Science.gov

Sample records for phototransduction gene knockout

  1. Transcriptome survey of phototransduction and clock genes in marine bivalves.

    PubMed

    Sun, X J; Zhou, L Q; Tian, J T; Liu, Z H; Wu, B; Dong, Y H; Yang, A G; Ma, W M

    2016-10-24

    Marine animals exhibit a variety of biological rhythms, such as solar and lunar-related cycles; however, our current molecular understanding of biological rhythms in marine animals is quite limited. Identifying and understanding the expression patterns of clock genes from available transcriptomes will help elucidate biological rhythms in marine species. Here, we perform a comprehensive survey of phototransduction and circadian genes using the mantle transcriptome of the scallop Patinopecten yessoensis and compare the results with those from three other bivalves. The comparison reveals the presence of transcripts for most of the core members of the phototransduction and circadian networks seen in terrestrial model species in the four marine bivalves. Matches were found for all 37 queried genes, and the expressed transcripts from the deep sequencing data matched 8 key insect and mammalian circadian genes. This demonstrates the high level of conservation of the timekeeping mechanism from terrestrial species to marine bivalves. The results provide a valuable gene resource for studies of "marine rhythms" and also further our understanding of the diversification and evolution of rhythms in marine species.

  2. Phototransduction and clock gene expression in the troglobiont beetle Ptomaphagus hirtus of Mammoth cave.

    PubMed

    Friedrich, Markus; Chen, Rui; Daines, Bryce; Bao, Riyue; Caravas, Jason; Rai, Puneet K; Zagmajster, Maja; Peck, Stewart B

    2011-11-01

    Obligatory cave species exhibit dramatic trait modifications such as eye reduction, loss of pigmentation and an increase in touch receptors. As molecular studies of cave adaptation have largely concentrated on vertebrate models, it is not yet possible to probe for genetic universalities underlying cave adaptation. We have therefore begun to study the strongly cave-adapted small carrion beetle Ptomaphagus hirtus. For over 100 years, this flightless signature inhabitant of Mammoth Cave, the world's largest known cave system, has been considered blind despite the presence of residual lens structures. By deep sequencing of the adult head transcriptome, we discovered the transcripts of all core members of the phototransduction protein machinery. Combined with the absence of transcripts of select structural photoreceptor and eye pigmentation genes, these data suggest a reduced but functional visual system in P. hirtus. This conclusion was corroborated by a negative phototactic response of P. hirtus in light/dark choice tests. We further detected the expression of the complete circadian clock gene network in P. hirtus, raising the possibility of a role of light sensation in the regulation of oscillating processes. We speculate that P. hirtus is representative of a large number of animal species with highly reduced but persisting visual capacities in the twilight zone of the subterranean realm. These can now be studied on a broad comparative scale given the efficiency of transcript discovery by next-generation sequencing.

  3. Method for determining gene knockouts

    DOEpatents

    Maranas, Costas D.; Burgard, Anthony R.; Pharkya, Priti

    2011-09-27

    A method for determining candidates for gene deletions and additions using a model of a metabolic network associated with an organism, the model includes a plurality of metabolic reactions defining metabolite relationships, the method includes selecting a bioengineering objective for the organism, selecting at least one cellular objective, forming an optimization problem that couples the at least one cellular objective with the bioengineering objective, and solving the optimization problem to yield at least one candidate.

  4. Method for determining gene knockouts

    DOEpatents

    Maranas, Costa D; Burgard, Anthony R; Pharkya, Priti

    2013-06-04

    A method for determining candidates for gene deletions and additions using a model of a metabolic network associated with an organism, the model includes a plurality of metabolic reactions defining metabolite relationships, the method includes selecting a bioengineering objective for the organism, selecting at least one cellular objective, forming an optimization problem that couples the at least one cellular objective with the bioengineering objective, and solving the optimization problem to yield at least one candidate.

  5. Eye-independent, light-activated chromatophore expansion (LACE) and expression of phototransduction genes in the skin of Octopus bimaculoides.

    PubMed

    Ramirez, M Desmond; Oakley, Todd H

    2015-05-15

    Cephalopods are renowned for changing the color and pattern of their skin for both camouflage and communication. Yet, we do not fully understand how cephalopods control the pigmented chromatophore organs in their skin and change their body pattern. Although these changes primarily rely on eyesight, we found that light causes chromatophores to expand in excised pieces of Octopus bimaculoides skin. We call this behavior light-activated chromatophore expansion (or LACE). To uncover how octopus skin senses light, we used antibodies against r-opsin phototransduction proteins to identify sensory neurons that express r-opsin in the skin. We hypothesized that octopus LACE relies on the same r-opsin phototransduction cascade found in octopus eyes. By creating an action spectrum for the latency to LACE, we found that LACE occurred most quickly in response to blue light. We fit our action spectrum data to a standard opsin curve template and estimated the λmax of LACE to be 480 nm. Consistent with our hypothesis, the maximum sensitivity of the light sensors underlying LACE closely matches the known spectral sensitivity of opsin from octopus eyes. LACE in isolated preparations suggests that octopus skin is intrinsically light sensitive and that this dispersed light sense might contribute to their unique and novel patterning abilities. Finally, our data suggest that a common molecular mechanism for light detection in eyes may have been co-opted for light sensing in octopus skin and then used for LACE.

  6. Eye-independent, light-activated chromatophore expansion (LACE) and expression of phototransduction genes in the skin of Octopus bimaculoides

    PubMed Central

    Ramirez, M. Desmond; Oakley, Todd H.

