Mice

PubMed Central

Brielmaier, Jennifer; Senerth, Julia M.; Silverman, Jill L.; Matteson, Paul G.; Millonig, James H.; DiCicco-Bloom, Emanuel; Crawley, Jacqueline N.

2014-01-01

Engrailed-2 (En2) is a homeobox transcription factor that regulates neurodevelopmental processes including neuronal connectivity and elaboration of monoaminergic neurons in the ventral hindbrain. We previously reported abnormalities in brain noradrenergic concentrations in En2 null mutant mice that were accompanied by increased immobility in the forced swim test, relevant to depression. An EN2 genetic polymorphism has been associated with autism spectrum disorders (ASD), and mice with a deletion in En2 display social abnormalities and cognitive deficits that may be relevant to multiple neuropsychiatric conditions. The present study evaluated the ability of chronic treatment with desipramine (DMI), a selective norepinephrine reuptake inhibitor and classical antidepressant, to reverse behavioral abnormalities in En2 −/− mice. DMI treatment significantly reduced immobility in the tail suspension and forced swim tests, restored sociability in the three-chambered social approach task, and reversed impairments in contextual fear conditioning in En2 −/− mice. Our findings indicate that modulation of brain noradrenergic systems rescues the depression-related phenotype in En2 −/− mice and suggest new roles for norepinephrine in the pathophysiology of the social and cognitive deficits seen in neuropsychiatric disorders such as autism or schizophrenia. PMID:24730055

TIF-IA: An oncogenic target of pre-ribosomal RNA synthesis.

PubMed

Jin, Rui; Zhou, Wei

2016-12-01

Cancer cells devote the majority of their energy consumption to ribosome biogenesis, and pre-ribosomal RNA transcription accounts for 30-50% of all transcriptional activity. This aberrantly elevated biological activity is an attractive target for cancer therapeutic intervention if approaches can be developed to circumvent the development of side effects in normal cells. TIF-IA is a transcription factor that connects RNA polymerase I with the UBF/SL-1 complex to initiate the transcription of pre-ribosomal RNA. Its function is conserved in eukaryotes from yeast to mammals, and its activity is promoted by the phosphorylation of various oncogenic kinases in cancer cells. The depletion of TIF-IA induces cell death in lung cancer cells and mouse embryonic fibroblasts but not in several other normal tissue types evaluated in knock-out studies. Furthermore, the nuclear accumulation of TIF-IA under UTP down-regulated conditions requires the activity of LKB1 kinase, and LKB1-inactivated cancer cells are susceptible to cell death under such stress conditions. Therefore, TIF-IA may be a unique target to suppress ribosome biogenesis without significantly impacting the survival of normal tissues. Copyright © 2016 Elsevier B.V. All rights reserved.

Conditional knockout of N-WASP in mouse fibroblast caused keratinocyte hyper proliferation and enhanced wound closure

PubMed Central

Jain, Neeraj; Kalailingam, Pazhanichamy; Tan, Kai Wei; Tan, Hui Bing; Sng, Ming Keat; Chan, Jeremy Soon Kiat; Tan, Nguan Soon; Thanabalu, Thirumaran

2016-01-01

Neural-Wiskott Aldrich Syndrome Protein (N-WASP) is expressed ubiquitously, regulates actin polymerization and is essential during mouse development. We have previously shown that N-WASP is critical for cell-ECM adhesion in fibroblasts. To characterize the role of N-WASP in fibroblast for skin development, we generated a conditional knockout mouse model in which fibroblast N-WASP was ablated using the Cre recombinase driven by Fibroblast Specific Protein promoter (Fsp-Cre). N-WASPFKO (N-WASPfl/fl; Fsp-cre) were born following Mendelian genetics, survived without any visible abnormalities for more than 1 year and were sexually reproductive, suggesting that expression of N-WASP in fibroblast is not critical for survival under laboratory conditions. Histological sections of N-WASPFKO mice skin (13 weeks old) showed thicker epidermis with higher percentage of cells staining for proliferation marker (PCNA), suggesting that N-WASP deficient fibroblasts promote keratinocyte proliferation. N-WASPFKO mice skin had elevated collagen content, elevated expression of FGF7 (keratinocyte growth factor) and TGFβ signaling proteins. Wound healing was faster in N-WASPFKO mice compared to control mice and N-WASP deficient fibroblasts were found to have enhanced collagen gel contraction properties. These results suggest that N-WASP deficiency in fibroblasts improves wound healing by growth factor-mediated enhancement of keratinocyte proliferation and increased wound contraction in mice. PMID:27909303

Hepatic changes in metabolic gene expression in old ghrelin and ghrelin receptor knockout mice

USDA-ARS?s Scientific Manuscript database

Ghrelin knockout (GKO) and ghrelin receptor (growth hormone secretagogue receptor) knockout (GHSRKO) mice exhibit enhanced insulin sensitivity, but the mechanism is unclear. Insulin sensitivity declines with age and is inversely associated with accumulation of lipid in liver, a key glucoregulatory ...

Ataxin-2 (Atxn2)-Knock-Out Mice Show Branched Chain Amino Acids and Fatty Acids Pathway Alterations.

PubMed

Meierhofer, David; Halbach, Melanie; Şen, Nesli Ece; Gispert, Suzana; Auburger, Georg

2016-05-01

Human Ataxin-2 (ATXN2) gene locus variants have been associated with obesity, diabetes mellitus type 1,and hypertension in genome-wide association studies, whereas mouse studies showed the knock-out of Atxn2 to lead to obesity, insulin resistance, and dyslipidemia. Intriguingly, the deficiency of ATXN2 protein orthologs in yeast and flies rescues the neurodegeneration process triggered by TDP-43 and Ataxin-1 toxicity. To understand the molecular effects of ATXN2 deficiency by unbiased approaches, we quantified the global proteome and metabolome of Atxn2-knock-out mice with label-free mass spectrometry. In liver tissue, significant downregulations of the proteins ACADS, ALDH6A1, ALDH7A1, IVD, MCCC2, PCCA, OTC, together with bioinformatic enrichment of downregulated pathways for branched chain and other amino acid metabolism, fatty acids, and citric acid cycle were observed. Statistical trends in the cerebellar proteome and in the metabolomic profiles supported these findings. They are in good agreement with recent claims that PBP1, the yeast ortholog of ATXN2, sequestrates the nutrient sensor TORC1 in periods of cell stress. Overall, ATXN2 appears to modulate nutrition and metabolism, and its activity changes are determinants of growth excess or cell atrophy. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

CRISPR/Cas9-mediated 2-sgRNA cleavage facilitates pseudorabies virus editing.

PubMed

Tang, Yan-Dong; Guo, Jin-Chao; Wang, Tong-Yun; Zhao, Kuan; Liu, Ji-Ting; Gao, Jia-Cong; Tian, Zhi-Jun; An, Tong-Qing; Cai, Xue-Hui

2018-03-06

Several groups have used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) for DNA virus editing. In most cases, one single-guide RNA (sgRNA) is used, which produces inconsistencies in gene editing. In this study, we used a swine herpesvirus, pseudorabies virus, as a model to systematically explore the application of CRISPR/Cas9 in DNA virus editing. In our current report, we demonstrated that cotransfection of 2 sgRNAs and a viral genome resulted in significantly better knockout efficiency than the transfection-infection-based approach. This method could result in 100% knockout of ≤3500 bp of viral nonessential large fragments. Furthermore, knockin efficiency was significantly improved by using 2 sgRNAs and was also correlated with the number of background viruses. We also demonstrated that the background viruses were all 2-sgRNA-mediated knockout mutants. Finally, this study demonstrated that the efficacy of gene knockin is determined by the replicative kinetics of background viruses. We propose that CRISPR/Cas9 coupled with 2 sgRNAs creates a powerful tool for DNA virus editing and offers great potential for future applications.-Tang, Y.-D., Guo, J.-C., Wang, T.-Y., Zhao, K., Liu, J.-T., Gao, J.-C., Tian, Z.-J., An, T.-Q., Cai, X.-H. CRISPR/Cas9-mediated 2-sgRNA cleavage facilitates pseudorabies virus editing.

Eliminating Xenoantigen Expression on Swine RBC.

PubMed

Wang, Zheng-Yu; Martens, Gregory R; Blankenship, Ross L; Sidner, Richard A; Li, Ping; Estrada, Jose L; Tector, Matthew; Tector, A Joseph

2017-03-01

The rapidly improving tools of genetic engineering may make it possible to overcome the humoral immune barrier that prevents xenotransplantation. We hypothesize that levels of human antibody binding to donor tissues from swine must approximate the antibody binding occurring in allotransplantation. It is uncertain if this is an attainable goal. Here we perform an initial analysis of this issue by comparing human antibody binding to red blood cells (RBC) isolated from knockout swine and to allogeneic or autologous human RBC. Human sera were incubated with RBC isolated from various genetically engineered swine or from humans. The level of IgG and IgM binding to these cells were compared using either flow cytometry or a novel mass spectrometric assay. Mass spectroscopic quantitation of human antibody binding demonstrated that as few as 3 gene inactivations can reduce the levels human antibody binding to swine RBC that is as low as autologous human RBC. Flow cytometry showed that RBC from 2-gene knockout swine exhibited less human antibody binding than human blood group O allogeneic RBC in 22% of tested sera. Deletion of a third gene from pigs resulted in 30% of human samples having less IgG and IgM RBC xenoreactivity than alloreactivity. Xenoantigenicity of swine RBC can be eliminated via gene disruption. These results suggest that the gene knockout approach may be able reduce antigenicity in other pig tissues to levels that enable the xenotransplantation humoral barrier to be overcome.

Alkaloid Cluster Gene ccsA of the Ergot Fungus Claviceps purpurea Encodes Chanoclavine I Synthase, a Flavin Adenine Dinucleotide-Containing Oxidoreductase Mediating the Transformation of N-Methyl-Dimethylallyltryptophan to Chanoclavine I ▿

PubMed Central

Lorenz, Nicole; Olšovská, Jana; Šulc, Miroslav; Tudzynski, Paul

2010-01-01

Ergot alkaloids are indole-derived secondary metabolites synthesized by the phytopathogenic ascomycete Claviceps purpurea. In wild-type strains, they are exclusively produced in the sclerotium, a hibernation structure; for biotechnological applications, submerse production strains have been generated by mutagenesis. It was shown previously that the enzymes specific for alkaloid biosynthesis are encoded by a gene cluster of 68.5 kb. This ergot alkaloid cluster consists of 14 genes coregulated and expressed under alkaloid-producing conditions. Although the role of some of the cluster genes in alkaloid biosynthesis could be confirmed by a targeted knockout approach, further functional analyses are needed, especially concerning the early pathway-specific steps up to the production of clavine alkaloids. Therefore, the gene ccsA, originally named easE and preliminarily annotated as coding for a flavin adenine dinucleotide-containing oxidoreductase, was deleted in the C. purpurea strain P1, which is able to synthesize ergot alkaloids in axenic culture. Five independent knockout mutants were analyzed with regard to alkaloid-producing capability. Thin-layer chromatography (TLC), ultrapressure liquid chromatography (UPLC), and mass spectrometry (MS) analyses revealed accumulation of N-methyl-dimethylallyltryptophan (Me-DMAT) and traces of dimethylallyltryptophan (DMAT), the first pathway-specific intermediate. Since other alkaloid intermediates could not be detected, we conclude that deletion of ccsA led to a block in alkaloid biosynthesis beyond Me-DMAT formation. Complementation with a ccsA/gfp fusion construct restored alkaloid biosynthesis. These data indicate that ccsA encodes the chanoclavine I synthase or a component thereof catalyzing the conversion of N-methyl-dimethylallyltryptophan to chanoclavine I. PMID:20118373

Alkaloid cluster gene ccsA of the ergot fungus Claviceps purpurea encodes chanoclavine I synthase, a flavin adenine dinucleotide-containing oxidoreductase mediating the transformation of N-methyl-dimethylallyltryptophan to chanoclavine I.

PubMed

Lorenz, Nicole; Olsovská, Jana; Sulc, Miroslav; Tudzynski, Paul

2010-03-01

Ergot alkaloids are indole-derived secondary metabolites synthesized by the phytopathogenic ascomycete Claviceps purpurea. In wild-type strains, they are exclusively produced in the sclerotium, a hibernation structure; for biotechnological applications, submerse production strains have been generated by mutagenesis. It was shown previously that the enzymes specific for alkaloid biosynthesis are encoded by a gene cluster of 68.5 kb. This ergot alkaloid cluster consists of 14 genes coregulated and expressed under alkaloid-producing conditions. Although the role of some of the cluster genes in alkaloid biosynthesis could be confirmed by a targeted knockout approach, further functional analyses are needed, especially concerning the early pathway-specific steps up to the production of clavine alkaloids. Therefore, the gene ccsA, originally named easE and preliminarily annotated as coding for a flavin adenine dinucleotide-containing oxidoreductase, was deleted in the C. purpurea strain P1, which is able to synthesize ergot alkaloids in axenic culture. Five independent knockout mutants were analyzed with regard to alkaloid-producing capability. Thin-layer chromatography (TLC), ultrapressure liquid chromatography (UPLC), and mass spectrometry (MS) analyses revealed accumulation of N-methyl-dimethylallyltryptophan (Me-DMAT) and traces of dimethylallyltryptophan (DMAT), the first pathway-specific intermediate. Since other alkaloid intermediates could not be detected, we conclude that deletion of ccsA led to a block in alkaloid biosynthesis beyond Me-DMAT formation. Complementation with a ccsA/gfp fusion construct restored alkaloid biosynthesis. These data indicate that ccsA encodes the chanoclavine I synthase or a component thereof catalyzing the conversion of N-methyl-dimethylallyltryptophan to chanoclavine I.

Genome engineering via homologous recombination in mouse embryonic stem (ES) cells: an amazingly versatile tool for the study of mammalian biology.

PubMed

Babinet, C; Cohen-Tannoudji, M

2001-09-01

The ability to introduce genetic modifications in the germ line of complex organisms has been a long-standing goal of those who study developmental biology. In this regard, the mouse, a favorite model for the study of the mammals, is unique: indeed not only is it possible since the late seventies, to add genes to the mouse genome like in several other complex organisms but also to perform gene replacement and modification. This has been made possible via two technological breakthroughs: 1) the isolation and culture of embryonic stem cells (ES), which have the unique ability to colonize all the tissues of an host embryo including its germ line; 2) the development of methods allowing homologous recombination between an incoming DNA and its cognate chromosomal sequence (gene "targeting"). As a result, it has become possible to create mice bearing null mutations in any cloned gene (knock-out mice). Such a possibility has revolutionized the genetic approach of almost all aspects of the biology of the mouse. In recent years, the scope of gene targeting has been widened even more, due to the refinement of the knock-out technology: other types of genetic modifications may now be created, including subtle mutations (point mutations, micro deletions or insertions, etc.) and chromosomal rearrangements such as large deletions, duplications and translocations. Finally, methods have been devised which permit the creation of conditional mutations, allowing the study of gene function throughout the life of an animal, when gene inactivation entails embryonic lethality. In this paper, we present an overview of the methods and scenarios used for the programmed modification of mouse genome, and we underline their enormous interest for the study of mammalian biology.

Involvement of respiratory processes in the transient knockout of net CO2 uptake in Mimosa pudica upon heat stimulation.

PubMed

Lautner, Silke; Stummer, Michaela; Matyssek, Rainer; Fromm, Jörg; Grams, Thorsten E E

2014-01-01

Leaf photosynthesis of the sensitive plant Mimosa pudica displays a transient knockout in response to electrical signals induced by heat stimulation. This study aims at clarifying the underlying mechanisms, in particular, the involvement of respiration. To this end, leaf gas exchange and light reactions of photosynthesis were assessed under atmospheric conditions largely eliminating photorespiration by either elevated atmospheric CO2 or lowered O2 concentration (i.e. 2000 μmol mol(-1) or 1%, respectively). In addition, leaf gas exchange was studied in the absence of light. Under darkness, heat stimulation caused a transient increase of respiratory CO2 release simultaneously with stomatal opening, hence reflecting direct involvement of respiratory stimulation in the drop of the net CO2 uptake rate. However, persistence of the transient decline in net CO2 uptake rate under illumination and elevated CO2 or 1% O2 makes it unlikely that photorespiration is the metabolic origin of the respiratory CO2 release. In conclusion, the transient knockout of net CO2 uptake is at least partially attributed to an increased CO2 release through mitochondrial respiration as stimulated by electrical signals. Putative CO2 limitation of Rubisco due to decreased activity of carbonic anhydrase was ruled out as the photosynthesis effect was not prevented by elevated CO2 . © 2013 John Wiley & Sons Ltd.

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.

NMDA Receptors Are Not Required for Pattern Completion During Associative Memory Recall

PubMed Central

Gu, Yiran; Cui, Zhenzhong; Tsien, Joe Z.

2011-01-01

Pattern completion, the ability to retrieve complete memories initiated by subsets of external cues, has been a major focus of many computation models. A previously study reports that such pattern completion requires NMDA receptors in the hippocampus. However, such a claim was derived from a non-inducible gene knockout experiment in which the NMDA receptors were absent throughout all stages of memory processes as well as animal's adult life. This raises the critical question regarding whether the previously described results were truly resulting from the requirement of the NMDA receptors in retrieval. Here, we have examined the role of the NMDA receptors in pattern completion via inducible knockout of NMDA receptors limited to the memory retrieval stage. By using two independent mouse lines, we found that inducible knockout mice, lacking NMDA receptor in either forebrain or hippocampus CA1 region at the time of memory retrieval, exhibited normal recall of associative spatial reference memory regardless of whether retrievals took place under full-cue or partial-cue conditions. Moreover, systemic antagonism of NMDA receptor during retention tests also had no effect on full-cue or partial-cue recall of spatial water maze memories. Thus, both genetic and pharmacological experiments collectively demonstrate that pattern completion during spatial associative memory recall does not require the NMDA receptor in the hippocampus or forebrain. PMID:21559402

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

Immune malfunction in the GPR39 zinc receptor of knockout mice: Its relationship to depressive disorder.

PubMed

Młyniec, Katarzyna; Trojan, Ewa; Ślusarczyk, Joanna; Głombik, Katarzyna; Basta-Kaim, Agnieszka; Budziszewska, Bogusława; Skrzeszewski, Jakub; Siwek, Agata; Holst, Birgitte; Nowak, Gabriel

2016-02-15

Depression is a serious psychiatric disorder affecting not only the monaminergic, glutamatergic, and GABAergic neurosystems, but also the immune system. Patients suffering from depression show disturbance in the immune parameters as well as increased susceptibility to infections. Zinc is well known as an anti-inflammatory agent, and its link with depression has been proved, zinc deficiency causing depression- and anxiety-like behavior with immune malfunction. It has been discovered that trace-element zinc acts as a neurotransmitter in the central nervous system via zinc receptor GPR39. In this study we investigated whether GPR39 knockout would cause depressive-like behavior as measured by the forced swim test, and whether these changes would coexist with immune malfunction. In GPR39 knockout mice versus a wild-type control we found: i) depressive-like behavior; ii) significantly reduced thymus weight; (iii) reduced cell viability of splenocytes; iv) reduced proliferative response of splenocytes; and v) increased IL-6 production of splenocytes after ConA stimulation and decreased IL-1b and IL-6 release after LPS stimulation. The results indicate depressive-like behavior in GPR39 KO animals with an immune response similar to that observed in depressive disorder. Here for the first time we show immunological changes under GPR39-deficient conditions. Copyright © 2015 Elsevier B.V. All rights reserved.

Multi-Scale Molecular Deconstruction of the Serotonin Neuron System.

PubMed

Okaty, Benjamin W; Freret, Morgan E; Rood, Benjamin D; Brust, Rachael D; Hennessy, Morgan L; deBairos, Danielle; Kim, Jun Chul; Cook, Melloni N; Dymecki, Susan M

2015-11-18

Serotonergic (5HT) neurons modulate diverse behaviors and physiology and are implicated in distinct clinical disorders. Corresponding diversity in 5HT neuronal phenotypes is becoming apparent and is likely rooted in molecular differences, yet a comprehensive approach characterizing molecular variation across the 5HT system is lacking, as is concomitant linkage to cellular phenotypes. Here we combine intersectional fate mapping, neuron sorting, and genome-wide RNA-seq to deconstruct the mouse 5HT system at multiple levels of granularity-from anatomy, to genetic sublineages, to single neurons. Our unbiased analyses reveal principles underlying system organization, 5HT neuron subtypes, constellations of differentially expressed genes distinguishing subtypes, and predictions of subtype-specific functions. Using electrophysiology, subtype-specific neuron silencing, and conditional gene knockout, we show that these molecularly defined 5HT neuron subtypes are functionally distinct. Collectively, this resource classifies molecular diversity across the 5HT system and discovers sertonergic subtypes, markers, organizing principles, and subtype-specific functions with potential disease relevance. Copyright © 2015 Elsevier Inc. All rights reserved.

Protein kinase D1 drives pancreatic acinar cell reprogramming and progression to intraepithelial neoplasia

NASA Astrophysics Data System (ADS)

Liou, Geou-Yarh; Döppler, Heike; Braun, Ursula B.; Panayiotou, Richard; Scotti Buzhardt, Michele; Radisky, Derek C.; Crawford, Howard C.; Fields, Alan P.; Murray, Nicole R.; Wang, Q. Jane; Leitges, Michael; Storz, Peter

2015-02-01

The transdifferentiation of pancreatic acinar cells to a ductal phenotype (acinar-to-ductal metaplasia, ADM) occurs after injury or inflammation of the pancreas and is a reversible process. However, in the presence of activating Kras mutations or persistent epidermal growth factor receptor (EGF-R) signalling, cells that underwent ADM can progress to pancreatic intraepithelial neoplasia (PanIN) and eventually pancreatic cancer. In transgenic animal models, ADM and PanINs are initiated by high-affinity ligands for EGF-R or activating Kras mutations, but the underlying signalling mechanisms are not well understood. Here, using a conditional knockout approach, we show that protein kinase D1 (PKD1) is sufficient to drive the reprogramming process to a ductal phenotype and progression to PanINs. Moreover, using 3D explant culture of primary pancreatic acinar cells, we show that PKD1 acts downstream of TGFα and Kras, to mediate formation of ductal structures through activation of the Notch pathway.

Modeling familial British and Danish dementia.

PubMed

Garringer, Holly J; Murrell, Jill; D'Adamio, Luciano; Ghetti, Bernardino; Vidal, Ruben

2010-03-01

Familial British dementia (FBD) and familial Danish dementia (FDD) are two autosomal dominant neurodegenerative diseases caused by mutations in the BRI ( 2 ) gene. FBD and FDD are characterized by widespread cerebral amyloid angiopathy (CAA), parenchymal amyloid deposition, and neurofibrillary tangles. Transgenic mice expressing wild-type and mutant forms of the BRI(2) protein, Bri ( 2 ) knock-in mutant mice, and Bri ( 2 ) gene knock-out mice have been developed. Transgenic mice expressing a human FDD-mutated form of the BRI ( 2 ) gene have partially reproduced the neuropathological lesions observed in FDD. These mice develop extensive CAA, parenchymal amyloid deposition, and neuroinflammation in the central nervous system. These animal models allow the study of the molecular mechanism(s) underlying the neuronal dysfunction in these diseases and allow the development of potential therapeutic approaches for these and related neurodegenerative conditions. In this review, a comprehensive account of the advances in the development of animal models for FBD and FDD and of their relevance to the study of Alzheimer disease is presented.

Emerging Role of Spinal Dynorphin in Chronic Pain, a Therapeutic Perspective

PubMed Central

Podvin, Sonia; Yaksh, Tony; Hook, Vivian

2016-01-01

Notable findings point to the significance of the dynorphin peptide neurotransmitter in chronic pain. Spinal dynorphin neuropeptide levels are elevated during development of chronic pain. Importantly, knockout of the dynorphin gene prevents development of chronic pain in mice, but acute nociception is unaffected. Intrathecal (IT) administration of opioid and non-opioid dynorphin peptides initiate allodynia through a non-opioid receptor mechanism; furthermore, anti-dynorphin antibodies administered by the IT route attenuate chronic pain. Thus, this review presents the compelling evidence in the field supporting the role of dynorphin in facilitating the development of a persistent pain state. These observations raise the question of the control mechanisms responsible for the upregulation of spinal dynorphin leading to chronic pain development. Also, spinal dynorphin regulation of downstream signaling molecules may be implicated in hyperpathic states. Therapeutic strategies to reduce spinal dynorphin may provide a non-addictive approach to improve the devastating condition of chronic pain that occurs in numerous human diseases. PMID:26738478

Allosteric ligands for the pharmacologically dark receptors GPR68 and GPR65

PubMed Central

Huang, Xi-Ping; Karpiak, Joel; Kroeze, Wesley K.; Zhu, Hu; Chen, Xin; Moy, Sheryl S.; Saddoris, Kara A.; Nikolova, Viktoriya; Farrell, Martilias S.; Wang, Sheng; Mangano, Thomas J.; Deshpande, Deepak A.; Jiang, Alice; Penn, Raymond B.; Jin, Jian; Koller, Beverly H.; Kenakin, Terry; Shoichet, Brian K.; Roth, Bryan L.

2016-01-01

At least 120 non-olfactory G protein-coupled receptors in the human genome are ”orphans” for which endogenous ligands are unknown, and many have no selective ligands, hindering elucidation of their biological functions and clinical relevance. Among these is GPR68, a proton receptor that lacks small molecule modulators for probing its biology. Yeast-based screens against GPR68 identified the benzodiazepine drug lorazepam as a non-selective GPR68 positive allosteric modulator. Over 3000 GPR68 homology models were refined to recognize lorazepam in a putative allosteric site. Docking 3.1 million molecules predicted new GPR68 modulators many of which were confirmed in functional assays. One potent GPR68 modulator—ogerin– suppressed recall in fear conditioning in wild-type, but not in GPR68 knockout mice. The same approach led to the discovery of allosteric agonists and negative allosteric modulators for GPR65. Combining physical and structure-based screening may be broadly useful for ligand discovery for understudied and orphan GPCRs. PMID:26550826

Integrin Alpha 8 Recessive Mutations Are Responsible for Bilateral Renal Agenesis in Humans

PubMed Central

Humbert, Camille; Silbermann, Flora; Morar, Bharti; Parisot, Mélanie; Zarhrate, Mohammed; Masson, Cécile; Tores, Frédéric; Blanchet, Patricia; Perez, Marie-José; Petrov, Yuliya; Khau Van Kien, Philippe; Roume, Joelle; Leroy, Brigitte; Gribouval, Olivier; Kalaydjieva, Luba; Heidet, Laurence; Salomon, Rémi; Antignac, Corinne; Benmerah, Alexandre; Saunier, Sophie; Jeanpierre, Cécile

2014-01-01

Renal hypodysplasia (RHD) is a heterogeneous condition encompassing a spectrum of kidney development defects including renal agenesis, hypoplasia, and (cystic) dysplasia. Heterozygous mutations of several genes have been identified as genetic causes of RHD with various severity. However, these genes and mutations are not associated with bilateral renal agenesis, except for RET mutations, which could be involved in a few cases. The pathophysiological mechanisms leading to total absence of kidney development thus remain largely elusive. By using a whole-exome sequencing approach in families with several fetuses with bilateral renal agenesis, we identified recessive mutations in the integrin α8-encoding gene ITGA8 in two families. Itga8 homozygous knockout in mice is known to result in absence of kidney development. We provide evidence of a damaging effect of the human ITGA8 mutations. These results demonstrate that mutations of ITGA8 are a genetic cause of bilateral renal agenesis and that, at least in some cases, bilateral renal agenesis is an autosomal-recessive disease. PMID:24439109

Beclin-1 deficiency in the murine ovary results in the reduction of progesterone production to promote preterm labor.

PubMed

Gawriluk, Thomas R; Ko, CheMyong; Hong, Xiaoman; Christenson, Lane K; Rucker, Edmund B

2014-10-07

Autophagy is an important cellular process that serves as a companion pathway to the ubiquitin-proteasome system to degrade long-lived proteins and organelles to maintain cell homeostasis. Although initially characterized in yeast, autophagy is being realized as an important regulator of development and disease in mammals. Beclin1 (Becn1) is a putative tumor suppressor gene that has been shown to undergo a loss of heterozygosity in 40-75% of human breast, ovarian, and prostate cancers. Because Becn1 is a key regulator of autophagy, we sought to investigate its role in female reproduction by using a conditional knockout approach in mice. We find that pregnant females lacking Becn1 in the ovarian granulosa cell population have a defect in progesterone production and a subsequent preterm labor phenotype. Luteal cells in this model exhibit defective autophagy and a failure to accumulate lipid droplets needed for steroidogenesis. Collectively, we show that Becn1 provides essential functions in the ovary that are essential for mammalian reproduction.

Multi-Scale Molecular Deconstruction of the Serotonin Neuron System

PubMed Central

Okaty, Benjamin W.; Freret, Morgan E.; Rood, Benjamin D.; Brust, Rachael D.; Hennessy, Morgan L.; deBairos, Danielle; Kim, Jun Chul; Cook, Melloni N.; Dymecki, Susan M.

2016-01-01

Summary Serotonergic (5HT) neurons modulate diverse behaviors and physiology and are implicated in distinct clinical disorders. Corresponding diversity in 5HT neuronal phenotypes is becoming apparent and is likely rooted in molecular differences, yet a comprehensive approach characterizing molecular variation across the 5HT system is lacking, as is concomitant linkage to cellular phenotypes. Here we combine intersectional fate mapping, neuron sorting, and genome-wide RNA-Seq to deconstruct the mouse 5HT system at multiple levels of granularity—from anatomy, to genetic sublineages, to single neurons. Our unbiased analyses reveal: principles underlying system organization, novel 5HT neuron subtypes, constellations of differentially expressed genes distinguishing subtypes, and predictions of subtype-specific functions. Using electrophysiology, subtype-specific neuron silencing, and conditional gene knockout, we show that these molecularly defined 5HT neuron subtypes are functionally distinct. Collectively, this resource classifies molecular diversity across the 5HT system and discovers new subtypes, markers, organizing principles, and subtype-specific functions with potential disease relevance. PMID:26549332

The 5-HT[subscript 3A] Receptor Is Essential for Fear Extinction

ERIC Educational Resources Information Center

Kondo, Makoto; Nakamura, Yukiko; Ishida, Yusuke; Yamada, Takahiro; Shimada, Shoichi

2014-01-01

The 5-HT [subscript 3] receptor, the only ionotropic 5-HT receptor, is expressed in limbic regions, including the hippocampus, amygdala, and cortex. However, it is not known whether it has a role in fear memory processes. Analysis of 5-HT [subscript 3A] receptor knockout mice in fear conditioning paradigms revealed that the 5-HT [subscript 3A]…

Mapping Mammary Epithelial Cell Transformation in BRCA1 Mutant Mice

DTIC Science & Technology

2006-07-01

Transformation in BRCA1 Mutant Mice PRINCIPAL INVESTIGATOR: Gerburg M. Wulf CONTRACTING ORGANIZATION: Beth Israel Deaconess Medical...REPORT NUMBER Beth Israel Deaconess Medical Center Boston, MA 02215 9. SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES...and whether it allowed us to analyze the early steps of tumor formation. For this purpose transgenic and conditional knock-out mice (mutant p53 or

Zinc Transporter 3 Is Involved in Learned Fear and Extinction, but Not in Innate Fear

ERIC Educational Resources Information Center

Martel, Guillaume; Hevi, Charles; Friebely, Olivia; Baybutt, Trevor; Shumyatsky, Gleb P.

2010-01-01

Synaptically released Zn[superscript 2+] is a potential modulator of neurotransmission and synaptic plasticity in fear-conditioning pathways. Zinc transporter 3 (ZnT3) knock-out (KO) mice are well suited to test the role of zinc in learned fear, because ZnT3 is colocalized with synaptic zinc, responsible for its transport to synaptic vesicles,…

Global Gene Expression Profiling in PAI-1 Knockout Murine Heart and Kidney: Molecular Basis of Cardiac-Selective Fibrosis

PubMed Central

Ghosh, Asish K.; Murphy, Sheila B.; Kishore, Raj; Vaughan, Douglas E.

2013-01-01

Fibrosis is defined as an abnormal matrix remodeling due to excessive synthesis and accumulation of extracellular matrix proteins in tissues during wound healing or in response to chemical, mechanical and immunological stresses. At present, there is no effective therapy for organ fibrosis. Previous studies demonstrated that aged plasminogen activator inhibitor-1(PAI-1) knockout mice develop spontaneously cardiac-selective fibrosis without affecting any other organs. We hypothesized that differential expressions of profibrotic and antifibrotic genes in PAI-1 knockout hearts and unaffected organs lead to cardiac selective fibrosis. In order to address this prediction, we have used a genome-wide gene expression profiling of transcripts derived from aged PAI-1 knockout hearts and kidneys. The variations of global gene expression profiling were compared within four groups: wildtype heart vs. knockout heart; wildtype kidney vs. knockout kidney; knockout heart vs. knockout kidney and wildtype heart vs. wildtype kidney. Analysis of illumina-based microarray data revealed that several genes involved in different biological processes such as immune system processing, response to stress, cytokine signaling, cell proliferation, adhesion, migration, matrix organization and transcriptional regulation were affected in hearts and kidneys by the absence of PAI-1, a potent inhibitor of urokinase and tissue-type plasminogen activator. Importantly, the expressions of a number of genes, involved in profibrotic pathways including Ankrd1, Pi16, Egr1, Scx, Timp1, Timp2, Klf6, Loxl1 and Klotho, were deregulated in PAI-1 knockout hearts compared to wildtype hearts and PAI-1 knockout kidneys. While the levels of Ankrd1, Pi16 and Timp1 proteins were elevated during EndMT, the level of Timp4 protein was decreased. To our knowledge, this is the first comprehensive report on the influence of PAI-1 on global gene expression profiling in the heart and kidney and its implication in fibrogenesis and several other biological processes. The significance of these observations in the light of heart-specific profibrotic signaling and fibrogenesis are discussed. PMID:23724005

Conditional ablation of glycogen synthase kinase 3β in postnatal mouse kidney.

PubMed

Ge, Yan; Si, Jin; Tian, Li; Zhuang, Shougang; Dworkin, Lance D; Gong, Rujun

2011-01-01

Glycogen synthase kinase (GSK)3 is a ubiquitously expressed serine/threonine kinase existing in two isoforms, namely GSK3α and GSK3β. Aside from the long-recognized role in insulin signal transduction and glycogen biosynthesis, GSK3β has been recently coined as a master control molecule in nuclear factor-κB activation and inflammatory kidney injury. Nevertheless, previous studies are less conclusive because they relied greatly on small molecule inhibitors, which lack selectivity and barely distinguish between the GSK3 isoforms. In addition, early embryonic lethality after global knockout of GSK3β precludes interrogation of the biological role of GSK3β in the adult kidney. To circumvent these issues, the Cre/loxP system was used to generate a conditional knockout mouse model in which the GSK3β gene was specifically deleted in kidney cortical tubules at postnatal mature stage. Kidney-specific ablation of GSK3β resulted in a phenotype no different from control littermates. Knockout mice (KO) were viable and exhibited normal development and normal kidney physiology in terms of kidney function, urine albumin excretion, and urine-concentrating ability. It is noteworthy that apart from normal glomerular and tubulointerstitial morphology, the kidneys from KO demonstrated more glycogen accumulation in the renal cortical tubules as assessed by both periodic acid-Schiff staining for light microscopy and direct biochemical assay, consistent with an elevated glycogen synthetic activity as evidenced by diminished inhibitory phosphorylation of glycogen synthase that occurred subsequent to GSK3β ablation. This finding was further validated by electron microscopic observations of increased deposition of glycogen particles in the renal tubules of KO, suggesting that GSK3α could not fully compensate for the loss of GSK3β in regulating glycogen metabolism in the kidney. Collectively, our study suggests that kidney-specific ablation of GSK3β barely affects kidney function and histology under normal circumstances. Extended examinations of these KO under diseased conditions are merited to understand the role of GSK3β in renal pathophysiology.

Describing the role of Drosophila melanogaster ABC transporters in insecticide biology using CRISPR-Cas9 knockouts.

PubMed

Denecke, Shane; Fusetto, Roberto; Batterham, Philip

2017-12-01

ABC transporters have a well-established role in drug resistance, effluxing xenobiotics from cells and tissues within the organism. More recently, research has been dedicated to understanding the role insect ABC transporters play in insecticide toxicity, but progress in understanding the contribution of specific transporters has been hampered by the lack of functional genetic tools. Here, we report knockouts of three Drosophila melanogaster ABC transporter genes, Mdr49, Mdr50, and Mdr65, that are homologous to the well-studied mammalian ABCB1 (P-glycoprotein). Each knockout mutant was created in the same wild type background and tested against a panel of insecticides representing different chemical classes. Mdr65 knockouts were more susceptible to all neuroactive insecticides tested, but Mdr49 and Mdr50 knockouts showed increased susceptibility or resistance depending on the insecticide used. Mdr65 was chosen for further analysis. Calculation of LC 50 values for the Mdr65 knockout allowed the substrate specificity of this transporter to be examined. No obvious distinguishing structural features were shared among MDR65 substrates. A role for Mdr65 in insecticide transport was confirmed by testing the capacity of the knockout to synergize with the ABC inhibitor verapamil and by measuring the levels of insecticide retained in the body of knockout flies. These data unambiguously establish the influence of ABC transporters on the capacity of wild type D. melanogaster to tolerate insecticide exposure and suggest that both tissue and substrate specificity underpin this capacity. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

Reduced Extinction of Hippocampal-Dependent Memories in CPEB Knockout Mice

ERIC Educational Resources Information Center

Zearfoss, N. Ruth; Richter, Joel D.; Berger-Sweeney, Joanne

2006-01-01

CPEB is a sequence-specific RNA binding protein that regulates translation at synapses. In neurons of CPEB knockout mice, synaptic efficacy is reduced. Here, we have performed a battery of behavioral tests and find that relative to wild-type animals, CPEB knockout mice, although similar on many baseline behaviors, have reduced extinction of…

Adaptive bi-level programming for optimal gene knockouts for targeted overproduction under phenotypic constraints

PubMed Central

2013-01-01

Background Optimization procedures to identify gene knockouts for targeted biochemical overproduction have been widely in use in modern metabolic engineering. Flux balance analysis (FBA) framework has provided conceptual simplifications for genome-scale dynamic analysis at steady states. Based on FBA, many current optimization methods for targeted bio-productions have been developed under the maximum cell growth assumption. The optimization problem to derive gene knockout strategies recently has been formulated as a bi-level programming problem in OptKnock for maximum targeted bio-productions with maximum growth rates. However, it has been shown that knockout mutants in fact reach the steady states with the minimization of metabolic adjustment (MOMA) from the corresponding wild-type strains instead of having maximal growth rates after genetic or metabolic intervention. In this work, we propose a new bi-level computational framework--MOMAKnock--which can derive robust knockout strategies under the MOMA flux distribution approximation. Methods In this new bi-level optimization framework, we aim to maximize the production of targeted chemicals by identifying candidate knockout genes or reactions under phenotypic constraints approximated by the MOMA assumption. Hence, the targeted chemical production is the primary objective of MOMAKnock while the MOMA assumption is formulated as the inner problem of constraining the knockout metabolic flux to be as close as possible to the steady-state phenotypes of wide-type strains. As this new inner problem becomes a quadratic programming problem, a novel adaptive piecewise linearization algorithm is developed in this paper to obtain the exact optimal solution to this new bi-level integer quadratic programming problem for MOMAKnock. Results Our new MOMAKnock model and the adaptive piecewise linearization solution algorithm are tested with a small E. coli core metabolic network and a large-scale iAF1260 E. coli metabolic network. The derived knockout strategies are compared with those from OptKnock. Our preliminary experimental results show that MOMAKnock can provide improved targeted productions with more robust knockout strategies. PMID:23368729

Adaptive bi-level programming for optimal gene knockouts for targeted overproduction under phenotypic constraints.

PubMed

Ren, Shaogang; Zeng, Bo; Qian, Xiaoning

2013-01-01

Optimization procedures to identify gene knockouts for targeted biochemical overproduction have been widely in use in modern metabolic engineering. Flux balance analysis (FBA) framework has provided conceptual simplifications for genome-scale dynamic analysis at steady states. Based on FBA, many current optimization methods for targeted bio-productions have been developed under the maximum cell growth assumption. The optimization problem to derive gene knockout strategies recently has been formulated as a bi-level programming problem in OptKnock for maximum targeted bio-productions with maximum growth rates. However, it has been shown that knockout mutants in fact reach the steady states with the minimization of metabolic adjustment (MOMA) from the corresponding wild-type strains instead of having maximal growth rates after genetic or metabolic intervention. In this work, we propose a new bi-level computational framework--MOMAKnock--which can derive robust knockout strategies under the MOMA flux distribution approximation. In this new bi-level optimization framework, we aim to maximize the production of targeted chemicals by identifying candidate knockout genes or reactions under phenotypic constraints approximated by the MOMA assumption. Hence, the targeted chemical production is the primary objective of MOMAKnock while the MOMA assumption is formulated as the inner problem of constraining the knockout metabolic flux to be as close as possible to the steady-state phenotypes of wide-type strains. As this new inner problem becomes a quadratic programming problem, a novel adaptive piecewise linearization algorithm is developed in this paper to obtain the exact optimal solution to this new bi-level integer quadratic programming problem for MOMAKnock. Our new MOMAKnock model and the adaptive piecewise linearization solution algorithm are tested with a small E. coli core metabolic network and a large-scale iAF1260 E. coli metabolic network. The derived knockout strategies are compared with those from OptKnock. Our preliminary experimental results show that MOMAKnock can provide improved targeted productions with more robust knockout strategies.

Tissue Specific Expression Of Sprouty1 In Mice Protects Against High Fat Diet Induced Fat Accumulation, Bone Loss, And Metabolic Dysfunction

PubMed Central

Urs, Sumithra; Henderson, Terry; Le, Phuong; Rosen, Clifford J.; Liaw, Lucy

2012-01-01

We recently characterized Sprouty1 (Spry1), a growth factor signaling inhibitor as a regulator of marrow progenitor cells promoting osteoblast differentiation at the expense of adipocytes. Adipose tissue specific Spry1 expression in mice resulted in increased bone mass and reduced body fat while conditional knockout of Spry1 had the opposite effect with decreased bone and increased body fat. Because Spry1 suppresses normal fat development, we tested the hypothesis that Spry1 expression prevents high fat diet-induced obesity, bone loss, and associated lipid abnormalities and demonstrate that Spry1 has a long-term protective effect on mice fed a high caloric diet. We studied diet-induced obesity in mice with fatty acid binding promoter (aP2)-driven expression or conditional knockout of Spry1 in adipocytes. Phenotyping was performed by whole body dual-energy X-ray absorptiometry, microCT, histology and blood analysis. In conditional Spry1 null mice, high fat diet increased body fat by 40%, impaired glucose regulation, and led to liver steatosis. However, over-expression of Spry1 led to 35% lower body fat, reduced bone loss, and normal metabolic function compared to single transgenics. This protective phenotype was associated with decreased circulating insulin (70%) and leptin (54%) compared to controls on a high fat diet. Additionally, Spry1 expression decreased adipose tissue inflammation by 45%. We show that conditional Spry1 expression in adipose tissue protects against high fat diet-induced obesity and associated bone loss. PMID:22142492

Tissue-specific expression of Sprouty1 in mice protects against high-fat diet-induced fat accumulation, bone loss and metabolic dysfunction.

PubMed

Urs, Sumithra; Henderson, Terry; Le, Phuong; Rosen, Clifford J; Liaw, Lucy

2012-09-28

We recently characterised Sprouty1 (Spry1), a growth factor signalling inhibitor as a regulator of marrow progenitor cells promoting osteoblast differentiation at the expense of adipocytes. Adipose tissue-specific Spry1 expression in mice resulted in increased bone mass and reduced body fat, while conditional knockout of Spry1 had the opposite effect with decreased bone mass and increased body fat. Because Spry1 suppresses normal fat development, we tested the hypothesis that Spry1 expression prevents high-fat diet-induced obesity, bone loss and associated lipid abnormalities, and demonstrate that Spry1 has a long-term protective effect on mice fed a high-energy diet. We studied diet-induced obesity in mice with fatty acid binding promoter-driven expression or conditional knockout of Spry1 in adipocytes. Phenotyping was performed by whole-body dual-energy X-ray absorptiometry, microCT, histology and blood analysis. In conditional Spry1-null mice, a high-fat diet increased body fat by 40 %, impaired glucose regulation and led to liver steatosis. However, overexpression of Spry1 led to 35 % (P < 0·05) lower body fat, reduced bone loss and normal metabolic function compared with single transgenics. This protective phenotype was associated with decreased circulating insulin (70 %) and leptin (54 %; P < 0·005) compared with controls on a high-fat diet. Additionally, Spry1 expression decreased adipose tissue inflammation by 45 %. We show that conditional Spry1 expression in adipose tissue protects against high-fat diet-induced obesity and associated bone loss.

Continuum analyzing power for 4He(p-->,p') at 100 MeV

NASA Astrophysics Data System (ADS)

Lawrie, J. J.; Whittal, D. M.; Cowley, A. A.

1990-08-01

Distorted-wave impulse approximation calculations of the continuum analyzing power for the inclusive reaction 4He(p-->,p') at an incident energy of 100 MeV are presented. In addition to the quasifree knockout of nucleons, contributions from the knockout of deuteron, triton, and helion clusters are taken into account, together with a breakup component. Whereas nucleon knockout by itself does not account for the experimentally observed analyzing power, the inclusion of clusters has a large effect. Thus a simple knockout model is able to provide a reasonable description of the experimental continuum analyzing power.

Genetic deletion of CB1 receptors improves non-associative learning.

PubMed

Degroot, Aldemar; Salhoff, Craig; Davis, Richard J; Nomikos, George G

2005-07-01

Habituation (a form of non-associative learning) was measured by assessing locomotion in novel activity monitors in CB1 receptor knockout mice and juxtaposed to habituation measured in muscarinic M2, M4, and double M2/M4 receptor knockout mice. M2 and M2/M4, but not M4, receptor knockout mice appeared to have an impaired ability to habituate, whereas CB1 receptor knockout mice showed enhanced habituation compared to wild-type animals. We conclude that CB1 receptor gene invalidation improves habituation tentatively through an increase in cholinergic neurotransmission.

Reduced extinction of hippocampal-dependent memories in CPEB knockout mice.

PubMed

Berger-Sweeney, Joanne; Zearfoss, N Ruth; Richter, Joel D

2006-01-01

CPEB is a sequence-specific RNA binding protein that regulates translation at synapses. In neurons of CPEB knockout mice, synaptic efficacy is reduced. Here, we have performed a battery of behavioral tests and find that relative to wild-type animals, CPEB knockout mice, although similar on many baseline behaviors, have reduced extinction of memories on two hippocampal-dependent tasks. A corresponding microarray analysis reveals that about 0.14% of hippocampal genes have an altered expression in the CPEB knockout mouse. These data suggest that CPEB-dependent local protein synthesis may be an important cellular mechanism underlying extinction of hippocampal-dependent memories.

CRISPR/Cas9-mediated gene knockout is insensitive to target copy number but is dependent on guide RNA potency and Cas9/sgRNA threshold expression level

PubMed Central

Yuen, Garmen; Khan, Fehad J.; Gao, Shaojian; Stommel, Jayne M.; Batchelor, Eric; Wu, Xiaolin

2017-01-01

Abstract CRISPR/Cas9 is a powerful gene editing tool for gene knockout studies and functional genomic screens. Successful implementation of CRISPR often requires Cas9 to elicit efficient target knockout in a population of cells. In this study, we investigated the role of several key factors, including variation in target copy number, inherent potency of sgRNA guides, and expression level of Cas9 and sgRNA, in determining CRISPR knockout efficiency. Using isogenic, clonal cell lines with variable copy numbers of an EGFP transgene, we discovered that CRISPR knockout is relatively insensitive to target copy number, but is highly dependent on the potency of the sgRNA guide sequence. Kinetic analysis revealed that most target mutation occurs between 5 and 10 days following Cas9/sgRNA transduction, while sgRNAs with different potencies differ by their knockout time course and by their terminal-phase knockout efficiency. We showed that prolonged, low level expression of Cas9 and sgRNA often fails to elicit target mutation, particularly if the potency of the sgRNA is also low. Our findings provide new insights into the behavior of CRISPR/Cas9 in mammalian cells that could be used for future improvement of this platform. PMID:29036671

CRISPR/Cas9-mediated gene knockout is insensitive to target copy number but is dependent on guide RNA potency and Cas9/sgRNA threshold expression level.

PubMed

Yuen, Garmen; Khan, Fehad J; Gao, Shaojian; Stommel, Jayne M; Batchelor, Eric; Wu, Xiaolin; Luo, Ji

2017-11-16

CRISPR/Cas9 is a powerful gene editing tool for gene knockout studies and functional genomic screens. Successful implementation of CRISPR often requires Cas9 to elicit efficient target knockout in a population of cells. In this study, we investigated the role of several key factors, including variation in target copy number, inherent potency of sgRNA guides, and expression level of Cas9 and sgRNA, in determining CRISPR knockout efficiency. Using isogenic, clonal cell lines with variable copy numbers of an EGFP transgene, we discovered that CRISPR knockout is relatively insensitive to target copy number, but is highly dependent on the potency of the sgRNA guide sequence. Kinetic analysis revealed that most target mutation occurs between 5 and 10 days following Cas9/sgRNA transduction, while sgRNAs with different potencies differ by their knockout time course and by their terminal-phase knockout efficiency. We showed that prolonged, low level expression of Cas9 and sgRNA often fails to elicit target mutation, particularly if the potency of the sgRNA is also low. Our findings provide new insights into the behavior of CRISPR/Cas9 in mammalian cells that could be used for future improvement of this platform. Published by Oxford University Press on behalf of Nucleic Acids Research 2017.

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.

SIRT6-mediated transcriptional suppression of Txnip is critical for pancreatic beta cell function and survival in mice.

PubMed

Qin, Kunhua; Zhang, Ning; Zhang, Zhao; Nipper, Michael; Zhu, Zhenxin; Leighton, Jake; Xu, Kexin; Musi, Nicolas; Wang, Pei

2018-04-01

Better understanding of how genetic and epigenetic components control beta cell differentiation and function is key to the discovery of novel therapeutic approaches to prevent beta cell dysfunction and failure in the progression of type 2 diabetes. Our goal was to elucidate the role of histone deacetylase sirtuin 6 (SIRT6) in beta cell development and homeostasis. Sirt6 endocrine progenitor cell conditional knockout and beta cell-specific knockout mice were generated using the Cre-loxP system. Mice were assayed for islet morphology, glucose tolerance, glucose-stimulated insulin secretion and susceptibility to streptozotocin. Transcriptional regulatory functions of SIRT6 in primary islets were evaluated by RNA-Seq analysis. Reverse transcription-quantitative (RT-q)PCR and immunoblot were used to verify and investigate the gene expression changes. Chromatin occupancies of SIRT6, H3K9Ac, H3K56Ac and active RNA polymerase II were evaluated by chromatin immunoprecipitation. Deletion of Sirt6 in pancreatic endocrine progenitor cells did not affect endocrine morphology, beta cell mass or insulin production but did result in glucose intolerance and defective glucose-stimulated insulin secretion in mice. Conditional deletion of Sirt6 in adult beta cells reproduced the insulin secretion defect. Loss of Sirt6 resulted in aberrant upregulation of thioredoxin-interacting protein (TXNIP) in beta cells. SIRT6 deficiency led to increased acetylation of histone H3 lysine residue at 9 (H3K9Ac), acetylation of histone H3 lysine residue at 56 (H3K56Ac) and active RNA polymerase II at the promoter region of Txnip. SIRT6-deficient beta cells exhibited a time-dependent increase in H3K9Ac, H3K56Ac and TXNIP levels. Finally, beta cell-specific SIRT6-deficient mice showed increased sensitivity to streptozotocin. Our results reveal that SIRT6 suppresses Txnip expression in beta cells via deacetylation of histone H3 and plays a critical role in maintaining beta cell function and viability. Sequence data have been deposited in the National Institutes of Health (NIH) Gene Expression Omnibus (GEO) with the accession code GSE104161.

Functional toxicogenomic assessment of triclosan in human ...

EPA Pesticide Factsheets

Thousands of chemicals for which limited toxicological data are available are used and then detected in humans and the environment. Rapid and cost-effective approaches for assessing the toxicological properties of chemicals are needed. We used CRISPR-Cas9 functional genomic screening to identify potential molecular mechanism of a widely used antimicrobial triclosan (TCS) in HepG2 cells. Resistant genes (whose knockout gives potential resistance) at IC50 (50% Inhibition concentration of cell viability) were significantly enriched in adherens junction pathway, MAPK signaling pathway and PPAR signaling pathway, suggesting a potential molecular mechanism in TCS induced cytotoxicity. Evaluation of top-ranked resistant genes, FTO (encoding an mRNA demethylase) and MAP2K3 (a MAP kinase kinase family gene), revealed that their loss conferred resistance to TCS. In contrast, sensitive genes (whose knockout enhances potential sensitivity) at IC10 and IC20 were specifically enriched in pathways involved with immune responses, which was concordant with the transcriptomic profiling of TCS at concentrations

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

Object recognition impairment in Fmr1 knockout mice is reversed by amphetamine: involvement of dopamine in the medial prefrontal cortex.

PubMed

Ventura, R; Pascucci, T; Catania, M V; Musumeci, S A; Puglisi-Allegra, S

2004-09-01

Fragile X syndrome is an X-linked form of mental retardation including, among others, symptoms such as stereotypic behaviour, hyperactivity, hyperarousal, and cognitive deficits. We hypothesized that hyperactivity and/or compromised attentional, cognitive functions may lead to impaired performance in cognitive tasks in Fmr1 knockout mice, the most widely used animal model of fragile X syndrome, and suggested that psychostimulant treatment may improve performance by acting on one or both components. Since hyperactivity and cognitive functions have been suggested to depend on striatal and prefrontal cortex dopaminergic dysfunction, we assessed whether amphetamine produced beneficial, positive effects by acting on dopaminergic corticostriatal systems. Our results show that Fmr1 knockout mice are not able to discriminate between a familiar object and a novel one in the object recognition test, thus showing a clear-cut cognitive impairment that, to date, has been difficult to demonstrate in other cognitive tasks. Amphetamine improved performance of Fmr1 knockout mice, leading to enhanced ability to discriminate novel versus familiar objects, without significantly affecting locomotor activity. In agreement with behavioural data, amphetamine produced a greater increase in dopamine release in the prefrontal cortex of Fmr1 knockout compared with the wild-type mice, while a weak striatal dopaminergic response was observed in Fmr1 knockout mice. Our data support the view that the psychostimulant ameliorates performance in Fmr1 knockout mice by improving merely cognitive functions through its action on prefrontal cortical dopamine, irrespective of its action on motor hyperactivity. These results indicate that prefrontal cortical dopamine plays a major role in cognitive impairments characterizing Fmr1 knockout mice, thus pointing to an important aetiological factor in the fragile X syndrome.

Phenotypic assessment of THC discriminative stimulus properties in fatty acid amide hydrolase knockout and wildtype mice.

PubMed

Walentiny, D Matthew; Vann, Robert E; Wiley, Jenny L

2015-06-01

A number of studies have examined the ability of the endogenous cannabinoid anandamide to elicit Δ(9)-tetrahydrocannabinol (THC)-like subjective effects, as modeled through the THC discrimination paradigm. In the present study, we compared transgenic mice lacking fatty acid amide hydrolase (FAAH), the enzyme primarily responsible for anandamide catabolism, to wildtype counterparts in a THC discrimination procedure. THC (5.6 mg/kg) served as a discriminative stimulus in both genotypes, with similar THC dose-response curves between groups. Anandamide fully substituted for THC in FAAH knockout, but not wildtype, mice. Conversely, the metabolically stable anandamide analog O-1812 fully substituted in both groups, but was more potent in knockouts. The CB1 receptor antagonist rimonabant dose-dependently attenuated THC generalization in both groups and anandamide substitution in FAAH knockouts. Pharmacological inhibition of monoacylglycerol lipase (MAGL), the primary catabolic enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG), with JZL184 resulted in full substitution for THC in FAAH knockout mice and nearly full substitution in wildtypes. Quantification of brain endocannabinoid levels revealed expected elevations in anandamide in FAAH knockout mice compared to wildtypes and equipotent dose-dependent elevations in 2-AG following JZL184 administration. Dual inhibition of FAAH and MAGL with JZL195 resulted in roughly equipotent increases in THC-appropriate responding in both groups. While the notable similarity in THC's discriminative stimulus effects across genotype suggests that the increased baseline brain anandamide levels (as seen in FAAH knockout mice) do not alter THC's subjective effects, FAAH knockout mice are more sensitive to the THC-like effects of pharmacologically induced increases in anandamide and MAGL inhibition (e.g., JZL184). Copyright © 2015 Elsevier Ltd. All rights reserved.

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.

The guinea pig ileum lacks the direct, high-potency, M(2)-muscarinic, contractile mechanism characteristic of the mouse ileum.

PubMed

Griffin, Michael T; Matsui, Minoru; Ostrom, Rennolds S; Ehlert, Frederick J

2009-10-01

We explored whether the M(2) muscarinic receptor in the guinea pig ileum elicits a highly potent, direct-contractile response, like that from the M(3) muscarinic receptor knockout mouse. First, we characterized the irreversible receptor-blocking activity of 4-DAMP mustard in ileum from muscarinic receptor knockout mice to verify its M(3) selectivity. Then, we used 4-DAMP mustard to inactivate M(3) responses in the guinea pig ileum to attempt to reveal direct, M(2) receptor-mediated contractions. The muscarinic agonist, oxotremorine-M, elicited potent contractions in ileum from wild-type, M(2) receptor knockout, and M(3) receptor knockout mice characterized by negative log EC(50) (pEC (50)) values +/- SEM of 6.75 +/- 0.03, 6.26 +/- 0.05, and 6.99 +/- 0.08, respectively. The corresponding E (max) values in wild-type and M(2) receptor knockout mice were approximately the same, but that in the M(3) receptor knockout mouse was only 36% of wild type. Following 4-DAMP mustard treatment, the concentration-response curve of oxotremorine-M in wild-type ileum resembled that of the M(3) knockout mouse in terms of its pEC (50), E (max), and inhibition by selective muscarinic antagonists. Thus, 4-DAMP mustard treatment appears to inactivate M(3) responses selectively and renders the muscarinic contractile behavior of the wild-type ileum similar to that of the M(3) knockout mouse. Following 4-DAMP mustard treatment, the contractile response of the guinea pig ileum to oxotremorine-M exhibited low potency and a competitive-antagonism profile consistent with an M(3) response. The guinea pig ileum, therefore, lacks a direct, highly potent, M(2)-contractile component but may have a direct, lower potency M(2) component.

Wip1 knockout inhibits the proliferation and enhances the migration of bone marrow mesenchymal stem cells

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

Tang, Yiting; State Key Laboratory of Animal Nutrition and Key Laboratory of Farm Animal Genetic Resources and Germplasm Innovation of Ministry of Agriculture, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193; Liu, Lan

2015-06-10

Mesenchymal stem cells (MSCs), a unique population of multipotent adult progenitor cells originally found in bone marrow (BM), are extremely useful for multifunctional therapeutic approaches. However, the growth arrest and premature senescence of MSCs in vitro prevent the in-depth characterization of these cells. In addition, the regulatory factors involved in MSCs migration remain largely unknown. Given that protein phosphorylation is associated with the processes of MSCs proliferation and migration, we focused on wild-type p53-inducible phosphatase-1 (Wip1), a well-studied modulator of phosphorylation, in this study. Our results showed that Wip1 knockout significantly inhibited MSCs proliferation and induced G2-phase cell-cycle arrest bymore » reducing cyclinB1 expression. Compared with WT-MSCs, Wip1{sup −/−} MSCs displayed premature growth arrest after six passages in culture. Transwell and scratch assays revealed that Wip1{sup −/−} MSCs migrate more effectively than WT-MSCs. Moreover, the enhanced migratory response of Wip1{sup −/−} MSCs may be attributed to increases in the induction of Rac1-GTP activity, the pAKT/AKT ratio, the rearrangement of filamentous-actin (f-actin), and filopodia formation. Based on these results, we then examined the effect of treatment with a PI3K/AKT and Rac1 inhibitor, both of which impaired the migratory activity of MSCs. Therefore, we propose that the PI3K/AKT/Rac1 signaling axis mediates the Wip1 knockout-induced migration of MSCs. Our findings indicate that the principal function of Wip1 in MSCs transformation is the maintenance of proliferative capacity. Nevertheless, knocking out Wip1 increases the migratory capacity of MSCs. This dual effect of Wip1 provides the potential for purposeful routing of MSCs. - Highlights: • Wip1 knockout inhibited MSCs proliferation through reducing cyclinB1 expression. • Wip1{sup −/−} MSCs displayed premature growth arrest in vitro after six passages. • Knocking out Wip1 increases the migratory capacity of MSCs. • The PI3K/AKT/Rac1 pathway mediates the Wip1-knockout-induced migration of MSCs. • Overexpression of Wip1 reversed premature senescence and migration of Wip1{sup −/−} MSCs.« less

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.

iNOS expression in CD4+ T cells limits Treg induction by repressing TGFβ1: combined iNOS inhibition and Treg depletion unmask endogenous antitumor immunity.

PubMed

Jayaraman, Padmini; Alfarano, Matthew G; Svider, Peter F; Parikh, Falguni; Lu, Geming; Kidwai, Sarah; Xiong, Huabao; Sikora, Andrew G

2014-12-15

Expression of inducible nitric oxide synthase (iNOS) in different cellular compartments may have divergent effects on immune function. We used a syngeneic tumor model to functionally characterize the role of iNOS in regulation of CD4(+)FOXP3(+) regulatory T cells (Treg), and optimize the beneficial effects of iNOS inhibition on antitumor immunity. Wild-type (WT) or iNOS knockout mice bearing established MT-RET-1 melanoma were treated with the small-molecule iNOS inhibitor L-NIL and/or cyclophosphamide alone or in combination. The effect of iNOS inhibition or knockout on induction of Treg from mouse and human CD4(+) T cells in ex vivo culture was determined in parallel in the presence or absence of TGFβ1-depleting antibodies, and TGFβ1 levels were assessed by ELISA. Whereas intratumoral myeloid-derived suppressor cells (MDSC) were suppressed by iNOS inhibition or knockout, systemic and intratumoral FOXP3(+) Treg levels increased in tumor-bearing mice. iNOS inhibition or knockout similarly enhanced induction of Treg from activated cultured mouse splenocytes or purified human or mouse CD4(+) T cells in a TGFβ1-dependent manner. Although either iNOS inhibition or Treg depletion with low-dose cyclophosphamide alone had little effect on growth of established MT-RET1 melanoma, combination treatment potently inhibited MDSC and Treg, boosted tumor-infiltrating CD8(+) T-cell levels, and arrested tumor growth in an immune-dependent fashion. iNOS expression in CD4(+) T cells suppresses Treg induction by inhibiting TGFβ1 production. Our data suggest that iNOS expression has divergent effects on induction of myeloid and lymphoid-derived regulatory populations, and strongly support development of combinatorial treatment approaches that target these populations simultaneously. ©2014 American Association for Cancer Research.

Production and characterization of murine models of classic and intermediate maple syrup urine disease

PubMed Central

Homanics, Gregg E; Skvorak, Kristen; Ferguson, Carolyn; Watkins, Simon; Paul, Harbhajan S

2006-01-01

Background Maple Syrup Urine Disease (MSUD) is an inborn error of metabolism caused by a deficiency of branched-chain keto acid dehydrogenase. MSUD has several clinical phenotypes depending on the degree of enzyme deficiency. Current treatments are not satisfactory and require new approaches to combat this disease. A major hurdle in developing new treatments has been the lack of a suitable animal model. Methods To create a murine model of classic MSUD, we used gene targeting and embryonic stem cell technologies to create a mouse line that lacked a functional E2 subunit gene of branched-chain keto acid dehydrogenase. To create a murine model of intermediate MSUD, we used transgenic technology to express a human E2 cDNA on the knockout background. Mice of both models were characterized at the molecular, biochemical, and whole animal levels. Results By disrupting the E2 subunit gene of branched-chain keto acid dehydrogenase, we created a gene knockout mouse model of classic MSUD. The homozygous knockout mice lacked branched-chain keto acid dehydrogenase activity, E2 immunoreactivity, and had a 3-fold increase in circulating branched-chain amino acids. These metabolic derangements resulted in neonatal lethality. Transgenic expression of a human E2 cDNA in the liver of the E2 knockout animals produced a model of intermediate MSUD. Branched-chain keto acid dehydrogenase activity was 5–6% of normal and was sufficient to allow survival, but was insufficient to normalize circulating branched-chain amino acids levels, which were intermediate between wildtype and the classic MSUD mouse model. Conclusion These mice represent important animal models that closely approximate the phenotype of humans with the classic and intermediate forms of MSUD. These animals provide useful models to further characterize the pathogenesis of MSUD, as well as models to test novel therapeutic strategies, such as gene and cellular therapies, to treat this devastating metabolic disease. PMID:16579849

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

A comparative study of the characterization of miR-155 in knockout mice

PubMed Central

Zhang, Dong; Cui, Yongchun; Li, Bin; Luo, Xiaokang; Li, Bo; Tang, Yue

2017-01-01

miR-155 is one of the most important miRNAs and plays a very important role in numerous biological processes. However, few studies have characterized this miRNA in mice under normal physiological conditions. We aimed to characterize miR-155 in vivo by using a comparative analysis. In our study, we compared miR-155 knockout (KO) mice with C57BL/6 wild type (WT) mice in order to characterize miR-155 in mice under normal physiological conditions using many evaluation methods, including a reproductive performance analysis, growth curve, ultrasonic estimation, haematological examination, and histopathological analysis. These analyses showed no significant differences between groups in the main evaluation indices. The growth and development were nearly normal for all mice and did not differ between the control and model groups. Using a comparative analysis and a summary of related studies published in recent years, we found that miR-155 was not essential for normal physiological processes in 8-week-old mice. miR-155 deficiency did not affect the development and growth of naturally ageing mice during the 42 days after birth. Thus, studying the complex biological functions of miR-155 requires the further use of KO mouse models. PMID:28278287

Cellular responses during morphological transformation in Azospirillum brasilense and Its flcA knockout mutant.

PubMed

Hou, Xingsheng; McMillan, Mary; Coumans, Joëlle V F; Poljak, Anne; Raftery, Mark J; Pereg, Lily

2014-01-01

FlcA is a response regulator controlling flocculation and the morphological transformation of Azospirillum cells from vegetative to cyst-like forms. To understand the cellular responses of Azospirillum to conditions that cause morphological transformation, proteins differentially expressed under flocculation conditions in A. brasilense Sp7 and its flcA knockout mutant were investigated. Comparison of 2-DE protein profiles of wild-type (Sp7) and a flcA deletion mutant (Sp7-flcAΔ) revealed a total of 33 differentially expressed 2-DE gel spots, with 22 of these spots confidently separated to allow protein identification. Analysis of these spots by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and MASCOT database searching identified 48 proteins (≥10% emPAI in each spot). The functional characteristics of these proteins included carbon metabolism (beta-ketothiolase and citrate synthase), nitrogen metabolism (Glutamine synthetase and nitric oxide synthase), stress tolerance (superoxide dismutase, Alkyl hydroperoxidase and ATP-dependent Clp protease proteolytic subunit) and morphological transformation (transducer coupling protein). The observed differences between Sp7 wild-type and flcA- strains enhance our understanding of the morphological transformation process and help to explain previous phenotypical observations. This work is a step forward in connecting the Azospirillum phenome and genome.

Cellular Responses during Morphological Transformation in Azospirillum brasilense and Its flcA Knockout Mutant

PubMed Central

Coumans, Joëlle V. F.; Poljak, Anne; Raftery, Mark J.; Pereg, Lily

2014-01-01

FlcA is a response regulator controlling flocculation and the morphological transformation of Azospirillum cells from vegetative to cyst-like forms. To understand the cellular responses of Azospirillum to conditions that cause morphological transformation, proteins differentially expressed under flocculation conditions in A. brasilense Sp7 and its flcA knockout mutant were investigated. Comparison of 2-DE protein profiles of wild-type (Sp7) and a flcA deletion mutant (Sp7-flcAΔ) revealed a total of 33 differentially expressed 2-DE gel spots, with 22 of these spots confidently separated to allow protein identification. Analysis of these spots by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and MASCOT database searching identified 48 proteins (≥10% emPAI in each spot). The functional characteristics of these proteins included carbon metabolism (beta-ketothiolase and citrate synthase), nitrogen metabolism (Glutamine synthetase and nitric oxide synthase), stress tolerance (superoxide dismutase, Alkyl hydroperoxidase and ATP-dependent Clp protease proteolytic subunit) and morphological transformation (transducer coupling protein). The observed differences between Sp7 wild-type and flcA − strains enhance our understanding of the morphological transformation process and help to explain previous phenotypical observations. This work is a step forward in connecting the Azospirillum phenome and genome. PMID:25502569

Incomplete penetrance and variable expressivity of a growth defect as a consequence of knocking out two K(+) transporters in the euascomycete fungus Podospora anserina.

PubMed Central

Lalucque, Hervé; Silar, Philippe

2004-01-01

We describe an example of incomplete penetrance and variable expressivity in the filamentous fungus Podospora anserina, two genetic properties classically associated with mutations in more complex organisms, such as green plants and animals. We show that the knockouts of two TRK-related K(+) transporters of this ascomycete present variability in their phenotype that cannot be attributed to fluctuations of the genetic background or the environment. Thalli of the knockout strains derived from independent monokaryotic ascospores or from a single monokaryotic ascospore and cultivated under standard growth conditions may or may not present impaired growth. When impaired, thalli exhibit a range of phenotypes. Environmental conditions control expressivity to a large extent and penetrance to a low extent. Restoration of functional potassium transport by heterologous expression of K(+) transporters from Neurospora crassa abolishes or strongly diminishes the growth impairment. These data show that incomplete penetrance and variable expressivity can be an intrinsic property of a single Mendelian loss-of-function mutation. They also show that such variability in the expression of a mutant phenotype can be promoted by a phenomenon not obviously related to the well-known chromatin structure modifications, i.e., potassium transport. They provide a framework to understand human channelopathies with similar properties. PMID:15020412

Incomplete penetrance and variable expressivity of a growth defect as a consequence of knocking out two K(+) transporters in the euascomycete fungus Podospora anserina.

PubMed

Lalucque, Hervé; Silar, Philippe

2004-01-01

We describe an example of incomplete penetrance and variable expressivity in the filamentous fungus Podospora anserina, two genetic properties classically associated with mutations in more complex organisms, such as green plants and animals. We show that the knockouts of two TRK-related K(+) transporters of this ascomycete present variability in their phenotype that cannot be attributed to fluctuations of the genetic background or the environment. Thalli of the knockout strains derived from independent monokaryotic ascospores or from a single monokaryotic ascospore and cultivated under standard growth conditions may or may not present impaired growth. When impaired, thalli exhibit a range of phenotypes. Environmental conditions control expressivity to a large extent and penetrance to a low extent. Restoration of functional potassium transport by heterologous expression of K(+) transporters from Neurospora crassa abolishes or strongly diminishes the growth impairment. These data show that incomplete penetrance and variable expressivity can be an intrinsic property of a single Mendelian loss-of-function mutation. They also show that such variability in the expression of a mutant phenotype can be promoted by a phenomenon not obviously related to the well-known chromatin structure modifications, i.e., potassium transport. They provide a framework to understand human channelopathies with similar properties.

Mutations in the murine homologue of TUBB5 cause microcephaly by perturbing cell cycle progression and inducing p53-associated apoptosis.

PubMed

Breuss, Martin; Fritz, Tanja; Gstrein, Thomas; Chan, Kelvin; Ushakova, Lyubov; Yu, Nuo; Vonberg, Frederick W; Werner, Barbara; Elling, Ulrich; Keays, David A

2016-04-01

Microtubules play a crucial role in the generation, migration and differentiation of nascent neurons in the developing vertebrate brain. Mutations in the constituents of microtubules, the tubulins, are known to cause an array of neurological disorders, including lissencephaly, polymicrogyria and microcephaly. In this study we explore the genetic and cellular mechanisms that cause TUBB5-associated microcephaly by exploiting two new mouse models: a conditional E401K knock-in, and a conditional knockout animal. These mice present with profound microcephaly due to a loss of upper-layer neurons that correlates with massive apoptosis and upregulation of p53. This phenotype is associated with a delay in cell cycle progression and ectopic DNA elements in progenitors, which is dependent on the dosage of functional Tubb5. Strikingly, we report ectopic Sox2-positive progenitors and defects in spindle orientation in our knock-in mouse line, which are absent in knockout animals. This work sheds light on the functional repertoire of Tubb5, reveals that the E401K mutation acts by a complex mechanism, and demonstrates that the cellular pathology driving TUBB5-associated microcephaly is cell death. © 2016. Published by The Company of Biologists Ltd.

Transcription factor YY1 can control AID-mediated mutagenesis in mice.

PubMed

Zaprazna, Kristina; Basu, Arindam; Tom, Nikola; Jha, Vibha; Hodawadekar, Suchita; Radova, Lenka; Malcikova, Jitka; Tichy, Boris; Pospisilova, Sarka; Atchison, Michael L

2018-02-01

Activation-induced cytidine deminase (AID) is crucial for controlling the immunoglobulin (Ig) diversification processes of somatic hypermutation (SHM) and class switch recombination (CSR). AID initiates these processes by deamination of cytosine, ultimately resulting in mutations or double strand DNA breaks needed for SHM and CSR. Levels of AID control mutation rates, and off-target non-Ig gene mutations can contribute to lymphomagenesis. Therefore, factors that control AID levels in the nucleus can regulate SHM and CSR, and may contribute to disease. We previously showed that transcription factor YY1 can regulate the level of AID in the nucleus and Ig CSR. Therefore, we hypothesized that conditional knock-out of YY1 would lead to reduction in AID localization at the Ig locus, and reduced AID-mediated mutations. Using mice that overexpress AID (IgκAID yy1 f/f ) or that express normal AID levels (yy1 f/f ), we found that conditional knock-out of YY1 results in reduced AID nuclear levels, reduced localization of AID to the Sμ switch region, and reduced AID-mediated mutations. We find that the mechanism of YY1 control of AID nuclear accumulation is likely due to YY1-AID physical interaction which blocks AID ubiquitination. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Attenuation of neurodegenerative phenotypes in Alzheimer-like presenilin 1/presenilin 2 conditional double knockout mice by EUK1001, a promising derivative of xanomeline.

PubMed

Wang, Dong; Yang, Liguo; Su, Jingjing; Niu, Yan; Lei, Xiaoping; Xiong, Juan; Cao, Xiaohua; Hu, Yinghe; Mei, Bing; Hu, Jin-Feng

2011-07-01

The M1/M4-preferring muscarinic agonist xanomeline was found to have some benefit in the treatment of the memory impairment of Alzheimer's disease (AD), but side effects precluded further development. EUK1001, a fluorinated derivative of xanomeline, because of greater affinity for M1 muscarinic receptors, is likely to have a significantly better side effect profile than xanomeline. We have now studied the effects of 3-month chronic administration of EUK1001 and xanomeline (0.5mg/kg/day) in AD-like presenilin 1/presenilin 2 conditional double knockout (PS cDKO) mice. Only EUK1001 was found to significantly ameliorate the deficit in recognition memory. Histological analysis demonstrated partial attenuation of the brain atrophy in EUK1001-treated PS cDKO mice and minimal effect in the xanomeline-treated mice. Both compounds effectively suppressed the elevation of brain tau phosphorylation in the PS cDKO mice, but neither inhibited the increased inflammatory responses. These results indicate that EUK1001 showed superiority to xanomeline with regard to attenuation of several AD-like neurodegenerative phenotypes in PS cDKO mice. These results suggest further investigation of the development of EUK1001 for the treatment of AD is indicated. Copyright © 2011 Elsevier Inc. All rights reserved.

A Conditional Knockout Mouse Model Reveals a Critical Role of PKD1 in Osteoblast Differentiation and Bone Development

PubMed Central

Li, Shao; Xu, Wanfu; Xing, Zhe; Qian, Jiabi; Chen, Liping; Gu, Ruonan; Guo, Wenjing; Lai, Xiaoju; Zhao, Wanlu; Li, Songyu; Wang, Yaodong; Wang, Q. Jane; Deng, Fan

2017-01-01

The protein kinase D family of serine/threonine kinases, particularly PKD1, has been implicated in the regulation of a complex array of fundamental biological processes. However, its function and mechanism underlying PKD1-mediated the bone development and osteoblast differentiation are not fully understood. Here we demonstrate that loss of PKD1 function led to impaired bone development and osteoblast differentiation through STAT3 and p38 MAPK signaling using in vitro and in vivo bone-specific conditional PKD1-knockout (PKD1-KO) mice models. These mice developed markedly craniofacial dysplasia, scapula dysplasia, long bone length shortage and body weight decrease compared with wild-type littermates. Moreover, deletion of PKD1 in vivo reduced trabecular development and activity of osteoblast development, confirmed by Micro-CT and histological staining as well as expression of osteoblastic marker (OPN, Runx2 and OSX). Mechanistically, loss of PKD1 mediated the downregulation of osteoblast markers and impaired osteoblast differentiation through STAT3 and p38 MAPK signaling pathways. Taken together, these results demonstrated that PKD1 contributes to the osteoblast differentiation and bone development via elevation of osteoblast markers through activation of STAT3 and p38 MAPK signaling pathways. PMID:28084409

Metabolic alterations due to caloric restriction and every other day feeding in normal and growth hormone receptor knockout mice.

PubMed

Westbrook, Reyhan; Bonkowski, Michael S; Arum, Oge; Strader, April D; Bartke, Andrzej

2014-01-01

Mutations causing decreased somatotrophic signaling are known to increase insulin sensitivity and extend life span in mammals. Caloric restriction and every other day (EOD) dietary regimens are associated with similar improvements to insulin signaling and longevity in normal mice; however, these interventions fail to increase insulin sensitivity or life span in growth hormone receptor knockout (GHRKO) mice. To investigate the interactions of the GHRKO mutation with caloric restriction and EOD dietary interventions, we measured changes in the metabolic parameters oxygen consumption (VO2) and respiratory quotient produced by either long-term caloric restriction or EOD in male GHRKO and normal mice. GHRKO mice had increased VO2, which was unaltered by diet. In normal mice, EOD diet caused a significant reduction in VO2 compared with ad libitum (AL) mice during fed and fasted conditions. In normal mice, caloric restriction increased both the range of VO2 and the difference in minimum VO2 between fed and fasted states, whereas EOD diet caused a relatively static VO2 pattern under fed and fasted states. No diet significantly altered the range of VO2 of GHRKO mice under fed conditions. This provides further evidence that longevity-conferring diets cause major metabolic changes in normal mice, but not in GHRKO mice.

Targeted Inactivation of a Developmentally Regulated Neural Plectin Isoform (Plectin 1c) in Mice Leads to Reduced Motor Nerve Conduction Velocity*

PubMed Central

Fuchs, Peter; Zörer, Michael; Reipert, Siegfried; Rezniczek, Günther A.; Propst, Friedrich; Walko, Gernot; Fischer, Irmgard; Bauer, Jan; Leschnik, Michael W.; Lüscher, Bernhard; Thalhammer, Johann G.; Lassmann, Hans; Wiche, Gerhard

2009-01-01

Cytolinker proteins stabilize cells mechanically, regulate cytoskeleton dynamics, and provide scaffolds for signaling molecules. For plectin, the prototype of these proteins, an unusual diversity of isoforms has been reported, which show distinct expression patterns, subcellular localizations, and functions. Plectin has been shown to have important functions in skin and muscle, but little is known about its role in neural cells. To address this issue, we generated two knock-out mouse lines, one which was selectively lacking plectin 1c (P1c), the major isoform expressed in neural cells, and another in which plectin was conditionally deleted in neuronal precursor cells. Using isoform-specific antibodies, we found P1c to be expressed late in development and to associate with postsynaptic dendrites of central nervous system neurons, motorneurons of spinal cord, sciatic nerve axons, and Schwann cells. Motor nerve conduction velocity was found significantly reduced in sciatic nerve from P1c-deficient as well as from conditional knock-out mice. This defect was traceable to an increased number of motor nerve fibers with small cross-sectional areas; the thicknesses of axons and of myelin sheaths were unaffected. This is the first report demonstrating an important role of plectin in a major nerve function. PMID:19625254

Persistent Low Level of Osterix Accelerates Interleukin-6 Production and Impairs Regeneration after Tissue Injury

PubMed Central

Baek, Wook-Young; Park, Seung-Yoon; Kim, Yeo Hyang; Lee, Min-A; Kwon, Tae-Hwan; Park, Kwon-Moo; de Crombrugghe, Benoit; Kim, Jung-Eun

2013-01-01

Osterix (Osx) is an essential transcription factor for osteoblast differentiation and bone formation. Osx knockout show a complete absence of bone formation, whereas Osx conditional knockout in osteoblasts produce an osteopenic phenotype after birth. Here, we questioned whether Osx has a potential role in regulating physiological homeostasis. In Osx heterozygotes expressing low levels of Osx in bones, the expression levels of pro-inflammatory cytokines were significantly elevated, indicating that reduced Osx expression may reflect an inflammatory-prone state. In particular, the expression of interleukin-6, a key mediator of chronic inflammation, was increased in Osx heterozygotes and decreased in Osx overexpressing osteoblasts, and transcriptionally down-regulated by Osx. Although no significant differences were revealed in renal morphology and function between Osx heterozygotes and wild-type under normoxic conditions, recovery of kidneys after ischemic damage was remarkably delayed in Osx heterozygotes, as indicated by elevated blood urea nitrogen and creatinine levels, and by morphological alterations consistent with acute tubular necrosis. Eventually, protracted low Osx expression level caused an inflammatory-prone state in the body, resulting in the enhanced susceptibility to renal injury and the delayed renal repair after ischemia/reperfusion. This study suggests that the maintenance of Osx expression in bone is important in terms of preventing the onset of an inflammatory-prone state. PMID:23922826

Septin9 is involved in septin filament formation and cellular stability.