    2015-01-01

    ABSTRACT Cephalopods are renowned for changing the color and pattern of their skin for both camouflage and communication. Yet, we do not fully understand how cephalopods control the pigmented chromatophore organs in their skin and change their body pattern. Although these changes primarily rely on eyesight, we found that light causes chromatophores to expand in excised pieces of Octopus bimaculoides skin. We call this behavior light-activated chromatophore expansion (or LACE). To uncover how octopus skin senses light, we used antibodies against r-opsin phototransduction proteins to identify sensory neurons that express r-opsin in the skin. We hypothesized that octopus LACE relies on the same r-opsin phototransduction cascade found in octopus eyes. By creating an action spectrum for the latency to LACE, we found that LACE occurred most quickly in response to blue light. We fit our action spectrum data to a standard opsin curve template and estimated the λmax of LACE to be 480 nm. Consistent with our hypothesis, the maximum sensitivity of the light sensors underlying LACE closely matches the known spectral sensitivity of opsin from octopus eyes. LACE in isolated preparations suggests that octopus skin is intrinsically light sensitive and that this dispersed light sense might contribute to their unique and novel patterning abilities. Finally, our data suggest that a common molecular mechanism for light detection in eyes may have been co-opted for light sensing in octopus skin and then used for LACE. PMID:25994633

  7. Exploring the opioid system by gene knockout.

    PubMed

    Kieffer, Brigitte L; Gavériaux-Ruff, Claire

    2002-04-01

    The endogenous opioid system consists of three opioid peptide precursor genes encoding enkephalins (preproenkephalin, Penk), dynorphins (preprodynorphin, Pdyn) and beta-endorphin (betaend), proopiomelanocortin (POMC) and three receptor genes encoding mu-opiod receptor (MOR), delta-opiod receptor (DOR) and kappa-opiod receptor (KOR). In the past years, all six genes have been inactivated in mice by homologous recombination. The analysis of spontaneous behavior in mutant mice has demonstrated significant and distinct roles of each gene in modulating locomotion, pain perception and emotional behaviors. The observation of opposing phenotypes of MOR- and DOR-deficient mice in several behaviors highlights unexpected roles for DOR to be further explored genetically and using more specific delta compounds. The analysis of responses of mutant mice to exogenous opiates has definitely clarified the essential role of MOR in both morphine analgesia and addiction, and demonstrated that DOR and KOR remain promising targets for pain treatment. These studies also show that prototypic DOR agonists partially require MOR for their biological activity and provide some support for the postulated mu-delta interactions in vivo. Finally, data confirm and define a role for several genes of the opioid system in responses to other drugs of abuse, and the triple opioid receptor knockout mutant allows exploring non-classical opioid pharmacology. In summary, the study of null mutant mice has extended our previous knowledge of the opioid system by identifying the molecular players in opioid pharmacology and physiology. Future studies should involve parallel behavioral analysis of mice lacking receptors and peptides and will benefit from more sophisticated gene targeting approaches, including site-directed and anatomically-restricted mutations.

  8. Inhibition of SULT4A1 expression induces up-regulation of phototransduction gene expression in 72-hour postfertilization zebrafish larvae.

    PubMed

    Crittenden, Frank; Thomas, Holly; Ethen, Cheryl M; Wu, Zhengliang L; Chen, Dongquan; Kraft, Timothy W; Parant, John M; Falany, Charles N

    2014-05-01

    Sulfotransferase (SULT) 4A1 is an orphan enzyme that shares distinct structure and sequence similarities with other cytosolic SULTs. SULT4A1 is primarily expressed in neuronal tissue and is also the most conserved SULT, having been identified in every vertebrate investigated to date. Certain haplotypes of the SULT4A1 gene are correlated with higher baseline psychopathology in schizophrenic patients, but no substrate or function for SULT4A1 has yet been identified despite its high level of sequence conservation. In this study, deep RNA sequencing was used to search for alterations in gene expression in 72-hour postfertilization zebrafish larvae following transient SULT4A1 knockdown (KD) utilizing splice blocking morpholino oligonucleotides. This study demonstrates that transient inhibition of SULT4A1 expression in developing zebrafish larvae results in the up-regulation of several genes involved in phototransduction. SULT4A1 KD was verified by immunoblot analysis and quantitative real-time polymerase chain reaction (qPCR). Gene regulation changes identified by deep RNA sequencing were validated by qPCR. This study is the first identification of a cellular process whose regulation appears to be associated with SULT4A1 expression.

  9. Inhibition of SULT4A1 Expression Induces Up-Regulation of Phototransduction Gene Expression in 72-Hour Postfertilization Zebrafish Larvae

    PubMed Central

    Crittenden, Frank; Thomas, Holly; Ethen, Cheryl M.; Wu, Zhengliang L.; Chen, Dongquan; Kraft, Timothy W.; Parant, John M.

    2014-01-01

    Sulfotransferase (SULT) 4A1 is an orphan enzyme that shares distinct structure and sequence similarities with other cytosolic SULTs. SULT4A1 is primarily expressed in neuronal tissue and is also the most conserved SULT, having been identified in every vertebrate investigated to date. Certain haplotypes of the SULT4A1 gene are correlated with higher baseline psychopathology in schizophrenic patients, but no substrate or function for SULT4A1 has yet been identified despite its high level of sequence conservation. In this study, deep RNA sequencing was used to search for alterations in gene expression in 72-hour postfertilization zebrafish larvae following transient SULT4A1 knockdown (KD) utilizing splice blocking morpholino oligonucleotides. This study demonstrates that transient inhibition of SULT4A1 expression in developing zebrafish larvae results in the up-regulation of several genes involved in phototransduction. SULT4A1 KD was verified by immunoblot analysis and quantitative real-time polymerase chain reaction (qPCR). Gene regulation changes identified by deep RNA sequencing were validated by qPCR. This study is the first identification of a cellular process whose regulation appears to be associated with SULT4A1 expression. PMID:24553382

  10. The biology of novel animal genes: Mouse APEX gene knockout

    SciTech Connect

    MacInnes, M.; Altherr, M.R.; Ludwig, D.; Pedersen, R.; Mold, C.

    1997-07-01

    This is the final report of a one-year, Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). The controlled breeding of novel genes into mice, including the gene knockout (KO), or conversely by adding back transgenes provide powerful genetic technologies that together suffice to determine in large part the biological role(s) of novel genes. Inbred mouse remains the best understood and most useful mammalian experimental system available for tackling the biology of novel genes. The major mammalian apurinic/apyrimidinic (AP) endonuclease (APE), is involved in a key step in the repair of spontaneous and induced AP sites in DNA. Efficient repair of these lesions is imperative to prevent the stable incorporation of mutations into the cellular genome which may lead to cell death or transformation. Loss or modulation of base excison repair activity in vivo may elevate the spontaneous mutation rate in cells, and may lead to a substantial increase in the incidence of cancer. Despite extensive biochemical analysis, however, the significance of these individual APE functions in vivo has not been elucidated. Mouse embryonic stem (ES) cells heterozygous for a deletion mutation in APE have been generated and whole animals containing the APE mutation have been derived from these ES cells. Animals homozygous for the APE null mutation die early in gestation, underscoring the biological significance of this DNA repair gene.

  11. Why Drosophila to Study Phototransduction?

    PubMed Central

    Pak, William L.

    2010-01-01

    This review recounts the early history of Drosophila phototransduction genetics, covering the period between approximately 1966 to 1979. Early in this period, the author felt that there was an urgent need for a new approach in phototransduction research. Through inputs from a number of colleagues, he was led to consider isolating Drosophila mutants that are defective in the electroretinogram. Thanks to the efforts of dedicated associates and technical staff, by the end of this period, he was able to accumulate a large number of such mutants. Particularly important in this effort was the use of the mutant assay protocol based on the “prolonged depolarizing afterpotential.” This collection of mutants formed the basis of the subsequent intensive investigations of the Drosophila phototransduction cascade by many investigators. PMID:20536286

  12. Gene profiling of postnatal Mfrprd6 mutant eyes reveals differential accumulation of Prss56, visual cycle and phototransduction mRNAs.

    PubMed

    Soundararajan, Ramani; Won, Jungyeon; Stearns, Timothy M; Charette, Jeremy R; Hicks, Wanda L; Collin, Gayle B; Naggert, Jürgen K; Krebs, Mark P; Nishina, Patsy M

    2014-01-01

    Mutations in the membrane frizzled-related protein (MFRP/Mfrp) gene, specifically expressed in the retinal pigment epithelium (RPE) and ciliary body, cause nanophthalmia or posterior microphthalmia with retinitis pigmentosa in humans, and photoreceptor degeneration in mice. To better understand MFRP function, microarray analysis was performed on eyes of homozygous Mfrprd6 and C57BL/6J mice at postnatal days (P) 0 and P14, prior to photoreceptor loss. Data analysis revealed no changes at P0 but significant differences in RPE and retina-specific transcripts at P14, suggesting a postnatal influence of the Mfrprd6 allele. A subset of these transcripts was validated by quantitative real-time PCR (qRT-PCR). In Mfrprd6 eyes, a significant 1.5- to 2.0-fold decrease was observed among transcripts of genes linked to retinal degeneration, including those involved in visual cycle (Rpe65, Lrat, Rgr), phototransduction (Pde6a, Guca1b, Rgs9), and photoreceptor disc morphogenesis (Rpgrip1 and Fscn2). Levels of RPE65 were significantly decreased by 2.0-fold. Transcripts of Prss56, a gene associated with angle-closure glaucoma, posterior microphthalmia and myopia, were increased in Mfrprd6 eyes by 17-fold. Validation by qRT-PCR indicated a 3.5-, 14- and 70-fold accumulation of Prss56 transcripts relative to controls at P7, P14 and P21, respectively. This trend was not observed in other RPE or photoreceptor mutant mouse models with similar disease progression, suggesting that Prss56 upregulation is a specific attribute of the disruption of Mfrp. Prss56 and Glul in situ hybridization directly identified Müller glia in the inner nuclear layer as the cell type expressing Prss56. In summary, the Mfrprd6 allele causes significant postnatal changes in transcript and protein levels in the retina and RPE. The link between Mfrp deficiency and Prss56 up-regulation, together with the genetic association of human MFRP or PRSS56 variants and ocular size, raises the possibility that these genes

  13. Gene Profiling of Postnatal Mfrprd6 Mutant Eyes Reveals Differential Accumulation of Prss56, Visual Cycle and Phototransduction mRNAs

    PubMed Central

    Soundararajan, Ramani; Won, Jungyeon; Stearns, Timothy M.; Charette, Jeremy R.; Hicks, Wanda L.; Collin, Gayle B.; Naggert, Jürgen K.; Krebs, Mark P.; Nishina, Patsy M.