PubMed

Füchtbauer, Annette; Lassen, Louise B; Jensen, Astrid B; Howard, Jennifer; Quiroga, Adán de Salas; Warming, Søren; Sørensen, Annette B; Pedersen, Finn S; Füchtbauer, Ernst-Martin

2011-08-01

Septin9 (Sept9) is a member of the filament-forming septin family of structural proteins and is associated with a variety of cancers and with hereditary neuralgic amyotrophy. We have generated mice with constitutive and conditional Sept9 knockout alleles. Homozygous deletion of Sept9 results in embryonic lethality around day 10 of gestation whereas mice homozygous for the conditional allele develop normally. Here we report the consequences of homozygous loss of Sept9 in immortalized murine embryonic fibroblasts. Proliferation rate was not changed but cells without Sept9 had an altered morphology compared to normal cells, particularly under low serum stress. Abnormal, fragmented, and multiple nuclei were more frequent in cells without Sept9. Cell migration, as measured by gap-filling and filter-invasion assays, was impaired, but individual cells did not move less than wild-type cells. Sept9 knockout cells showed a reduced resistance to hypo-osmotic stress. Stress fiber and vinculin staining at focal adhesion points was less prominent. Long septin filaments stained for Sept7 disappeared. Instead, staining was found in short, often curved filaments and rings. Furthermore, Sept7 was no longer localized to the mitotic spindle. Together, these data reveal the importance of Sept9 for septin filament formation and general cell stability.

ADAM10 is essential for cranial neural crest-derived maxillofacial bone development.

PubMed

Tan, Yu; Fu, Runqing; Liu, Jiaqiang; Wu, Yong; Wang, Bo; Jiang, Ning; Nie, Ping; Cao, Haifeng; Yang, Zhi; Fang, Bing

2016-07-08

Growth disorders of the craniofacial bones may lead to craniofacial deformities. The majority of maxillofacial bones are derived from cranial neural crest cells via intramembranous bone formation. Any interruption of the craniofacial skeleton development process might lead to craniofacial malformation. A disintegrin and metalloprotease (ADAM)10 plays an essential role in organ development and tissue integrity in different organs. However, little is known about its function in craniofacial bone formation. Therefore, we investigated the role of ADAM10 in the developing craniofacial skeleton, particularly during typical mandibular bone development. First, we showed that ADAM10 was expressed in a specific area of the craniofacial bone and that the expression pattern dynamically changed during normal mouse craniofacial development. Then, we crossed wnt1-cre transgenic mice with adam10-flox mice to generate ADAM10 conditional knockout mice. The stereomicroscopic, radiographic, and von Kossa staining results showed that conditional knockout of ADAM10 in cranial neural crest cells led to embryonic death, craniofacial dysmorphia and bone defects. Furthermore, we demonstrated that impaired mineralization could be triggered by decreased osteoblast differentiation, increased cell death. Overall, these findings show that ADAM10 plays an essential role in craniofacial bone development. Copyright © 2016 Elsevier Inc. All rights reserved.

Polarization of macrophages in the tumor microenvironment is influenced by EGFR signaling within colon cancer cells

PubMed Central

Zhang, Weina; Chen, Lechuang; Ma, Kai; Zhao, Yahui; Liu, Xianghe; Wang, Yu; Liu, Mei; Liang, Shufang; Zhu, Hongxia; Xu, Ningzhi

2016-01-01

Epidermal growth factor receptor (EGFR) is a target of colon cancer therapy, but the effects of this therapy on the tumor microenvironment remain poorly understood. Our in vivo studies showed that cetuximab, an anti-EGFR monoclonal antibody, effectively inhibited AOM/DSS-induced, colitis-associated tumorigenesis, downregulated M2-related markers, and decreased F4/80+/CD206+ macrophage populations. Treatment with conditioned medium of colon cancer cells increased macrophage expression of the M2-related markers arginase-1 (Arg1), CCL17, CCL22, IL-10 and IL-4. By contrast, conditioned medium of EGFR knockout colon cancer cells inhibited expression of these M2-related markers and induced macrophage expression of the M1-related markers inducible nitric oxide synthase (iNOS), IL-12, TNF-α and CCR7. EGFR knockout in colon cancer cells inhibited macrophage-induced promotion of xenograft tumor growth. Moreover, colon cancer-derived insulin-like growth factor-1 (IGF-1) increased Arg1 expression, and treatment with the IGF1R inhibitor AG1024 inhibited that increase. These results suggest that inhibition of EGFR signaling in colon cancer cells modulates cytokine secretion (e.g. IGF-1) and prevents M1-to-M2 macrophage polarization, thereby inhibiting cancer cell growth. PMID:27683110

Na+, HCO3--cotransporter NBCn1 increases pHi gradients, filopodia, and migration of smooth muscle cells and promotes arterial remodelling.

PubMed

Boedtkjer, Ebbe; Bentzon, Jacob F; Dam, Vibeke S; Aalkjaer, Christian

2016-08-01

Arterial remodelling can cause luminal narrowing and obstruct blood flow. We tested the hypothesis that cellular acid-base transport facilitates proliferation and migration of vascular smooth muscle cells (VSMCs) and enhances remodelling of conduit arteries. [Formula: see text]-cotransport via NBCn1 (Slc4a7) mediates net acid extrusion and controls steady-state intracellular pH (pHi) in VSMCs of mouse carotid arteries and primary aortic explants. Carotid arteries undergo hypertrophic inward remodelling in response to partial or complete ligation in vivo, but the increase in media area and thickness and reduction in lumen diameter are attenuated in arteries from NBCn1 knock-out compared with wild-type mice. With [Formula: see text] present, gradients for pHi (∼0.2 units magnitude) exist along the axis of VSMC migration in primary explants from wild-type but not NBCn1 knock-out mice. Knock-out or pharmacological inhibition of NBCn1 also reduces filopodia and lowers initial rates of VSMC migration after scratch-wound infliction. Interventions to reduce H(+)-buffer mobility (omission of [Formula: see text] or inhibition of carbonic anhydrases) re-establish axial pHi gradients, filopodia, and migration rates in explants from NBCn1 knock-out mice. The omission of [Formula: see text] also lowers global pHi and inhibits proliferation in primary explants. Under physiological conditions (i.e. with [Formula: see text] present), NBCn1-mediated [Formula: see text] uptake raises VSMC pHi and promotes filopodia, VSMC migration, and hypertrophic inward remodelling. We propose that axial pHi gradients enhance VSMC migration whereas global acidification inhibits VSMC proliferation and media hypertrophy after carotid artery ligation. These findings support a key role of acid-base transport, particularly via NBCn1, for development of occlusive artery disease. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For permissions please email: journals.permissions@oup.com.

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

Cortical Gene Expression After a Conditional Knockout of 67 kDa Glutamic Acid Decarboxylase in Parvalbumin Neurons.

PubMed

Georgiev, Danko; Yoshihara, Toru; Kawabata, Rika; Matsubara, Takurou; Tsubomoto, Makoto; Minabe, Yoshio; Lewis, David A; Hashimoto, Takanori

2016-07-01

In the cortex of subjects with schizophrenia, expression of glutamic acid decarboxylase 67 (GAD67), the enzyme primarily responsible for cortical GABA synthesis, is reduced in the subset of GABA neurons that express parvalbumin (PV). This GAD67 deficit is accompanied by lower cortical levels of other GABA-associated transcripts, including GABA transporter-1, PV, brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinase B, somatostatin, GABAA receptor α1 subunit, and KCNS3 potassium channel subunit mRNAs. In contrast, messenger RNA (mRNA) levels for glutamic acid decarboxylase 65 (GAD65), another enzyme for GABA synthesis, are not altered. We tested the hypothesis that this pattern of GABA-associated transcript levels is secondary to the GAD67 deficit in PV neurons by analyzing cortical levels of these GABA-associated mRNAs in mice with a PV neuron-specific GAD67 knockout. Using in situ hybridization, we found that none of the examined GABA-associated transcripts had lower cortical expression in the knockout mice. In contrast, PV, BDNF, KCNS3, and GAD65 mRNA levels were higher in the homozygous mice. In addition, our behavioral test battery failed to detect a change in sensorimotor gating or working memory, although the homozygous mice exhibited increased spontaneous activities. These findings suggest that reduced GAD67 expression in PV neurons is not an upstream cause of the lower levels of GABA-associated transcripts, or of the characteristic behaviors, in schizophrenia. In PV neuron-specific GAD67 knockout mice, increased levels of PV, BDNF, and KCNS3 mRNAs might be the consequence of increased neuronal activity secondary to lower GABA synthesis, whereas increased GAD65 mRNA might represent a compensatory response to increase GABA synthesis. © The Author 2016. Published by Oxford University Press on behalf of the Maryland Psychiatric Research Center. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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

Jang, Insook; Park, Sujin; Cho, Jin Won

12/15-lipoxygenase (12/15-LOX) is involved in organelle homeostasis by degrading mitochondria in maturing red blood cells and by eliminating excess peroxisomes in liver. Furthermore, 12/15-LOX contributes to diseases by exacerbating oxidative stress-related injury, notably in stroke. Nonetheless, it is unclear what the consequences are of abolishing 12/15-LOX activity. Mice in which the alox15 gene has been ablated do not show an obvious phenotype, and LOX enzyme inhibition is not overtly detrimental. We show here that liver histology is also unremarkable. However, electron microscopy demonstrated that 12/15-LOX knockout surprisingly leads to increased macroautophagy in the liver. Not only macroautophagy but also mitophagymore » and pexophagy were increased in hepatocytes, which otherwise showed unaltered fine structure and organelle morphology. These findings were substantiated by immunofluorescence showing significantly increased number of LC3 puncta and by Western blotting demonstrating a significant increase for LC3-II protein in both liver and brain homogenates of 12/15-LOX knockout mice. Inhibition of 12/15-LOX activity by treatment with four structurally different inhibitors had similar effects in cultured HepG2 hepatoma cells and SH-SY5Y neuroblastoma cells with significantly increased autophagy discernable already after 2 hours. Hence, our study reveals a link between ablation or inhibition of 12/15-LOX and stimulation of macroautophagy. The enhanced macroautophagy may be related to the known tissue-protective effects of LOX ablation or inhibition under various diseased conditions caused by oxidative stress and ischemia. This could provide an important cleaning mechanism of cells and tissues to prevent accumulation of damaged mitochondria and other cellular components. - Highlights: • A relationship between lipoxygenases and autophagy is disclosed. • 12/15-lipoxygenase knockout increases autophagy in mice liver and brain. • Lipoxygenase inhibition boosts autophagy in human hepatoma and neuroblastoma cells. • Lipoxygenase knockout or inhibition triggers selective autophagy.« less

Sucrose-conditioned flavor preferences in sweet ageusic T1r3 and Calhm1 knockout mice.

PubMed

Sclafani, Anthony; Marambaud, Philippe; Ackroff, Karen

2014-03-14

The present study compared the ability of sweet ageusic T1r3 knockout (KO) and Calhm1 KO mice to acquire preferences for a sucrose-paired flavor as well as for unflavored sucrose. The KO and wildtype (WT) mice were given 24-h one-bottle access to 8% sucrose containing one flavor CS+, e.g., grape) and to water containing a different flavor (CS-, e.g., cherry) over 4 training days. In subsequent two-bottle tests with the flavors in water only, the T1r3 KO and Calhm1 KO mice, like WT mice, preferred the CS+ to the CS-. After training with flavored solutions, both KO groups also preferred unflavored 8% sucrose to water although Calhm1 KO mice required more sugar experience to match the preference of the T1r3 KO mice. These findings demonstrate that Calhm1 KO mice, like T1r3 KO mice and WT mice, are sensitive to the post-oral preference conditioning actions of sucrose and can discriminate sugar from water. Yet, despite their acquired sucrose preferences, the Calhm1 KO and T1r3 KO mice consumed only half as much sugar per day as did WT mice. Thus, sweet taste signaling elements are not needed in the gut for sugar conditioning, but sweet taste signaling in the mouth is essential for the full expression of sugar appetite. Copyright © 2013 Elsevier Inc. All rights reserved.

Mosaic partial deletion of PTPN12 in a child with interrupted aortic arch type A.

PubMed

Duffy, Elizabeth A; Pretorius, Pamela R; Lerach, Stephanie; Lohr, Jamie L; Hirsch, Betsy; Souza, Cleiton M; Veillette, André; Schimmenti, Lisa A

2015-11-01

Congenital heart malformations, including those of the great vessels, are among the most common human birth defects. The goal of this study was to identify the significance of a de novo mosaic PTPN12 partial deletion identified in a newborn with an interrupted aortic arch type A, ventricular septal defect, and pyloric stenosis. PTPN12, a downstream target of the RAS pathway, has a known role in endothelial cell adhesion and migration. Neither genetic nor genomic variants in PTPN12 have been described in a human patient; therefore, we evaluated the effect of ptpn12 in a mouse conditional knockout and zebrafish knockdown model to determine the significance of a loss in gene expression. Observed loss of ptpn12 expression in zebrafish resulted in abnormal branchial arch and tail vasculature patterns, with reduced blood flow throughout the animal. This phenotype was supported by anomalous vasculature in a conditional Ptpn12 mouse knockout. Given the novel co-occurrence of interrupted aortic arch type A, ventricular septal defect, and partial deletion of PTPN12 in the patient, as well as vascular phenotypes in Ptpn12 mouse and ptpn12 zebrafish models, it is likely that PTPN12 has a significant role in cardiovascular development and vessel formation during human embryonic development. Furthermore, the partial deletion of PTPN12 lead to interrupted aortic arch type A in this child and may represent a novel condition caused by a null mutation in the RAS pathway. © 2015 Wiley Periodicals, Inc.

Deletion of Numb/Numblike in glutamatergic neurons leads to anxiety-like behavior in mice.

PubMed

Qian, Wenyu; Hong, Yang; Zhu, Minyan; Zhou, Liang; Li, Hongchang; Li, Huashun

2017-06-15

Endocytic adaptor protein Numb is the first identified cell fate determinant in Drosophila melanogaster. It has been implicated in Notch signaling pathway and regulation of neural stem cells proliferation in the central nervous system. Numb is also expressed in postmitotic neurons, in vitro studies showed that Numb is involved in neuronal morphologic development, such as neurite growth, axonal growth and spine development. However, in vivo functions of Numb in the postmitotic neurons are largely unknown. Here we show that deletion of Numb/Numblike in glutamatergic neurons causes anxiety-like behavior in mouse. In this study, we conditionally deleted Numb and its homologous gene Numblike in the glutamatergic neurons in dorsal forebrain, and thoroughly characterized the behavioral phenotypes of mutant mice. On a battery of tests for anxiety-like behavior, the conditional double knockout mice showed increased anxiety-like behavior on light/dark exploration and novel open field tests, but not on elevated zero maze tests. The conditional double knockout mice also displayed novelty induced hyperactivity in novel open field test. Control measures of general health, motor functions, startle response, sensorimotor gating, depression-related behaviors did not show differences between genotypes. Our present findings provide new insight into the indispensable functions of Numb/Numblike in the brain and behavior, and suggest that Numb/Numblike may play a role in mediating neuronal functions that underlie behaviors related to anxiety. Copyright © 2017. Published by Elsevier B.V.

Grainyhead-like 3 (Grhl3) deficiency in brain leads to altered locomotor activity and decreased anxiety-like behaviors in aged mice.

PubMed

Dworkin, Sebastian; Auden, Alana; Partridge, Darren D; Daglas, Maria; Medcalf, Robert L; Mantamadiotis, Theo; Georgy, Smitha R; Darido, Charbel; Jane, Stephen M; Ting, Stephen B

2017-06-01

The highly conserved Grainyhead-like (Grhl) family of transcription factors, comprising three members in vertebrates (Grhl1-3), play critical regulatory roles during embryonic development, cellular proliferation, and apoptosis. Although loss of Grhl function leads to multiple neural abnormalities in numerous animal models, a comprehensive analysis of Grhl expression and function in the mammalian brain has not been reported. Here they show that only Grhl3 expression is detectable in the embryonic mouse brain; particularly within the habenula, an organ known to modulate repressive behaviors. Using both Grhl3-knockout mice (Grhl3 -/- ), and brain-specific conditional deletion of Grhl3 in adult mice (Nestin-Cre/Grhl3 flox/flox ), they performed histological expression analyses and behavioral tests to assess long-term effects of Grhl3 loss on motor co-ordination, spatial memory, anxiety, and stress. They found that complete deletion of Grhl3 did not lead to noticeable structural or cell-intrinsic defects in the embryonic brain; however, aged Grhl3 conditional knockout (cKO) mice showed enlarged lateral ventricles and displayed marked changes in motor function and behaviors suggestive of decreased fear and anxiety. They conclude that loss of Grhl3 in the brain leads to significant alterations in locomotor activity and decreased self-inhibition, and as such, these mice may serve as a novel model of human conditions of impulsive behavior or hyperactivity. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 775-788, 2017. © 2017 Wiley Periodicals, Inc.

Uterine epithelial cell proliferation and endometrial hyperplasia: evidence from a mouse model

PubMed Central

Gao, Yang; Li, Shu; Li, Qinglei

2014-01-01

In the uterus, epithelial cell proliferation changes during the estrous cycle and pregnancy. Uncontrolled epithelial cell proliferation results in implantation failure and/or cancer development. Transforming growth factor-β (TGF-β) signaling is a fundamental regulator of diverse biological processes and is indispensable for multiple reproductive functions. However, the in vivo role of TGF-β signaling in uterine epithelial cells remains poorly defined. We have shown that in the uterus, conditional deletion of the Type 1 receptor for TGF-β (Tgfbr1) using anti-Müllerian hormone receptor type 2 (Amhr2) Cre leads to myometrial defects. Here, we describe enhanced epithelial cell proliferation by immunostaining of Ki67 in the uteri of these mice. The aberration culminated in endometrial hyperplasia in aged females. To exclude the potential influence of ovarian steroid hormones, the proliferative status of uterine epithelial cells was assessed following ovariectomy. Increased uterine epithelial cell proliferation was also revealed in ovariectomized Tgfbr1 Amhr2-Cre conditional knockout mice. We further demonstrated that transcript levels for fibroblast growth factor 10 (Fgf10) were markedly up-regulated in Tgfbr1 Amhr2-Cre conditional knockout uteri. Consistently, treatment of primary uterine stromal cells with TGF-β1 significantly reduced Fgf10 mRNA expression. Thus, our findings suggest a potential involvement of TGFBR1-mediated signaling in the regulation of uterine epithelial cell proliferation, and provide genetic evidence supporting the role of uterine epithelial cell proliferation in the pathogenesis of endometrial hyperplasia. PMID:24770950

Conditional Deletion of the Pten Gene in the Mouse Prostate Induces Prostatic Intraepithelial Neoplasms at Early Ages but a Slow Progression to Prostate Tumors

PubMed Central

Zhu, Chunfang; Lee, Suk Hyung; Ye, Ding-Wei; Luong, Richard; Sun, Zijie

2013-01-01

The PTEN tumor suppressor gene is frequently inactivated in human prostate cancer. Using Osr1 (odd skipped related 1)-Cre mice, we generated a novel conditional Pten knockout mouse strain, PtenLoxP:Osr1-Cre. Conditional biallelic and monoallelic Pten knockout mice were viable. Deletion of Pten expression was detected in the prostate of PtenLoxP/LoxP:Osr1-Cre mice as early as 2 weeks of age. Intriguingly, PtenLoxP/LoxP:Osr1-Cre mice develop high-grade prostatic intraepithelial neoplasms (PINs) with high penetrance as early as one-month of age, and locally invasive prostatic tumors after 12-months of age. PtenLoxP/+:Osr1-Cre mice show only mild oncogenic changes after 8-weeks of age. Castration of PtenLoxP/LoxP:Osr1-Cre mice shows no significant regression of prostate tumors, although a shift of androgen receptor (AR) staining from the nuclei to cytoplasm is observed in Pten null tumor cells of castrated mice. Enhanced Akt activity is observed in Pten null tumor cells of castrated PtenLoxP/LoxP:Osr1-Cre. This study provides a novel mouse model that can be used to investigate a primary role of Pten in initiating oncogenic transformation in the prostate and to examine other genetic and epigenetic changes that are required for tumor progression in the mouse prostate. PMID:23308230

Enhanced serotonin response in the hippocampus of Galphaz protein knock-out mice.

PubMed

Oleskevich, Sharon; Leck, Kwong-Joo; Matthaei, Klaus; Hendry, Ian A

2005-06-21

The serotonin-1A [5-hydroxytryptamine 1A (5HT1A)] receptor is important for emotional and homeostatic processes in the central nervous system. In the hippocampus, the 5HT1A receptor couples to inhibitory Gi/o proteins to decrease pyramidal cell excitability. Here we investigate the 5HT1A receptor in a mouse deficient in the alpha-subunit of Gz protein (Galphaz knock-out). Behavioural tests showed heightened anxiety and depression-like behaviour in the Galphaz knock-out mice. Whole-cell recording in CA1 pyramidal neurons showed a significantly greater 5HT1A receptor-mediated potassium current in Galphaz knock-out mice. The effect was independent of 5HT4 receptors as the slow after-hyperpolarization was unaffected and a slow depolarization was absent in the Galphaz knock-out mice. Other receptors linked to Gi/o proteins [gamma-aminobutyric acid type B receptor (GABAB), adenosine A1 and muscarinic acetylcholine receptors] were not affected in Galphaz knock-out mice. These results suggest that the 5HT1A receptor may be linked to Galphaz protein, as reported previously in cell culture but shown here in an intact neural network.

Loss of CDKL5 in Glutamatergic Neurons Disrupts Hippocampal Microcircuitry and Leads to Memory Impairment in Mice

PubMed Central

Wang, I-Ting Judy; Yue, Cuiyong; Takano, Hajime; Terzic, Barbara

2017-01-01

Cyclin-dependent kinase-like 5 (CDKL5) deficiency is a neurodevelopmental disorder characterized by epileptic seizures, severe intellectual disability, and autistic features. Mice lacking CDKL5 display multiple behavioral abnormalities reminiscent of the disorder, but the cellular origins of these phenotypes remain unclear. Here, we find that ablating CDKL5 expression specifically from forebrain glutamatergic neurons impairs hippocampal-dependent memory in male conditional knock-out mice. Hippocampal pyramidal neurons lacking CDKL5 show decreased dendritic complexity but a trend toward increased spine density. This morphological change is accompanied by an increase in the frequency of spontaneous miniature EPSCs and interestingly, miniature IPSCs. Using voltage-sensitive dye imaging to interrogate the evoked response of the CA1 microcircuit, we find that CA1 pyramidal neurons lacking CDKL5 show hyperexcitability in their dendritic domain that is constrained by elevated inhibition in a spatially and temporally distinct manner. These results suggest a novel role for CDKL5 in the regulation of synaptic function and uncover an intriguing microcircuit mechanism underlying impaired learning and memory. SIGNIFICANCE STATEMENT Cyclin-dependent kinase-like 5 (CDKL5) deficiency is a severe neurodevelopmental disorder caused by mutations in the CDKL5 gene. Although Cdkl5 constitutive knock-out mice have recapitulated key aspects of human symptomatology, the cellular origins of CDKL5 deficiency-related phenotypes are unknown. Here, using conditional knock-out mice, we show that hippocampal-dependent learning and memory deficits in CDKL5 deficiency have origins in glutamatergic neurons of the forebrain and that loss of CDKL5 results in the enhancement of synaptic transmission and disruptions in neural circuit dynamics in a spatially and temporally specific manner. Our findings demonstrate that CDKL5 is an important regulator of synaptic function in glutamatergic neurons and serves a critical role in learning and memory. PMID:28674172

Loss of CDKL5 in Glutamatergic Neurons Disrupts Hippocampal Microcircuitry and Leads to Memory Impairment in Mice.

PubMed

Tang, Sheng; Wang, I-Ting Judy; Yue, Cuiyong; Takano, Hajime; Terzic, Barbara; Pance, Katarina; Lee, Jun Y; Cui, Yue; Coulter, Douglas A; Zhou, Zhaolan

2017-08-02

Cyclin-dependent kinase-like 5 (CDKL5) deficiency is a neurodevelopmental disorder characterized by epileptic seizures, severe intellectual disability, and autistic features. Mice lacking CDKL5 display multiple behavioral abnormalities reminiscent of the disorder, but the cellular origins of these phenotypes remain unclear. Here, we find that ablating CDKL5 expression specifically from forebrain glutamatergic neurons impairs hippocampal-dependent memory in male conditional knock-out mice. Hippocampal pyramidal neurons lacking CDKL5 show decreased dendritic complexity but a trend toward increased spine density. This morphological change is accompanied by an increase in the frequency of spontaneous miniature EPSCs and interestingly, miniature IPSCs. Using voltage-sensitive dye imaging to interrogate the evoked response of the CA1 microcircuit, we find that CA1 pyramidal neurons lacking CDKL5 show hyperexcitability in their dendritic domain that is constrained by elevated inhibition in a spatially and temporally distinct manner. These results suggest a novel role for CDKL5 in the regulation of synaptic function and uncover an intriguing microcircuit mechanism underlying impaired learning and memory. SIGNIFICANCE STATEMENT Cyclin-dependent kinase-like 5 (CDKL5) deficiency is a severe neurodevelopmental disorder caused by mutations in the CDKL5 gene. Although Cdkl5 constitutive knock-out mice have recapitulated key aspects of human symptomatology, the cellular origins of CDKL5 deficiency-related phenotypes are unknown. Here, using conditional knock-out mice, we show that hippocampal-dependent learning and memory deficits in CDKL5 deficiency have origins in glutamatergic neurons of the forebrain and that loss of CDKL5 results in the enhancement of synaptic transmission and disruptions in neural circuit dynamics in a spatially and temporally specific manner. Our findings demonstrate that CDKL5 is an important regulator of synaptic function in glutamatergic neurons and serves a critical role in learning and memory. Copyright © 2017 the authors 0270-6474/17/377420-18$15.00/0.

High susceptibility to liver injury in IL-27 p28 conditional knockout mice involves intrinsic interferon-γ dysregulation of CD4+ T cells.

PubMed

Zhang, Song; Liang, Ruifang; Luo, Wei; Liu, Chang; Wu, Xiaoli; Gao, Yanan; Hao, Jianlei; Cao, Guangchao; Chen, Xi; Wei, Jun; Xia, Siyuan; Li, Zheng; Wen, Ti; Wu, Yunyun; Zhou, Xinglong; Wang, Puyue; Zhao, Liqing; Wu, Zhengzhou; Xiong, Sidong; Gao, Xiaoming; Gao, Xiang; Chen, Yongyan; Ge, Qing; Tian, Zhigang; Yin, Zhinan

2013-04-01

Interleukin (IL)-27, a newly discovered IL-12 family cytokine, is composed of p28 and EBI3. In this study, CD11c-p28(f/f) conditional knockout mice were generated to delete p28 specifically in dendritic cells (DCs). We demonstrated that in the absence of DC-derived p28, these mice were highly susceptible to both low and higher concentrations of concanavalin A (ConA) (5 mg/kg or 10 mg/kg), with extremely early and steady high levels of interferon-γ (IFN-γ) in sera. Neutralizing IFN-γ prevented ConA-induced liver damage in these mice, indicating a critical role of IFN-γ in this pathological process. Interestingly, the main source of the increased IFN-γ in CD11c-p28(f/f) mice was CD4+ T cells, but not natural killer T (NKT) cells. Depletion of CD4+ , but not NK1.1+ , cells completely abolished liver damage, whereas transferring CD4+ T cells from CD11c-p28(f/f) mice, but not from wild-type mice or CD11c-p28(f/f) -IFN-γ(-/-) double knockout mice to CD4(-/-) mice, restored the increased liver damage. Further studies defined higher levels of IFN-γ and T-bet messenger RNA in naïve CD4+ T cells from CD11c-p28(f/f) mice, and these CD4+ T cells were highly responsive to both low and higher concentrations of anti-CD3, indicating a programmed functional alternation of CD4+ T cells. We provide a unique model for studying the pathology of CD4+ T cell-mediated liver injury and reveal a novel function of DC-derived p28 on ConA-induced fulminant hepatitis through regulation of the intrinsic ability for IFN-γ production by CD4+ T cells. Copyright © 2012 American Association for the Study of Liver Diseases.

SHH-dependent knockout of HIF-1 alpha accelerates the degenerative process in mouse intervertebral disc.

PubMed

Wu, W J; Zhang, X K; Zheng, X F; Yang, Y H; Jiang, S D; Jiang, L S

2013-01-01

Hypoxia-inducible factor-1alpha (HIF-1 alpha) has been reported to have an important role in the metabolism and synthesis of extracellular matrix of the nucleus pulposus cells (NPCs) and was assumed to be involved in the process of intervertebral disc degeneration. The objective of this study was to investigate the role of HIF-1alpha in disc degeneration in vivo using a conditional HIF-1alpha knockout (KO) mouse model. ShhCre transgenic mice were mated with HIF-1 alpha fl/fl mice to generate conditional HIF-1alpha KO mice (HIF-1alpha fl/fl-ShhCre+). Three mice of each genotype (Wide-type and HIF-1alpha KO) at the age of 3 days, 6, and 12 weeks were sacrificed after genotyping. Five lumbar disc samples were harvested from each mouse, with a total of 45 disc samples for each genotype. In situ hybridization and immunohistochemical analysis were used to check the efficacy of HIF-1alpha knockout. Histological grading of the disc degeneration was performed according to the classification system proposed by Boos et al. Picro-sirius red staining, Safranine O/fast green staining and immunohistochemical study were used to evaluate the expression of aggrecan, type-II collagen and vascular endothelial growth factor (VEGF). Histologic analysis revealed more NPC deaths and signs of degeneration in HIF-1alpha KO mice and the degeneration scores of HIF-1alpha KO mice were significantly higher than those of the Wide-type mice at the age of 6 weeks and 12 weeks. There were less expressions of aggrecan, type-II collagen and VEGF in the intervertebral discs of HIF1-alpha KO mice than in those of wild-type mice. Taken together, the results of our study indicated that HIF-1alpha is a pivotal contributor to NPC survival and the homeotasis of extracellular matrix through the HIF-1alpha/VEGF signaling pathway, and plays an important role in the development of disc degeneration.

A mouse model of DEPDC5-related epilepsy: Neuronal loss of Depdc5 causes dysplastic and ectopic neurons, increased mTOR signaling, and seizure susceptibility.

PubMed

Yuskaitis, Christopher J; Jones, Brandon M; Wolfson, Rachel L; Super, Chloe E; Dhamne, Sameer C; Rotenberg, Alexander; Sabatini, David M; Sahin, Mustafa; Poduri, Annapurna

2018-03-01

DEPDC5 is a newly identified epilepsy-related gene implicated in focal epilepsy, brain malformations, and Sudden Unexplained Death in Epilepsy (SUDEP). In vitro, DEPDC5 negatively regulates amino acid sensing by the mTOR complex 1 (mTORC1) pathway, but the role of DEPDC5 in neurodevelopment and epilepsy has not been described. No animal model of DEPDC5-related epilepsy has recapitulated the neurological phenotypes seen in patients, and germline knockout rodent models are embryonic lethal. Here, we establish a neuron-specific Depdc5 conditional knockout mouse by cre-recombination under the Synapsin1 promotor. Depdc5 flox/flox -Syn1 Cre (Depdc5cc+) mice survive to adulthood with a progressive neurologic phenotype that includes motor abnormalities (i.e., hind limb clasping) and reduced survival compared to littermate control mice. Depdc5cc+ mice have larger brains with increased cortical neuron size and dysplastic neurons throughout the cortex, comparable to the abnormal neurons seen in human focal cortical dysplasia specimens. Depdc5 results in constitutive mTORC1 hyperactivation exclusively in neurons as measured by the increased phosphorylation of the downstream ribosomal protein S6. Despite a lack of increased mTORC1 signaling within astrocytes, Depdc5cc+ brains show reactive astrogliosis. We observed two Depdc5cc+ mice to have spontaneous seizures, including a terminal seizure. We demonstrate that as a group Depdc5cc+ mice have lowered seizure thresholds, as evidenced by decreased latency to seizures after chemoconvulsant injection and increased mortality from pentylenetetrazole-induced seizures. In summary, our neuron-specific Depdc5 knockout mouse model recapitulates clinical, pathological, and biochemical features of human DEPDC5-related epilepsy and brain malformations. We thereby present an important model in which to study targeted therapeutic strategies for DEPDC5-related conditions. Copyright © 2017 Elsevier Inc. 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.

Construction of conditional acid ceramidase knockout mice and in vivo effects on oocyte development and fertility.

PubMed

Eliyahu, Efrat; Shtraizent, Nataly; Shalgi, Ruth; Schuchman, Edward H

2012-01-01

The number of resting follicles in the ovary and their successful maturation during development define the fertile female lifespan. Oocytes, enclosed within follicles, are subject to natural selection, and the majority will undergo apoptosis during prenatal life through adulthood. Our previous studies revealed high levels of the lipid hydrolase, acid ceramidase (AC), in human and mouse oocytes, follicular fluid and cumulus cells. In addition, supplementation of in vitro fertilization media with recombinant AC enhanced the survival of oocytes and preimplantation embryos. Herein we constructed and used a conditional knockout mouse model of AC deficiency (cACKO) to further investigate the role of this enzyme in oocyte survival in vivo. Immunohistochemical staining, activity assays, and western blot analysis revealed that AC expression was high in the ovaries of normal mice, particularly in the theca cells. After induction of the AC gene knockout with tamoxifen (TM), AC levels decreased in ovaries, and ceramide was correspondingly elevated. A novel immunostaining method was developed to visualize follicles at various stages, and together with light microscopic examination, the transition of the follicle from the secondary to antral stage was found to be defective in the absence of AC. Western blot analysis showed elevated BAX and PARP expression in TM-treated cACKO mouse ovaries compared to control animals. In parallel, the levels of BCL-2 and anti-Mullerian hormone, a marker of ovarian reserve, were decreased. In addition to the above, there was a significant decrease in fertility observed in the TM-treated cACKO mice. Together, these data suggest that AC plays an important role in the preservation of fertility by maintaining low ceramide levels and preventing apoptosis of theca cells, thereby promoting survival of the follicle during the transition from the secondary to antral stage. Copyright © 2012 S. Karger AG, Basel.

Cold Shock as a Screen for Genes Involved in Cold Acclimatization in Neurospora crassa

PubMed Central

Watters, Michael K.; Manzanilla, Victor; Howell, Holly; Mehreteab, Alexander; Rose, Erik; Walters, Nicole; Seitz, Nicholas; Nava, Jacob; Kekelik, Sienna; Knuth, Laura; Scivinsky, Brianna

2018-01-01

When subjected to rapid drops of temperature (cold shock), Neurospora responds with a temporary shift in its morphology. This report is the first to examine this response genetically. We report here the results of a screen of selected mutants from the Neurospora knockout library for alterations in their morphological response to cold shock. Three groups of knockouts were selected to be subject to this screen: genes previously suspected to be involved in hyphal development as well as knockouts resulting in morphological changes; transcription factors; and genes homologous to E. coli genes known to alter their expression in response to cold shock. A total of 344 knockout strains were subjected to cold shock. Of those, 118 strains were identified with altered responses. We report here the cold shock morphologies and GO categorizations of strains subjected to this screen. Of strains with knockouts in genes associated with hyphal growth or morphology, 33 of 131 tested (25%) showed an altered response to cold shock. Of strains with knockouts in transcription factor genes, 30 of 145 (20%) showed an altered response to cold shock. Of strains with knockouts in genes homologous to E. coli genes which display altered levels of transcription in response to cold shock, a total of 55 of 68 tested (81%) showed an altered cold shock response. This suggests that the response to cold shock in these two organisms is largely shared in common. PMID:29563189

YY1 Controls Immunoglobulin Class Switch Recombination and Nuclear Activation-Induced Deaminase Levels

PubMed Central

Zaprazna, Kristina

2012-01-01

Activation-induced deaminase (AID) is an enzyme required for class switch recombination (CSR) and somatic hypermutation (SHM), processes that ensure antibody maturation and expression of different immunoglobulin isotypes. AID function is tightly regulated by tissue- and stage-specific expression, nuclear localization, and protein stability. Transcription factor YY1 is crucial for early B cell development, but its function at late B cell stages is unknown. Here, we show that YY1 conditional knockout in activated splenic B cells interferes with CSR. Knockout of YY1 did not affect B cell proliferation, transcription of the AID and IgM genes, or levels of various switch region germ line transcripts. However, we show that YY1 physically interacts with AID and controls the accumulation of nuclear AID, at least in part, by increasing nuclear AID stability. We show for the first time that YY1 plays a novel role in CSR and controls nuclear AID protein levels. PMID:22290437

Knockout of the PKN Family of Rho Effector Kinases Reveals a Non-redundant Role for PKN2 in Developmental Mesoderm Expansion

PubMed Central

Quétier, Ivan; Marshall, Jacqueline J.T.; Spencer-Dene, Bradley; Lachmann, Sylvie; Casamassima, Adele; Franco, Claudio; Escuin, Sarah; Worrall, Joseph T.; Baskaran, Priththivika; Rajeeve, Vinothini; Howell, Michael; Copp, Andrew J.; Stamp, Gordon; Rosewell, Ian; Cutillas, Pedro; Gerhardt, Holger; Parker, Peter J.; Cameron, Angus J.M.

2016-01-01

Summary In animals, the protein kinase C (PKC) family has expanded into diversely regulated subgroups, including the Rho family-responsive PKN kinases. Here, we describe knockouts of all three mouse PKN isoforms and reveal that PKN2 loss results in lethality at embryonic day 10 (E10), with associated cardiovascular and morphogenetic defects. The cardiovascular phenotype was not recapitulated by conditional deletion of PKN2 in endothelial cells or the developing heart. In contrast, inducible systemic deletion of PKN2 after E7 provoked collapse of the embryonic mesoderm. Furthermore, mouse embryonic fibroblasts, which arise from the embryonic mesoderm, depend on PKN2 for proliferation and motility. These cellular defects are reflected in vivo as dependence on PKN2 for mesoderm proliferation and neural crest migration. We conclude that failure of the mesoderm to expand in the absence of PKN2 compromises cardiovascular integrity and development, resulting in lethality. PMID:26774483

Knockout of the PKN Family of Rho Effector Kinases Reveals a Non-redundant Role for PKN2 in Developmental Mesoderm Expansion.

PubMed

Quétier, Ivan; Marshall, Jacqueline J T; Spencer-Dene, Bradley; Lachmann, Sylvie; Casamassima, Adele; Franco, Claudio; Escuin, Sarah; Worrall, Joseph T; Baskaran, Priththivika; Rajeeve, Vinothini; Howell, Michael; Copp, Andrew J; Stamp, Gordon; Rosewell, Ian; Cutillas, Pedro; Gerhardt, Holger; Parker, Peter J; Cameron, Angus J M

2016-01-26

In animals, the protein kinase C (PKC) family has expanded into diversely regulated subgroups, including the Rho family-responsive PKN kinases. Here, we describe knockouts of all three mouse PKN isoforms and reveal that PKN2 loss results in lethality at embryonic day 10 (E10), with associated cardiovascular and morphogenetic defects. The cardiovascular phenotype was not recapitulated by conditional deletion of PKN2 in endothelial cells or the developing heart. In contrast, inducible systemic deletion of PKN2 after E7 provoked collapse of the embryonic mesoderm. Furthermore, mouse embryonic fibroblasts, which arise from the embryonic mesoderm, depend on PKN2 for proliferation and motility. These cellular defects are reflected in vivo as dependence on PKN2 for mesoderm proliferation and neural crest migration. We conclude that failure of the mesoderm to expand in the absence of PKN2 compromises cardiovascular integrity and development, resulting in lethality. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Single-neuron labeling with inducible cre-mediated knockout in transgenic mice

PubMed Central

Young, Paul; Qiu, Li; Wang, Dongqing; Zhao, Shengli; Gross, James; Feng, Guoping

2011-01-01

To facilitate functional analysis of neuronal connectivity in a mammalian nervous system tightly packed with billions of cells, we developed a new technique that allows inducible genetic manipulations within fluorescently labeled single neurons in mice. We term this technique SLICK for Single-neuron Labeling with Inducible Cre-mediated Knockout. SLICK is achieved by co-expressing a drug-inducible form of cre recombinase and a fluorescent protein within the same small subsets of neurons. Thus, SLICK combines the powerful cre recombinase system for conditional genetic manipulation and the fluorescent labeling of single neurons for imaging. We demonstrate efficient inducible genetic manipulation in several types of neurons using SLICK. Furthermore, we apply SLICK to eliminate synaptic transmission in a small subset of neuromuscular junctions. Our results provide evidence for the long-term stability of inactive neuromuscular synapses in adult animals. More broadly, these studies demonstrate a cre-LoxP compatible system for dissecting gene functions in single identifiable neurons. PMID:18454144

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.

TRPV1 SUMOylation regulates nociceptive signaling in models of inflammatory pain.

PubMed

Wang, Yan; Gao, Yingwei; Tian, Quan; Deng, Qi; Wang, Yangbo; Zhou, Tian; Liu, Qiang; Mei, Kaidi; Wang, Yingping; Liu, Huiqing; Ma, Ruining; Ding, Yuqiang; Rong, Weifang; Cheng, Jinke; Yao, Jing; Xu, Tian-Le; Zhu, Michael X; Li, Yong

2018-04-18

Although TRPV1 channels represent a key player of noxious heat sensation, the precise mechanisms for thermal hyperalgesia remain unknown. We report here that conditional knockout of deSUMOylation enzyme, SENP1, in mouse dorsal root ganglion (DRG) neurons exacerbated thermal hyperalgesia in both carrageenan- and Complete Freund's adjuvant-induced inflammation models. TRPV1 is SUMOylated at a C-terminal Lys residue (K822), which specifically enhances the channel sensitivity to stimulation by heat, but not capsaicin, protons or voltage. TRPV1 SUMOylation is decreased by SENP1 but upregulated upon peripheral inflammation. More importantly, the reduced ability of TRPV1 knockout mice to develop inflammatory thermal hyperalgesia was rescued by viral infection of lumbar 3/4 DRG neurons of wild-type TRPV1, but not its SUMOylation-deficient mutant, K822R. These data suggest that TRPV1 SUMOylation is essential for the development of inflammatory thermal hyperalgesia, through a mechanism that involves sensitization of the channel response specifically to thermal stimulation.

A STAT-1 Knockout Mouse Model for Machupo Virus Pathogenesis

DTIC Science & Technology

2011-06-14

hemorrhagic fever viruses, including Ebola, Marburg, Junín, and Crimean - Congo Hemorrhagic Fever viruses [11-14...Akerstrom S, Klingstrom J, Mirazimi A: Crimean - Congo hemorrhagic fever virus infection is lethal for adult type I interferon receptor-knockout mice. J...Shieh WJ, Camus G, Stroher U, Zaki S, Jones SM: Pathogenesis and immune response of Crimean - Congo hemorrhagic fever virus in a STAT-1 knockout

Embryonic Lethality Due to Arrested Cardiac Development in Psip1/Hdgfrp2 Double-Deficient Mice.

PubMed

Wang, Hao; Shun, Ming-Chieh; Dickson, Amy K; Engelman, Alan N

2015-01-01

Hepatoma-derived growth factor (HDGF) related protein 2 (HRP2) and lens epithelium-derived growth factor (LEDGF)/p75 are closely related members of the HRP2 protein family. LEDGF/p75 has been implicated in numerous human pathologies including cancer, autoimmunity, and infectious disease. Knockout of the Psip1 gene, which encodes for LEDGF/p75 and the shorter LEDGF/p52 isoform, was previously shown to cause perinatal lethality in mice. The function of HRP2 was by contrast largely unknown. To learn about the role of HRP2 in development, we knocked out the Hdgfrp2 gene, which encodes for HRP2, in both normal and Psip1 knockout mice. Hdgfrp2 knockout mice developed normally and were fertile. By contrast, the double deficient mice died at approximate embryonic day (E) 13.5. Histological examination revealed ventricular septal defect (VSD) associated with E14.5 double knockout embryos. To investigate the underlying molecular mechanism(s), RNA recovered from ventricular tissue was subjected to RNA-sequencing on the Illumina platform. Bioinformatic analysis revealed several genes and biological pathways that were significantly deregulated by the Psip1 knockout and/or Psip1/Hdgfrp2 double knockout. Among the dozen genes known to encode for LEDGF/p75 binding factors, only the expression of Nova1, which encodes an RNA splicing factor, was significantly deregulated by the knockouts. However the expression of other RNA splicing factors, including the LEDGF/p52-interacting protein ASF/SF2, was not significantly altered, indicating that deregulation of global RNA splicing was not a driving factor in the pathology of the VSD. Tumor growth factor (Tgf) β-signaling, which plays a key role in cardiac morphogenesis during development, was the only pathway significantly deregulated by the double knockout as compared to control and Psip1 knockout samples. We accordingly speculate that deregulated Tgf-β signaling was a contributing factor to the VSD and prenatal lethality of Psip1/Hdgfrp2 double-deficient mice.

Differential cytokine expression in skin graft healing in inducible nitric oxide synthase knockout mice.

PubMed

Most, D; Efron, D T; Shi, H P; Tantry, U S; Barbul, A

2001-10-01

Inducible nitric oxide synthase (iNOS) and its product, nitric oxide, have been shown to play important roles in wound biology. The present study was performed to investigate the role of iNOS in modulating the cytokine cascade during the complex process of skin graft wound healing.Fifteen iNOS-knockout mice and 15 wild-type C57BL/6J mice were subjected to autogenous 1-cm2 intrascapular full-thickness skin grafts. Three animals in each group were killed on postoperative days 3, 5, 7, 10, and 14. Specimens were then analyzed using nonisotopic in situ hybridization versus mRNA of tumor growth factor-beta1, vascular endothelial growth factor, iNOS, endothelial nitric oxide synthase (eNOS), tumor necrosis factor-alpha, and basic fibroblast growth factor, as well as positive and negative control probes. Positive cells in both grafts and wound beds were counted using a Leica microgrid. Scar thickness was measured with a Leica micrometer. Data were analyzed using the unpaired Student's t test. Expression of iNOS was 2- to 4-fold higher in knockout mice than in wild-type mice on postoperative days 5, 7, and 14. Expression of eNOS was 2- to 2.5-fold higher in knockout mice than in wild-type mice on postoperative days 5 and 7. Tumor necrosis factor-alpha expression was 2- to 7-fold higher in knockout mice than in wild-type mice on all postoperative days. In contrast, expression levels of angiogenic/fibrogenic cytokines (vascular endothelial growth factor, basis fibroblast growth factor, and tumor growth factor-beta1) were 2.5- to 4-fold higher in wild-type mice than in knockout mice. Scars were 1.5- to 2.5-fold thicker in knockout mice than in wild-type mice at all time points. All of the above results represent statistically significant differences (p < 0.05). Significantly different patterns of cytokine expression were seen in knockout and wild-type mice. Although the scar layer was thicker in knockout mice, it showed much greater infiltration with inflammatory cells. These data further delineate the modulatory effect of iNOS and nitric oxide in healing skin grafts.

Functional Genomic Screening Approaches in Mechanistic Toxicology and Potential Future Applications of CRISPR-Cas9

PubMed Central

Shen, Hua; McHale, Cliona M.; Smith, Martyn T; Zhang, Luoping

2015-01-01

Characterizing variability in the extent and nature of responses to environmental exposures is a critical aspect of human health risk assessment. Chemical toxicants act by many different mechanisms, however, and the genes involved in adverse outcome pathways (AOPs) and AOP networks are not yet characterized. Functional genomic approaches can reveal both toxicity pathways and susceptibility genes, through knockdown or knockout of all non-essential genes in a cell of interest, and identification of genes associated with a toxicity phenotype following toxicant exposure. Screening approaches in yeast and human near-haploid leukemic KBM7 cells, have identified roles for genes and pathways involved in response to many toxicants but are limited by partial homology among yeast and human genes and limited relevance to normal diploid cells. RNA interference (RNAi) suppresses mRNA expression level but is limited by off-target effects (OTEs) and incomplete knockdown. The recently developed gene editing approach called clustered regularly interspaced short palindrome repeats-associated nuclease (CRISPR)-Cas9, can precisely knock-out most regions of the genome at the DNA level with fewer OTEs than RNAi, in multiple human cell types, thus overcoming the limitations of the other approaches. It has been used to identify genes involved in the response to chemical and microbial toxicants in several human cell types and could readily be extended to the systematic screening of large numbers of environmental chemicals. CRISPR-Cas9 can also repress and activate gene expression, including that of non-coding RNA, with near-saturation, thus offering the potential to more fully characterize AOPs and AOP networks. Finally, CRISPR-Cas9 can generate complex animal models in which to conduct preclinical toxicity testing at the level of individual genotypes or haplotypes. Therefore, CRISPR-Cas9 is a powerful and flexible functional genomic screening approach that can be harnessed to provide unprecedented mechanistic insight in the field of modern toxicology. PMID:26041264

Mouse Model for Human Arginase Deficiency

PubMed Central

Iyer, Ramaswamy K.; Yoo, Paul K.; Kern, Rita M.; Rozengurt, Nora; Tsoa, Rosemarie; O'Brien, William E.; Yu, Hong; Grody, Wayne W.; Cederbaum, Stephen D.

2002-01-01

Deficiency of liver arginase (AI) causes hyperargininemia (OMIM 207800), a disorder characterized by progressive mental impairment, growth retardation, and spasticity and punctuated by sometimes fatal episodes of hyperammonemia. We constructed a knockout mouse strain carrying a nonfunctional AI gene by homologous recombination. Arginase AI knockout mice completely lacked liver arginase (AI) activity, exhibited severe symptoms of hyperammonemia, and died between postnatal days 10 and 14. During hyperammonemic crisis, plasma ammonia levels of these mice increased >10-fold compared to those for normal animals. Livers of AI-deficient animals showed hepatocyte abnormalities, including cell swelling and inclusions. Plasma amino acid analysis showed the mean arginine level in knockouts to be approximately fourfold greater than that for the wild type and threefold greater than that for heterozygotes; the mean proline level was approximately one-third and the ornithine level was one-half of the proline and ornithine levels, respectively, for wild-type or heterozygote mice—understandable biochemical consequences of arginase deficiency. Glutamic acid, citrulline, and histidine levels were about 1.5-fold higher than those seen in the phenotypically normal animals. Concentrations of the branched-chain amino acids valine, isoleucine, and leucine were 0.4 to 0.5 times the concentrations seen in phenotypically normal animals. In summary, the AI-deficient mouse duplicates several pathobiological aspects of the human condition and should prove to be a useful model for further study of the disease mechanism(s) and to explore treatment options, such as pharmaceutical administration of sodium phenylbutyrate and/or ornithine and development of gene therapy protocols. PMID:12052859

Transferrin Receptor 1 in Chronic Hypoxia-Induced Pulmonary Vascular Remodeling.

PubMed

Naito, Yoshiro; Hosokawa, Manami; Sawada, Hisashi; Oboshi, Makiko; Hirotani, Shinichi; Iwasaku, Toshihiro; Okuhara, Yoshitaka; Morisawa, Daisuke; Eguchi, Akiyo; Nishimura, Koichi; Soyama, Yuko; Fujii, Kenichi; Mano, Toshiaki; Ishihara, Masaharu; Tsujino, Takeshi; Masuyama, Tohru

2016-06-01

Iron is associated with the pathophysiology of several cardiovascular diseases, including pulmonary hypertension (PH). In addition, disrupted pulmonary iron homeostasis has been reported in several chronic lung diseases. Transferrin receptor 1 (TfR1) plays a key role in cellular iron transport. However, the role of TfR1 in the pathophysiology of PH has not been well characterized. In this study, we investigate the role of TfR1 in the development of hypoxia-induced pulmonary vascular remodeling. PH was induced by exposing wild-type (WT) mice and TfR1 hetero knockout mice to hypoxia for 4 weeks and evaluated via assessment of pulmonary vascular remodeling, right ventricular (RV) systolic pressure, and RV hypertrophy. In addition, we assessed the functional role of TfR1 in pulmonary artery smooth muscle cells in vitro. The morphology of pulmonary arteries did not differ between WT mice and TfR1 hetero knockout mice under normoxic conditions. In contrast, TfR1 hetero knockout mice exposed to 4 weeks hypoxia showed attenuated pulmonary vascular remodeling, RV systolic pressure, and RV hypertrophy compared with WT mice. In addition, the depletion of TfR1 by RNA interference attenuated human pulmonary artery smooth muscle cells proliferation induced by platelet-derived growth factor-BB (PDGF-BB) in vitro. These results suggest that TfR1 plays an important role in the development of hypoxia-induced pulmonary vascular remodeling. © American Journal of Hypertension, Ltd 2015. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

[Future directions of molecular bone cell biology].

PubMed

Yoneda, T

2001-01-01

Introduction of genetic approaches using knockout and/or transgenic mice has produced many pieces of information that can't be obtained by conventional cell biological studies and profoundly advanced our understanding of bone biology and metabolism. Here, the author will first briefly summarize the current findings in the recent bone research and subsequently attempt to predict future directions to which bone research is going to proceed with a special emphasis of osteoclast and osteoblast biology.

Equilibrative nucleoside transporter 1 (ENT1) regulates postischemic blood flow during acute kidney injury in mice

PubMed Central

Grenz, Almut; Bauerle, Jessica D.; Dalton, Julee H.; Ridyard, Douglas; Badulak, Alexander; Tak, Eunyoung; McNamee, Eóin N.; Clambey, Eric; Moldovan, Radu; Reyes, German; Klawitter, Jost; Ambler, Kelly; Magee, Kristann; Christians, Uwe; Brodsky, Kelley S.; Ravid, Katya; Choi, Doo-Sup; Wen, Jiaming; Lukashev, Dmitriy; Blackburn, Michael R.; Osswald, Hartmut; Coe, Imogen R.; Nürnberg, Bernd; Haase, Volker H.; Xia, Yang; Sitkovsky, Michail; Eltzschig, Holger K.

2012-01-01

A complex biologic network regulates kidney perfusion under physiologic conditions. This system is profoundly perturbed following renal ischemia, a leading cause of acute kidney injury (AKI) — a life-threatening condition that frequently complicates the care of hospitalized patients. Therapeutic approaches to prevent and treat AKI are extremely limited. Better understanding of the molecular pathways promoting postischemic reflow could provide new candidate targets for AKI therapeutics. Due to its role in adapting tissues to hypoxia, we hypothesized that extracellular adenosine has a regulatory function in the postischemic control of renal perfusion. Consistent with the notion that equilibrative nucleoside transporters (ENTs) terminate adenosine signaling, we observed that pharmacologic ENT inhibition in mice elevated renal adenosine levels and dampened AKI. Deletion of the ENTs resulted in selective protection in Ent1–/– mice. Comprehensive examination of adenosine receptor–knockout mice exposed to AKI demonstrated that renal protection by ENT inhibitors involves the A2B adenosine receptor. Indeed, crosstalk between renal Ent1 and Adora2b expressed on vascular endothelia effectively prevented a postischemic no-reflow phenomenon. These studies identify ENT1 and adenosine receptors as key to the process of reestablishing renal perfusion following ischemic AKI. If translatable from mice to humans, these data have important therapeutic implications. PMID:22269324

Distribution of Nidogen in the Murine Eye and Ocular Phenotype of the Nidogen-1 Knockout Mouse

PubMed Central

May, Christian Albrecht

2012-01-01

Distribution and lack of nidogen-1, part of numerous basement membranes, were studied in the mouse eye. For that purpose, eyes of C57BL/6 and nidogen-1 knockout mice were stained immunohistochemically for nidogen-1, and intraocular pressure measurements and light- and electron microscopy were used to study the nidogen-1 knockout animals. In normal mice, nidogen-1 was present in many basement membranes, but showed irregularities underneath the corneal epithelium, in Bruch's membrane and in the iris. Homozygous knockout of nidogen-1 in the mouse showed only mild pathological changes. In the anterior eye segment, small interruptions were noted in the nonpigmented ciliary epithelium without further consequences. In the posterior eye segment, interruptions of the inner limiting membrane led to small retinal ectopias and subsequent changes in the optic nerve. In summary, the knockout of nidogen-1 showed mild but significant morphological changes pointing to the importance of this protein which can in part, but not completely; be replaced by nidogen-2. PMID:24555126

Dcdc2 knockout mice display exacerbated developmental disruptions following knockdown of Dcx

PubMed Central

Wang, Yu; Yin, Xiuyin; Rosen, Glenn; Gabel, Lisa; Guadiana, Sarah M.; Sarkisian, Matthew R; Galaburda, Albert M.; LoTurco, Joseph J.

2011-01-01

The dyslexia-associated gene DCDC2 is a member of the DCX family of genes known to play roles in neurogenesis, neuronal migration and differentiation. Here we report the first phenotypic analysis of a Dcdc2 knockout mouse. Comparisons between Dcdc2 knockout mice and wild type littermates revealed no significant differences in neuronal migration, neocortical lamination, neuronal cilliogenesis or dendritic differentiation. Considering previous studies showing genetic interactions and potential functional redundancy among members of the DCX family, we tested whether decreasing Dcx expression by RNAi would differentially impair neurodevelopment in Dcdc2 knockouts and wild type mice. Consistent with this hypothesis, we found that deficits in neuronal migration, and dendritic growth caused by RNAi of Dcx were more severe in Dcdc2 knockouts than in wild type mice with the same transfection. These results indicate that Dcdc2 is not required for neurogenesis, neuronal migration or differentiation in mice, but may have partial functional redundancy with Dcx. PMID:21689730

Gene Therapy to Extend Lifespan of Tsc1 Conditional Brain Knockouts

DTIC Science & Technology

2015-07-01

TSC) is an autosomal genetic disorder which affects about 1 in 6,000 newborns. The disease is caused by inactivating mutations in either of two...are seen in the majority (>90%) of TSC patients, and disrupt neuronal architecture causing epilepsy and obstruction of ventricles, respectively (Short...an autosomal genetic disorder which affects about 1 in 6,000 newborns. The disease is caused by inactivating mutations in either of two related tumor

Rab10 Disruption Results in Delayed OPC Maturation.

PubMed

Zhang, Zhao-Huan; Zhao, Wei-Qian; Ma, Fan-Fei; Zhang, Hui; Xu, Xiao-Hui

2017-10-01

Oligodendrocyte precursor cell (OPC) maturation requires membrane addition for myelin sheath formation. Since the Rab system has been shown to contribute to membrane addition in other cell types, in this study, we explored the role of Rab in OPC maturation. SiRNA and shRNA techniques and conditional knockout mice provided in vitro and in vivo evidence that Rab10 is involved in OPC maturation and may affect myelination during OPC development.

Age-related NMDA signaling alterations in SOD2 deficient mice.

PubMed

Carvajal, Francisco J; Mira, Rodrigo G; Rovegno, Maximiliano; Minniti, Alicia N; Cerpa, Waldo

2018-06-01

Oxidative stress affects the survival and function of neurons. Hence, they have a complex and highly regulated machinery to handle oxidative changes. The dysregulation of this antioxidant machinery is associated with a wide range of neurodegenerative conditions. Therefore, we evaluated signaling alterations, synaptic properties and behavioral performance in 2 and 6-month-old heterozygous manganese superoxide dismutase knockout mice (SOD2 +/- mice). We found that their low antioxidant capacity generated direct oxidative damage in proteins, lipids, and DNA. However, only 6-month-old heterozygous knockout mice presented behavioral impairments. On the other hand, synaptic plasticity, synaptic strength and NMDA receptor (NMDAR) dependent postsynaptic potentials were decreased in an age-dependent manner. We also analyzed the phosphorylation state of the NMDAR subunit GluN2B. We found that while the levels of GluN2B phosphorylated on tyrosine 1472 (synaptic form) remain unchanged, we detected increased levels of GluN2B phosphorylated on tyrosine 1336 (extrasynaptic form), establishing alterations in the synaptic/extrasynaptic ratio of GluN2B. Additionally, we found increased levels of two phosphatases associated with dephosphorylation of p-1472: striatal-enriched protein tyrosine phosphatase (STEP) and phosphatase and tensin homolog deleted on chromosome Ten (PTEN). Moreover, we found decreased levels of p-CREB, a master transcription factor activated by synaptic stimulation. In summary, we describe mechanisms by which glutamatergic synapses are altered under oxidative stress conditions. Our results uncovered new putative therapeutic targets for conditions where NMDAR downstream signaling is altered. This work also contributes to our understanding of processes such as synapse formation, learning, and memory in neuropathological conditions. Copyright © 2018 Elsevier B.V. All rights reserved.

2-cysteine peroxiredoxins and thylakoid ascorbate peroxidase create a water-water cycle that is essential to protect the photosynthetic apparatus under high light stress conditions.

PubMed

Awad, Jasmin; Stotz, Henrik U; Fekete, Agnes; Krischke, Markus; Engert, Cornelia; Havaux, Michel; Berger, Susanne; Mueller, Martin J

2015-04-01

Different peroxidases, including 2-cysteine (2-Cys) peroxiredoxins (PRXs) and thylakoid ascorbate peroxidase (tAPX), have been proposed to be involved in the water-water cycle (WWC) and hydrogen peroxide (H2O2)-mediated signaling in plastids. We generated an Arabidopsis (Arabidopsis thaliana) double-mutant line deficient in the two plastid 2-Cys PRXs (2-Cys PRX A and B, 2cpa 2cpb) and a triple mutant deficient in 2-Cys PRXs and tAPX (2cpa 2cpb tapx). In contrast to wild-type and tapx single-knockout plants, 2cpa 2cpb double-knockout plants showed an impairment of photosynthetic efficiency and became photobleached under high light (HL) growth conditions. In addition, double-mutant plants also generated elevated levels of superoxide anion radicals, H2O2, and carbonylated proteins but lacked anthocyanin accumulation under HL stress conditions. Under HL conditions, 2-Cys PRXs seem to be essential in maintaining the WWC, whereas tAPX is dispensable. By comparison, this HL-sensitive phenotype was more severe in 2cpa 2cpb tapx triple-mutant plants, indicating that tAPX partially compensates for the loss of functional 2-Cys PRXs by mutation or inactivation by overoxidation. In response to HL, H2O2- and photooxidative stress-responsive marker genes were found to be dramatically up-regulated in 2cpa 2cpb tapx but not 2cpa 2cpb mutant plants, suggesting that HL-induced plastid to nucleus retrograde photooxidative stress signaling takes place after loss or inactivation of the WWC enzymes 2-Cys PRX A, 2-Cys PRX B, and tAPX. © 2015 American Society of Plant Biologists. All Rights Reserved.

PERCEPTION OF SWEET TASTE IS IMPORTANT FOR VOLUNTARY ALCOHOL CONSUMPTION IN MICE

PubMed Central

Blednov, Y.A.; Walker, D.; Martinez, M.; Levine, M.; Damak, S.; Margolskee, R.F.

2012-01-01

To directly evaluate the association between taste perception and alcohol intake, we used three different mutant mice, each lacking a gene expressed in taste buds and critical to taste transduction: α-gustducin (Gnat3), Tas1r3 or Trpm5. Null mutant mice lacking any of these three genes showed lower preference score for alcohol and consumed less alcohol in a two-bottle choice test, as compared with wild-type littermates. These null mice also showed lower preference score for saccharin solutions than did wild-type littermates. In contrast, avoidance of quinine solutions was less in Gnat3 or Trpm5 knockout mice than in wild type mice, whereas Tas1r3 null mice were not different from wild-type in their response to quinine solutions. There were no differences in null vs. wild-type mice in their consumption of sodium chloride solutions. To determine the cause for reduction of ethanol intake, we studied other ethanol-induced behaviors known to be related to alcohol consumption. There were no differences between null and wild-type mice in ethanol-induced loss of righting reflex, severity of acute ethanol withdrawal or conditioned place preference for ethanol. Weaker conditioned taste aversion to alcohol in null mice may have been caused by weaker rewarding value of the conditioned stimulus (saccharin). When saccharin was replaced by sodium chloride, no differences in conditioned taste aversion to alcohol between knockout and wild-type mice were seen. Thus, deletion of any one of three different genes involved in detection of sweet taste leads to a substantial reduction of alcohol intake without any changes in pharmacological actions of ethanol. PMID:17376151

Methionine Sulfoxide Reductase A Knockout Mice Show Progressive Hearing Loss and Sensitivity to Acoustic Trauma.

PubMed

Alqudah, Safa; Chertoff, Mark; Durham, Dianne; Moskovitz, Jackob; Staecker, Hinrich; Peppi, Marcello

2018-06-21

Methionine sulfoxide reductases (MsrA and MsrB) protect the biological activity of proteins from oxidative modifications to methionine residues and are important for protecting against the pathological effects of neurodegenerative diseases. In the current study, we characterized the auditory phenotype of the MsrA knockout mouse. Young MsrA knockout mice showed small high-frequency threshold elevations for auditory brainstem response and distortion product otoacoustic emission compared to those of wild-type mice, which progressively worsened in older MsrA knockout mice. MsrA knockout mice showed an increased sensitivity to noise at young and older ages, suggesting that MsrA is part of a mechanism that protects the cochlea from acoustic damage. MsrA mRNA in the cochlea was increased following acoustic stimulation. Finally, expression of mRNA MsrB1 was compromised at 6 months old, but not in younger MsrA knockout mice (compared to controls). The identification of MsrA in the cochlea as a protective mediator from both early onset hearing loss and acoustic trauma expands our understanding of the pathways that may induce protection from acoustic trauma and foster further studies on how to prevent the damaging effect of noise exposure through Msr-based therapy. © 2018 S. Karger AG, Basel.

Alterations of amino acids and monoamine metabolism in male Fmr1 knockout mice: a putative animal model of the human fragile X mental retardation syndrome.

PubMed

Gruss, M; Braun, K

2001-01-01

The Fragile X syndrome, a common form of mental retardation in humans, is caused by silencing the fragile X mental retardation (FMR1) gene leading to the absence of the encoded fragile X mental retardation protein 1 (FMRP). We describe morphological and behavioral abnormalities for both affected humans and Fmr1 knockout mice, a putative animal model for the human Fragile X syndrome. The aim of the present study was to identify possible neurochemical abnormalities in Fmr1 knockout mice, with particular focus on neurotransmission. Significant region-specific differences of basal neurotransmitter and metabolite levels were found between wildtype and Fmr1 knockout animals, predominantly in juveniles (post-natal days 28 to 31). Adults (postnatal days 209 to 221) showed only few abnormalities as compared with the wildtype. In juvenile knockout mice, aspartate and taurine were especially increased in cortical regions, striatum, hippocampus, cerebellum, and brainstem. In addition, juveniles showed an altered balance between excitatory and inhibitory amino acids in the caudal cortex, hippocampus, and brainstem. We detected very few differences in monoamine turnover in both age stages. The results presented here provide the first evidence that lack of FMRP expression in FMRP knockout mice is accompanied by age-dependent, region-specific alterations in neurotransmission.

Knockouts of high-ranking males have limited impact on baboon social networks.

PubMed

Franz, Mathias; Altmann, Jeanne; Alberts, Susan C

Social network structures can crucially impact complex social processes such as collective behaviour or the transmission of information and diseases. However, currently it is poorly understood how social networks change over time. Previous studies on primates suggest that `knockouts' (due to death or dispersal) of high-ranking individuals might be important drivers for structural changes in animal social networks. Here we test this hypothesis using long-term data on a natural population of baboons, examining the effects of 29 natural knockouts of alpha or beta males on adult female social networks. We investigated whether and how knockouts affected (1) changes in grooming and association rates among adult females, and (2) changes in mean degree and global clustering coefficient in these networks. The only significant effect that we found was a decrease in mean degree in grooming networks in the first month after knockouts, but this decrease was rather small, and grooming networks rebounded to baseline levels by the second month after knockouts. Taken together our results indicate that the removal of high-ranking males has only limited or no lasting effects on social networks of adult female baboons. This finding calls into question the hypothesis that the removal of high-ranking individuals has a destabilizing effect on social network structures in social animals.

Disease phenotype of a ferret CFTR-knockout model of cystic fibrosis

PubMed Central

Sun, Xingshen; Sui, Hongshu; Fisher, John T.; Yan, Ziying; Liu, Xiaoming; Cho, Hyung-Ju; Joo, Nam Soo; Zhang, Yulong; Zhou, Weihong; Yi, Yaling; Kinyon, Joann M.; Lei-Butters, Diana C.; Griffin, Michelle A.; Naumann, Paul; Luo, Meihui; Ascher, Jill; Wang, Kai; Frana, Timothy; Wine, Jeffrey J.; Meyerholz, David K.; Engelhardt, John F.

2010-01-01

Cystic fibrosis (CF) is a recessive disease that affects multiple organs. It is caused by mutations in CFTR. Animal modeling of this disease has been challenging, with species- and strain-specific differences in organ biology and CFTR function influencing the emergence of disease pathology. Here, we report the phenotype of a CFTR-knockout ferret model of CF. Neonatal CFTR-knockout ferrets demonstrated many of the characteristics of human CF disease, including defective airway chloride transport and submucosal gland fluid secretion; variably penetrant meconium ileus (MI); pancreatic, liver, and vas deferens disease; and a predisposition to lung infection in the early postnatal period. Severe malabsorption by the gastrointestinal (GI) tract was the primary cause of death in CFTR-knockout kits that escaped MI. Elevated liver function tests in CFTR-knockout kits were corrected by oral administration of ursodeoxycholic acid, and the addition of an oral proton-pump inhibitor improved weight gain and survival. To overcome the limitations imposed by the severe intestinal phenotype, we cloned 4 gut-corrected transgenic CFTR-knockout kits that expressed ferret CFTR specifically in the intestine. One clone passed feces normally and demonstrated no detectable ferret CFTR expression in the lung or liver. The animals described in this study are likely to be useful tools for dissecting CF disease pathogenesis and developing treatments. PMID:20739752

Disease phenotype of a ferret CFTR-knockout model of cystic fibrosis.

PubMed

Sun, Xingshen; Sui, Hongshu; Fisher, John T; Yan, Ziying; Liu, Xiaoming; Cho, Hyung-Ju; Joo, Nam Soo; Zhang, Yulong; Zhou, Weihong; Yi, Yaling; Kinyon, Joann M; Lei-Butters, Diana C; Griffin, Michelle A; Naumann, Paul; Luo, Meihui; Ascher, Jill; Wang, Kai; Frana, Timothy; Wine, Jeffrey J; Meyerholz, David K; Engelhardt, John F

2010-09-01

Cystic fibrosis (CF) is a recessive disease that affects multiple organs. It is caused by mutations in CFTR. Animal modeling of this disease has been challenging, with species- and strain-specific differences in organ biology and CFTR function influencing the emergence of disease pathology. Here, we report the phenotype of a CFTR-knockout ferret model of CF. Neonatal CFTR-knockout ferrets demonstrated many of the characteristics of human CF disease, including defective airway chloride transport and submucosal gland fluid secretion; variably penetrant meconium ileus (MI); pancreatic, liver, and vas deferens disease; and a predisposition to lung infection in the early postnatal period. Severe malabsorption by the gastrointestinal (GI) tract was the primary cause of death in CFTR-knockout kits that escaped MI. Elevated liver function tests in CFTR-knockout kits were corrected by oral administration of ursodeoxycholic acid, and the addition of an oral proton-pump inhibitor improved weight gain and survival. To overcome the limitations imposed by the severe intestinal phenotype, we cloned 4 gut-corrected transgenic CFTR-knockout kits that expressed ferret CFTR specifically in the intestine. One clone passed feces normally and demonstrated no detectable ferret CFTR expression in the lung or liver. The animals described in this study are likely to be useful tools for dissecting CF disease pathogenesis and developing treatments.

Trpc2 Depletion Protects RBC from Oxidative Stress-Induced Hemolysis

PubMed Central

Hirschler-Laszkiewicz, Iwona; Zhang, Wenyi; Keefer, Kerry; Conrad, Kathleen; Tong, Qin; Chen, Shu-jen; Bronson, Sarah; Cheung, Joseph Y.; Miller, Barbara A.

2011-01-01

Transient receptor potential channels Trpc2 and Trpc3 are expressed on normal murine erythroid precursors, and erythropoietin stimulates an increase in intracellular calcium ([Ca2+]i) through TRPC2 and TRPC3. Because modulation of [Ca2+]i is an important signaling pathway in erythroid proliferation and differentiation, Trpc2, Trpc3, and Trpc2/Trpc3 double knockout mice were utilized to explore the roles of these channels in erythropoiesis. Trpc2, Trpc3, and Trpc2/Trpc3 double knockout mice were not anemic, and had similar red blood cell counts, hemoglobins, and reticulocyte counts as wild type littermate controls. Although the erythropoietin induced increase in [Ca2+]i was reduced, these knockout mice showed no defects in red cell production. The major phenotypic difference at steady state was that the mean corpuscular volume, mean corpuscular hemoglobin, and hematocrit of red cells were significantly greater in Trpc2 and Trpc2/Trpc3 double knockout mice, and mean corpuscular hemoglobin concentration was significantly reduced. All hematological parameters in Trpc3 knockout mice were similar to controls. When exposed to phenyhydrazine, unlike the Trpc3 knockouts, Trpc2 and Trpc2/Trpc3 double knockout mice showed significant resistance to hemolysis. This was associated with significant reduction in hydrogen peroxide-induced calcium influx in erythroblasts. While erythropoietin induced calcium influx through TRPC2 or TRPC3 is not critical for erythroid production, these data demonstrate that TRPC2 plays an important role in oxidative stress-induced hemolysis which may be related to reduced calcium entry in red cells in the presence of Trpc2 depletion. PMID:21924222

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.

Decreased APOE-containing HDL subfractions and cholesterol efflux capacity of serum in mice lacking Pcsk9

PubMed Central

2013-01-01

Background Studies in animals showed that PCSK9 is involved in HDL metabolism. We investigated the molecular mechanism by which PCSK9 regulates HDL cholesterol concentration and also whether Pcsk9 inactivation might affect cholesterol efflux capacity of serum and atherosclerotic fatty streak volume. Methods Mass spectrometry and western blot were used to analyze the level of apolipoprotein E (APOE) and A1 (APOA1). A mouse model overexpressing human LDLR was used to test the effect of high levels of liver LDLR on the concentration of HDL cholesterol and APOE-containing HDL subfractions. Pcsk9 knockout males lacking LDLR and APOE were used to test whether LDLR and APOE are necessary for PCSK9-mediated HDL cholesterol regulation. We also investigated the effects of Pcsk9 inactivation on cholesterol efflux capacity of serum using THP-1 and J774.A1 macrophage foam cells and atherosclerotic fatty streak volume in the aortic sinus of Pcsk9 knockout males fed an atherogenic diet. Results APOE and APOA1 were reduced in the same HDL subfractions of Pcsk9 knockout and human LDLR transgenic male mice. In Pcsk9/Ldlr double-knockout mice, HDL cholesterol concentration was lower than in Ldlr knockout mice and higher than in wild-type controls. In Pcsk9/Apoe double-knockout mice, HDL cholesterol concentration was similar to that of Apoe knockout males. In Pcsk9 knockout males, THP-1 macrophage cholesterol efflux capacity of serum was reduced and the fatty streak lesion volume was similar to wild-type controls. Conclusions In mice, LDLR and APOE are important factors for PCSK9-mediated HDL regulation. Our data suggest that, although LDLR plays a major role in PCSK9-mediated regulation of HDL cholesterol concentration, it is not the only mechanism and that, regardless of mechanism, APOE is essential. Pcsk9 inactivation decreases the HDL cholesterol concentration and cholesterol efflux capacity in serum, but does not increase atherosclerotic fatty streak volume. PMID:23883163

Fragile X syndrome: a preclinical review on metabotropic glutamate receptor 5 (mGluR5) antagonists and drug development.

PubMed

Pop, Andreea S; Gomez-Mancilla, Baltazar; Neri, Giovanni; Willemsen, Rob; Gasparini, Fabrizio

2014-03-01

Fragile X syndrome (FXS) is considered the leading inherited cause of intellectual disability and autism. In FXS, the fragile X mental retardation 1 (FMR1) gene is silenced and the fragile X mental retardation protein (FMRP) is not expressed, resulting in the characteristic features of the syndrome. Despite recent advances in understanding the pathophysiology of FXS, there is still no cure for this condition; current treatment is symptomatic. Preclinical research is essential in the development of potential therapeutic agents. This review provides an overview of the preclinical evidence supporting metabotropic glutamate receptor 5 (mGluR5) antagonists as therapeutic agents for FXS. According to the mGluR theory of FXS, the absence of FMRP leads to enhanced glutamatergic signaling via mGluR5, which leads to increased protein synthesis and defects in synaptic plasticity including enhanced long-term depression. As such, efforts to develop agents that target the underlying pathophysiology of FXS have focused on mGluR5 modulation. Animal models, particularly the Fmr1 knockout mouse model, have become invaluable in exploring therapeutic approaches on an electrophysiological, behavioral, biochemical, and neuroanatomical level. Two direct approaches are currently being investigated for FXS treatment: reactivating the FMR1 gene and compensating for the lack of FMRP. The latter approach has yielded promising results, with mGluR5 antagonists showing efficacy in clinical trials. Targeting mGluR5 is a valid approach for the development of therapeutic agents that target the underlying pathophysiology of FXS. Several compounds are currently in development, with encouraging results.

The rewarding action of acute cocaine is reduced in β-endorphin deficient but not in μ opioid receptor knockout mice.

PubMed

Nguyen, Alexander T; Marquez, Paul; Hamid, Abdul; Kieffer, Brigitte; Friedman, Theodore C; Lutfy, Kabirullah

2012-07-05

We have previously shown that β-endorphin plays a functional role in the rewarding effect of acute cocaine. Considering that β-endorphin has high affinity for the μ opioid receptor, we determined the role of this receptor in the rewarding action of acute cocaine. For comparison, we assessed the role of the μ opioid receptor in the rewarding effect of acute morphine. We also examined the effect of intracerebroventricular (i.c.v.) administration of β-funaltrexamine (β-FNA), an irreversible μ opioid receptor antagonist, on the rewarding action of acute cocaine as well as that of morphine. Using the conditioned place preference (CPP) paradigm as an animal model of reward, we first assessed the rewarding action of cocaine in mice lacking β-endorphin or the μ opioid receptor and their respective wild-type littermates/controls. Mice were tested for preconditioning place preference on day 1, conditioned once daily with saline/cocaine (30mg/kg, i.p.) or cocaine/saline on days 2 and 3, and then tested for postconditioning place preference on day 4. We next studied the rewarding action of acute morphine in μ knockout mice and their wild-type controls. The CPP was induced by single alternate-day saline/morphine (10mg/kg, s.c.) or morphine/saline conditioning. We finally determined the effect of β-FNA on CPP induced by cocaine or morphine in wild-type mice, in which mice were treated with saline or β-FNA (9ug/3μl; i.c.v.) a day prior to the preconditioning test day. Our results revealed that morphine induced a robust CPP in wild-type mice but not in mice lacking the μ opioid receptor or in wild-type mice treated with β-FNA. In contrast, cocaine induced CPP in μ knockout mice as well as in wild-type mice treated with β-FNA. On the other hand, cocaine failed to induce CPP in mice lacking β-endorphin. These results illustrate that β-endorphin is essential for the rewarding action of acute cocaine, but the μ opioid receptor may not mediate the regulatory action of endogenous β-endorphin. Copyright © 2012 Elsevier B.V. All rights reserved.