    2014-01-01

    Mutations in the membrane frizzled-related protein (MFRP/Mfrp) gene, specifically expressed in the retinal pigment epithelium (RPE) and ciliary body, cause nanophthalmia or posterior microphthalmia with retinitis pigmentosa in humans, and photoreceptor degeneration in mice. To better understand MFRP function, microarray analysis was performed on eyes of homozygous Mfrprd6 and C57BL/6J mice at postnatal days (P) 0 and P14, prior to photoreceptor loss. Data analysis revealed no changes at P0 but significant differences in RPE and retina-specific transcripts at P14, suggesting a postnatal influence of the Mfrprd6 allele. A subset of these transcripts was validated by quantitative real-time PCR (qRT-PCR). In Mfrprd6 eyes, a significant 1.5- to 2.0-fold decrease was observed among transcripts of genes linked to retinal degeneration, including those involved in visual cycle (Rpe65, Lrat, Rgr), phototransduction (Pde6a, Guca1b, Rgs9), and photoreceptor disc morphogenesis (Rpgrip1 and Fscn2). Levels of RPE65 were significantly decreased by 2.0-fold. Transcripts of Prss56, a gene associated with angle-closure glaucoma, posterior microphthalmia and myopia, were increased in Mfrprd6 eyes by 17-fold. Validation by qRT-PCR indicated a 3.5-, 14- and 70-fold accumulation of Prss56 transcripts relative to controls at P7, P14 and P21, respectively. This trend was not observed in other RPE or photoreceptor mutant mouse models with similar disease progression, suggesting that Prss56 upregulation is a specific attribute of the disruption of Mfrp. Prss56 and Glul in situ hybridization directly identified Müller glia in the inner nuclear layer as the cell type expressing Prss56. In summary, the Mfrprd6 allele causes significant postnatal changes in transcript and protein levels in the retina and RPE. The link between Mfrp deficiency and Prss56 up-regulation, together with the genetic association of human MFRP or PRSS56 variants and ocular size, raises the possibility that these genes

  14. Visual responses in mice lacking critical components of all known retinal phototransduction cascades.

    PubMed

    Allen, Annette E; Cameron, Morven A; Brown, Timothy M; Vugler, Anthony A; Lucas, Robert J

    2010-11-29

    The mammalian visual system relies upon light detection by outer-retinal rod/cone photoreceptors and melanopsin-expressing retinal ganglion cells. Gnat1(-/-);Cnga3(-/-);Opn4(-/-) mice lack critical elements of each of these photoreceptive mechanisms via targeted disruption of genes encoding rod α transducin (Gnat1); the cone-specific α3 cyclic nucleotide gated channel subunit (Cnga3); and melanopsin (Opn4). Although assumed blind, we show here that these mice retain sufficiently widespread retinal photoreception to drive a reproducible flash electroretinogram (ERG). The threshold sensitivity of this ERG is similar to that of cone-based responses, however it is lost under light adapted conditions. Its spectral efficiency is consistent with that of rod opsin, but not cone opsins or melanopsin, indicating that it originates with light absorption by the rod pigment. The TKO light response survives intravitreal injection of U73122 (a phospholipase C antagonist), but is inhibited by a missense mutation of cone α transducin (Gnat2(cpfl3)), suggesting Gnat2-dependence. Visual responses in TKO mice extend beyond the retina to encompass the lateral margins of the lateral geniculate nucleus and components of the visual cortex. Our data thus suggest that a Gnat1-independent phototransduction mechanism downstream of rod opsin can support relatively widespread responses in the mammalian visual system. This anomalous rod opsin-based vision should be considered in experiments relying upon Gnat1 knockout to silence rod phototransduction.

  15. Evolution of Vertebrate Phototransduction: Cascade Activation

    PubMed Central

    Lamb, Trevor D.; Patel, Hardip; Chuah, Aaron; Natoli, Riccardo C.; Davies, Wayne I. L.; Hart, Nathan S.; Collin, Shaun P.; Hunt, David M.

    2016-01-01

    We applied high-throughput sequencing to eye tissue from several species of basal vertebrates (a hagfish, two species of lamprey, and five species of gnathostome fish), and we analyzed the mRNA sequences for the proteins underlying activation of the phototransduction cascade. The molecular phylogenies that we constructed from these sequences are consistent with the 2R WGD model of two rounds of whole genome duplication. Our analysis suggests that agnathans retain an additional representative (that has been lost in gnathostomes) in each of the gene families we studied; the evidence is strong for the G-protein α subunit (GNAT) and the cGMP phosphodiesterase (PDE6), and indicative for the cyclic nucleotide-gated channels (CNGA and CNGB). Two of the species (the hagfish Eptatretus cirrhatus and the lamprey Mordacia mordax) possess only a single class of photoreceptor, simplifying deductions about the composition of cascade protein isoforms utilized in their photoreceptors. For the other lamprey, Geotria australis, analysis of the ratios of transcript levels in downstream and upstream migrant animals permits tentative conclusions to be drawn about the isoforms used in four of the five spectral classes of photoreceptor. Overall, our results suggest that agnathan rod-like photoreceptors utilize the same GNAT1 as gnathostomes, together with a homodimeric PDE6 that may be agnathan-specific, whereas agnathan cone-like photoreceptors utilize a GNAT that may be agnathan-specific, together with the same PDE6C as gnathostomes. These findings help elucidate the evolution of the vertebrate phototransduction cascade from an ancestral chordate phototransduction cascade that existed prior to the vertebrate radiation. PMID:27189541

  16. Timing Is Everything: GTPase Regulation in Phototransduction

    PubMed Central

    Arshavsky, Vadim Y.; Wensel, Theodore G.