Proximal Gut Mucosal Epithelial Homeostasis in Aged IL-1 Type I Receptor Knockout Mice After Starvation

DTIC Science & Technology

2011-08-01

increases whole-body lean mass and insulin sensitivity in elderly subjects with sarcopenia . Am J Cardiol. 2008; 101:69E. [PubMed: 18157968] 11. Iwakiri R...nutritional deficiencies in the elderly can be corrected by nutritional supplementation [5-7], especially among patients who are fed enterally [8-10...mechanistic approach regarding intestinal cell dysfunction in the elderly . Starvation causes mucosal atrophy and loss of mucosal height [32], and glutamine

The Streptococcus agalactiae Stringent Response Enhances Virulence and Persistence in Human Blood

PubMed Central

Hooven, Thomas A.; Catomeris, Andrew J.; Bonakdar, Maryam; Tallon, Luke J.; Santana-Cruz, Ivette; Ott, Sandra; Daugherty, Sean C.; Tettelin, Hervé

2017-01-01

ABSTRACT Streptococcus agalactiae (group B Streptococcus [GBS]) causes serious infections in neonates. We previously reported a transposon sequencing (Tn-seq) system for performing genomewide assessment of gene fitness in GBS. In order to identify molecular mechanisms required for GBS to transition from a mucosal commensal lifestyle to bloodstream invasion, we performed Tn-seq on GBS strain A909 with human whole blood. Our analysis identified 16 genes conditionally essential for GBS survival in blood, of which 75% were members of the capsular polysaccharide (cps) operon. Among the non-cps genes identified as conditionally essential was relA, which encodes an enzyme whose activity is central to the bacterial stringent response—a conserved adaptation to environmental stress. We used blood coincubation studies of targeted knockout strains to confirm the expected growth defects of GBS deficient in capsule or stringent response activation. Unexpectedly, we found that the relA knockout strains demonstrated decreased expression of β-hemolysin/cytolysin, an important cytotoxin implicated in facilitating GBS invasion. Furthermore, chemical activation of the stringent response with serine hydroxamate increased β-hemolysin/cytolysin expression. To establish a mechanism by which the stringent response leads to increased cytotoxicity, we performed transcriptome sequencing (RNA-seq) on two GBS strains grown under stringent response or control conditions. This revealed a conserved decrease in the expression of genes in the arginine deiminase pathway during stringent response activation. Through coincubation with supplemental arginine and the arginine antagonist canavanine, we show that arginine availability is a determinant of GBS cytotoxicity and that the pathway between stringent response activation and increased virulence is arginine dependent. PMID:29109175

The L-type voltage-gated calcium channel CaV1.2 mediates fear extinction and modulates synaptic tone in the lateral amygdala.

PubMed

Temme, Stephanie J; Murphy, Geoffrey G

2017-11-01

L-type voltage-gated calcium channels (LVGCCs) have been implicated in both the formation and the reduction of fear through Pavlovian fear conditioning and extinction. Despite the implication of LVGCCs in fear learning and extinction, studies of the individual LVGCC subtypes, Ca V 1.2 and Ca V 1.3, using transgenic mice have failed to find a role of either subtype in fear extinction. This discontinuity between the pharmacological studies of LVGCCs and the studies investigating individual subtype contributions could be due to the limited neuronal deletion pattern of the Ca V 1.2 conditional knockout mice previously studied to excitatory neurons in the forebrain. To investigate the effects of deletion of Ca V 1.2 in all neuronal populations, we generated Ca V 1.2 conditional knockout mice using the synapsin1 promoter to drive Cre recombinase expression. Pan-neuronal deletion of Ca V 1.2 did not alter basal anxiety or fear learning. However, pan-neuronal deletion of Ca V 1.2 resulted in a significant deficit in extinction of contextual fear, implicating LVGCCs, specifically Ca V 1.2, in extinction learning. Further exploration on the effects of deletion of Ca V 1.2 on inhibitory and excitatory input onto the principle neurons of the lateral amygdala revealed a significant shift in inhibitory/excitatory balance. Together these data illustrate an important role of Ca V 1.2 in fear extinction and the synaptic regulation of activity within the amygdala. © 2017 Temme and Murphy; Published by Cold Spring Harbor Laboratory Press.

Glutaminyl Cyclase Knock-out Mice Exhibit Slight Hypothyroidism but No Hypogonadism

PubMed Central

Schilling, Stephan; Kohlmann, Stephanie; Bäuscher, Christoph; Sedlmeier, Reinhard; Koch, Birgit; Eichentopf, Rico; Becker, Andreas; Cynis, Holger; Hoffmann, Torsten; Berg, Sabine; Freyse, Ernst-Joachim; von Hörsten, Stephan; Rossner, Steffen; Graubner, Sigrid; Demuth, Hans-Ulrich

2011-01-01

Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamate (pGlu) residues at the N terminus of peptides and proteins. Hypothalamic pGlu hormones, such as thyrotropin-releasing hormone and gonadotropin-releasing hormone are essential for regulation of metabolism and fertility in the hypothalamic pituitary thyroid and gonadal axes, respectively. Here, we analyzed the consequences of constitutive genetic QC ablation on endocrine functions and on the behavior of adult mice. Adult homozygous QC knock-out mice are fertile and behave indistinguishably from wild type mice in tests of motor function, cognition, general activity, and ingestion behavior. The QC knock-out results in a dramatic drop of enzyme activity in the brain, especially in hypothalamus and in plasma. Other peripheral organs like liver and spleen still contain QC activity, which is most likely caused by its homolog isoQC. The serum gonadotropin-releasing hormone, TSH, and testosterone concentrations were not changed by QC depletion. The serum thyroxine was decreased by 24% in homozygous QC knock-out animals, suggesting a mild hypothyroidism. QC knock-out mice were indistinguishable from wild type with regard to blood glucose and glucose tolerance, thus differing from reports of thyrotropin-releasing hormone knock-out mice significantly. The results suggest a significant formation of the hypothalamic pGlu hormones by alternative mechanisms, like spontaneous cyclization or conversion by isoQC. The different effects of QC depletion on the hypothalamic pituitary thyroid and gonadal axes might indicate slightly different modes of substrate conversion of both enzymes. The absence of significant abnormalities in QC knock-out mice suggests the presence of a therapeutic window for suppression of QC activity in current drug development. PMID:21330373

Hyperactivity of newborn Pten knock-out neurons results from increased excitatory synaptic drive.

PubMed

Williams, Michael R; DeSpenza, Tyrone; Li, Meijie; Gulledge, Allan T; Luikart, Bryan W

2015-01-21

Developing neurons must regulate morphology, intrinsic excitability, and synaptogenesis to form neural circuits. When these processes go awry, disorders, including autism spectrum disorder (ASD) or epilepsy, may result. The phosphatase Pten is mutated in some patients having ASD and seizures, suggesting that its mutation disrupts neurological function in part through increasing neuronal activity. Supporting this idea, neuronal knock-out of Pten in mice can cause macrocephaly, behavioral changes similar to ASD, and seizures. However, the mechanisms through which excitability is enhanced following Pten depletion are unclear. Previous studies have separately shown that Pten-depleted neurons can drive seizures, receive elevated excitatory synaptic input, and have abnormal dendrites. We therefore tested the hypothesis that developing Pten-depleted neurons are hyperactive due to increased excitatory synaptogenesis using electrophysiology, calcium imaging, morphological analyses, and modeling. This was accomplished by coinjecting retroviruses to either "birthdate" or birthdate and knock-out Pten in granule neurons of the murine neonatal dentate gyrus. We found that Pten knock-out neurons, despite a rapid onset of hypertrophy, were more active in vivo. Pten knock-out neurons fired at more hyperpolarized membrane potentials, displayed greater peak spike rates, and were more sensitive to depolarizing synaptic input. The increased sensitivity of Pten knock-out neurons was due, in part, to a higher density of synapses located more proximal to the soma. We determined that increased synaptic drive was sufficient to drive hypertrophic Pten knock-out neurons beyond their altered action potential threshold. Thus, our work contributes a developmental mechanism for the increased activity of Pten-depleted neurons. Copyright © 2015 the authors 0270-6474/15/350943-17$15.00/0.

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.

Vasoactive intestinal peptide-null mice demonstrate enhanced sweet taste preference, dysglycemia, and reduced taste bud leptin receptor expression.

PubMed

Martin, Bronwen; Shin, Yu-Kyong; White, Caitlin M; Ji, Sunggoan; Kim, Wook; Carlson, Olga D; Napora, Joshua K; Chadwick, Wayne; Chapter, Megan; Waschek, James A; Mattson, Mark P; Maudsley, Stuart; Egan, Josephine M

2010-05-01

It is becoming apparent that there is a strong link between taste perception and energy homeostasis. Recent evidence implicates gut-related hormones in taste perception, including glucagon-like peptide 1 and vasoactive intestinal peptide (VIP). We used VIP knockout mice to investigate VIP's specific role in taste perception and connection to energy regulation. Body weight, food intake, and plasma levels of multiple energy-regulating hormones were measured and pancreatic morphology was determined. In addition, the immunocytochemical profile of taste cells and gustatory behavior were examined in wild-type and VIP knockout mice. VIP knockout mice demonstrate elevated plasma glucose, insulin, and leptin levels, with no islet beta-cell number/topography alteration. VIP and its receptors (VPAC1, VPAC2) were identified in type II taste cells of the taste bud, and VIP knockout mice exhibit enhanced taste preference to sweet tastants. VIP knockout mouse taste cells show a significant decrease in leptin receptor expression and elevated expression of glucagon-like peptide 1, which may explain sweet taste preference of VIP knockout mice. This study suggests that the tongue can play a direct role in modulating energy intake to correct peripheral glycemic imbalances. In this way, we could view the tongue as a sensory mechanism that is bidirectionally regulated and thus forms a bridge between available foodstuffs and the intricate hormonal balance in the animal itself.

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

Studies of UCP2 transgenic and knockout mice reveal that liver UCP2 is not essential for the antiobesity effects of fish oil.

PubMed

Tsuboyama-Kasaoka, Nobuyo; Sano, Kayo; Shozawa, Chikako; Osaka, Toshimasa; Ezaki, Osamu

2008-03-01

Uncoupling protein 2 (UCP2) is a possible target molecule for energy dissipation. Many dietary fats, including safflower oil and lard, induce obesity in C57BL/6 mice, whereas fish oil does not. Fish oil increases UCP2 expression in hepatocytes and may enhance UCP2 activity by activating the UCP2 molecule or altering the lipid bilayer environment. To examine the role of liver UCP2 in obesity, we created transgenic mice that overexpressed human UCP2 in hepatocytes and examined whether UCP2 transgenic mice showed less obesity when fed a high-fat diet (safflower oil or lard). In addition, we examined whether fish oil had antiobesity effects in UCP2 knockout mice. UCP2 transgenic and wild-type mice fed a high-fat diet (safflower oil or lard) developed obesity to a similar degree. UCP2 knockout and wild-type mice fed fish oil had lower rates of obesity than mice fed safflower oil. Remarkably, safflower oil did not induce obesity in female UCP2 knockout mice, an unexpected phenotype for which we presently have no explanation. However, this unexpected effect was not observed in male UCP2 knockout mice or in UCP2 knockout mice fed a high-lard diet. These data indicate that liver UCP2 is not essential for fish oil-induced decreases in body fat.

A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer.

PubMed

Merenda, Alessandra; Andersson-Rolf, Amanda; Mustata, Roxana C; Li, Taibo; Kim, Hyunki; Koo, Bon-Kyoung

2017-07-12

CRISPR/Cas9 technology has greatly improved the feasibility and speed of loss-of-function studies that are essential in understanding gene function. In higher eukaryotes, paralogous genes can mask a potential phenotype by compensating the loss of a gene, thus limiting the information that can be obtained from genetic studies relying on single gene knockouts. We have developed a novel, rapid cloning method for guide RNA (gRNA) concatemers in order to create multi-gene knockouts following a single round of transfection in mouse small intestinal organoids. Our strategy allows for the concatemerization of up to four individual gRNAs into a single vector by performing a single Golden Gate shuffling reaction with annealed gRNA oligos and a pre-designed retroviral vector. This allows either the simultaneous knockout of up to four different genes, or increased knockout efficiency following the targeting of one gene by multiple gRNAs. In this protocol, we show in detail how to efficiently clone multiple gRNAs into the retroviral CRISPR-concatemer vector and how to achieve highly efficient electroporation in intestinal organoids. As an example, we show that simultaneous knockout of two pairs of genes encoding negative regulators of the Wnt signaling pathway (Axin1/2 and Rnf43/Znrf3) renders intestinal organoids resistant to the withdrawal of key growth factors.

Genome editing using CRISPR/Cas9-targeted mutagenesis: An opportunity for yield improvements of crop plants grown under environmental stresses.

PubMed

Abdelrahman, Mostafa; Al-Sadi, Abdullah M; Pour-Aboughadareh, Alireza; Burritt, David J; Tran, Lam-Son Phan

2018-03-12

Developing more crops able to sustainably produce high yields when grown under biotic/abiotic stresses is an important goal, if crop production and food security are to be guaranteed in the face of ever-increasing human population and unpredictable global climatic conditions. However, conventional crop improvement, through random mutagenesis or genetic recombination, is time-consuming and cannot keep pace with increasing food demands. Targeted genome editing (GE) technologies, especially clustered regularly interspaced short palindromic repeats (CRISPR)/(CRISPR)-associated protein 9 (Cas9), have great potential to aid in the breeding of crops that are able to produce high yields under conditions of biotic/abiotic stress. This is due to their high efficiency, accuracy and low risk of off-target effects, compared with conventional random mutagenesis methods. The use of CRISPR/Cas9 system has grown very rapidly in recent years with numerous examples of targeted mutagenesis in crop plants, including gene knockouts, modifications, and the activation and repression of target genes. The potential of the GE approach for crop improvement has been clearly demonstrated. However, the regulation and social acceptance of GE crops still remain a challenge. In this review, we evaluate the recent applications of the CRISPR/Cas9-mediated GE, as a means to produce crop plants with greater resilience to the stressors they encounter when grown under increasing stressful environmental conditions. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Candidate gene prioritization by network analysis of differential expression using machine learning approaches

PubMed Central

2010-01-01

Background Discovering novel disease genes is still challenging for diseases for which no prior knowledge - such as known disease genes or disease-related pathways - is available. Performing genetic studies frequently results in large lists of candidate genes of which only few can be followed up for further investigation. We have recently developed a computational method for constitutional genetic disorders that identifies the most promising candidate genes by replacing prior knowledge by experimental data of differential gene expression between affected and healthy individuals. To improve the performance of our prioritization strategy, we have extended our previous work by applying different machine learning approaches that identify promising candidate genes by determining whether a gene is surrounded by highly differentially expressed genes in a functional association or protein-protein interaction network. Results We have proposed three strategies scoring disease candidate genes relying on network-based machine learning approaches, such as kernel ridge regression, heat kernel, and Arnoldi kernel approximation. For comparison purposes, a local measure based on the expression of the direct neighbors is also computed. We have benchmarked these strategies on 40 publicly available knockout experiments in mice, and performance was assessed against results obtained using a standard procedure in genetics that ranks candidate genes based solely on their differential expression levels (Simple Expression Ranking). Our results showed that our four strategies could outperform this standard procedure and that the best results were obtained using the Heat Kernel Diffusion Ranking leading to an average ranking position of 8 out of 100 genes, an AUC value of 92.3% and an error reduction of 52.8% relative to the standard procedure approach which ranked the knockout gene on average at position 17 with an AUC value of 83.7%. Conclusion In this study we could identify promising candidate genes using network based machine learning approaches even if no knowledge is available about the disease or phenotype. PMID:20840752

Mechanisms of Abnormal Growth Regulation in Prostatic Adenocarcinoma Using Abi1/Hssh3bp1 Conditional Knockout Mouse Model

DTIC Science & Technology

2009-06-01

density lipoprotein uptake and cholesterol accumulation by macrophages differentiated from human monocytes with macrophage-colony-stimulating factor (M...model will likely have high impact on the prostate cancer research field including development of novel potential drug therapies. Our model will...Shin, and B.J. Mayer. 2007. High -throughput phosphotyrosine profiling using SH2 domains. Mol Cell. 26:899-915. Macoska, J.A., J. Xu, D. Ziemnicka, T.S

A-Kinase Anchor Protein 12 Is Required for Oligodendrocyte Differentiation in Adult White Matter.

PubMed

Maki, Takakuni; Choi, Yoon Kyung; Miyamoto, Nobukazu; Shindo, Akihiro; Liang, Anna C; Ahn, Bum Ju; Mandeville, Emiri T; Kaji, Seiji; Itoh, Kanako; Seo, Ji Hae; Gelman, Irwin H; Lok, Josephine; Takahashi, Ryosuke; Kim, Kyu-Won; Lo, Eng H; Arai, Ken

2018-05-01

Oligodendrocyte precursor cells (OPCs) give rise to oligodendrocytes in cerebral white matter. However, the underlying mechanisms that regulate this process remain to be fully defined, especially in adult brains. Recently, it has been suggested that signaling via A-kinase anchor protein 12 (AKAP12), a scaffolding protein that associates with intracellular molecules such as protein kinase A, may be involved in Schwann cell homeostasis and peripheral myelination. Here, we asked whether AKAP12 also regulates the mechanisms of myelination in the CNS. AKAP12 knockout mice were compared against wild-type (WT) mice in a series of neurochemical and behavioral assays. Compared with WTs, 2-months old AKAP12 knockout mice exhibited loss of myelin in white matter of the corpus callosum, along with perturbations in working memory as measured by a standard Y-maze test. Unexpectedly, very few OPCs expressed AKAP12 in the corpus callosum region. Instead, pericytes appeared to be one of the major AKAP12-expressing cells. In a cell culture model system, conditioned culture media from normal pericytes promoted in-vitro OPC maturation. However, conditioned media from AKAP12-deficient pericytes did not support the OPC function. These findings suggest that AKAP12 signaling in pericytes may be required for OPC-to-oligodendrocyte renewal to maintain the white matter homeostasis in adult brain. Stem Cells 2018;36:751-760. © AlphaMed Press 2018.

Ibuprofen partially attenuates neurodegenerative symptoms in presenilin conditional double-knockout mice.

PubMed

Dong, Z; Yan, L; Huang, G; Zhang, L; Mei, B; Meng, B

2014-06-13

Ibuprofen is a widely used nonsteroidal anti-inflammatory drug that reportedly reduces the risk of Alzheimer's disease (AD) development. The anti-inflammatory effect of ibuprofen occurred via inhibition of cyclooxygenases and anti-amyloidogenesis through modulation of γ-secretase. Presenilin 1 and 2 conditional double-knockout (cDKO) mice exhibited age-dependent memory impairment and forebrain degeneration without elevation of amyloid β deposition. Therefore, cDKO mice can be an ideal animal model on which to independently test the effects of ibuprofen anti-inflammatory properties on the prevention of AD. Three- and six-month-old cDKO mice were fed diet containing 375 ppm ibuprofen for six months. After multiple, well-validated behavioral tests, treatment with ibuprofen improved cognition-related behavioral performance, and drug efficacy was correlated with the timing of administration. Ibuprofen was more effective on six-month-old than on three-month-old cDKO mice. Biochemical analysis demonstrated that the effects of ibuprofen on glial fibrillary acidic protein and CD68 expression levels were uneven in different brain regions of cDKO mice and that age also influenced such effects. Tau hyperphosphorylation and the cleavage of caspase-3 decreased after ibuprofen treatment, and this effect was more significant in the older than the younger group of mice, which was consistent with the results of behavioral tests. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

Association between the SPRY1 gene polymorphism and obesity-related traits and osteoporosis in Korean women.

PubMed

Jin, Hyun-Seok; Kim, Bo-Young; Kim, Jeonghyun; Hong, Kyung-Won; Jung, Suk-Yul; Lee, Yun-Seok; Huh, Dam; Oh, Bermseok; Chung, Yoon-Sok; Jeong, Seon-Yong

2013-01-01

Emerging evidence has revealed a close relationship between obesity and osteoporosis. It was reported recently that conditional knockout of the Spry1 gene in mice adipocytes causes an increase in body fat and a decrease in bone mass, and that these phenotypes are rescued by Spry1 overexpression in adipose tissue. In this study, we investigated whether genetic variation in the human SPRY1 gene is associated with obesity-related phenotypes and/or osteoporosis in humans. We performed a candidate gene association analysis between the four single nucleotide polymorphisms (SNPs) and 14 imputed SNPs in the SPRY1 gene and obesity-related traits and osteoporosis in a Korean women cohort (3013 subjects). All four SPRY1 gene SNPs were significantly associated with either obesity-related traits or osteoporosis. The TGCC haplotype in the SRPY1 gene showed simultaneous association with an increased risk for obesity-related traits, percentage body fat (p=0.0087) and percentage abdominal fat (p=0.047), and osteoporosis (odds ratio=1.50; p=0.025) in the recessive genetic model. Our results support a previous finding in conditional Spry1 gene knockout mice and suggest that the SPRY1 gene is an important genetic factor for determining the risk of both obesity and osteoporosis in humans. Copyright © 2012 Elsevier Inc. All rights reserved.

Loss of Smad4 in Sertoli and Leydig Cells Leads to Testicular Dysgenesis and Hemorrhagic Tumor Formation in Mice1

PubMed Central

Archambeault, Denise R.; Yao, Humphrey Hung-Chang

2014-01-01

ABSTRACT As the central component of canonical TGFbeta superfamily signaling, SMAD4 is a critical regulator of organ development, patterning, tumorigenesis, and many other biological processes. Because numerous TGFbeta superfamily ligands are expressed in developing testes, there may exist specific requirements for SMAD4 in individual testicular cell types. Previously, we reported that expansion of the fetal testis cords requires expression of SMAD4 by the Sertoli cell lineage. To further uncover the role of Smad4 in murine testes, we produced conditional knockout mice lacking Smad4 in either Leydig cells or in both Sertoli and Leydig cells simultaneously. Loss of Smad4 concomitantly in Sertoli and Leydig cells led to underdevelopment of the testis cords during fetal life and mild testicular dysgenesis in young adulthood (decreased testis size, partially dysgenic seminiferous tubules, and low sperm production). When the Sertoli/Leydig cell Smad4 conditional knockout mice aged (56- to 62-wk old), the testis phenotypes became exacerbated with the appearance of hemorrhagic tumors, Leydig cell adenomas, and a complete loss of spermatogenesis. In contrast, loss of Smad4 in Leydig cells alone did not appreciably alter fetal and adult testis development. Our findings support a cell type-specific requirement of Smad4 in testis development and suppression of testicular tumors. PMID:24501173

The Role of Glutamate Signalling in the Pathogenesis and Treatment of Obsessive-Compulsive Disorder

PubMed Central

Wu, Ke; Hanna, Gregory L.; Rosenberg, David R.; Arnold, Paul D

2011-01-01

Obsessive-compulsive disorder (OCD) is a common and often debilitating neuropsychiatric condition characterized by persistent intrusive thoughts (obsessions), repetitive ritualistic behaviours (compulsions) and excessive anxiety. While the neurobiology and etiology of OCD has not been fully elucidated, there is growing evidence that disrupted neurotransmission of glutamate within corticalstriatal-thalamocortical (CSTC) circuitry plays a role in OCD pathogenesis. This review summarizes the findings from neuroimaging, animal model, candidate gene and treatment studies in the context of glutamate signalling dysfunction in OCD. First, studies using magnetic resonance spectroscopy are reviewed demonstrating altered glutamate concentrations in the caudate and anterior cingulate cortex of patients with OCD. Second, knockout mouse models, particularly the DLGAP3 and Slitrk5 knockout mouse models, display remarkably similar phenotypes of compulsive grooming behaviour associated with glutamate signalling dysfunction. Third, candidate gene studies have identified associations between variants in glutamate system genes and OCD, particularly for SLC1A1 which has been shown to be associated with OCD in five independent studies. This converging evidence for a role of glutamate in OCD has led to the development of novel treatment strategies involving glutamatergic compounds, particularly riluzole and memantine. We conclude the review by outlining a glutamate hypothesis for OCD, which we hope will inform further research into etiology and treatment for this severe neuropsychiatric condition. PMID:22024159

Body water balance and body temperature in vasopressin V1b receptor knockout mice.

PubMed

Daikoku, R; Kunitake, T; Kato, K; Tanoue, A; Tsujimoto, G; Kannan, H

2007-10-30

In an attempt to determine whether there is a specific vasopressin receptor (V(1b)) subtype involved in the regulation of body water balance and temperature, vasopressin V(1b) receptor knockout mice were used. Daily drinking behavior and renal excretory function were examined in V(1b)-deficient (V(1b)(-/-)) and control (V(1b)(+/+)) mice under the basal and stress-induced condition. In addition, body temperature and locomotor activity were measured with a biotelemetry system. The baseline daily water intake and urine volume were larger in V(1b)(-/-) mice than in V(1b)(+/+) mice. V(1b)(-/-) mice (V(1b)(-/-)) had significantly higher locomotor activity than wild-type, whereas the body temperature and oxygen consumption were lower in V(1b)(-/-) than in the V(1b)(+/+) mice. Next, the V(1b)(-/-) and V(1b)(+/+) mice were subjected to water deprivation for 48 hr. Under this condition, their body temperature decreased with the time course, which was significantly larger for V(1b)(-/-) than for V(1b)(+/+) mice. Central vasopressin has been reported to elicit drinking behavior and antipyretic action, and the V(1b) receptor has been reported to be located in the kidney. Thus, the findings suggest that the V(1b) receptor may be, at least in part, involved in body water balance and body temperature regulation.

Multiple strategies to prevent oxidative stress in Arabidopsis plants lacking the malate valve enzyme NADP-malate dehydrogenase

PubMed Central

Hebbelmann, Inga; Selinski, Jennifer; Wehmeyer, Corinna; Goss, Tatjana; Voss, Ingo; Mulo, Paula; Kangasjärvi, Saijaliisa; Aro, Eva-Mari; Oelze, Marie-Luise; Dietz, Karl-Josef; Nunes-Nesi, Adriano; Do, Phuc T.; Fernie, Alisdair R.; Talla, Sai K.; Raghavendra, Agepati S.; Linke, Vera; Scheibe, Renate

2012-01-01

The nuclear-encoded chloroplast NADP-dependent malate dehydrogenase (NADP-MDH) is a key enzyme controlling the malate valve, to allow the indirect export of reducing equivalents. Arabidopsis thaliana (L.) Heynh. T-DNA insertion mutants of NADP-MDH were used to assess the role of the light-activated NADP-MDH in a typical C3 plant. Surprisingly, even when exposed to high-light conditions in short days, nadp-mdh knockout mutants were phenotypically indistinguishable from the wild type. The photosynthetic performance and typical antioxidative systems, such as the Beck–Halliwell–Asada pathway, were barely affected in the mutants in response to high-light treatment. The reactive oxygen species levels remained low, indicating the apparent absence of oxidative stress, in the mutants. Further analysis revealed a novel combination of compensatory mechanisms in order to maintain redox homeostasis in the nadp-mdh plants under high-light conditions, particularly an increase in the NTRC/2-Cys peroxiredoxin (Prx) system in chloroplasts. There were indications of adjustments in extra-chloroplastic components of photorespiration and proline levels, which all could dissipate excess reducing equivalents, sustain photosynthesis, and prevent photoinhibition in nadp-mdh knockout plants. Such metabolic flexibility suggests that the malate valve acts in concert with other NADPH-consuming reactions to maintain a balanced redox state during photosynthesis under high-light stress in wild-type plants. PMID:22140244

Cognitive abnormalities and hippocampal alterations in monoamine oxidase A and B knockout mice

PubMed Central

Singh, Chanpreet; Bortolato, Marco; Bali, Namrata; Godar, Sean C.; Scott, Anna L.; Chen, Kevin; Thompson, Richard F.; Shih, Jean C.

2013-01-01

The monoamine oxidase isoenzymes (MAOs) A and B play important roles in the homeostasis of monoaminergic neurotransmitters. The combined deficiency of MAO A and B results in significantly elevated levels of serotonin (5-hydroxytryptamine), norepinephrine, dopamine, and β-phenylethylamine; in humans and mice, these neurochemical changes are accompanied by neurodevelopmental perturbations as well as autistic-like responses. Ample evidence indicates that normal levels of monoamines in the hippocampus, amygdala, frontal cortex, and cerebellum are required for the integrity of learning and memory. Thus, in the present study, the cognitive status of MAO A/B knockout (KO) mice was examined with a wide array of behavioral tests. In comparison with male wild-type littermates, MAO A/B KO mice exhibited abnormally high and overgeneralized fear conditioning and enhanced eye-blink conditioning. These alterations were accompanied by significant increases in hippocampal long-term potentiation and alterations in the relative expression of NMDA glutamate receptor subunits. Our data suggest that chronic elevations of monoamines, because of the absence of MAO A and MAO B, cause functional alterations that are accompanied with changes in the cellular mechanisms underlying learning and memory. The characteristics exhibited by MAO A/B KO mice highlight the potential of these animals as a useful tool to provide further insight into the molecular bases of disorders associated with abnormal monoaminergic profiles. PMID:23858446

Cognitive abnormalities and hippocampal alterations in monoamine oxidase A and B knockout mice.

PubMed

Singh, Chanpreet; Bortolato, Marco; Bali, Namrata; Godar, Sean C; Scott, Anna L; Chen, Kevin; Thompson, Richard F; Shih, Jean C

2013-07-30

The monoamine oxidase isoenzymes (MAOs) A and B play important roles in the homeostasis of monoaminergic neurotransmitters. The combined deficiency of MAO A and B results in significantly elevated levels of serotonin (5-hydroxytryptamine), norepinephrine, dopamine, and β-phenylethylamine; in humans and mice, these neurochemical changes are accompanied by neurodevelopmental perturbations as well as autistic-like responses. Ample evidence indicates that normal levels of monoamines in the hippocampus, amygdala, frontal cortex, and cerebellum are required for the integrity of learning and memory. Thus, in the present study, the cognitive status of MAO A/B knockout (KO) mice was examined with a wide array of behavioral tests. In comparison with male wild-type littermates, MAO A/B KO mice exhibited abnormally high and overgeneralized fear conditioning and enhanced eye-blink conditioning. These alterations were accompanied by significant increases in hippocampal long-term potentiation and alterations in the relative expression of NMDA glutamate receptor subunits. Our data suggest that chronic elevations of monoamines, because of the absence of MAO A and MAO B, cause functional alterations that are accompanied with changes in the cellular mechanisms underlying learning and memory. The characteristics exhibited by MAO A/B KO mice highlight the potential of these animals as a useful tool to provide further insight into the molecular bases of disorders associated with abnormal monoaminergic profiles.

The Multiple Functions of Common Microbial Carbon Polymers, Glycogen and PHB, during Stress Responses in the Non-Diazotrophic Cyanobacterium Synechocystis sp. PCC 6803.

PubMed

Damrow, Ramon; Maldener, Iris; Zilliges, Yvonne

2016-01-01

Classical microbial carbon polymers such as glycogen and polyhydroxybutyrate (PHB) have a crucial impact as both a sink and a reserve under macronutrient stress conditions. Most microbial species exclusively synthesize and degrade either glycogen or PHB. A few bacteria such as the phototrophic model organism Synechocystis sp. PCC 6803 surprisingly produce both physico-chemically different polymers under conditions of high C to N ratios. For the first time, the function and interrelation of both carbon polymers in non-diazotrophic cyanobacteria are analyzed in a comparative physiological study of single- and double-knockout mutants (ΔglgC; ΔphaC; ΔglgC/ΔphaC), respectively. Most of the observed phenotypes are explicitly related to the knockout of glycogen synthesis, highlighting the metabolic, energetic, and structural impact of this process whenever cells switch from an active, photosynthetic 'protein status' to a dormant 'glycogen status'. The carbon flux regulation into glycogen granules is apparently crucial for both phycobilisome degradation and thylakoid layer disassembly in the presence of light. In contrast, PHB synthesis is definitely not involved in this primary acclimation response. Moreover, the very weak interrelations between the two carbon-polymer syntheses indicate that the regulation and role of PHB synthesis in Synechocystis sp. PCC 6803 is different from glycogen synthesis.

Exacerbated febrile responses to LPS, but not turpentine, in TNF double receptor-knockout mice.

PubMed

Leon, L R; Kozak, W; Peschon, J; Kluger, M J

1997-02-01

We examined the effects of injections of systemic [lipopolysaccharide (LPS), 2.5 mg/kg or 50 pg/kg ip] or local (turpentine, 100 microl sc) inflammatory stimuli on fever, motor activity, body weight, and food intake in tumor necrosis factor (TNF) double receptor (TNFR)-knockout mice. A high dose of LPS resulted in exacerbated fevers in TNFR-knockout mice compared with wild-type mice for the early phase of fever (3-15 h); the late phase of fever (16-24 h) and fevers to a low dose of LPS were similar in both groups. Motor activity, body weight, and food intake were similarly reduced in both groups of mice after LPS administration. In response to turpentine, TNFR-knockout and wild-type mice developed virtually identical responses to all variables monitored. These results suggest that 1) TNF modulates fevers to LPS dose dependently, 2) TNF does not modulate fevers to a subcutaneous injection of turpentine, and 3) knockout mice may develop cytokine redundancy in the regulation of the acute phase response to intraperitoneally injected LPS or subcutaneously injected turpentine.

The bcl-2 knockout mouse exhibits marked changes in osteoblast phenotype and collagen deposition in bone as well as a mild growth plate phenotype

PubMed Central

BOOT-HANDFORD, R. P.; MICHAELIDIS, T. M.; HILLARBY, M. C.; ZAMBELLI, A.; DENTON, J.; HOYLAND, J. A.; FREEMONT, A. J.; GRANT, M. E.; WALLIS, G. A.

1998-01-01

Histological examination of long bones from 1-day-old bcl-2 knockout and age-matched control mice revealed no obvious differences in length of bone, growth plate architecture or stage of endochondral ossification. In 35-day-old bcl-2 knockout mice that are growth retarded or ‘dwarfed’, the proliferative zone of the growth plate appeared slightly thinner and the secondary centres of ossification less well developed than their age-matched wild-type controls. The most marked histological effects of bcl-2 ablation were on osteoblasts and bone. 35-day-old knockout mouse bones exhibited far greater numbers of osteoblasts than controls and the osteoblasts had a cuboidal phenotype in comparison with the normal flattened cell appearance. In addition, the collagen deposited by the osteoblasts in the bcl-2 knockout mouse bone was disorganized in comparison with control tissue and had a pseudo-woven appearance. The results suggest an important role for Bcl-2 in controlling osteoblast phenotype and bone deposition in vivo. PMID:10193316

MONOAMINE OXIDASE: From Genes to Behavior

PubMed Central

Shih, J. C.; Chen, K.; Ridd, M. J.

2010-01-01

Cloning of MAO (monoamine oxidase) A and B has demonstrated unequivocally that these enzymes are made up of different polypeptides, and our understanding of MAO structure, regulation, and function has been significantly advanced by studies using their cDNA. MAO A and B genes are located on the X-chromosome (Xp11.23) and comprise 15 exons with identical intron-exon organization, which suggests that they are derived from the same ancestral gene. MAO A and B knockout mice exhibit distinct differences in neurotransmitter metabolism and behavior. MAO A knock-out mice have elevated brain levels of serotonin, norephinephrine, and dopamine and manifest aggressive behavior similar to human males with a deletion of MAO A. In contrast, MAO B knock-out mice do not exhibit aggression and only levels of phenylethylamine are increased. Mice lacking MAO B are resistant to the Parkinsongenic neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine. Both MAO A and B knock-out mice show increased reactivity to stress. These knock-out mice are valuable models for investigating the role of monoamines in psychoses and neurodegenerative and stress-related disorders. PMID:10202537

Lentiviral silencing of GSK-3β in adult dentate gyrus impairs contextual fear memory and synaptic plasticity

PubMed Central

Chew, Benjamin; Ryu, Jae Ryun; Ng, Teclise; Ma, Dongliang; Dasgupta, Ananya; Neo, Sin Hui; Zhao, Jing; Zhong, Zhong; Bichler, Zoë; Sajikumar, Sreedharan; Goh, Eyleen L. K.

2015-01-01

Attempts have been made to use glycogen synthase kinase-3 beta (GSK3β) inhibitors for prophylactic treatment of neurocognitive conditions. However the use of lithium, a non-specific inhibitor of GSK3β results in mild cognitive impairment in humans. The effects of global GSK3β inhibition or knockout on learning and memory in healthy adult mice are also inconclusive. Our study aims to better understand the role of GSK3β in learning and memory through a more regionally, targeted approach, specifically performing lentiviral-mediated knockdown of GSK3β within the dentate gyrus (DG). DG-GSK3β-silenced mice showed impaired contextual fear memory retrieval. However, cue fear memory, spatial memory, locomotor activity and anxiety levels were similar to control. These GSK3β-silenced mice also showed increased induction and maintenance of DG long-term potentiation (DG-LTP) compared to control animals. Thus, this region-specific, targeted knockdown of GSK3β in the DG provides better understanding on the role of GSK3β in learning and memory. PMID:26157370

α/β-Hydrolase Domain 6 in the Ventromedial Hypothalamus Controls Energy Metabolism Flexibility.

PubMed

Fisette, Alexandre; Tobin, Stephanie; Décarie-Spain, Léa; Bouyakdan, Khalil; Peyot, Marie-Line; Madiraju, S R Murthy; Prentki, Marc; Fulton, Stephanie; Alquier, Thierry

2016-10-25

α/β-Hydrolase domain 6 (ABHD6) is a monoacylglycerol hydrolase that degrades the endocannabinoid 2-arachidonoylglycerol (2-AG). Although complete or peripheral ABHD6 loss of function is protective against diet-induced obesity and insulin resistance, the role of ABHD6 in the central control of energy balance is unknown. Using a viral-mediated knockout approach, targeted endocannabinoid measures, and pharmacology, we discovered that mice lacking ABHD6 from neurons of the ventromedial hypothalamus (VMH KO ) have higher VMH 2-AG levels in conditions of endocannabinoid recruitment and fail to physiologically adapt to key metabolic challenges. VMH KO mice exhibited blunted fasting-induced feeding and reduced food intake, energy expenditure, and adaptive thermogenesis in response to cold exposure, high-fat feeding, and dieting (transition to a low-fat diet). Our findings identify ABHD6 as a regulator of the counter-regulatory responses to major metabolic shifts, including fasting, nutrient excess, cold, and dieting, thereby highlighting the importance of ABHD6 in the VMH in mediating energy metabolism flexibility. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Selective Deletion of GRK2 Alters Psychostimulant-Induced Behaviors and Dopamine Neurotransmission

PubMed Central

Daigle, Tanya L; Ferris, Mark J; Gainetdinov, Raul R; Sotnikova, Tatyana D; Urs, Nikhil M; Jones, Sara R; Caron, Marc G

2014-01-01

GRK2 is a G protein-coupled receptor kinase (GRK) that is broadly expressed and is known to regulate diverse types of receptors. GRK2 null animals exhibit embryonic lethality due to a severe developmental heart defect, which has precluded the study of this kinase in the adult brain. To elucidate the specific role of GRK2 in the brain dopamine (DA) system, we used a conditional gene knockout approach to selectively delete GRK2 in DA D1 receptor (D1R)-, DA D2 receptor (D2R)-, adenosine 2A receptor (A2AR)-, or DA transporter (DAT)-expressing neurons. Here we show that select GRK2-deficient mice display hyperactivity, hyposensitivity, or hypersensitivity to the psychomotor effects of cocaine, altered striatal signaling, and DA release and uptake. Mice with GRK2 deficiency in D2R-expressing neurons also exhibited increased D2 autoreceptor activity. These findings reveal a cell-type-specific role for GRK2 in the regulation of normal motor behavior, sensitivity to psychostimulants, dopamine neurotransmission, and D2 autoreceptor function. PMID:24776686

Critical role of dendritic cell-derived IL-27 in antitumor immunity through regulating the recruitment and activation of NK and NKT cells.