    2013-01-01

    As the molecular mechanisms of vertebrate phototransduction became increasingly clear in the 1980s, a persistent problem was the discrepancy between the slow GTP hydrolysis catalyzed by the phototransduction G protein, transducin, and the much more rapid physiological recovery of photoreceptor cells from light stimuli. Beginning with a report published in 1989, a series of studies revealed that transducin GTPase activity could approach the rate needed to explain physiological recovery kinetics in the presence of one or more factors present in rod outer segment membranes. One by one, these factors were identified, beginning with PDEγ, the inhibitory subunit of the cGMP phosphodiesterase activated by transducin. There followed the discovery of the crucial role played by the regulator of G protein signaling, RGS9, a member of a ubiquitous family of GTPase-accelerating proteins, or GAPs, for heterotrimeric G proteins. Soon after, the G protein β isoform Gβ5 was identified as an obligate partner subunit, followed by the discovery or R9AP, a transmembrane protein that anchors the RGS9 GAP complex to the disk membrane, and is essential for the localization, stability, and activity of this complex in vivo. The physiological importance of all of the members of this complex was made clear first by knockout mouse models, and then by the discovery of a human visual defect, bradyopsia, caused by an inherited deficiency in one of the GAP components. Further insights have been gained by high-resolution crystal structures of subcomplexes, and by extensive mechanistic studies both in vitro and in animal models. PMID:24265205

  17. Generation of Gene Knockout Mice by ES Cell Microinjection

    PubMed Central

    Longenecker, Glenn; Kulkarni, Ashok B

    2009-01-01

    This unit lists and describes protocols used in the production of chimeric mice leading to the generation of gene knockout mice. These protocols include the collection of blastocyst embryos, ES cell injection, and uterine transfer of injected blastocysts. Support protocols in the superovulation of blastocyst donor mice, generation of pseudopregnant recipients, fabrication of glass pipettes, and generation of germline mice are also included. Practical tips and solutions are mentioned to help troubleshoot problems that may occur. PMID:19731226

  18. HFE gene knockout produces mouse model of hereditary hemochromatosis

    PubMed Central

    Zhou, Xiao Yan; Tomatsu, Shunji; Fleming, Robert E.; Parkkila, Seppo; Waheed, Abdul; Jiang, Jinxing; Fei, Ying; Brunt, Elizabeth M.; Ruddy, David A.; Prass, Cynthia E.; Schatzman, Randall C.; O’Neill, Rosemary; Britton, Robert S.; Bacon, Bruce R.; Sly, William S.

    1998-01-01

    Hereditary hemochromatosis (HH) is a common autosomal recessive disease characterized by increased iron absorption and progressive iron storage that results in damage to major organs in the body. Recently, a candidate gene for HH called HFE encoding a major histocompatibility complex class I-like protein was identified by positional cloning. Nearly 90% of Caucasian HH patients have been found to be homozygous for the same mutation (C282Y) in the HFE gene. To test the hypothesis that the HFE gene is involved in regulation of iron homeostasis, we studied the effects of a targeted disruption of the murine homologue of the HFE gene. The HFE-deficient mice showed profound differences in parameters of iron homeostasis. Even on a standard diet, by 10 weeks of age, fasting transferrin saturation was significantly elevated compared with normal littermates (96 ± 5% vs. 77 ± 3%, P < 0.007), and hepatic iron concentration was 8-fold higher than that of wild-type littermates (2,071 ± 450 vs. 255 ± 23 μg/g dry wt, P < 0.002). Stainable hepatic iron in the HFE mutant mice was predominantly in hepatocytes in a periportal distribution. Iron concentrations in spleen, heart, and kidney were not significantly different. Erythroid parameters were normal, indicating that the anemia did not contribute to the increased iron storage. This study shows that the HFE protein is involved in the regulation of iron homeostasis and that mutations in this gene are responsible for HH. The knockout mouse model of HH will facilitate investigation into the pathogenesis of increased iron accumulation in HH and provide opportunities to evaluate therapeutic strategies for prevention or correction of iron overload. PMID:9482913

  19. Health and population effects of rare gene knockouts in adult humans with related parents.

    PubMed

    Narasimhan, Vagheesh M; Hunt, Karen A; Mason, Dan; Baker, Christopher L; Karczewski, Konrad J; Barnes, Michael R; Barnett, Anthony H; Bates, Chris; Bellary, Srikanth; Bockett, Nicholas A; Giorda, Kristina; Griffiths, Christopher J; Hemingway, Harry; Jia, Zhilong; Kelly, M Ann; Khawaja, Hajrah A; Lek, Monkol; McCarthy, Shane; McEachan, Rosie; O'Donnell-Luria, Anne; Paigen, Kenneth; Parisinos, Constantinos A; Sheridan, Eamonn; Southgate, Laura; Tee, Louise; Thomas, Mark; Xue, Yali; Schnall-Levin, Michael; Petkov, Petko M; Tyler-Smith, Chris; Maher, Eamonn R; Trembath, Richard C; MacArthur, Daniel G; Wright, John; Durbin, Richard; van Heel, David A

    2016-04-22

    Examining complete gene knockouts within a viable organism can inform on gene function. We sequenced the exomes of 3222 British adults of Pakistani heritage with high parental relatedness, discovering 1111 rare-variant homozygous genotypes with predicted loss of function (knockouts) in 781 genes. We observed 13.7% fewer homozygous knockout genotypes than we expected, implying an average load of 1.6 recessive-lethal-equivalent loss-of-function (LOF) variants per adult. When genetic data were linked to the individuals' lifelong health records, we observed no significant relationship between gene knockouts and clinical consultation or prescription rate. In this data set, we identified a healthy PRDM9-knockout mother and performed phased genome sequencing on her, her child, and control individuals. Our results show that meiotic recombination sites are localized away from PRDM9-dependent hotspots. Thus, natural LOF variants inform on essential genetic loci and demonstrate PRDM9 redundancy in humans.

  20. Health and population effects of rare gene knockouts in adult humans with related parents

    PubMed Central

    Narasimhan, Vagheesh M.; Hunt, Karen A.; Mason, Dan; Baker, Christopher L.; Karczewski, Konrad J.; Barnes, Michael R.; Barnett, Anthony H.; Bates, Chris; Bellary, Srikanth; Bockett, Nicholas A.; Giorda, Kristina; Griffiths, Christopher J.; Hemingway, Harry; Jia, Zhilong; Kelly, M. Ann; Khawaja, Hajrah A.; Lek, Monkol; McCarthy, Shane; McEachan, Rosie; O’Donnell-Luria, Anne; Paigen, Kenneth; Parisinos, Constantinos A.; Sheridan, Eamonn; Southgate, Laura; Tee, Louise; Thomas, Mark; Xue, Yali; Schnall-Levin, Michael; Petkov, Petko M.; Tyler-Smith, Chris; Maher, Eamonn R.; Trembath, Richard C.; MacArthur, Daniel G.; Wright, John; Durbin, Richard; van Heel, David A.

    2016-01-01

    Examining complete gene knockouts within a viable organism can inform on gene function. We sequenced the exomes of 3,222 British Pakistani-heritage adults with high parental relatedness, discovering 1,111 rare-variant homozygous genotypes with predicted loss of gene function (knockouts) in 781 genes. We observed 13.7% fewer than expected homozygous knockout genotypes, implying an average load of 1.6 recessive-lethal-equivalent LOF variants per adult. Linking genetic data to lifelong health records, knockouts were not associated with clinical consultation or prescription rate. In this dataset we identified a healthy PRDM9 knockout mother, and performed phased genome sequencing on her, her child and controls, which showed meiotic recombination sites localised away from PRDM9-dependent hotspots. Thus, natural LOF variants inform upon essential genetic loci, and demonstrate PRDM9 redundancy in humans. PMID:26940866

  1. Functional genomics identifies regulators of the phototransduction machinery in the Drosophila larval eye and adult ocelli.

    PubMed

    Mishra, Abhishek Kumar; Bargmann, Bastiaan O R; Tsachaki, Maria; Fritsch, Cornelia; Sprecher, Simon G

    2016-02-15

    Sensory perception of light is mediated by specialized Photoreceptor neurons (PRs) in the eye. During development all PRs are genetically determined to express a specific Rhodopsin (Rh) gene and genes mediating a functional phototransduction pathway. While the genetic and molecular mechanisms of PR development is well described in the adult compound eye, it remains unclear how the expression of Rhodopsins and the phototransduction cascade is regulated in other visual organs in Drosophila, such as the larval eye and adult ocelli. Using transcriptome analysis of larval PR-subtypes and ocellar PRs we identify and study new regulators required during PR differentiation or necessary for the expression of specific signaling molecules of the functional phototransduction pathway. We found that the transcription factor Krüppel (Kr) is enriched in the larval eye and controls PR differentiation by promoting Rh5 and Rh6 expression. We also identified Camta, Lola, Dve and Hazy as key genes acting during ocellar PR differentiation. Further we show that these transcriptional regulators control gene expression of the phototransduction cascade in both larval eye and adult ocelli. Our results show that PR cell type-specific transcriptome profiling is a powerful tool to identify key transcriptional regulators involved during several aspects of PR development and differentiation. Our findings greatly contribute to the understanding of how combinatorial action of key transcriptional regulators control PR development and the regulation of a functional phototransduction pathway in both larval eye and adult ocelli.

  2. Xenobiotic transporters: ascribing function from gene knockout and mutation studies.

    PubMed

    Klaassen, Curtis D; Lu, Hong

    2008-02-01

    Transporter-mediated absorption, secretion, and reabsorption of chemicals are increasingly recognized as important determinants in the biological activities of many xenobiotics. In recent years, the rapid progress in generating and characterizing mice with targeted deletion of transporters has greatly increased our knowledge of the functions of transporters in the pharmacokinetics/toxicokinetics of xenobiotics. In this introduction, we focus on functions of transporters learned from experiments on knockout mice as well as humans and rodents with natural mutations of these transporters. We limit our discussion to transporters that either directly transport xenobiotics or are important in biliary excretion or cellular defenses, namely multidrug resistance, multidrug resistance-associated proteins, breast cancer resistance protein, organic anion transporting polypeptides, organic anion transporters, organic cation transporters, nucleoside transporters, peptide transporters, bile acid transporters, cholesterol transporters, and phospholipid transporters, as well as metal transporters. Efflux transporters in intestine, liver, kidney, brain, testes, and placenta can efflux xenobiotics out of cells and serve as barriers against the entrance of xenobiotics into cells, whereas many xenobiotics enter the biological system via uptake transporters. The functional importance of a given transporter in each tissue depends on its substrate specificity, expression level, and the presence/absence of other transporters with overlapping substrate preferences. Nevertheless, a transporter may affect a tissue independent of its local expression by altering systemic metabolism. Further studies on the gene regulation and function of transporters, as well as the interrelationship between transporters and phase I/II xenobiotic-metabolizing enzymes, will provide a complete framework for developing novel strategies to protect us from xenobiotic insults.