PubMed

Wei, Jun; Xia, Siyuan; Sun, Huayan; Zhang, Song; Wang, Jingya; Zhao, Huiyuan; Wu, Xiaoli; Chen, Xi; Hao, Jianlei; Zhou, Xinglong; Zhu, Zhengmao; Gao, Xiang; Gao, Jian-xin; Wang, Puyue; Wu, Zhenzhou; Zhao, Liqing; Yin, Zhinan

2013-07-01

Critical roles of IL-27 in autoimmune diseases and infections have been reported; however, the contribution of endogenous IL-27 to tumor progression remains elusive. In this study, by using IL-27p28 conditional knockout mice, we demonstrate that IL-27 is critical in protective immune response against methyl-cholanthrene-induced fibrosarcoma and transplanted B16 melanoma, and dendritic cells (DCs) are the primary source. DC-derived IL-27 is required for shaping tumor microenvironment by inducing CXCL-10 expression in myeloid-derived suppressor cells and regulating IL-12 production from DCs, which lead to the recruitment and activation of NK and NKT cells resulting in immunological control of tumors. Indeed, reconstitution of IL-27 or CXCL-10 in tumor site significantly inhibits tumor growth and restores the number and activation of NK and NKT cells. In summary, our study identifies a previous unknown critical role of DC-derived IL-27 in NK and NKT cell-dependent antitumor immunity through shaping tumor microenvironment, and sheds light on developing novel therapeutic approaches based on IL-27.

Beclin-1 deficiency in the murine ovary results in the reduction of progesterone production to promote preterm labor

PubMed Central

Gawriluk, Thomas R.; Ko, CheMyong; Hong, Xiaoman; Christenson, Lane K.; Rucker, Edmund B.

2014-01-01

Autophagy is an important cellular process that serves as a companion pathway to the ubiquitin-proteasome system to degrade long-lived proteins and organelles to maintain cell homeostasis. Although initially characterized in yeast, autophagy is being realized as an important regulator of development and disease in mammals. Beclin1 (Becn1) is a putative tumor suppressor gene that has been shown to undergo a loss of heterozygosity in 40–75% of human breast, ovarian, and prostate cancers. Because Becn1 is a key regulator of autophagy, we sought to investigate its role in female reproduction by using a conditional knockout approach in mice. We find that pregnant females lacking Becn1 in the ovarian granulosa cell population have a defect in progesterone production and a subsequent preterm labor phenotype. Luteal cells in this model exhibit defective autophagy and a failure to accumulate lipid droplets needed for steroidogenesis. Collectively, we show that Becn1 provides essential functions in the ovary that are essential for mammalian reproduction. PMID:25246579

Probucol prevents early coronary heart disease and death in the high-density lipoprotein receptor SR-BI/apolipoprotein E double knockout mouse

PubMed Central

Braun, Anne; Zhang, Songwen; Miettinen, Helena E.; Ebrahim, Shamsah; Holm, Teresa M.; Vasile, Eliza; Post, Mark J.; Yoerger, Danita M.; Picard, Michael H.; Krieger, Joshua L.; Andrews, Nancy C.; Simons, Michael; Krieger, Monty

2003-01-01

Mice with homozygous null mutations in the high-density lipoprotein receptor SR-BI (scavenger receptor class B, type I) and apolipoprotein E genes fed a low-fat diet exhibit a constellation of pathologies shared with human atherosclerotic coronary heart disease (CHD): hypercholesterolemia, occlusive coronary atherosclerosis, myocardial infarctions, cardiac dysfunction (heart enlargement, reduced systolic function and ejection fraction, and ECG abnormalities), and premature death (mean age 6 weeks). They also exhibit a block in RBC maturation and abnormally high plasma unesterified-to-total cholesterol ratio (0.8) with associated abnormal lipoprotein morphology (lamellar/vesicular and stacked discoidal particles reminiscent of those in lecithin/cholesterol acyltransferase deficiency and cholestasis). Treatment with the lipid-lowering, antiatherosclerosis, and antioxidation drug probucol extended life to as long as 60 weeks (mean 36 weeks), and at 5–6 weeks of age, virtually completely reversed the cardiac and most RBC pathologies and corrected the unesterified to total cholesterol ratio (0.3) and associated distinctive abnormal lipoprotein morphologies. Manipulation of the timing of administration and withdrawal of probucol could control the onset of death and suggested that critical pathological changes usually occurred in untreated double knockout mice between ≈3 (weaning) and 5 weeks of age and that probucol delayed heart failure even after development of substantial CHD. The ability of probucol treatment to modulate pathophysiology in the double knockout mice enhances the potential of this murine system for analysis of the pathophysiology of CHD and preclinical testing of new approaches for the prevention and treatment of cardiovascular disease. PMID:12771386

Role of the Norrie disease pseudoglioma gene in sprouting angiogenesis during development of the retinal vasculature.

PubMed

Luhmann, Ulrich F O; Lin, Jihong; Acar, Niyazi; Lammel, Stefanie; Feil, Silke; Grimm, Christian; Seeliger, Mathias W; Hammes, Hans-Peter; Berger, Wolfgang

2005-09-01

To characterize developmental defects and the time course of Norrie disease in retinal and hyaloid vasculature during retinal development and to identify underlying molecular angiogenic pathways that may be affected in Norrie disease, exudative vitreoretinopathy, retinopathy of prematurity, and Coats' disease. Norrie disease pseudoglioma homologue (Ndph)-knockout mice were studied during retinal development at early postnatal (p) stages (p5, p10, p15, and p21). Histologic techniques, quantitative RT-PCR, ELISA, and Western blot analyses provided molecular data, and scanning laser ophthalmoscopy (SLO) angiography and electroretinography (ERG) were used to obtain in vivo data. The data showed that regression of the hyaloid vasculature of Ndph-knockout mice occurred but was drastically delayed. The development of the superficial retinal vasculature was strongly delayed, whereas the deep retinal vasculature did not form because of the blockage of vessel outgrowth into the deep retinal layers. Subsequently, microaneurysm-like lesions formed. Several angiogenic factors were differentially transcribed during retinal development. Increased levels of hypoxia inducible factor-1alpha (HIF1alpha) and VEGFA, as well as a characteristic ERG pattern, confirmed hypoxic conditions in the inner retina of the Ndph-knockout mouse. These data provide evidence for a crucial role of Norrin in hyaloid vessel regression and in sprouting angiogenesis during retinal vascular development, especially in the development of the deep retinal capillary networks. They also suggest an early and a late phase of Norrie disease and may provide an explanation for similar phenotypic features of allelic retinal diseases in mice and patients as secondary consequences of pathologic hypoxia.

The role of platelet and endothelial GARP in thrombosis and hemostasis.

PubMed

Vermeersch, Elien; Denorme, Frederik; Maes, Wim; De Meyer, Simon F; Vanhoorelbeke, Karen; Edwards, Justin; Shevach, Ethan M; Unutmaz, Derya; Fujii, Hodaka; Deckmyn, Hans; Tersteeg, Claudia

2017-01-01

Glycoprotein-A Repetitions Predominant protein (GARP or LRRC32) is present on among others human platelets and endothelial cells. Evidence for its involvement in thrombus formation was suggested by full knockout of GARP in zebrafish. To evaluate the role of GARP in platelet physiology and in thrombus formation using platelet and endothelial conditional GARP knock out mice. Platelet and endothelial specific GARP knockout mice were generated using the Cre-loxP recombination system. The function of platelets without GARP was measured by flow cytometry, spreading analysis and aggregometry using PAR4-activating peptide and collagen related peptide. Additionally, clot retraction and collagen-induced platelet adhesion and aggregation under flow were analyzed. Finally, in vivo tail bleeding time, occlusion time of the mesenteric and carotid artery after FeCl3-induced thrombosis were determined in platelet and endothelial specific GARP knock out mice. Platelet specific GARP knockout mice had normal surface GPIb, GPVI and integrin αIIb glycoprotein expression. Although GARP expression was increased upon platelet activation, platelets without GARP displayed normal agonist induced activation, spreading on fibrinogen and aggregation responses. Furthermore, absence of GARP on platelets did not influence clot retraction and had no impact on thrombus formation on collagen-coated surfaces under flow. In line with this, neither the tail bleeding time nor the occlusion time in the carotid- and mesenteric artery after FeCl3-induced thrombus formation in platelet or endothelial specific GARP knock out mice were affected. Evidence is provided that platelet and endothelial GARP are not important in hemostasis and thrombosis in mice.

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.

The role of platelet and endothelial GARP in thrombosis and hemostasis

PubMed Central

Vermeersch, Elien; Denorme, Frederik; Maes, Wim; De Meyer, Simon F.; Vanhoorelbeke, Karen; Edwards, Justin; Shevach, Ethan M.; Unutmaz, Derya; Fujii, Hodaka; Deckmyn, Hans; Tersteeg, Claudia

2017-01-01

Background Glycoprotein-A Repetitions Predominant protein (GARP or LRRC32) is present on among others human platelets and endothelial cells. Evidence for its involvement in thrombus formation was suggested by full knockout of GARP in zebrafish. Objectives To evaluate the role of GARP in platelet physiology and in thrombus formation using platelet and endothelial conditional GARP knock out mice. Methods Platelet and endothelial specific GARP knockout mice were generated using the Cre-loxP recombination system. The function of platelets without GARP was measured by flow cytometry, spreading analysis and aggregometry using PAR4-activating peptide and collagen related peptide. Additionally, clot retraction and collagen-induced platelet adhesion and aggregation under flow were analyzed. Finally, in vivo tail bleeding time, occlusion time of the mesenteric and carotid artery after FeCl3-induced thrombosis were determined in platelet and endothelial specific GARP knock out mice. Results Platelet specific GARP knockout mice had normal surface GPIb, GPVI and integrin αIIb glycoprotein expression. Although GARP expression was increased upon platelet activation, platelets without GARP displayed normal agonist induced activation, spreading on fibrinogen and aggregation responses. Furthermore, absence of GARP on platelets did not influence clot retraction and had no impact on thrombus formation on collagen-coated surfaces under flow. In line with this, neither the tail bleeding time nor the occlusion time in the carotid- and mesenteric artery after FeCl3-induced thrombus formation in platelet or endothelial specific GARP knock out mice were affected. Conclusions Evidence is provided that platelet and endothelial GARP are not important in hemostasis and thrombosis in mice. PMID:28278197

A Knock-in Mouse Model of Human PHD2 Gene-associated Erythrocytosis Establishes a Haploinsufficiency Mechanism*

PubMed Central

Arsenault, Patrick R.; Pei, Fei; Lee, Rebecca; Kerestes, Heddy; Percy, Melanie J.; Keith, Brian; Simon, M. Celeste; Lappin, Terence R. J.; Khurana, Tejvir S.; Lee, Frank S.

2013-01-01

The central pathway for controlling red cell mass is the PHD (prolyl hydroxylase domain protein):hypoxia-inducible factor (HIF) pathway. HIF, which is negatively regulated by PHD, activates numerous genes, including ones involved in erythropoiesis, such as the ERYTHROPOIETIN (EPO) gene. Recent studies have implicated PHD2 as the key PHD isoform regulating red cell mass. Studies of humans have identified erythrocytosis-associated, heterozygous point mutations in the PHD2 gene. A key question concerns the mechanism by which human mutations lead to phenotypes. In the present report, we generated and characterized a mouse line in which a P294R knock-in mutation has been introduced into the mouse Phd2 locus to model the first reported human PHD2 mutation (P317R). Phd2P294R/+ mice display a degree of erythrocytosis equivalent to that seen in Phd2+/− mice. The Phd2P294R/+-associated erythrocytosis is reversed in a Hif2a+/−, but not a Hif1a+/− background. Additional studies using various conditional knock-outs of Phd2 reveal that erythrocytosis can be induced by homozygous and heterozygous knock-out of Phd2 in renal cortical interstitial cells using a Pax3-Cre transgene or by homozygous knock-out of Phd2 in hematopoietic progenitors driven by a Vav1-Cre transgene. These studies formally prove that a missense mutation in PHD2 is the cause of the erythrocytosis, show that this occurs through haploinsufficiency, and point to multifactorial control of red cell mass by PHD2. PMID:24121508

Loss of Notch3 Signaling in Vascular Smooth Muscle Cells Promotes Severe Heart Failure Upon Hypertension.

PubMed

Ragot, Hélène; Monfort, Astrid; Baudet, Mathilde; Azibani, Fériel; Fazal, Loubina; Merval, Régine; Polidano, Evelyne; Cohen-Solal, Alain; Delcayre, Claude; Vodovar, Nicolas; Chatziantoniou, Christos; Samuel, Jane-Lise

2016-08-01

Hypertension, which is a risk factor of heart failure, provokes adaptive changes at the vasculature and cardiac levels. Notch3 signaling plays an important role in resistance arteries by controlling the maturation of vascular smooth muscle cells. Notch3 deletion is protective in pulmonary hypertension while deleterious in arterial hypertension. Although this latter phenotype was attributed to renal and cardiac alterations, the underlying mechanisms remained unknown. To investigate the role of Notch3 signaling in the cardiac adaptation to hypertension, we used mice with either constitutive Notch3 or smooth muscle cell-specific conditional RBPJκ knockout. At baseline, both genotypes exhibited a cardiac arteriolar rarefaction associated with oxidative stress. In response to angiotensin II-induced hypertension, the heart of Notch3 knockout and SM-RBPJκ knockout mice did not adapt to pressure overload and developed heart failure, which could lead to an early and fatal acute decompensation of heart failure. This cardiac maladaptation was characterized by an absence of media hypertrophy of the media arteries, the transition of smooth muscle cells toward a synthetic phenotype, and an alteration of angiogenic pathways. A subset of mice exhibited an early fatal acute decompensated heart failure, in which the same alterations were observed, although in a more rapid timeframe. Altogether, these observations indicate that Notch3 plays a major role in coronary adaptation to pressure overload. These data also show that the hypertrophy of coronary arterial media on pressure overload is mandatory to initially maintain a normal cardiac function and is regulated by the Notch3/RBPJκ pathway. © 2016 American Heart Association, Inc.

Muscle Glycogen Remodeling and Glycogen Phosphate Metabolism following Exhaustive Exercise of Wild Type and Laforin Knockout Mice*

PubMed Central

Irimia, Jose M.; Tagliabracci, Vincent S.; Meyer, Catalina M.; Segvich, Dyann M.; DePaoli-Roach, Anna A.; Roach, Peter J.

2015-01-01

Glycogen, the repository of glucose in many cell types, contains small amounts of covalent phosphate, of uncertain function and poorly understood metabolism. Loss-of-function mutations in the laforin gene cause the fatal neurodegenerative disorder, Lafora disease, characterized by increased glycogen phosphorylation and the formation of abnormal deposits of glycogen-like material called Lafora bodies. It is generally accepted that the phosphate is removed by the laforin phosphatase. To study the dynamics of skeletal muscle glycogen phosphorylation in vivo under physiological conditions, mice were subjected to glycogen-depleting exercise and then monitored while they resynthesized glycogen. Depletion of glycogen by exercise was associated with a substantial reduction in total glycogen phosphate and the newly resynthesized glycogen was less branched and less phosphorylated. Branching returned to normal on a time frame of days, whereas phosphorylation remained suppressed over a longer period of time. We observed no change in markers of autophagy. Exercise of 3-month-old laforin knock-out mice caused a similar depletion of glycogen but no loss of glycogen phosphate. Furthermore, remodeling of glycogen to restore the basal branching pattern was delayed in the knock-out animals. From these results, we infer that 1) laforin is responsible for glycogen dephosphorylation during exercise and acts during the cytosolic degradation of glycogen, 2) excess glycogen phosphorylation in the absence of laforin delays the normal remodeling of the branching structure, and 3) the accumulation of glycogen phosphate is a relatively slow process involving multiple cycles of glycogen synthesis-degradation, consistent with the slow onset of the symptoms of Lafora disease. PMID:26216881

Evidence for a role of dystroglycan regulating the membrane architecture of astroglial endfeet

PubMed Central

Noell, Susan; Wolburg-Buchholz, Karen; Mack, Andreas F; Beedle, Aaron M; Satz, Jakob S; Campbell, Kevin P; Wolburg, Hartwig; Fallier-Becker, Petra

2011-01-01

The dystrophin–dystroglycan complex (DDC) is a molecular array of proteins in muscle and brain cells. The central component of the DDC is dystroglycan, which comprises α- and β-subunits. α-Dystroglycan (α-DG) binds to extracellular matrix components such as agrin, whereas β-dystroglycan (β-DG) is a membrane-spanning protein linking α-DG to the cytoskeleton and other intracellular components such as α-syntrophin. In astrocytes, α-syntrophin binds to the water channel protein aquaporin-4 (AQP4). Recently, it has been shown that AQP4 expression is unaltered in agrin-knockout mice, but that formation of orthogonal arrays of particles (OAPs), consisting of AQP4, is abnormal. As the brain-selective deletion of the DG gene causes a disorganization of the astroglial endfeet, we investigated whether DG deletion has an impact on AQP4. Western blotting revealed reduced AQP4 in the parenchymal but not in the superficial compartment of the astrocyte-conditioned DG-knockout mouse brain. Accordingly, immunohistochemical stainings of AQP4 revealed a selective loss of AQP4 in perivascular but not in superficial astroglial endfeet. In both superficial and perivascular endfeet of the DG-knockout brain, we observed a loss of OAPs. We conclude that in the absence of DG the majority of superficial AQP4 molecules did not form OAPs, and that expression of AQP4 in perivascular endfeet is compromised. However, the decreased number of perivascular AQP4 molecules obviously did form a few OAPs, even in the absence of DG. PMID:21501259

Computing smallest intervention strategies for multiple metabolic networks in a boolean model.

PubMed

Lu, Wei; Tamura, Takeyuki; Song, Jiangning; Akutsu, Tatsuya

2015-02-01

This article considers the problem whereby, given two metabolic networks N1 and N2, a set of source compounds, and a set of target compounds, we must find the minimum set of reactions whose removal (knockout) ensures that the target compounds are not producible in N1 but are producible in N2. Similar studies exist for the problem of finding the minimum knockout with the smallest side effect for a single network. However, if technologies of external perturbations are advanced in the near future, it may be important to develop methods of computing the minimum knockout for multiple networks (MKMN). Flux balance analysis (FBA) is efficient if a well-polished model is available. However, that is not always the case. Therefore, in this article, we study MKMN in Boolean models and an elementary mode (EM)-based model. Integer linear programming (ILP)-based methods are developed for these models, since MKMN is NP-complete for both the Boolean model and the EM-based model. Computer experiments are conducted with metabolic networks of clostridium perfringens SM101 and bifidobacterium longum DJO10A, respectively known as bad bacteria and good bacteria for the human intestine. The results show that larger networks are more likely to have MKMN solutions. However, solving for these larger networks takes a very long time, and often the computation cannot be completed. This is reasonable, because small networks do not have many alternative pathways, making it difficult to satisfy the MKMN condition, whereas in large networks the number of candidate solutions explodes. Our developed software minFvskO is available online.

Population of positive-parity states in {sup 53}Sc through one-proton knockout.

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

McDaniel, S.; Gade, A.; Janssens, R. V. F.

2010-02-01

The one-proton knockout reaction {sup 9}Be({sup 54}Ti,{sup 53}Sc+{gamma})X at 72 MeV/nucleon has been measured. The location of the first 3/2{sup -} state at 2110(3) keV was confirmed, and new {gamma}-ray transitions were observed at 1111(2), 1273(2), 1539(4), and 2495(5) keV. Large spectroscopic strength to excited states in {sup 53}Sc was found and attributed to the knockout of sd-shell protons.

Population of positive-parity states in {sup 53}Sc through one-proton knockout

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

McDaniel, S.; Gade, A.; Brown, B. A.

2010-02-15

The one-proton knockout reaction {sup 9}Be({sup 54}Ti,{sup 53}Sc+{gamma})X at 72 MeV/nucleon has been measured. The location of the first 3/2{sup -} state at 2110(3) keV was confirmed, and new {gamma}-ray transitions were observed at 1111(2), 1273(2), 1539(4), and 2495(5) keV. Large spectroscopic strength to excited states in {sup 53}Sc was found and attributed to the knockout of sd-shell protons.

Conditional Deletion of the L-Type Calcium Channel Cav1.2 in NG2-Positive Cells Impairs Remyelination in Mice.

PubMed

Santiago González, Diara A; Cheli, Veronica T; Zamora, Norma N; Lama, Tenzing N; Spreuer, Vilma; Murphy, Geoffrey G; Paez, Pablo M

2017-10-18

Exploring the molecular mechanisms that drive the maturation of oligodendrocyte progenitor cells (OPCs) during the remyelination process is essential to developing new therapeutic tools to intervene in demyelinating diseases such as multiple sclerosis. To determine whether L-type voltage-gated calcium channels (L-VGCCs) are required for OPC development during remyelination, we generated an inducible conditional knock-out mouse in which the L-VGCC isoform Cav1.2 was deleted in NG2-positive OPCs (Cav1.2 KO ). Using the cuprizone (CPZ) model of demyelination and mice of either sex, we establish that Cav1.2 deletion in OPCs leads to less efficient remyelination of the adult brain. Specifically, Cav1.2 KO OPCs mature slower and produce less myelin than control oligodendrocytes during the recovery period after CPZ intoxication. This reduced remyelination was accompanied by an important decline in the number of myelinating oligodendrocytes and in the rate of OPC proliferation. Furthermore, during the remyelination phase of the CPZ model, the corpus callosum of Cav1.2 KO animals presented a significant decrease in the percentage of myelinated axons and a substantial increase in the mean g-ratio of myelinated axons compared with controls. In addition, in a mouse line in which the Cav1.2 KO OPCs were identified by a Cre reporter, we establish that Cav1.2 KO OPCs display a reduced maturational rate through the entire remyelination process. These results suggest that Ca 2+ influx mediated by L-VGCCs in oligodendroglial cells is necessary for normal remyelination and is an essential Ca 2+ channel for OPC maturation during the remyelination of the adult brain. SIGNIFICANCE STATEMENT Ion channels implicated in oligodendrocyte differentiation and maturation may induce positive signals for myelin recovery. Voltage-gated Ca 2+ channels (VGCCs) are important for normal myelination by acting at several critical steps during oligodendrocyte progenitor cell (OPC) development. To determine whether voltage Ca 2+ entry is involved in oligodendrocyte differentiation and remyelination, we used a conditional knockout mouse for VGCCs in OPCs. Our results indicate that VGCCs can modulate oligodendrocyte maturation in the demyelinated brain and suggest that voltage-gated Ca 2+ influx in OPCs is critical for remyelination. These findings could lead to novel approaches for obtaining a better understanding of the factors that control OPC maturation in order to stimulate this pool of progenitors to replace myelin in demyelinating diseases. Copyright © 2017 the authors 0270-6474/17/3710038-14$15.00/0.

Repair of dentin defects from DSPP knockout mice by PILP mineralization

PubMed Central

Nurrohman, H.; Saeki, K.; Carneiro, K.; Chien, Y.C.; Djomehri, S.; Ho, S.P.; Qin, C.; Marshall, S.J.; Gower, L.B.; Marshall, G.W.; Habelitz, S.

2016-01-01

Dentinogenesis imperfecta type II (DGI-II) lacks intrafibrillar mineral with severe compromise of dentin mechanical properties. A Dspp knockout (Dspp−/−) mouse, with a phenotype similar to that of human DGI-II, was used to determine if poly-L-aspartic acid [poly(ASP)] in the “polymer-induced liquid-precursor” (PILP) system can restore its mechanical properties. Dentin from six-week old Dspp−/− and wild-type mice was treated with CaP solution containing poly(ASP) for up to 14 days. Elastic modulus and hardness before and after treatment were correlated with mineralization from Micro x-ray computed tomography (Micro-XCT). Transmission electron microscopy (TEM)/Selected area electron diffraction (SAED) were used to compare matrix mineralization and crystallography. Mechanical properties of the Dspp−/− dentin were significantly less than wild-type dentin and recovered significantly (P < 0.05) after PILP-treatment, reaching values comparable to wild-type dentin. Micro-XCT showed mineral recovery similar to wild-type dentin after PILP-treatment. TEM/SAED showed repair of patchy mineralization and complete mineralization of defective dentin. This approach may lead to new strategies for hard tissue repair. PMID:27239097

Genetic background can result in a marked or minimal effect of gene knockout (GPR55 and CB2 receptor) in experimental autoimmune encephalomyelitis models of multiple sclerosis.

PubMed

Sisay, Sofia; Pryce, Gareth; Jackson, Samuel J; Tanner, Carolyn; Ross, Ruth A; Michael, Gregory J; Selwood, David L; Giovannoni, Gavin; Baker, David

2013-01-01

Endocannabinoids and some phytocannabinoids bind to CB1 and CB2 cannabinoid receptors, transient receptor potential vanilloid one (TRPV1) receptor and the orphan G protein receptor fifty-five (GPR55). Studies using C57BL/10 and C57BL/6 (Cnr2 (tm1Zim)) CB2 cannabinoid receptor knockout mice have demonstrated an immune-augmenting effect in experimental autoimmune encephalomyelitis (EAE) models of multiple sclerosis. However, other EAE studies in Biozzi ABH mice often failed to show any treatment effect of either CB2 receptor agonism or antagonism on inhibition of T cell autoimmunity. The influence of genetic background on the induction of EAE in endocannabinoid system-related gene knockout mice was examined. It was found that C57BL/6.GPR55 knockout mice developed less severe disease, notably in female mice, following active induction with myelin oligodendrocyte glycoprotein 35-55 peptide. In contrast C57BL/6.CB2 (Cnr2 (Dgen)) receptor knockout mice developed augmented severity of disease consistent with the genetically and pharmacologically-distinct, Cnr2 (tm1Zim) mice. However, when the knockout gene was bred into the ABH mouse background and EAE induced with spinal cord autoantigens the immune-enhancing effect of CB2 receptor deletion was lost. Likewise CB1 receptor and transient receptor potential vanilloid one knockout mice on the ABH background demonstrated no alteration in immune-susceptibility, in terms of disease incidence and severity of EAE, in contrast to that reported in some C57BL/6 mouse studies. Furthermore the immune-modulating influence of GPR55 was marginal on the ABH mouse background. Whilst sedative doses of tetrahydrocannabinol could induce immunosuppression, this was associated with a CB1 receptor rather than a CB2 receptor-mediated effect. These data support the fact that non-psychoactive doses of medicinal cannabis have a marginal influence on the immune response in MS. Importantly, it adds a note of caution for the translational value of some transgenic/gene knockout and other studies on low-EAE susceptibility backgrounds with inconsistent disease course and susceptibility.

Genetic Background Can Result in a Marked or Minimal Effect of Gene Knockout (GPR55 and CB2 Receptor) in Experimental Autoimmune Encephalomyelitis Models of Multiple Sclerosis

PubMed Central

Jackson, Samuel J.; Tanner, Carolyn; Ross, Ruth A.; Michael, Gregory J.; Selwood, David L.; Giovannoni, Gavin; Baker, David

2013-01-01

Endocannabinoids and some phytocannabinoids bind to CB1 and CB2 cannabinoid receptors, transient receptor potential vanilloid one (TRPV1) receptor and the orphan G protein receptor fifty-five (GPR55). Studies using C57BL/10 and C57BL/6 (Cnr2 tm1Zim) CB2 cannabinoid receptor knockout mice have demonstrated an immune-augmenting effect in experimental autoimmune encephalomyelitis (EAE) models of multiple sclerosis. However, other EAE studies in Biozzi ABH mice often failed to show any treatment effect of either CB2 receptor agonism or antagonism on inhibition of T cell autoimmunity. The influence of genetic background on the induction of EAE in endocannabinoid system-related gene knockout mice was examined. It was found that C57BL/6.GPR55 knockout mice developed less severe disease, notably in female mice, following active induction with myelin oligodendrocyte glycoprotein 35-55 peptide. In contrast C57BL/6.CB2 (Cnr2 Dgen) receptor knockout mice developed augmented severity of disease consistent with the genetically and pharmacologically-distinct, Cnr2 tm1Zim mice. However, when the knockout gene was bred into the ABH mouse background and EAE induced with spinal cord autoantigens the immune-enhancing effect of CB2 receptor deletion was lost. Likewise CB1 receptor and transient receptor potential vanilloid one knockout mice on the ABH background demonstrated no alteration in immune-susceptibility, in terms of disease incidence and severity of EAE, in contrast to that reported in some C57BL/6 mouse studies. Furthermore the immune-modulating influence of GPR55 was marginal on the ABH mouse background. Whilst sedative doses of tetrahydrocannabinol could induce immunosuppression, this was associated with a CB1 receptor rather than a CB2 receptor-mediated effect. These data support the fact that non-psychoactive doses of medicinal cannabis have a marginal influence on the immune response in MS. Importantly, it adds a note of caution for the translational value of some transgenic/gene knockout and other studies on low-EAE susceptibility backgrounds with inconsistent disease course and susceptibility. PMID:24130809

The Essential Role of Mbd5 in the Regulation of Somatic Growth and Glucose Homeostasis in Mice

PubMed Central

Du, Yarui; Liu, Bo; Guo, Fan; Xu, Guifang; Ding, Yuqiang; Liu, Yong; Sun, Xin; Xu, Guoliang

2012-01-01

Methyl-CpG binding domain protein 5 (MBD5) belongs to the MBD family proteins, which play central roles in transcriptional regulation and development. The significance of MBD5 function is highlighted by recent studies implicating it as a candidate gene involved in human 2q23.1 microdeletion syndrome. To investigate the physiological role of Mbd5, we generated knockout mice. The Mbd5-deficient mice showed growth retardation, wasting and pre-weaning lethality. The observed growth retardation was associated with the impairment of GH/IGF-1 axis in Mbd5-null pups. Conditional knockout of Mbd5 in the brain resulted in the similar phenotypes as whole body deletion, indicating that Mbd5 functions in the nervous system to regulate postnatal growth. Moreover, the mutant mice also displayed enhanced glucose tolerance and elevated insulin sensitivity as a result of increased insulin signaling, ultimately resulting in disturbed glucose homeostasis and hypoglycemia. These results indicate Mbd5 as an essential factor for mouse postnatal growth and maintenance of glucose homeostasis. PMID:23077600

The essential role of Mbd5 in the regulation of somatic growth and glucose homeostasis in mice.

PubMed

Du, Yarui; Liu, Bo; Guo, Fan; Xu, Guifang; Ding, Yuqiang; Liu, Yong; Sun, Xin; Xu, Guoliang

2012-01-01

Methyl-CpG binding domain protein 5 (MBD5) belongs to the MBD family proteins, which play central roles in transcriptional regulation and development. The significance of MBD5 function is highlighted by recent studies implicating it as a candidate gene involved in human 2q23.1 microdeletion syndrome. To investigate the physiological role of Mbd5, we generated knockout mice. The Mbd5-deficient mice showed growth retardation, wasting and pre-weaning lethality. The observed growth retardation was associated with the impairment of GH/IGF-1 axis in Mbd5-null pups. Conditional knockout of Mbd5 in the brain resulted in the similar phenotypes as whole body deletion, indicating that Mbd5 functions in the nervous system to regulate postnatal growth. Moreover, the mutant mice also displayed enhanced glucose tolerance and elevated insulin sensitivity as a result of increased insulin signaling, ultimately resulting in disturbed glucose homeostasis and hypoglycemia. These results indicate Mbd5 as an essential factor for mouse postnatal growth and maintenance of glucose homeostasis.

Interval timing in genetically modified mice: a simple paradigm

PubMed Central

Balci, F.; Papachristos, E. B.; Gallistel, C. R.; Brunner, D.; Gibson, J.; Shumyatsky, G. P.

2009-01-01

We describe a behavioral screen for the quantitative study of interval timing and interval memory in mice. Mice learn to switch from a short-latency feeding station to a long-latency station when the short latency has passed without a feeding. The psychometric function is the cumulative distribution of switch latencies. Its median measures timing accuracy and its interquartile interval measures timing precision. Next, using this behavioral paradigm, we have examined mice with a gene knockout of the receptor for gastrin-releasing peptide that show enhanced (i.e. prolonged) freezing in fear conditioning. We have tested the hypothesis that the mutants freeze longer because they are more uncertain than wild types about when to expect the electric shock. The knockouts however show normal accuracy and precision in timing, so we have rejected this alternative hypothesis. Last, we conduct the pharmacological validation of our behavioral screen using D-amphetamine and methamphetamine. We suggest including the analysis of interval timing and temporal memory in tests of genetically modified mice for learning and memory and argue that our paradigm allows this to be done simply and efficiently. PMID:17696995

Interval timing in genetically modified mice: a simple paradigm.

PubMed

Balci, F; Papachristos, E B; Gallistel, C R; Brunner, D; Gibson, J; Shumyatsky, G P

2008-04-01

We describe a behavioral screen for the quantitative study of interval timing and interval memory in mice. Mice learn to switch from a short-latency feeding station to a long-latency station when the short latency has passed without a feeding. The psychometric function is the cumulative distribution of switch latencies. Its median measures timing accuracy and its interquartile interval measures timing precision. Next, using this behavioral paradigm, we have examined mice with a gene knockout of the receptor for gastrin-releasing peptide that show enhanced (i.e. prolonged) freezing in fear conditioning. We have tested the hypothesis that the mutants freeze longer because they are more uncertain than wild types about when to expect the electric shock. The knockouts however show normal accuracy and precision in timing, so we have rejected this alternative hypothesis. Last, we conduct the pharmacological validation of our behavioral screen using d-amphetamine and methamphetamine. We suggest including the analysis of interval timing and temporal memory in tests of genetically modified mice for learning and memory and argue that our paradigm allows this to be done simply and efficiently.

Requirement for Dot1l in murine postnatal hematopoiesis and leukemogenesis by MLL translocation

PubMed Central

Jo, Stephanie Y.; Granowicz, Eric M.; Maillard, Ivan; Thomas, Dafydd

2011-01-01

Disruptor of telomeric silencing 1-like (Dot1l) is a histone 3 lysine 79 methyltransferase. Studies of constitutive Dot1l knockout mice show that Dot1l is essential for embryonic development and prenatal hematopoiesis. DOT1L also interacts with translocation partners of Mixed Lineage Leukemia (MLL) gene, which is commonly translocated in human leukemia. However, the requirement of Dot1l in postnatal hematopoiesis and leukemogenesis of MLL translocation proteins has not been conclusively shown. With a conditional Dot1l knockout mouse model, we examined the consequences of Dot1l loss in postnatal hematopoiesis and MLL translocation leukemia. Deletion of Dot1l led to pancytopenia and failure of hematopoietic homeostasis, and Dot1l-deficient cells minimally reconstituted recipient bone marrow in competitive transplantation experiments. In addition, MLL-AF9 cells required Dot1l for oncogenic transformation, whereas cells with other leukemic oncogenes, such as Hoxa9/Meis1 and E2A-HLF, did not. These findings illustrate a crucial role of Dot1l in normal hematopoiesis and leukemogenesis of specific oncogenes. PMID:21398221

Construction and phenotypic characterization of M68, an RruI quorum sensing knockout mutant of the photosynthetic alphaproteobacterium Rhodospirillum rubrum.

PubMed

Condori, Sandra; Atkinson, Steve; Leys, Natalie; Wattiez, Ruddy; Mastroleo, Felice

2016-06-01

Many bacterial species communicate using a complex system known as quorum sensing (QS) in which gene expression is controlled in response to cell density. In this study an N-acylhomoserine lactone (AHL) synthase (Rru_A3396) knockout mutant (M68) of Rhodospirillum rubrum S1H (WT) was constructed and characterized phenotypically under light anaerobic conditions. Results showed that R. rubrum WT produces unsubstituted, 3-OH and 3-oxo-substituted AHLs with acyl chains ranging from 4 to 14 carbons, with 3-OH-C8 being the most abundant. Growth, pigment content and swimming motility were found to be under the control of this LuxI-type QS system. In addition, cultivation in a low shear environment put forward the aggregative phenotype of M68 and linked biofilm formation to QS in R. rubrum S1H. Interestingly, QS-mutant M68 continued to produce decreased levels of 3-OH-C8-HSL, probably due to the presence of an extra HdtS-type AHL synthase. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Key Enzymes of the Semiphosphorylative Entner-Doudoroff Pathway in the Haloarchaeon Haloferax volcanii: Characterization of Glucose Dehydrogenase, Gluconate Dehydratase, and 2-Keto-3-Deoxy-6-Phosphogluconate Aldolase.

PubMed

Sutter, Jan-Moritz; Tästensen, Julia-Beate; Johnsen, Ulrike; Soppa, Jörg; Schönheit, Peter

2016-08-15

The halophilic archaeon Haloferax volcanii has been proposed to degrade glucose via the semiphosphorylative Entner-Doudoroff (spED) pathway. So far, the key enzymes of this pathway, glucose dehydrogenase (GDH), gluconate dehydratase (GAD), and 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (KDPGA), have not been characterized, and their functional involvement in glucose degradation has not been demonstrated. Here we report that the genes HVO_1083 and HVO_0950 encode GDH and KDPGA, respectively. The recombinant enzymes show high specificity for glucose and KDPG and did not convert the corresponding C4 epimers galactose and 2-keto-3-deoxy-6-phosphogalactonate at significant rates. Growth studies of knockout mutants indicate the functional involvement of both GDH and KDPGA in glucose degradation. GAD was purified from H. volcanii, and the encoding gene, gad, was identified as HVO_1488. GAD catalyzed the specific dehydration of gluconate and did not utilize galactonate at significant rates. A knockout mutant of GAD lost the ability to grow on glucose, indicating the essential involvement of GAD in glucose degradation. However, following a prolonged incubation period, growth of the Δgad mutant on glucose was recovered. Evidence is presented that under these conditions, GAD was functionally replaced by xylonate dehydratase (XAD), which uses both xylonate and gluconate as substrates. Together, the characterization of key enzymes and analyses of the respective knockout mutants present conclusive evidence for the in vivo operation of the spED pathway for glucose degradation in H. volcanii The work presented here describes the identification and characterization of the key enzymes glucose dehydrogenase, gluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase and their encoding genes of the proposed semiphosphorylative Entner-Doudoroff pathway in the haloarchaeon Haloferax volcanii The functional involvement of the three enzymes was proven by analyses of the corresponding knockout mutants. These results provide evidence for the in vivo operation of the semiphosphorylative Entner-Doudoroff pathway in haloarchaea and thus expand our understanding of the unusual sugar degradation pathways in the domain Archaea. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Ethylene production with engineered Synechocystis sp PCC 6803 strains.

PubMed

Veetil, Vinod Puthan; Angermayr, S Andreas; Hellingwerf, Klaas J

2017-02-23

Metabolic engineering and synthetic biology of cyanobacteria offer a promising sustainable alternative approach for fossil-based ethylene production, by using sunlight via oxygenic photosynthesis, to convert carbon dioxide directly into ethylene. Towards this, both well-studied cyanobacteria, i.e., Synechocystis sp PCC 6803 and Synechococcus elongatus PCC 7942, have been engineered to produce ethylene by introducing the ethylene-forming enzyme (Efe) from Pseudomonas syringae pv. phaseolicola PK2 (the Kudzu strain), which catalyzes the conversion of the ubiquitous tricarboxylic acid cycle intermediate 2-oxoglutarate into ethylene. This study focuses on Synechocystis sp PCC 6803 and shows stable ethylene production through the integration of a codon-optimized version of the efe gene under control of the Ptrc promoter and the core Shine-Dalgarno sequence (5'-AGGAGG-3') as the ribosome-binding site (RBS), at the slr0168 neutral site. We have increased ethylene production twofold by RBS screening and further investigated improving ethylene production from a single gene copy of efe, using multiple tandem promoters and by putting our best construct on an RSF1010-based broad-host-self-replicating plasmid, which has a higher copy number than the genome. Moreover, to raise the intracellular amounts of the key Efe substrate, 2-oxoglutarate, from which ethylene is formed, we constructed a glycogen-synthesis knockout mutant (ΔglgC) and introduced the ethylene biosynthetic pathway in it. Under nitrogen limiting conditions, the glycogen knockout strain has increased intracellular 2-oxoglutarate levels; however, surprisingly, ethylene production was lower in this strain than in the wild-type background. Making use of different RBS sequences, production of ethylene ranging over a 20-fold difference has been achieved. However, a further increase of production through multiple tandem promoters and a broad-host plasmid was not achieved speculating that the transcription strength and the gene copy number are not the limiting factors in our system.

The phosphatase JKAP/DUSP22 inhibits T-cell receptor signalling and autoimmunity by inactivating Lck.

PubMed

Li, Ju-Pi; Yang, Chia-Yu; Chuang, Huai-Chia; Lan, Joung-Liang; Chen, Der-Yuan; Chen, Yi-Ming; Wang, Xiaohong; Chen, Alice J; Belmont, John W; Tan, Tse-Hua

2014-04-09

JNK pathway-associated phosphatase (JKAP, also known as DUSP22 or JSP-1) is a JNK activator. The in vivo role of JKAP in immune regulation remains unclear. Here we report that JKAP directly inactivates Lck by dephosphorylating tyrosine-394 residue during T-cell receptor (TCR) signalling. JKAP-knockout T cells display enhanced cell proliferation and cytokine production. JKAP-knockout mice show enhanced T-cell-mediated immune responses and are more susceptible to experimental autoimmune encephalomyelitis (EAE). In addition, the recipient mice that are adoptively transferred with JKAP-knockout T cells show exacerbated EAE symptoms. Aged JKAP-knockout mice spontaneously develop inflammation and autoimmunity. Thus, our results indicate that JKAP is an important phosphatase that inactivates Lck in the TCR signalling turn-off stage, leading to suppression of T-cell-mediated immunity and autoimmunity.

IL-6-Type Cytokine Signaling in Adipocytes Induces Intestinal GLP-1 Secretion.

PubMed

Wueest, Stephan; Laesser, Céline I; Böni-Schnetzler, Marianne; Item, Flurin; Lucchini, Fabrizio C; Borsigova, Marcela; Müller, Werner; Donath, Marc Y; Konrad, Daniel

2018-01-01

We recently showed that interleukin (IL)-6-type cytokine signaling in adipocytes induces free fatty acid release from visceral adipocytes, thereby promoting obesity-induced hepatic insulin resistance and steatosis. In addition, IL-6-type cytokines may increase the release of leptin from adipocytes and by those means induce glucagon-like peptide 1 (GLP-1) secretion. We thus hypothesized that IL-6-type cytokine signaling in adipocytes may regulate insulin secretion. To this end, mice with adipocyte-specific knockout of gp130, the signal transducer protein of IL-6, were fed a high-fat diet for 12 weeks. Compared with control littermates, knockout mice showed impaired glucose tolerance and circulating leptin, GLP-1, and insulin levels were reduced. In line, leptin release from isolated adipocytes was reduced, and intestinal proprotein convertase subtilisin/kexin type 1 ( Pcsk1 ) expression, the gene encoding PC1/3, which controls GLP-1 production, was decreased in knockout mice. Importantly, treatment with the GLP-1 receptor antagonist exendin 9-39 abolished the observed difference in glucose tolerance between control and knockout mice. Ex vivo, supernatant collected from isolated adipocytes of gp130 knockout mice blunted Pcsk1 expression and GLP-1 release from GLUTag cells. In contrast, glucose- and GLP-1-stimulated insulin secretion was not affected in islets of knockout mice. In conclusion, adipocyte-specific IL-6 signaling induces intestinal GLP-1 release to enhance insulin secretion, thereby counteracting insulin resistance in obesity. © 2017 by the American Diabetes Association.

Knockout mutations of insulin-like peptide genes enhance sexual receptivity in Drosophila virgin females.

PubMed

Watanabe, Kazuki; Sakai, Takaomi

2016-01-01

In the fruitfly Drosophila melanogaster, females take the initiative to mate successfully because they decide whether to mate or not. However, little is known about the molecular and neuronal mechanisms regulating sexual receptivity in virgin females. Genetic tools available in Drosophila are useful for identifying molecules and neural circuits involved in the regulation of sexual receptivity. We previously demonstrated that insulin-producing cells (IPCs) in the female brain are critical to the regulation of female sexual receptivity. Ablation and inactivation of IPCs enhance female sexual receptivity, suggesting that neurosecretion from IPCs inhibits female sexual receptivity. IPCs produce and release insulin-like peptides (Ilps) that modulate various biological processes such as metabolism, growth, lifespan and behaviors. Here, we report a novel role of the Ilps in sexual behavior in Drosophila virgin females. Compared with wild-type females, females with knockout mutations of Ilps showed a high mating success rate toward wild-type males, whereas wild-type males courted wild-type and Ilp-knockout females to the same extent. Wild-type receptive females retard their movement during male courtship and this reduced female mobility allows males to copulate. Thus, it was anticipated that knockout mutations of Ilps would reduce general locomotion. However, the locomotor activity in Ilp-knockout females was significantly higher than that in wild-type females. Thus, our findings indicate that the high mating success rate in Ilp-knockout females is caused by their enhanced sexual receptivity, but not by improvement of their sex appeal or by general sluggishness.

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.

The role of system Xc- in methamphetamine-induced dopaminergic neurotoxicity in mice.

PubMed

Dang, Duy-Khanh; Shin, Eun-Joo; Tran, Hai-Quyen; Kim, Dae-Joong; Jeong, Ji Hoon; Jang, Choon-Gon; Nah, Seung-Yeol; Sato, Hideyo; Nabeshima, Toshitaka; Yoneda, Yukio; Kim, Hyoung-Chun

2017-09-01

The cystine/glutamate antiporter (system Xc - , Sxc) transports cystine into cell in exchange for glutamate. Since xCT is a specific subunit of Sxc, we employed xCT knockout mice and investigated whether this antiporter affected methamphetamine (MA)-induced dopaminergic neurotoxicity. MA treatment significantly increased striatal oxidative burdens in wild type mice. xCT inhibitor [i.e., S-4-carboxy-phenylglycine (CPG), sulfasalazine] or an xCT knockout significantly protected against these oxidative burdens. MA-induced increases in Iba-1 expression and Iba-1-labeled microglial immunoreactivity (Iba-1-IR) were significantly attenuated by CPG or sulfasalazine administration or xCT knockout. CPG or sulfasalazine significantly attenuated MA-induced TUNEL-positive cell populations in the striatum of Taconic ICR mice. The decrease in excitatory amino acid transporter-2 (or glutamate transporter-1) expression and increase in glutamate release were attenuated by CPG, sulfasalazine or xCT knockout. In addition, CPG, sulfasalazine or xCT knockout significantly protected against dopaminergic loss (i.e., decreases in tyrosine hydroxylase expression and immunoreactivity, and an increase in dopamine turnover rate) induced by MA. However, CPG, sulfasalazine or xCT knockout did not significantly affect the impaired glutathione system [i.e., decrease in reduced glutathione (GSH) and increase in oxidized glutathione (GSSG)] induced by MA. Our results suggest that Sxc mediates MA-induced neurotoxicity via facilitating oxidative stress, microgliosis, proapoptosis, and glutamate-related toxicity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cathepsin K knockout alleviates aging-induced cardiac dysfunction

PubMed Central

Hua, Yinan; Robinson, Timothy J; Cao, Yongtao; Shi, Guo-Ping; Ren, Jun; Nair, Sreejayan

2015-01-01

Aging is a major risk factor for cardiovascular disease. It has previously been shown that protein levels of cathepsin K, a lysosomal cysteine protease, are elevated in the failing heart and that genetic ablation of cathepsin K protects against pressure overload-induced cardiac hypertrophy and contractile dysfunction. Here we test the hypothesis that cathepsin K knockout alleviates age-dependent decline in cardiac function. Cardiac geometry, contractile function, intracellular Ca2+ properties, and cardiomyocyte apoptosis were evaluated using echocardiography, fura-2 technique, immunohistochemistry, Western blot and TUNEL staining, respectively. Aged (24-month-old) mice exhibited significant cardiac remodeling (enlarged chamber size, wall thickness, myocyte cross-sectional area, and fibrosis), decreased cardiac contractility, prolonged relengthening along with compromised intracellular Ca2+ release compared to young (6-month-old) mice, which were attenuated in the cathepsin K knockout mice. Cellular markers of senescence, including cardiac lipofuscin, p21 and p16, were lower in the aged-cathepsin K knockout mice compared to their wild-type counterpart. Mechanistically, cathepsin K knockout mice attenuated an age-induced increase in cardiomyocyte apoptosis and nuclear translocation of mitochondrial apoptosis-inducing factor (AIF). In cultured H9c2 cells, doxorubicin stimulated premature senescence and apoptosis. Silencing of cathepsin K blocked the doxorubicin-induced translocation of AIF from the mitochondria to the nuclei. Collectively, these results suggest that cathepsin K knockout attenuates age-related decline in cardiac function via suppressing caspase-dependent and caspase-independent apoptosis. PMID:25692548

Abrogated Freud-1/Cc2d1a Repression of 5-HT1A Autoreceptors Induces Fluoxetine-Resistant Anxiety/Depression-Like Behavior.

PubMed

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

2017-12-06

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 knock-out 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 knock-out ( cF1/1A dko ) to disrupt both Freud-1 and 5-HT1A genes in 5-HT neurons, no increase in anxiety- or depression-like behavior was seen upon knock-out of Freud-1 on the 5-HT1A autoreceptor-negative background; rather, a reduction in depression-like behavior emerged. These studies implicate transcriptional dysregulation 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, such as Freud-1, to restore transcriptional balance may augment response to antidepressant treatment. SIGNIFICANCE STATEMENT Altered regulation of the 5-HT1A autoreceptor has been implicated in human anxiety, major depression, suicide, and resistance to antidepressants. This study uniquely identifies a single transcription factor, Freud-1, as crucial for 5-HT1A autoreceptor expression in vivo Disruption of Freud-1 in serotonin neurons in mice links upregulation of 5-HT1A autoreceptors to anxiety/depression-like behavior and provides a new model of antidepressant resistance. Treatment strategies to reestablish transcriptional regulation of 5-HT1A autoreceptors could provide a more robust and sustained antidepressant response. Copyright © 2017 the authors 0270-6474/17/3711967-12$15.00/0.

Quantitation of NAD+ biosynthesis from the salvage pathway in Saccharomyces cerevisiae

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

Sporty, J; Lin, S; Kato, M

2009-02-18

Nicotinamide adenine dinucleotide (NAD{sup +}) is synthesized via two major pathways in prokaryotic and eukaryotic systems: the de novo biosynthesis pathway from tryptophan precursors, or by the salvage biosynthesis pathway from either extracellular nicotinic acid or various intracellular NAD{sup +} decomposition products. NAD{sup +} biosynthesis via the salvage pathway has been linked to an increase in yeast replicative lifespan under calorie restriction (CR). However, the relative contribution of each pathway to NAD{sup +} biosynthesis under both normal and CR conditions is not known. Here, we have performed lifespan, NAD{sup +} and NADH (the reduced form of NAD{sup +}) analyses onmore » BY4742 wild type, NAD+ salvage pathway knockout (npt1{Delta}), and NAD+ de novo pathway knockout (qpt1{Delta}) yeast strains cultured in media containing either 2% glucose (normal growth) or 0.5% glucose (CR). We have utilized {sup 14}C labeled nicotinic acid in the culture media combined with HPLC speciation and both UV and {sup 14}C detection to quantitate the total amounts of NAD{sup +} and NADH and the amounts derived from the salvage pathway. We observe that wild type and qpt1{Delta} yeast exclusively utilize extracellular nicotinic acid for NAD{sup +} and NADH biosynthesis under both the 2% and 0.5% glucose growth conditions suggesting that the de novo pathway plays little role if a functional salvage pathway is present. We also observe that NAD{sup +} concentrations decrease in all three strains under CR. However, unlike the wild type strain, NADH concentrations do not decrease and NAD{sup +}:NADH ratios do not increase under CR for either knockout strain. Lifespan analyses reveal that CR results in a lifespan increase of approximately 25% for the wild type and qpt1{Delta} strains, while no increase in lifespan is observed for the npt1{Delta} strain. In combination these data suggest that having a functional salvage pathway is more important than the absolute levels of NAD{sup +} or NADH for lifespan extension under CR.« less

Manifestation of α clustering in 10Be via α -knockout reaction

NASA Astrophysics Data System (ADS)

Lyu, Mengjiao; Yoshida, Kazuki; Kanada-En'yo, Yoshiko; Ogata, Kazuyuki

2018-04-01

Background: Proton-induced α -knockout reactions may allow direct experimental observation of α clustering in nuclei. This is obtained by relating the theoretical descriptions of clustering states to the experimental reaction observables. It is desired to introduce microscopic structure models into the theoretical frameworks for α -knockout reactions. Purpose: Our goal is to probe the α clustering in the 10Be nucleus by proton-induced α -knockout reaction observables. Method: We adopt an extended version of the Tohsaki-Horiuchi-Schuck-Röpke wave function of 10Be and integrate it with the distorted-wave impulse approximation framework for the calculation of (p ,p α ) -knockout reactions. Results: We make the first calculation for the 10Be(p ,p α )6He reaction at 250 MeV by implementing a microscopic α -cluster wave function, and we predict the triple-differential cross section (TDX). Furthermore, by constructing artificial states of the target nucleus 10Be with compact or dilute spatial distributions, the TDX is found to be highly sensitive to the extent of clustering in the target nuclei. Conclusions: These results provide reliable manifestation of α clustering in 10Be.

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.

Different roles of axon guidance cues and patterned spontaneous activity in establishing receptive fields in the mouse superior colliculus.

PubMed

Liu, Mingna; Wang, Lupeng; Cang, Jianhua

2014-01-01

Visual neurons in the superior colliculus (SC) respond to both bright (On) and dark (Off) stimuli in their receptive fields. This receptive field property is due to proper convergence of On- and Off-centered retinal ganglion cells to their target cells in the SC. In this study, we have compared the receptive field structure of individual SC neurons in two lines of mutant mice that are deficient in retinotopic mapping: the ephrin-A knockouts that lack important retinocollicular axonal guidance cues and the nAChR-β2 knockouts that have altered activity-dependent refinement of retinocollicular projections. We find that even though the receptive fields are much larger in the ephrin-A knockouts, their On-Off overlap remains unchanged. These neurons also display normal level of selectivity for stimulus direction and orientation. In contrast, the On-Off overlap is disrupted in the β2 knockouts. Together with the previous finding of disrupted direction and orientation selectivity in the β2 knockout mice, our results indicate that molecular guidance cues and activity-dependent processes play different roles in the development of receptive field properties in the SC.

Olivocochlear neuron central anatomy is normal in alpha 9 knockout mice.

PubMed

Brown, M Christian; Vetter, Douglas E

2009-03-01

Olivocochlear (OC) neurons were studied in a transgenic mouse with deletion of the alpha 9 nicotinic acetylcholine receptor subunit. In this alpha 9 knockout mouse, the peripheral effects of OC stimulation are lacking and the peripheral terminals of OC neurons under outer hair cells have abnormal morphology. To account for this mouse's apparently normal hearing, it has been proposed to have central compensation via collateral branches to the cochlear nucleus. We tested this idea by staining OC neurons for acetylcholinesterase and examining their morphology in knockout mice, wild-type mice of the same background strain, and CBA/CaJ mice. Knockout mice had normal OC systems in terms of numbers of OC neurons, dendritic patterns, and numbers of branches to the cochlear nucleus. The branch terminations were mainly to edge regions and to a lesser extent the core of the cochlear nucleus, and were similar among the strains in terms of the distribution and staining density. These data demonstrate that there are no obvious changes in the central morphology of the OC neurons in alpha 9 knockout mice and make less attractive the idea that there is central compensation for deletion of the peripheral receptor in these mice.

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).

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

Novel mTORC1 and 2 Signaling Pathways in Polycystic Kidney Disease (PKD)

DTIC Science & Technology

2017-09-01

AWARD NUMBER: W81XWH-16-1-0172 TITLE: Novel mTORC1 and 2 Signaling Pathways in Polycystic Kidney Disease (PKD) PRINCIPAL INVESTIGATOR: Charles...TITLE AND SUBTITLE 5a. CONTRACT NUMBER W81XWH-16-1-0172 Novel mTORC1 and 2 Signaling Pathways in Polycystic Kidney Disease (PKD) 5b. GRANT NUMBER 5c...investigate the effects of mTORC1 (Raptor) knockout, mTORC2 (Rictor) knockout or combined mTORC1 and 2 knockout on cyst growth and kidney function. The

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

Proton Knock-Out in Hall A

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

Kees de Jager

Proton knock-out is studied in a broad program in Hall A at Jefferson Lab. The first experiment performed in Hall A studied the {sup 16}O(e,e'p) reaction. Since then proton knock-out experiments have studied a variety of aspects of that reaction, from single-nucleon properties to its mechanism, such as final-state interactions and two-body currents, in nuclei from {sup 2}H to {sup 16}O. In this review the results of this program will be summarized and an outlook given of future accomplishments.

A Rapid Embryonic Stem Cell-Based Mouse Model for B-cell Lymphomas Driven by Epstein-Barr Virus Protein LMP1.

PubMed

Ba, Zhaoqing; Meng, Fei-Long; Gostissa, Monica; Huang, Pei-Yi; Ke, Qiang; Wang, Zhe; Dao, Mai N; Fujiwara, Yuko; Rajewsky, Klaus; Zhang, Baochun; Alt, Frederick W

2015-06-01

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) contributes to oncogenic human B-cell transformation. Mouse B cells conditionally expressing LMP1 are not predisposed to B-cell malignancies, as LMP1-expressing B cells are eliminated by T cells. However, mice with conditional B-cell LMP1 expression and genetic elimination of α/β and γ/δ T cells ("CLT" mice) die early in association with B-cell lymphoproliferation and lymphomagenesis. Generation of CLT mice involves in-breeding multiple independently segregating alleles. Thus, although introduction of additional activating or knockout mutations into the CLT model is desirable for further B-cell expansion and immunosurveillance studies, doing such experiments by germline breeding is time-consuming, expensive, and sometimes unfeasible. To generate a more tractable model, we generated clonal CLT embryonic stem (ES) cells from CLT embryos and injected them into RAG2-deficient blastocysts to generate chimeric mice, which, like germline CLT mice, harbor splenic CLT B cells and lack T cells. CLT chimeric mice generated by this RAG2-deficient blastocyst complementation ("RDBC") approach die rapidly in association with B-cell lymphoproliferation and lymphoma. Because CLT lymphomas routinely express the activation-induced cytidine deaminase (AID) antibody diversifier, we tested potential AID roles by eliminating the AID gene in CLT ES cells and testing them via RDBC. We found that CLT and AID-deficient CLT ES chimeras had indistinguishable phenotypes, showing that AID is not essential for LMP1-induced lymphomagenesis. Beyond expanding accessibility and utility of CLT mice as a cancer immunotherapy model, our studies provide a new approach for facilitating generation of genetically complex mouse cancer models. ©2015 American Association for Cancer Research.

A Rapid Embryonic Stem Cell-Based Mouse Model for B-cell Lymphomas Driven by Epstein-Barr Virus Protein LMP1

PubMed Central

Ba, Zhaoqing; Meng, Fei-Long; Gostissa, Monica; Huang, Pei-Yi; Ke, Qiang; Wang, Zhe; Dao, Mai N.; Fujiwara, Yuko; Rajewsky, Klaus; Baochun, Zhang; Alt, Frederick W.

2015-01-01

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) contributes to oncogenic human B-cell transformation. Mouse B cells conditionally expressing LMP1 are not predisposed to B-cell malignancies, as LMP1-expressing B cells are eliminated by T cells. However, mice with conditional B-cell LMP1 expression and genetic elimination of α/β and γ/δ T cells (“CLT” mice) die early in association with B-cell lymphoproliferation and lymphomagenesis. Generation of CLT mice involves in-breeding multiple independently segregating alleles. Thus, while introduction of additional activating or knock-out mutations into the CLT model is desirable for further B-cell expansion and immunosurveillance studies, doing such experiments by germline breeding is time-consuming, expensive and sometimes unfeasible. To generate a more tractable model, we generated clonal CLT ES cells from CLT embryos and injected them into RAG2-deficient blastocysts to generate chimeric mice, which like germline CLT mice harbor splenic CLT B cells and lack T cells. CLT chimeric mice generated by this RAG2-deficient blastocyst complementation (“RDBC”) approach die rapidly in association with B-cell lymphoproliferation and lymphoma. As CLT lymphomas routinely express the Activation-Induced Cytidine Deaminase (AID) antibody diversifier, we tested potential AID roles by eliminating the AID gene in CLT ES cells and testing them via RDBC. We found that CLT and AID-deficient CLT ES chimeras had indistinguishable phenotypes, showing that AID is not essential for LMP1-induced lymphomagenesis. Beyond expanding accessibility and utility of CLT mice as a cancer immunotherapy model, our studies provide a new approach for facilitating generation of genetically complex mouse cancer models. PMID:25934172

Blimp-1–mediated CD4 T cell exhaustion causes CD8 T cell dysfunction during chronic toxoplasmosis

PubMed Central

Cobb, Dustin A.; Bhadra, Rajarshi

2016-01-01

CD8, but not CD4, T cells are considered critical for control of chronic toxoplasmosis. Although CD8 exhaustion has been previously reported in Toxoplasma encephalitis (TE)–susceptible model, our current work demonstrates that CD4 not only become exhausted during chronic toxoplasmosis but this dysfunction is more pronounced than CD8 T cells. Exhausted CD4 population expressed elevated levels of multiple inhibitory receptors concomitant with the reduced functionality and up-regulation of Blimp-1, a transcription factor. Our data demonstrates for the first time that Blimp-1 is a critical regulator for CD4 T cell exhaustion especially in the CD4 central memory cell subset. Using a tamoxifen-dependent conditional Blimp-1 knockout mixed bone marrow chimera as well as an adoptive transfer approach, we show that CD4 T cell–intrinsic deletion of Blimp-1 reversed CD8 T cell dysfunction and resulted in improved pathogen control. To the best of our knowledge, this is a novel finding, which demonstrates the role of Blimp-1 as a critical regulator of CD4 dysfunction and links it to the CD8 T cell dysfunctionality observed in infected mice. The critical role of CD4-intrinsic Blimp-1 expression in mediating CD4 and CD8 T cell exhaustion may provide a rational basis for designing novel therapeutic approaches. PMID:27481131

Effect of knockout of α2δ-1 on action potentials in mouse sensory neurons.

PubMed

Margas, Wojciech; Ferron, Laurent; Nieto-Rostro, Manuela; Schwartz, Arnold; Dolphin, Annette C

2016-08-05

Gene deletion of the voltage-gated calcium channel auxiliary subunit α2δ-1 has been shown previously to have a cardiovascular phenotype, and a reduction in mechano- and cold sensitivity, coupled with delayed development of neuropathic allodynia. We have also previously shown that dorsal root ganglion (DRG) neuron calcium channel currents were significantly reduced in α2δ-1 knockout mice. To extend our findings in these sensory neurons, we have examined here the properties of action potentials (APs) in DRG neurons from α2δ-1 knockout mice in comparison to their wild-type (WT) littermates, in order to dissect how the calcium channels that are affected by α2δ-1 knockout are involved in setting the duration of individual APs and their firing frequency. Our main findings are that there is reduced Ca(2+) entry on single AP stimulation, particularly in the axon proximal segment, reduced AP duration and reduced firing frequency to a 400 ms stimulation in α2δ-1 knockout neurons, consistent with the expected role of voltage-gated calcium channels in these events. Furthermore, lower intracellular Ca(2+) buffering also resulted in reduced AP duration, and a lower frequency of AP firing in WT neurons, mimicking the effect of α2δ-1 knockout. By contrast, we did not obtain any consistent evidence for the involvement of Ca(2+)-activation of large conductance calcium-activated potassium (BK) and small conductance calcium-activated potassium (SK) channels in these events. In conclusion, the reduced Ca(2+) elevation as a result of single AP stimulation is likely to result from the reduced duration of the AP in α2δ-1 knockout sensory neurons.This article is part of the themed issue 'Evolution brings Ca(2+) and ATP together to control life and death'. © 2016 The Authors.

Changes in the expression of neurotransmitter receptors in Parkin and DJ-1 knockout mice--A quantitative multireceptor study.

PubMed

Cremer, J N; Amunts, K; Schleicher, A; Palomero-Gallagher, N; Piel, M; Rösch, F; Zilles, K

2015-12-17

Parkinson's disease (PD) is a well-characterized neurological disorder with regard to its neuropathological and symptomatic appearance. At the genetic level, mutations of particular genes, e.g. Parkin and DJ-1, were found in human hereditary PD with early onset. Neurotransmitter receptors constitute decisive elements in neural signal transduction. Furthermore, since they are often altered in neurological and psychiatric diseases, receptors have been successful targets for pharmacological agents. However, the consequences of PD-associated gene mutations on the expression of transmitter receptors are largely unknown. Therefore, we studied the expression of 16 different receptor binding sites of the neurotransmitters glutamate, GABA, acetylcholine, adrenaline, serotonin, dopamine and adenosine by means of quantitative receptor autoradiography in Parkin and DJ-1 knockout mice. These knockout mice exhibit electrophysiological and behavioral deficits, but do not show the typical dopaminergic cell loss. We demonstrated differential changes of binding site densities in eleven brain regions. Most prominently, we found an up-regulation of GABA(B) and kainate receptor densities in numerous cortical areas of Parkin and DJ-1 knockout mice, as well as increased NMDA but decreased AMPA receptor densities in different brain regions of the Parkin knockout mice. The alterations of three different glutamate receptor types may indicate the potential relevance of the glutamatergic system in the pathogenesis of PD. Furthermore, the cholinergic M1, M2 and nicotinic receptors as well as the adrenergic α2 and the adenosine A(2A) receptors showed differentially increased densities in Parkin and DJ-1 knockout mice. Taken together, knockout of the PD-associated genes Parkin or DJ-1 results in differential changes of neurotransmitter receptor densities, highlighting a possible role of altered non-dopaminergic, and in particular of glutamatergic neurotransmission in PD pathogenesis. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

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

Role of medial prefrontal cortex Narp in the extinction of morphine conditioned place preference.

PubMed

Blouin, Ashley M; Han, Sungho; Pearce, Anne M; Cheng, Kailun; Lee, Jongah J; Johnson, Alexander W; Wang, Chuansong; During, Matthew J; Holland, Peter C; Shaham, Yavin; Baraban, Jay M; Reti, Irving M

2013-01-15

Narp knockout (KO) mice demonstrate an impaired extinction of morphine conditioned place preference (CPP). Because the medial prefrontal cortex (mPFC) has been implicated in extinction learning, we tested whether Narp cells in this region play a role in the extinction of morphine CPP. We found that intracranial injections of adenoassociated virus (AAV) expressing wild-type (WT) Narp into the mPFC of Narp KO mice rescued the extinction and the injection of AAV expressing a dominant negative form of Narp (NarpN) into the mPFC of WT mice impaired the extinction of morphine CPP. These findings suggest that Narp in the mPFC mediates the extinction of morphine CPP.

Functional Divergence of Platelet Protein Kinase C (PKC) Isoforms in Thrombus Formation on Collagen*

PubMed Central

Gilio, Karen; Harper, Matthew T.; Cosemans, Judith M. E. M.; Konopatskaya, Olga; Munnix, Imke C. A.; Prinzen, Lenneke; Leitges, Michael; Liu, Qinghang; Molkentin, Jeffery D.; Heemskerk, Johan W. M.; Poole, Alastair W.

2010-01-01

Arterial thrombosis, a major cause of myocardial infarction and stroke, is initiated by activation of blood platelets by subendothelial collagen. The protein kinase C (PKC) family centrally regulates platelet activation, and it is becoming clear that the individual PKC isoforms play distinct roles, some of which oppose each other. Here, for the first time, we address all four of the major platelet-expressed PKC isoforms, determining their comparative roles in regulating platelet adhesion to collagen and their subsequent activation under physiological flow conditions. Using mouse gene knock-out and pharmacological approaches in human platelets, we show that collagen-dependent α-granule secretion and thrombus formation are mediated by the conventional PKC isoforms, PKCα and PKCβ, whereas the novel isoform, PKCθ, negatively regulates these events. PKCδ also negatively regulates thrombus formation but not α-granule secretion. In addition, we demonstrate for the first time that individual PKC isoforms differentially regulate platelet calcium signaling and exposure of phosphatidylserine under flow. Although platelet deficient in PKCα or PKCβ showed reduced calcium signaling and phosphatidylserine exposure, these responses were enhanced in the absence of PKCθ. In summary therefore, this direct comparison between individual subtypes of PKC, by standardized methodology under flow conditions, reveals that the four major PKCs expressed in platelets play distinct non-redundant roles, where conventional PKCs promote and novel PKCs inhibit thrombus formation on collagen. PMID:20479008

Functional divergence of platelet protein kinase C (PKC) isoforms in thrombus formation on collagen.

PubMed

Gilio, Karen; Harper, Matthew T; Cosemans, Judith M E M; Konopatskaya, Olga; Munnix, Imke C A; Prinzen, Lenneke; Leitges, Michael; Liu, Qinghang; Molkentin, Jeffery D; Heemskerk, Johan W M; Poole, Alastair W

2010-07-23

Arterial thrombosis, a major cause of myocardial infarction and stroke, is initiated by activation of blood platelets by subendothelial collagen. The protein kinase C (PKC) family centrally regulates platelet activation, and it is becoming clear that the individual PKC isoforms play distinct roles, some of which oppose each other. Here, for the first time, we address all four of the major platelet-expressed PKC isoforms, determining their comparative roles in regulating platelet adhesion to collagen and their subsequent activation under physiological flow conditions. Using mouse gene knock-out and pharmacological approaches in human platelets, we show that collagen-dependent alpha-granule secretion and thrombus formation are mediated by the conventional PKC isoforms, PKCalpha and PKCbeta, whereas the novel isoform, PKC, negatively regulates these events. PKCdelta also negatively regulates thrombus formation but not alpha-granule secretion. In addition, we demonstrate for the first time that individual PKC isoforms differentially regulate platelet calcium signaling and exposure of phosphatidylserine under flow. Although platelet deficient in PKCalpha or PKCbeta showed reduced calcium signaling and phosphatidylserine exposure, these responses were enhanced in the absence of PKC. In summary therefore, this direct comparison between individual subtypes of PKC, by standardized methodology under flow conditions, reveals that the four major PKCs expressed in platelets play distinct non-redundant roles, where conventional PKCs promote and novel PKCs inhibit thrombus formation on collagen.

Knock-Outs, Stick-Outs, Cut-Outs: Clipping Paths Separate Objects from Background.

ERIC Educational Resources Information Center

Wilson, Bradley

1998-01-01

Outlines a six-step process that allows computer operators, using Photoshop software, to create "knock-outs" to precisely define the path that will serve to separate the object from the background. (SR)

Generating gene knockout rats by homologous recombination in embryonic stem cells

PubMed Central

Tong, Chang; Huang, Guanyi; Ashton, Charles; Li, Ping; Ying, Qi-Long

2013-01-01

We describe here a detailed protocol for generating gene knockout rats by homologous recombination in embryonic stem (ES) cells. This protocol comprises the following procedures: derivation and expansion of rat ES cells, construction of gene-targeting vectors, generation of gene-targeted rat ES cells and, finally, production of gene-targeted rats. The major differences between this protocol and the classical mouse gene-targeting protocol include ES cell culture methods, drug selection scheme, colony picking and screening strategies. This ES cell–based gene-targeting technique allows sophisticated genetic modifications to be performed in the rat, as many laboratories have been doing in the mouse for the past two decades. Recently we used this protocol to generate Tp53 (also known as p53) gene knockout rats. The entire process requires ~1 year to complete, from derivation of ES cells to generation of knockout rats. PMID:21637202

CRISPR/Cas9-mediated gene knockout of NANOG and NANOGP8 decreases the malignant potential of prostate cancer cells.

PubMed

Kawamura, Norihiko; Nimura, Keisuke; Nagano, Hiromichi; Yamaguchi, Sohei; Nonomura, Norio; Kaneda, Yasufumi

2015-09-08

NANOG expression in prostate cancer is highly correlated with cancer stem cell characteristics and resistance to androgen deprivation. However, it is not clear whether NANOG or its pseudogenes contribute to the malignant potential of cancer. We established NANOG- and NANOGP8-knockout DU145 prostate cancer cell lines using the CRISPR/Cas9 system. Knockouts of NANOG and NANOGP8 significantly attenuated malignant potential, including sphere formation, anchorage-independent growth, migration capability, and drug resistance, compared to parental DU145 cells. NANOG and NANOGP8 knockout did not inhibit in vitro cell proliferation, but in vivo tumorigenic potential decreased significantly. These phenotypes were recovered in NANOG- and NANOGP8-rescued cell lines. These results indicate that NANOG and NANOGP8 proteins are expressed in prostate cancer cell lines, and NANOG and NANOGP8 equally contribute to the high malignant potential of prostate cancer.

Skeletal muscle-specific HMG-CoA reductase knockout mice exhibit rhabdomyolysis: A model for statin-induced myopathy.

PubMed

Osaki, Yoshinori; Nakagawa, Yoshimi; Miyahara, Shoko; Iwasaki, Hitoshi; Ishii, Akiko; Matsuzaka, Takashi; Kobayashi, Kazuto; Yatoh, Shigeru; Takahashi, Akimitsu; Yahagi, Naoya; Suzuki, Hiroaki; Sone, Hirohito; Ohashi, Ken; Ishibashi, Shun; Yamada, Nobuhiro; Shimano, Hitoshi

2015-10-23

HMG-CoA reductase (HMGCR) catalyzes the conversion of HMG-CoA to mevalonic acid (MVA); this is the rate-limiting enzyme of the mevalonate pathway that synthesizes cholesterol. Statins, HMGCR inhibitors, are widely used as cholesterol-reducing drugs. However, statin-induced myopathy is the most adverse side effect of statins. To eludicate the mechanisms underlying statin the myotoxicity and HMGCR function in the skeletal muscle, we developed the skeletal muscle-specific HMGCR knockout mice. Knockout mice exhibited postnatal myopathy with elevated serum creatine kinase levels and necrosis. Myopathy in knockout mice was completely rescued by the oral administration of MVA. These results suggest that skeletal muscle toxicity caused by statins is dependent on the deficiencies of HMGCR enzyme activity and downstream metabolites of the mevalonate pathway in skeletal muscles rather than the liver or other organs. Copyright © 2015 Elsevier Inc. All rights reserved.

Transgenic and gene knockout mice in gastric cancer research

PubMed Central

Jiang, Yannan; Yu, Yingyan

2017-01-01

Mouse models are useful tool for carcinogenic study. They will greatly enrich the understanding of pathogenesis and molecular mechanisms for gastric cancer. However, only few of mice could develop gastric cancer spontaneously. With the development and improvement of gene transfer technology, investigators created a variety of transgenic and knockout/knockin mouse models of gastric cancer, such as INS-GAS mice and gastrin knockout mice. Combined with helicobacter infection and carcinogens treatment, these transgenic/knockout/knockin mice developed precancerous or cancerous lesions, which are proper for gene function study or experimental therapy. Here we review the progression of genetically engineered mouse models on gastric cancer research, and emphasize the effects of chemical carcinogens or infectious factors on carcinogenesis of genetically modified mouse. We also emphasize the histological examination on mouse stomach. We expect to provide researchers with some inspirations on this field. PMID:27713138

Conditional knockout of the leptin receptor in the colonic epithelium revealed the local effects of leptin receptor signaling in the progression of colonic tumors in mice.

PubMed

Higurashi, Takuma; Endo, Hiroki; Uchiyama, Takashi; Uchiyama, Shiori; Yamada, Eiji; Ohkubo, Hidenori; Sakai, Eiji; Takahashi, Hirokazu; Maeda, Shin; Wada, Koichiro; Natsumeda, Yutaka; Hippo, Yoshitaka; Nakajima, Atsushi; Nakagama, Hitoshi

2014-09-01

Leptin, secreted by the adipose tissue and known to be related to obesity, is considered to be involved in the onset and progression of colorectal cancer. However, the exact role of leptin in colorectal carcinogenesis is still unclear, as several controversial reports have been published on the various systemic effects of leptin. The aim of this study was to clarify the local and precise roles of leptin receptor (LEPR)-mediated signaling in colonic carcinogenesis using intestinal epithelium-specific LEPRb conditional knockout (cKO) mice. We produced and used colonic epithelium-specific LEPRb cKO mice to investigate the carcinogen-induced formation of aberrant crypt foci (ACF) and tumors in the colon, using their littermates as control. There were no differences in the body weight or systemic condition between the control and cKO mice. The tumor sizes and number of large-sized tumors were significantly lower in the cKO mice as compared with those in the control mice. On the other hand, there was no significant difference in the proliferative activity of the normal colonic epithelial cells or ACF formation between the control and cKO mice. In the control mice, marked increase of the LEPRb expression level was observed in the colonic tumors as compared with that in the normal epithelium; furthermore, signal transducer and activator of transcription (STAT3) was activated in the tumor cells. These findings suggest that STAT3 is one of the important molecules downstream of LEPRb, and LEPRb/STAT3 signaling controls tumor cell proliferation. We demonstrated the importance of local/regional LEPR-mediated signaling in colorectal carcinogenesis. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Perception of sweet taste is important for voluntary alcohol consumption in mice.

PubMed

Blednov, Y A; Walker, D; Martinez, M; Levine, M; Damak, S; Margolskee, R F

2008-02-01

To directly evaluate the association between taste perception and alcohol intake, we used three different mutant mice, each lacking a gene expressed in taste buds and critical to taste transduction: alpha-gustducin (Gnat3), Tas1r3 or Trpm5. Null mutant mice lacking any of these three genes showed lower preference score for alcohol and consumed less alcohol in a two-bottle choice test, as compared with wild-type littermates. These null mice also showed lower preference score for saccharin solutions than did wild-type littermates. In contrast, avoidance of quinine solutions was less in Gnat3 or Trpm5 knockout mice than in wild-type mice, whereas Tas1r3 null mice were not different from wild type in their response to quinine solutions. There were no differences in null vs. wild-type mice in their consumption of sodium chloride solutions. To determine the cause for reduction of ethanol intake, we studied other ethanol-induced behaviors known to be related to alcohol consumption. There were no differences between null and wild-type mice in ethanol-induced loss of righting reflex, severity of acute ethanol withdrawal or conditioned place preference for ethanol. Weaker conditioned taste aversion (CTA) to alcohol in null mice may have been caused by weaker rewarding value of the conditioned stimulus (saccharin). When saccharin was replaced by sodium chloride, no differences in CTA to alcohol between knockout and wild-type mice were seen. Thus, deletion of any one of three different genes involved in detection of sweet taste leads to a substantial reduction of alcohol intake without any changes in pharmacological actions of ethanol.

Uterine epithelial cell proliferation and endometrial hyperplasia: evidence from a mouse model.

PubMed

Gao, Yang; Li, Shu; Li, Qinglei

2014-08-01

In the uterus, epithelial cell proliferation changes during the estrous cycle and pregnancy. Uncontrolled epithelial cell proliferation results in implantation failure and/or cancer development. Transforming growth factor-β (TGF-β) signaling is a fundamental regulator of diverse biological processes and is indispensable for multiple reproductive functions. However, the in vivo role of TGF-β signaling in uterine epithelial cells remains poorly defined. We have shown that in the uterus, conditional deletion of the Type 1 receptor for TGF-β (Tgfbr1) using anti-Müllerian hormone receptor type 2 (Amhr2) Cre leads to myometrial defects. Here, we describe enhanced epithelial cell proliferation by immunostaining of Ki67 in the uteri of these mice. The aberration culminated in endometrial hyperplasia in aged females. To exclude the potential influence of ovarian steroid hormones, the proliferative status of uterine epithelial cells was assessed following ovariectomy. Increased uterine epithelial cell proliferation was also revealed in ovariectomized Tgfbr1 Amhr2-Cre conditional knockout mice. We further demonstrated that transcript levels for fibroblast growth factor 10 (Fgf10) were markedly up-regulated in Tgfbr1 Amhr2-Cre conditional knockout uteri. Consistently, treatment of primary uterine stromal cells with TGF-β1 significantly reduced Fgf10 mRNA expression. Thus, our findings suggest a potential involvement of TGFBR1-mediated signaling in the regulation of uterine epithelial cell proliferation, and provide genetic evidence supporting the role of uterine epithelial cell proliferation in the pathogenesis of endometrial hyperplasia. © 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.

Behavioral characterization of cereblon forebrain-specific conditional null mice: A model for human non-syndromic intellectual disability

PubMed Central

Rajadhyaksha, Anjali M.; Ra, Stephen; Kishinevsky, Sarah; Lee, Anni S.; Romanienko, Peter; DuBoff, Mariel; Yang, Chingwen; Zupan, Bojana; Byrne, Maureen; Daruwalla, Zeeba R.; Mark, Willie; Kosofsky, Barry E.; Toth, Miklos; Higgins, Joseph J.