  3. Autophagy supports survival and phototransduction protein levels in rod photoreceptors

    PubMed Central

    Zhou, Z; Doggett, T A; Sene, A; Apte, R S; Ferguson, T A

    2015-01-01

    Damage and loss of the postmitotic photoreceptors is a leading cause of blindness in many diseases of the eye. Although the mechanisms of photoreceptor death have been extensively studied, few studies have addressed mechanisms that help sustain these non-replicating neurons for the life of an organism. Autophagy is an intracellular pathway where cytoplasmic constituents are delivered to the lysosomal pathway for degradation. It is not only a major pathway activated in response to cellular stress, but is also important for cytoplasmic turnover and to supply the structural and energy needs of cells. We examined the importance of autophagy in photoreceptors by deleting the essential autophagy gene Atg5 specifically in rods. Loss of autophagy led to progressive degeneration of rod photoreceptors beginning at 8 weeks of age such that by 44 weeks few rods remained. Cone photoreceptor numbers were only slightly diminished following rod degeneration but their function was significantly decreased. Rod cell death was apoptotic but was not dependent on daily light exposure or accelerated by intense light. Although the light-regulated translocation of the phototransduction proteins arrestin and transducin were unaffected in rods lacking autophagy, Atg5-deficient rods accumulated transducin-α as they degenerated suggesting autophagy might regulate the level of this protein. This was confirmed when the light-induced decrease in transducin was abolished in Atg5-deficient rods and the inhibition of autophagy in retinal explants cultures prevented its degradation. These results demonstrate that basal autophagy is essential to the long-term health of rod photoreceptors and a critical process for maintaining optimal levels of the phototransduction protein transducin-α. As the lack of autophagy is associated with retinal degeneration and altered phototransduction protein degradation in the absence of harmful gene products, this process may be a viable therapeutic target where rod

  4. Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases

    PubMed Central

    Santiago, Yolanda; Chan, Edmond; Liu, Pei-Qi; Orlando, Salvatore; Zhang, Lin; Urnov, Fyodor D.; Holmes, Michael C.; Guschin, Dmitry; Waite, Adam; Miller, Jeffrey C.; Rebar, Edward J.; Gregory, Philip D.; Klug, Aaron; Collingwood, Trevor N.

    2008-01-01

    Gene knockout is the most powerful tool for determining gene function or permanently modifying the phenotypic characteristics of a cell. Existing methods for gene disruption are limited by their efficiency, time to completion, and/or the potential for confounding off-target effects. Here, we demonstrate a rapid single-step approach to targeted gene knockout in mammalian cells, using engineered zinc-finger nucleases (ZFNs). ZFNs can be designed to target a chosen locus with high specificity. Upon transient expression of these nucleases the target gene is first cleaved by the ZFNs and then repaired by a natural—but imperfect—DNA repair process, nonhomologous end joining. This often results in the generation of mutant (null) alleles. As proof of concept for this approach we designed ZFNs to target the dihydrofolate reductase (DHFR) gene in a Chinese hamster ovary (CHO) cell line. We observed biallelic gene disruption at frequencies >1%, thus obviating the need for selection markers. Three new genetically distinct DHFR−/− cell lines were generated. Each new line exhibited growth and functional properties consistent with the specific knockout of the DHFR gene. Importantly, target gene disruption is complete within 2–3 days of transient ZFN delivery, thus enabling the isolation of the resultant DHFR−/− cell lines within 1 month. These data demonstrate further the utility of ZFNs for rapid mammalian cell line engineering and establish a new method for gene knockout with application to reverse genetics, functional genomics, drug discovery, and therapeutic recombinant protein production. PMID:18359850

  5. Gene Knockout Identification Using an Extension of Bees Hill Flux Balance Analysis

    PubMed Central

    Choon, Yee Wen; Mohamad, Mohd Saberi; Deris, Safaai; Chong, Chuii Khim; Omatu, Sigeru; Corchado, Juan Manuel

    2015-01-01

    Microbial strain optimisation for the overproduction of a desired phenotype has been a popular topic in recent years. Gene knockout is a genetic engineering technique that can modify the metabolism of microbial cells to obtain desirable phenotypes. Optimisation algorithms have been developed to identify the effects of gene knockout. However, the complexities of metabolic networks have made the process of identifying the effects of genetic modification on desirable phenotypes challenging. Furthermore, a vast number of reactions in cellular metabolism often lead to a combinatorial problem in obtaining optimal gene knockout. The computational time increases exponentially as the size of the problem increases. This work reports an extension of Bees Hill Flux Balance Analysis (BHFBA) to identify optimal gene knockouts to maximise the production yield of desired phenotypes while sustaining the growth rate. This proposed method functions by integrating OptKnock into BHFBA for validating the results automatically. The results show that the extension of BHFBA is suitable, reliable, and applicable in predicting gene knockout. Through several experiments conducted on Escherichia coli, Bacillus subtilis, and Clostridium thermocellum as model organisms, extension of BHFBA has shown better performance in terms of computational time, stability, growth rate, and production yield of desired phenotypes. PMID:25874200

  6. Generation of hypoxanthine phosphoribosyltransferase gene knockout rabbits by homologous recombination and gene trapping through somatic cell nuclear transfer.