2018-01-01

A nonsense mutation in the human cereblon gene (CRBN) causes a mild type of autosomal recessive non-syndromic intellectual disability (ID). Animal studies show that crbn is a cytosolic protein with abundant expression in the hippocampus (HPC) and neocortex (CTX). Its diverse functions include the developmental regulation of ion channels at the neuronal synapse, the mediation of developmental programs by ubiquitination, and a target for herpes simplex type I virus in HPC neurons. To test the hypothesis that anomalous CRBN expression leads to HPC-mediated memory and learning deficits, we generated germ-line crbn knock-out mice (crbn−/−). We also inactivated crbn in forebrain neurons in conditional knock-out mice in which crbn exons 3 and 4 are deleted by cre recombinase under the direction of the Ca2+/calmodulin-dependent protein kinase II alpha promoter (CamKIIcre/+, crbn−/−). crbn mRNA levels were negligible in the HPC, CTX, and cerebellum (CRBM) of the crbn−/− mice. In contrast, crbn mRNA levels were reduced 3- to 4-fold in the HPC, CTX but not in the CRBM in CamKIIcre/+, crbn−/− mice as compared to wild type (CamKIIcre/+, crbn+/+). Contextual fear conditioning showed a significant decrease in the percentage of freezing time in CamKIIcre/+, crbn−/− and crbn−/− mice while motor function, exploratory motivation, and anxiety-related behaviors were normal. These findings suggest that CamKIIcre/+, crbn−/− mice exhibit selective HPC-dependent deficits in associative learning and supports the use of these mice as in vivo models to study the functional consequences of CRBN aberrations on memory and learning in humans. PMID:21995942

FOXL2 impairment in human disease.

PubMed

Verdin, Hannah; De Baere, Elfride

2012-01-01

FOXL2 encodes a forkhead transcription factor that plays important roles in the ovary during development and in post-natal, adult life. Here, we focus on the clinical consequences of FOXL2 impairment in human disease. In line with other forkhead transcription factors, its constitutional genetic defects and a somatic mutation lead to developmental disease and cancer, respectively. More than 100 unique constitutional mutations and regulatory defects have been found in blepharophimosis syndrome (BPES), a complex eyelid malformation associated (type I) or not (type II) with premature ovarian failure (POF). In agreement with the BPES phenotype, FOXL2 is expressed in the developing eyelids and in fetal and adult ovaries. Two knock-out mice and at least one natural animal model, the Polled Intersex Syndrome goat, are known. They recapitulate the BPES phenotype and have provided many insights into the ovarian pathology. Only a few constitutional mutations have been described in nonsyndromic POF. Moreover, a recurrent somatic mutation p.C134W was found to be specific for adult ovarian granulo-sa cell tumors. Functional studies investigating the consequences of FOXL2 mutations or regulatory defects have shed light on the molecular pathogenesis of the aforementioned conditions, and contributed considerably to genotype-phenotype correlations. Recently, a conditional knock-out of Foxl2 in the mouse induced somatic transdifferentiation of ovary into testis in adult mice, suggesting that Foxl2 has an anti-testis function in the adult ovary. This changed our view on the ovary and testis as terminally differentiated organs in adult mammals. Finally, this might have potential implications for the understanding and treatment of frequent conditions such as POF and polycystic ovary syndrome. Copyright © 2012 S. Karger AG, Basel.

Suppressed translation and ULK1 degradation as potential mechanisms of autophagy limitation under prolonged starvation.

PubMed

Allavena, Giulia; Boyd, Caroline; Oo, Kyaw Soe; Maellaro, Emilia; Zhivotovsky, Boris; Kaminskyy, Vitaliy O

2016-11-01

Macroautophagy/autophagy is a well-organized process of intracellular degradation, which is rapidly activated under starvation conditions. Recent data demonstrate a transcriptional upregulation of several autophagy genes as a mechanism that controls autophagy in response to starvation. Here we report that despite the significant upregulation of mRNA of the essential autophagy initiation gene ULK1, its protein level is rapidly reduced under starvation. Although both autophagic and proteasomal systems contribute to the degradation of ULK1, under prolonged nitrogen deprivation, its level was still reduced in ATG7 knockout cells, and only initially stabilized in cells treated with the lysosomal or proteasomal inhibitors. We demonstrate that under starvation, protein translation is rapidly diminished and, similar to treatments with the proteosynthesis inhibitors cycloheximide or anisomycin, is associated with a significant reduction of ULK1. Furthermore, it was found that inhibition of the mitochondrial respiratory complexes or the mitochondrial ATP synthase function that could also take place in the absence of substrates, promote upregulation of ULK1 mRNA and protein expression in an AMPK-dependent manner in U1810 lung cancer cells growing in complete culture medium. These inhibitors could also drastically increase the ULK1 protein in U1810 cells with knockout of ATG13, where the ULK1 expression is significantly diminished. However, such upregulation of ULK1 protein is negligible under starvation conditions, further signifying the contribution of translation and suggesting that transcriptional upregulation of ULK1 protein will be diminished under such conditions. Thus, we propose a model where inhibition of protein translation, together with the degradation systems, limit autophagy during starvation.

Decreased Incentive Motivation Following Knockout or Acute Blockade of the Serotonin Transporter: Role of the 5-HT2C Receptor.

PubMed

Browne, Caleb J; Fletcher, Paul J

2016-09-01

Acute pharmacological elevation of serotonin (5-hydroxytryptamine; 5-HT) activity decreases operant responding for primary reinforcers, suggesting that 5-HT reduces incentive motivation. The mechanism by which 5-HT alters incentive motivation is unknown, but parallel evidence that 5-HT2C receptor agonists also reduce responding for primary reinforcers implicates this receptor as a potential candidate. These experiments examined whether chronic and acute disruptions of serotonin transporter (SERT) activity altered incentive motivation, and whether the 5-HT2C receptor mediated the effects of elevated 5-HT on behavior. To assess incentive motivation, we measured responding for three different reinforcers: a primary reinforcer (saccharin), a conditioned reinforcer (CRf), and an unconditioned sensory reinforcer (USRf). In the chronic condition, responding was compared between SERT knockout (SERT-KO) mice and their wild-type littermates. In the acute condition, responding was examined in wild-type mice following treatment with 10 or 20 mg/kg citalopram, or its vehicle. The ability of the selective 5-HT2C antagonist SB 242084 to prevent the effects of SERT-KO and citalopram on responding was subsequently examined. Both SERT-KO and citalopram reduced responding for saccharin, a CRf, and a USRf. Treatment with SB 242084 enhanced responding for a CRf and a USRf in SERT-KO mice and blocked the effects of citalopram on CRf and USRf responding. However, SB 242084 was unable to prevent the effects of SERT-KO or citalopram on responding for saccharin. These results support a powerful inhibitory function for 5-HT in the control of incentive motivation, and indicate that the 5-HT2C receptor mediates these effects of 5-HT in a reinforcer-dependent manner.

Proteomic Analysis of Mitochondria-Enriched Fraction Isolated from the Frontal Cortex and Hippocampus of Apolipoprotein E Knockout Mice Treated with Alda-1, an Activator of Mitochondrial Aldehyde Dehydrogenase (ALDH2).

PubMed

Stachowicz, Aneta; Olszanecki, Rafał; Suski, Maciej; Głombik, Katarzyna; Basta-Kaim, Agnieszka; Adamek, Dariusz; Korbut, Ryszard

2017-02-17

The role of different genotypes of apolipoprotein E (apoE) in the etiology of Alzheimer's disease is widely recognized. It has been shown that altered functioning of apoE may promote 4-hydroxynonenal modification of mitochondrial proteins, which may result in mitochondrial dysfunction, aggravation of oxidative stress, and neurodegeneration. Mitochondrial aldehyde dehydrogenase (ALDH2) is an enzyme considered to perform protective function in mitochondria by the detoxification of the end products of lipid peroxidation, such as 4-hydroxynonenal and other reactive aldehydes. The goal of our study was to apply a differential proteomics approach in concert with molecular and morphological techniques to elucidate the changes in the frontal cortex and hippocampus of apolipoprotein E knockout (apoE -/- ) mice upon treatment with Alda-1-a small molecular weight activator of ALDH2. Despite the lack of significant morphological changes in the brain of apoE -/- mice as compared to age-matched wild type animals, the proteomic and molecular approach revealed many changes in the expression of genes and proteins, indicating the impairment of energy metabolism, neuroplasticity, and neurogenesis in brains of apoE -/- mice. Importantly, prolonged treatment of apoE -/- mice with Alda-1 led to the beneficial changes in the expression of genes and proteins related to neuroplasticity and mitochondrial function. The pattern of alterations implies mitoprotective action of Alda-1, however, the accurate functional consequences of the revealed changes require further research.

Deficiency of eNOS exacerbates early-stage NAFLD pathogenesis by changing the fat distribution.

PubMed

Nozaki, Yuichi; Fujita, Koji; Wada, Koichiro; Yoneda, Masato; Shinohara, Yoshiyasu; Imajo, Kento; Ogawa, Yuji; Kessoku, Takaomi; Nakamuta, Makoto; Saito, Satoru; Masaki, Naohiko; Nagashima, Yoji; Terauchi, Yasuo; Nakajima, Atsushi

2015-12-17

Although many factors and molecules that are closely associated with non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH) have been reported, the role of endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) in the pathogenesis of NAFLD/NASH remains unclear. We therefore investigated the role of eNOS-derived NO in NAFLD pathogenesis using systemic eNOS-knockout mice fed a high-fat diet. eNOS-knockout and wild-type mice were fed a basal diet or a high-fat diet for 12 weeks. Lipid accumulation and inflammation were evaluated in the liver, and various factors that are closely associated with NAFLD/NASH and hepatic tissue blood flow were analyzed. Lipid accumulation and inflammation were more extensive in the liver and lipid accumulation was less extensive in the visceral fat tissue in eNOS-knockout mice, compared with wild-type mice, after 12 weeks of being fed a high-fat diet. While systemic insulin resistance was comparable between the eNOS-knockout and wild-type mice fed a high-fat diet, hepatic tissue blood flow was significantly suppressed in the eNOS-knockout mice, compared with the wild-type mice, in mice fed a high-fat diet. The microsomal triglyceride transfer protein activity was down-regulated in eNOS-knockout mice, compared with wild-type mice, in mice fed a high-fat diet. A deficiency of eNOS-derived NO may exacerbate the early-stage of NASH pathogenesis by changing the fat distribution in a mouse model via the regulation of hepatic tissue blood flow.

Decreased consumption of sweet fluids in mu opioid receptor knockout mice: a microstructural analysis of licking behavior

PubMed Central

Ostlund, Sean B.; Kosheleff, Alisa; Maidment, Nigel T.; Murphy, Niall P.

2013-01-01

Summary Rationale Evidence suggests that the palatability of food (i.e., the hedonic impact produced by its sensory features) can promote feeding and may underlie compulsive eating, leading to obesity. Pharmacological studies implicate opioid transmission in the hedonic control of feeding, though these studies often rely on agents lacking specificity for particular opioid receptors. Objectives Here, we investigated the role of mu opioid receptors (MORs) specifically in determining hedonic responses to palatable sweet stimuli. Methods In Experiment 1, licking microstructure when consuming sucrose solution (2 to 20 %) was compared in MOR knockout and wildtype mice as a function of sucrose concentration and level of food deprivation. In Experiment 2, a similar examination was conducted using the palatable but calorie-free stimulus sucralose (0.001 to 1%), allowing study of licking behavior independent of homeostatic variables. Results In Experiment 1, MOR knockout mice exhibited several alterations in sucrose licking. Although wildtype mice exhibited a two-fold increase in the burst length when food deprived, relative to the nondeprived test, this aspect of sucrose licking was generally insensitive to manipulations of food deprivation for MOR knockout mice. Furthermore, during concentration testing, their rate of sucrose licking was less than half that of wildtype mice. During sucralose testing (Experiment 2), MOR knockout mice licked at approximately half the wildtype rate, providing more direct evidence that MOR knockout mice were impaired in processing stimulus palatability. Conclusions These results suggest that transmission through MORs mediates hedonic responses to palatable stimuli, and therefore likely contributes to normal and pathological eating. PMID:23568577

Age- and region-specific imbalances of basal amino acids and monoamine metabolism in limbic regions of female Fmr1 knock-out mice.

PubMed

Gruss, Michael; Braun, Katharina

2004-07-01

The Fragile X syndrome, a common form of mental retardation in humans, originates from the loss of expression of the Fragile X mental retardation gene leading to the absence of the encoded Fragile X mental retardation protein 1 (FMRP). A broad pattern of morphological and behavioral abnormalities is well described for affected humans as well as Fmr1 knock-out mice, a transgenic animal model for the human Fragile X syndrome. In the present study, we examined neurochemical differences between female Fmr1 knock-out and wildtype mice with particular focus on neurotransmission. Significant age- and region-specific differences of basal tissue neurotransmitter and metabolite levels measured by high performance liquid chromatography were found. Those differences were more numerous in juvenile animals (postnatal day (PND) 28-31) compared to adults (postnatal day 209-221). In juvenile female knock-out mice, especially aspartate and taurine were increased in cortical regions, striatum, cerebellum, and brainstem. Furthermore, compared to the wildtype animals, the juvenile knock-out mice displayed an increased level of neuronal inhibition in the hippocampus and brainstem reflected by decreased ratios of (aspartate + glutamate)/(taurine + GABA), as well as an increased dopamine (DA) turnover in cortical regions, striatum, and hippocampus. These results provide the first evidence that the lack of FMRP expression in female Fmr1 knock-out mice is accompanied by age-dependent, region-specific alterations in brain amino acids, and monoamine turnover, which might be related to the reported synaptical and behavioural alterations in these animals.

Discrete change in volatile anesthetic sensitivity in mice with inactivated tandem pore potassium ion channel TRESK.

PubMed

Chae, Yun Jeong; Zhang, Jianan; Au, Paul; Sabbadini, Marta; Xie, Guo-Xi; Yost, C Spencer

2010-12-01

We investigated the role of tandem pore potassium ion channel (K2P) TRESK in neurobehavioral function and volatile anesthetic sensitivity in genetically modified mice. Exon III of the mouse TRESK gene locus was deleted by homologous recombination using a targeting vector. The genotype of bred mice (wild type, knockout, or heterozygote) was determined using polymerase chain reaction. Morphologic and behavioral evaluations of TRESK knockout mice were compared with wild-type littermates. Sensitivity of bred mice to isoflurane, halothane, sevoflurane, and desflurane were studied by determining the minimum alveolar concentration preventing movement to tail clamping in 50% of each genotype. With the exception of decreased number of inactive periods and increased thermal pain sensitivity (20% decrease in latency with hot plate test), TRESK knockout mice had healthy development and behavior. TRESK knockout mice showed a statistically significant 8% increase in isoflurane minimum alveolar concentration compared with wild-type littermates. Sensitivity to other volatile anesthetics was not significantly different. Spontaneous mortality of TRESK knockout mice after initial anesthesia testing was nearly threefold higher than that of wild-type littermates. TRESK alone is not critical for baseline central nervous system function but may contribute to the action of volatile anesthetics. The inhomogeneous change in anesthetic sensitivity corroborates findings in other K2P knockout mice and supports the theory that the mechanism of volatile anesthetic action involves multiple targets. Although it was not shown in this study, a compensatory effect by other K2P channels may also contribute to these observations.

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

Global Nav1.7 Knockout Mice Recapitulate the Phenotype of Human Congenital Indifference to Pain

PubMed Central

Gingras, Jacinthe; Smith, Sarah; Matson, David J.; Johnson, Danielle; Nye, Kim; Couture, Lauren; Feric, Elma; Yin, Ruoyuan; Moyer, Bryan D.; Peterson, Matthew L.; Rottman, James B.; Beiler, Rudolph J.; Malmberg, Annika B.; McDonough, Stefan I.

2014-01-01

Clinical genetic studies have shown that loss of Nav1.7 function leads to the complete loss of acute pain perception. The global deletion is reported lethal in mice, however, and studies of mice with promoter-specific deletions of Nav1.7 have suggested that the role of Nav1.7 in pain transduction depends on the precise form of pain. We developed genetic and animal husbandry strategies that overcame the neonatal-lethal phenotype and enabled construction of a global Nav1.7 knockout mouse. Knockouts were anatomically normal, reached adulthood, and had phenotype wholly analogous to human congenital indifference to pain (CIP): compared to littermates, knockouts showed no defects in mechanical sensitivity or overall movement yet were completely insensitive to painful tactile, thermal, and chemical stimuli and were anosmic. Knockouts also showed no painful behaviors resulting from peripheral injection of nonselective sodium channel activators, did not develop complete Freund’s adjuvant-induced thermal hyperalgesia, and were insensitive to intra-dermal histamine injection. Tetrodotoxin-sensitive sodium current recorded from cell bodies of isolated sensory neurons and the mechanically-evoked spiking of C-fibers in a skin-nerve preparation each were reduced but not eliminated in tissue from knockouts compared to littermates. Results support a role for Nav1.7 that is conserved between rodents and humans and suggest several possibly translatable biomarkers for the study of Nav1.7-targeted therapeutics. Results further suggest that Nav1.7 may retain its key role in persistent as well as acute forms of pain. PMID:25188265

Anaplerotic Triheptanoin Diet Enhances Mitochondrial Substrate Use to Remodel the Metabolome and Improve Lifespan, Motor Function, and Sociability in MeCP2-Null Mice

PubMed Central

Li, Qun; Degano, Alicia L.; Penati, Judith; Zhuo, Justin; Roe, Charles R.; Ronnett, Gabriele V.

2014-01-01

Rett syndrome (RTT) is an autism spectrum disorder (ASD) caused by mutations in the X-linked MECP2 gene that encodes methyl-CpG binding protein 2 (MeCP2). Symptoms range in severity and include psychomotor disabilities, seizures, ataxia, and intellectual disability. Symptom onset is between 6-18 months of age, a critical period of brain development that is highly energy-dependent. Notably, patients with RTT have evidence of mitochondrial dysfunction, as well as abnormal levels of the adipokines leptin and adiponectin, suggesting overall metabolic imbalance. We hypothesized that one contributor to RTT symptoms is energy deficiency due to defective nutrient substrate utilization by the TCA cycle. This energy deficit would lead to a metabolic imbalance, but would be treatable by providing anaplerotic substrates to the TCA cycle to enhance energy production. We show that dietary therapy with triheptanoin significantly increased longevity and improved motor function and social interaction in male mice hemizygous for Mecp2 knockout. Anaplerotic therapy in Mecp2 knockout mice also improved indicators of impaired substrate utilization, decreased adiposity, increased glucose tolerance and insulin sensitivity, decreased serum leptin and insulin, and improved mitochondrial morphology in skeletal muscle. Untargeted metabolomics of liver and skeletal muscle revealed increases in levels of TCA cycle intermediates with triheptanoin diet, as well as normalizations of glucose and fatty acid biochemical pathways consistent with the improved metabolic phenotype in Mecp2 knockout mice on triheptanoin. These results suggest that an approach using dietary supplementation with anaplerotic substrate is effective in improving symptoms and metabolic health in RTT. PMID:25299635

Activated ERK1/2 increases CD44 in glomerular parietal epithelial cells leading to matrix expansion

PubMed Central

Roeder, Sebastian S.; Barnes, Taylor J.; Lee, Jonathan S.; Kato, India; Eng, Diana G.; Kaverina, Natalya V.; Sunseri, Maria W.; Daniel, Christoph; Amann, Kerstin; Pippin, Jeffrey W.; Shankland, Stuart J.

2017-01-01

The glycoprotein CD44 is barely detected in normal mouse and human glomeruli, but is increased in glomerular parietal epithelial cells following podocyte injury in focal segmental glomerulosclerosis (FSGS). To determine the biological role and regulation of CD44 in these cells, we employed an in vivo and in vitro approach. Experimental FSGS was induced in CD44 knockout and wildtype mice with a cytotoxic podocyte antibody. Albuminuria, focal and global glomerulosclerosis (periodic acid-Schiff stain) and collagen IV staining were lower in CD44 knockout compared with wild type mice with FSGS. Parietal epithelial cells had lower migration from Bowman’s capsule to the glomerular tuft in CD44 knockout mice with disease compared with wild type mice. In cultured murine parietal epithelial cells, overexpressing CD44 with a retroviral vector encoding CD44 was accompanied by significantly increased collagen IV expression and parietal epithelial cells migration. Because our results showed de novo co-staining for activated ERK1/2 (pERK) in parietal epithelial cells in experimental FSGS, and also in biopsies from patients with FSGS, two in vitro strategies were employed to prove that pERK regulated CD44 levels. First, mouse parietal epithelial cells were infected with a retroviral vector for the upstream kinase MEK-DD to increase pERK, which was accompanied by increased CD44 levels. Second, in CD44 overexpressing parietal epithelial cells, decreasing pERK with U0126 was accompanied by reduced CD44. Finally, parietal epithelial cell migration was higher in cells with increased and reduced in cells with decreased pERK. Thus, pERK is a regulator of CD44 expression and increased CD44 expression leads to a pro-sclerotic and migratory parietal epithelial cells phenotype. PMID:27998643

TgTKL1 Is a Unique Plant-Like Nuclear Kinase That Plays an Essential Role in Acute Toxoplasmosis

PubMed Central

Varberg, Joseph M.; Coppens, Isabelle; Arrizabalaga, Gustavo

2018-01-01

ABSTRACT In the protozoan parasite Toxoplasma gondii, protein kinases have been shown to play key roles in regulating parasite motility, invasion, replication, egress, and survival within the host. The tyrosine kinase-like (TKL) family of proteins are an unexplored set of kinases in Toxoplasma. Of the eight annotated TKLs in the Toxoplasma genome, a recent genome-wide loss-of-function screen showed that six are important for tachyzoite fitness. By utilizing an endogenous tagging approach, we showed that these six T. gondii TKLs (TgTKLs) localize to various subcellular compartments, including the nucleus, the cytosol, the inner membrane complex, and the Golgi apparatus. To gain insight into the function of TKLs in Toxoplasma, we first characterized TgTKL1, which contains the plant-like enhanced disease resistance 1 (EDR1) domain and localizes to the nucleus. TgTKL1 knockout parasites displayed significant defects in progression through the lytic cycle; we show that the defects were due to specific impairment of host cell attachment. Transcriptomics analysis identified over 200 genes of diverse functions that were differentially expressed in TgTKL1 knockout parasites. Importantly, numerous genes implicated in host cell attachment and invasion were among those most significantly downregulated, resulting in defects in microneme secretion and processing. Significantly, all of the mice inoculated intraperitoneally with TgTKL1 knockout parasites survived the infection, suggesting that TgTKL1 plays an essential role in acute toxoplasmosis. Together, these findings suggest that TgTKL1 mediates a signaling pathway that regulates the expression of multiple factors required for parasite virulence, underscoring the potential of this kinase as a novel therapeutic target. PMID:29559568

Deoxynucleoside stress exacerbates the phenotype of a mouse model of mitochondrial neurogastrointestinal encephalopathy

PubMed Central

Garcia-Diaz, Beatriz; Garone, Caterina; Barca, Emanuele; Mojahed, Hamed; Gutierrez, Purification; Pizzorno, Giuseppe; Tanji, Kurenai; Arias-Mendoza, Fernando; Quinzii, Caterina M.

2014-01-01

Balanced pools of deoxyribonucleoside triphosphate precursors are required for DNA replication, and alterations of this balance are relevant to human mitochondrial diseases including mitochondrial neurogastrointestinal encephalopathy. In this disease, autosomal recessive TYMP mutations cause severe reductions of thymidine phosphorylase activity; marked elevations of the pyrimidine nucleosides thymidine and deoxyuridine in plasma and tissues, and somatic multiple deletions, depletion and site-specific point mutations of mitochondrial DNA. Thymidine phosphorylase and uridine phosphorylase double knockout mice recapitulated several features of these patients including thymidine phosphorylase activity deficiency, elevated thymidine and deoxyuridine in tissues, mitochondrial DNA depletion, respiratory chain defects and white matter changes. However, in contrast to patients with this disease, mutant mice showed mitochondrial alterations only in the brain. To test the hypothesis that elevated levels of nucleotides cause unbalanced deoxyribonucleoside triphosphate pools and, in turn, pathogenic mitochondrial DNA instability, we have stressed double knockout mice with exogenous thymidine and deoxyuridine, and assessed clinical, neuroradiological, histological, molecular, and biochemical consequences. Mutant mice treated with exogenous thymidine and deoxyuridine showed reduced survival, body weight, and muscle strength, relative to untreated animals. Moreover, in treated mutants, leukoencephalopathy, a hallmark of the disease, was enhanced and the small intestine showed a reduction of smooth muscle cells and increased fibrosis. Levels of mitochondrial DNA were depleted not only in the brain but also in the small intestine, and deoxyribonucleoside triphosphate imbalance was observed in the brain. The relative proportion, rather than the absolute amount of deoxyribonucleoside triphosphate, was critical for mitochondrial DNA maintenance. Thus, our results demonstrate that stress of exogenous pyrimidine nucleosides enhances the mitochondrial phenotype of our knockout mice. Our mouse studies provide insights into the pathogenic role of thymidine and deoxyuridine imbalance in mitochondrial neurogastrointestinal encephalopathy and an excellent model to study new therapeutic approaches. PMID:24727567

Abnormal Sleep/Wake Dynamics in Orexin Knockout Mice

PubMed Central

Diniz Behn, Cecilia G.; Klerman, Elizabeth B.; Mochizuki, Takatoshi; Lin, Shih-Chieh; Scammell, Thomas E.

2010-01-01

Study Objectives: Narcolepsy with cataplexy is caused by a loss of orexin (hypocretin) signaling, but the physiologic mechanisms that result in poor maintenance of wakefulness and fragmented sleep remain unknown. Conventional scoring of sleep cannot reveal much about the process of transitioning between states or the variations within states. We developed an EEG spectral analysis technique to determine whether the state instability in a mouse model of narcolepsy reflects abnormal sleep or wake states, faster movements between states, or abnormal transitions between states. Design: We analyzed sleep recordings in orexin knockout (OXKO) mice and wild type (WT) littermates using a state space analysis technique. This non-categorical approach allows quantitative and unbiased examination of sleep/wake states and state transitions. Measurements and Results: OXKO mice spent less time in deep, delta-rich NREM sleep and in active, theta-rich wake and instead spent more time near the transition zones between states. In addition, while in the midst of what should be stable wake, OXKO mice initiated rapid changes into NREM sleep with high velocities normally seen only in transition regions. Consequently, state transitions were much more frequent and rapid even though the EEG progressions during state transitions were normal. Conclusions: State space analysis enables visualization of the boundaries between sleep and wake and shows that narcoleptic mice have less distinct and more labile states of sleep and wakefulness. These observations provide new perspectives on the abnormal state dynamics resulting from disrupted orexin signaling and highlight the usefulness of state space analysis in understanding narcolepsy and other sleep disorders. Citation: Diniz Behn CG; Klerman EB; Mochizuki T; Lin S; Scammell TE. Abnormal sleep/wake dynamics in orexin knockout mice. SLEEP 2010;33(3):297-306. PMID:20337187

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.

T-Cell Mineralocorticoid Receptor Controls Blood Pressure by Regulating Interferon-Gamma.

PubMed

Sun, Xue-Nan; Li, Chao; Liu, Yuan; Du, Lin-Juan; Zeng, Meng-Ru; Zheng, Xiao-Jun; Zhang, Wu-Chang; Liu, Yan; Zhu, Mingjiang; Kong, Deping; Zhou, Li; Lu, Limin; Shen, Zhu-Xia; Yi, Yi; Du, Lili; Qin, Mu; Liu, Xu; Hua, Zichun; Sun, Shuyang; Yin, Huiyong; Zhou, Bin; Yu, Ying; Zhang, Zhiyuan; Duan, Sheng-Zhong

2017-05-12

Hypertension remains to be a global public health burden and demands novel intervention strategies such as targeting T cells and T-cell-derived cytokines. Mineralocorticoid receptor (MR) antagonists have been clinically used to treat hypertension. However, the function of T-cell MR in blood pressure (BP) regulation has not been elucidated. We aim to determine the role of T-cell MR in BP regulation and to explore the mechanism. Using T-cell MR knockout mouse in combination with angiotensin II-induced hypertensive mouse model, we demonstrated that MR deficiency in T cells strikingly decreased both systolic and diastolic BP and attenuated renal and vascular damage. Flow cytometric analysis showed that T-cell MR knockout mitigated angiotensin II-induced accumulation of interferon-gamma (IFN-γ)-producing T cells, particularly CD8 + population, in both kidneys and aortas. Similarly, eplerenone attenuated angiotensin II-induced elevation of BP and accumulation of IFN-γ-producing T cells in wild-type mice. In cultured CD8 + T cells, T-cell MR knockout suppressed IFN-γ expression whereas T-cell MR overexpression and aldosterone both enhanced IFN-γ expression. At the molecular level, MR interacted with NFAT1 (nuclear factor of activated T-cells 1) and activator protein-1 in T cells. Finally, T-cell MR overexpressing mice manifested more elevated BP compared with control mice after angiotensin II infusion and such difference was abolished by IFN-γ-neutralizing antibodies. MR may interact with NFAT1 and activator protein-1 to control IFN-γ in T cells and to regulate target organ damage and ultimately BP. Targeting MR in T cells specifically may be an effective novel approach for hypertension treatment. © 2017 American Heart Association, Inc.

Differential long-term effects of MDMA on the serotoninergic system and hippocampal cell proliferation in 5-HTT knock-out vs. wild-type mice.

PubMed

Renoir, Thibault; Païzanis, Eleni; El Yacoubi, Malika; Saurini, Françoise; Hanoun, Naïma; Melfort, Maxette; Lesch, Klaus Peter; Hamon, Michel; Lanfumey, Laurence

2008-12-01

Although numerous studies investigated the mechanisms underlying 3,4-methylenedioxymethamphetamine (MDMA)-induced neurotoxicity, little is known about its long-term functional consequences on 5-HT neurotransmission in mice. This led us to evaluate the delayed effects of MDMA exposure on the 5-HT system, using in-vitro and in-vivo approaches in both 5-HTT wild-type and knock-out mice. Acute MDMA in-vitro application on slices of the dorsal raphe nucleus (DRN) induced concentration-dependent 5-HT release and 5-HT cell firing inhibition. Four weeks after MDMA administration (20 mg/kg b.i.d for 4 d), a 2-fold increase in the potency of the 5-HT1A receptor agonist ipsapirone to inhibit the discharge of DRN 5-HT neurons and a larger hypothermic response to 8-OH-DPAT were observed in MDMA- compared to saline-treated mice. This adaptive 5-HT1A autoreceptor supersensitivity was associated with decreases in 5-HT levels but no changes of [3H]citalopram binding in brain. Long-term MDMA treatment also induced a 30% decrease in BrdU labelling of proliferating hippocampal cells and an increased immobility duration in the forced swim test suggesting a depressive-like behaviour induced by MDMA treatment. All these effects were abolished in 5-HTT-/- knock-out mice. These data indicated that, in mice, MDMA administration induced a delayed adaptive supersensitivity of 5-HT1A autoreceptors in the DRN, a deficit in hippocampal cell proliferation and a depressive-like behaviour. These 5-HTT-dependent effects, opposite to those of antidepressants, might contribute to MDMA-induced mood disorders.

Altered prostate epithelial development in mice lacking the androgen receptor in stromal fibroblasts.

PubMed

Yu, Shengqiang; Yeh, Chiuan-Ren; Niu, Yuanjie; Chang, Hong-Chiang; Tsai, Yu-Chieh; Moses, Harold L; Shyr, Chih-Rong; Chang, Chawnshang; Yeh, Shuyuan

2012-03-01

Androgens and the androgen receptor (AR) play important roles in the development of male urogenital organs. We previously found that mice with total AR knockout (ARKO) and epithelial ARKO failed to develop normal prostate with loss of differentiation. We have recently knocked out AR gene in smooth muscle cells and found the reduced luminal infolding and IGF-1 production in the mouse prostate. However, AR roles of stromal fibroblasts in prostate development remain unclear. To further probe the stromal fibroblast AR roles in prostate development, we generated tissue-selective knockout mice with the AR gene deleted in stromal fibroblasts (FSP-ARKO). We also used primary culture stromal cells to confirm the in vivo data and investigate mechanisms related to prostate development. The results showed cellular alterations in the FSP-ARKO mouse prostate with decreased epithelial proliferation, increased apoptosis, and decreased collagen composition. Further mechanistic studies demonstrated that FSP-ARKO mice have defects in the expression of prostate stromal growth factors. To further confirm these in vivo findings, we prepared primary cultured mouse prostate stromal cells and found knocking down the stromal AR could result in growth retardation of prostate stromal cells and co-cultured prostate epithelial cells, as well as decrease of some stromal growth factors. Our FSP-ARKO mice not only provide the first in vivo evidence in Cre-loxP knockout system for the requirement of stromal fibroblast AR to maintain the normal development of the prostate, but may also suggest the selective knockdown of stromal AR might become a potential therapeutic approach to battle prostate hyperplasia and cancer. Copyright © 2011 Wiley Periodicals, Inc.

ATP-Sensitive K+ Channel Knockout Induces Cardiac Proteome Remodeling Predictive of Heart Disease Susceptibility

PubMed Central

Arrell, D. Kent; Zlatkovic, Jelena; Kane, Garvan C.; Yamada, Satsuki; Terzic, Andre

2010-01-01

Forecasting disease susceptibility requires detection of maladaptive signatures prior to onset of overt symptoms. A case-in-point are cardiac ATP-sensitive K+ (KATP) channelopathies, for which the substrate underlying disease vulnerability remains to be identified. Resolving molecular pathobiology, even for single genetic defects, mandates a systems platform to reliably diagnose disease predisposition. High-throughput proteomic analysis was here integrated with network biology to decode consequences of Kir6.2 KATP channel pore deletion. Differential two-dimensional gel electrophoresis reproducibly resolved > 800 protein species from hearts of asymptomatic wild-type and Kir6.2-knockout counterparts. KATP channel ablation remodeled the cardiac proteome, significantly altering 71 protein spots, from which 102 unique identities were assigned following hybrid linear ion trap quadrupole-Orbitrap tandem mass spectrometry. Ontological annotation stratified the KATP channel-dependent protein cohort into a predominant bioenergetic module (63 resolved identities), with additional focused sets representing signaling molecules (6), oxidoreductases (8), chaperones (6), and proteins involved in catabolism (6), cytostructure (8), and transcription and translation (5). Protein interaction mapping, in conjunction with expression level changes, localized a KATP channel-associated subproteome within a nonstochastic scale-free network. Global assessment of the KATP channel deficient environment verified the primary impact on metabolic pathways and revealed overrepresentation of markers associated with cardiovascular disease. Experimental imposition of graded stress precipitated exaggerated structural and functional myocardial defects in the Kir6.2-knockout, decreasing survivorship and validating the forecast of disease susceptibility. Proteomic cartography thus provides an integral view of molecular remodeling in the heart induced by KATP channel deletion, establishing a systems approach that predicts outcome at a presymptomatic stage. PMID:19673485

Integrative approach for inference of gene regulatory networks using lasso-based random featuring and application to psychiatric disorders.

PubMed

Kim, Dongchul; Kang, Mingon; Biswas, Ashis; Liu, Chunyu; Gao, Jean

2016-08-10

Inferring gene regulatory networks is one of the most interesting research areas in the systems biology. Many inference methods have been developed by using a variety of computational models and approaches. However, there are two issues to solve. First, depending on the structural or computational model of inference method, the results tend to be inconsistent due to innately different advantages and limitations of the methods. Therefore the combination of dissimilar approaches is demanded as an alternative way in order to overcome the limitations of standalone methods through complementary integration. Second, sparse linear regression that is penalized by the regularization parameter (lasso) and bootstrapping-based sparse linear regression methods were suggested in state of the art methods for network inference but they are not effective for a small sample size data and also a true regulator could be missed if the target gene is strongly affected by an indirect regulator with high correlation or another true regulator. We present two novel network inference methods based on the integration of three different criteria, (i) z-score to measure the variation of gene expression from knockout data, (ii) mutual information for the dependency between two genes, and (iii) linear regression-based feature selection. Based on these criterion, we propose a lasso-based random feature selection algorithm (LARF) to achieve better performance overcoming the limitations of bootstrapping as mentioned above. In this work, there are three main contributions. First, our z score-based method to measure gene expression variations from knockout data is more effective than similar criteria of related works. Second, we confirmed that the true regulator selection can be effectively improved by LARF. Lastly, we verified that an integrative approach can clearly outperform a single method when two different methods are effectively jointed. In the experiments, our methods were validated by outperforming the state of the art methods on DREAM challenge data, and then LARF was applied to inferences of gene regulatory network associated with psychiatric disorders.

ADAM10 is essential for cranial neural crest-derived maxillofacial bone development

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

Tan, Yu, E-mail: tanyu2048@163.com; Fu, Runqing, E-mail: furunqing@sjtu.edu.cn; Liu, Jiaqiang, E-mail: liujqmj@163.com

Growth disorders of the craniofacial bones may lead to craniofacial deformities. The majority of maxillofacial bones are derived from cranial neural crest cells via intramembranous bone formation. Any interruption of the craniofacial skeleton development process might lead to craniofacial malformation. A disintegrin and metalloprotease (ADAM)10 plays an essential role in organ development and tissue integrity in different organs. However, little is known about its function in craniofacial bone formation. Therefore, we investigated the role of ADAM10 in the developing craniofacial skeleton, particularly during typical mandibular bone development. First, we showed that ADAM10 was expressed in a specific area of themore » craniofacial bone and that the expression pattern dynamically changed during normal mouse craniofacial development. Then, we crossed wnt1-cre transgenic mice with adam10-flox mice to generate ADAM10 conditional knockout mice. The stereomicroscopic, radiographic, and von Kossa staining results showed that conditional knockout of ADAM10 in cranial neural crest cells led to embryonic death, craniofacial dysmorphia and bone defects. Furthermore, we demonstrated that impaired mineralization could be triggered by decreased osteoblast differentiation, increased cell death. Overall, these findings show that ADAM10 plays an essential role in craniofacial bone development. -- Highlights: •We firstly reported that ADAM10 was essentially involved in maxillofacial bone development. •ADAM10 cKO mice present craniofacial dysmorphia and bone defects. •Impaired osteoblast differentiation,proliferation and apoptosis underlie the bone deformity.« less

Postnatal Loss of Mef2c Results in Dissociation of Effects on Synapse Number and Learning and Memory.

PubMed

Adachi, Megumi; Lin, Pei-Yi; Pranav, Heena; Monteggia, Lisa M

2016-07-15

Myocyte enhancer factor 2 (MEF2) transcription factors play critical roles in diverse cellular processes during central nervous system development. Studies attempting to address the role of MEF2 in brain have largely relied on overexpression of a constitutive MEF2 construct that impairs memory formation or knockdown of MEF2 function that increases spine numbers and enhances memory formation. Genetic deletion of individual MEF2 isoforms in brain during embryogenesis demonstrated that Mef2c loss negatively regulates spine numbers resulting in learning and memory deficits, possibly as a result of its essential role in development. To investigate MEF2C function in brain further, we genetically deleted Mef2c during postnatal development in mice. We characterized these conditional Mef2c knockout mice in an array of behavioral paradigms and examined the impact of postnatal loss of Mef2c on long-term potentiation. We observed increased spine numbers in hippocampus of the conditional Mef2c knockout mice. However, the postnatal loss of Mef2c did not impact learning and memory, long-term potentiation, or social and repetitive behaviors. Our findings demonstrate a critical role for MEF2C in the regulation of spine numbers with a dissociation of learning and memory, synaptic plasticity, and measures of autism-related behaviors in postnatal brain. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Detection of ultrastructural changes in genetically altered and exercised skeletal muscle using PS-OCT

NASA Astrophysics Data System (ADS)

Pasquesi, James J.; Schlachter, Simon C.; Boppart, Marni D.; Chaney, Eric; Kaufman, Stephen J.; Boppart, Stephen A.

2006-02-01

Birefringence of skeletal muscle has been associated with the ultrastructure of individual sarcomeres, specifically the arrangement of A-bands corresponding to the thick myosin filaments. Murine skeletal muscle (gastrocnemius) was imaged with a fiber-based PS-OCT imaging system to determine the level of birefringence present in the tissue under various conditions. In addition to muscle controls from wild-type mice, muscle from abnormal mice included: genetically-modified (mdx) mice which model human muscular dystrophy, transgenic mice exhibiting an overexpression of integrin (α7β1), and transgenic integrin (α7β1)knockout mice. Comparisons were also made between rested and exercised muscles to determine the effects of exercise on muscle birefringence for each of these normal and abnormal conditions. The PS-OCT images revealed that the presence of birefringence was similar in the rested muscle with dystrophy-like features (i.e., lacking the structural protein dystrophin - mdx) and in the integrin (α7β1)knockout muscle when compared to the normal (wild-type) control. However, exercising these abnormal muscle tissues drastically reduced the presence of birefringence detected by the PS-OCT system. The muscle exhibiting an overexpression of integrin (α7β1) remained heavily birefringent before and after exercise, similar to the normal (wild-type) muscle. These results suggest that there is a distinct relationship between the degree of birefringence detected using PS-OCT and the sarcomeric ultrastructure present within skeletal muscle.