    PubMed

    Yin, Mingru; Jiang, Weihua; Fang, Zhenfu; Kong, Pengcheng; Xing, Fengying; Li, Yao; Chen, Xuejin; Li, Shangang

    2015-11-02

    The rabbit is a common animal model that has been employed in studies on various human disorders, and the generation of genetically modified rabbit lines is highly desirable. Female rabbits have been successfully cloned from cumulus cells, and the somatic cell nuclear transfer (SCNT) technology is well established. The present study generated hypoxanthine phosphoribosyltransferase (HPRT) gene knockout rabbits using recombinant adeno-associated virus-mediated homologous recombination and SCNT. Gene trap strategies were employed to enhance the gene targeting rates. The male and female gene knockout fibroblast cell lines were derived by different strategies. When male HPRT knockout cells were used for SCNT, no live rabbits were obtained. However, when female HPRT(+/-) cells were used for SCNT, live, healthy rabbits were generated. The cloned HPRT(+/-) rabbits were fertile at maturity. We demonstrate a new technique to produce gene-targeted rabbits. This approach may also be used in the genetic manipulation of different genes or in other species.

  7. UV light phototransduction depolarizes human melanocytes.

    PubMed

    Bellono, Nicholas W; Oancea, Elena

    2013-01-01

    Exposure of human skin to low doses of solar UV radiation (UVR) causes increased pigmentation, while chronic exposure is a powerful risk factor for skin cancers. The mechanisms mediating UVR detection in skin, however, remain poorly understood. Our recent studies revealed that UVR activates a retinal-dependent G protein-coupled signaling pathway in melanocytes. This phototransduction pathway leads to the activation of transient receptor potential A1 (TRPA1) ion channels, elevation of intracellular calcium (Ca( 2+)) and rapid increase in cellular melanin content. Here we report that physiological doses of solar-like UVR elicit a retinal-dependent membrane depolarization in human epidermal melanocytes. This transient depolarization correlates with delayed inactivation time of the UVR-evoked photocurrent and with sustained Ca( 2+) responses required for early melanin synthesis. Thus, the cellular depolarization induced by UVR phototransduction in melanocytes is likely to be a critical signaling mechanism necessary for the protective response represented by increased melanin content.

  8. Generation of Recombinant Capripoxvirus Vectors for Vaccines and Gene Knockout Function Studies.

    PubMed

    Boshra, Hani; Cao, Jingxin; Babiuk, Shawn

    2016-01-01

    The ability to manipulate capripoxvirus through gene knockouts and gene insertions has become an increasingly valuable research tool in elucidating the function of individual genes of capripoxvirus, as well as in the development of capripoxvirus-based recombinant vaccines. The homologous recombination technique is used to generate capripoxvirus knockout viruses (KO), and is based on the targeting a particular viral gene of interest. This technique can also be used to insert a gene of interest. A protocol for the generation of a viral gene knockout is described. This technique involves the use of a plasmid which encodes the flanking sequences of the regions where the homologous recombination will occur, and will result in the insertion of an EGFP reporter gene for visualization of recombinant virus, as well as the E. coli gpt gene as a positive selection marker. If an additional gene is to be incorporated, this can be achieved by inserting a gene of interest for expression under a poxvirus promoter into the plasmid between the flanking regions for insertion. This chapter describes a protocol for generating such recombinant capripoxviruses.

  9. Selection-independent generation of gene knockout mouse embryonic stem cells using zinc-finger nucleases.

    PubMed

    Osiak, Anna; Radecke, Frank; Guhl, Eva; Radecke, Sarah; Dannemann, Nadine; Lütge, Fabienne; Glage, Silke; Rudolph, Cornelia; Cantz, Tobias; Schwarz, Klaus; Heilbronn, Regine; Cathomen, Toni

    2011-01-01

    Gene knockout in murine embryonic stem cells (ESCs) has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10(-6). In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs). Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells.

  10. Selection-Independent Generation of Gene Knockout Mouse Embryonic Stem Cells Using Zinc-Finger Nucleases

    PubMed Central

    Osiak, Anna; Radecke, Frank; Guhl, Eva; Radecke, Sarah; Dannemann, Nadine; Lütge, Fabienne; Glage, Silke; Rudolph, Cornelia; Cantz, Tobias; Schwarz, Klaus; Heilbronn, Regine; Cathomen, Toni

    2011-01-01

    Gene knockout in murine embryonic stem cells (ESCs) has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10−6. In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs). Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells. PMID:22194948

  11. Characterisation of iunH gene knockout strain from Mycobacterium tuberculosis

    PubMed Central

    Villela, Anne Drumond; Rodrigues, Valnês da Silva; Pinto, Antônio Frederico Michel; Wink, Priscila Lamb; Sánchez-Quitian, Zilpa Adriana; Petersen, Guilherme Oliveira; Campos, Maria Martha; Basso, Luiz Augusto; Santos, Diógenes Santiago

    2017-01-01

    BACKGROUND Tuberculosis (TB) is an infectious disease caused mainly by the bacillus Mycobacterium tuberculosis. The better understanding of important metabolic pathways from M. tuberculosis can contribute to the development of novel therapeutic and prophylactic strategies to combat TB. Nucleoside hydrolase (MtIAGU-NH), encoded by iunH gene (Rv3393), is an enzyme from purine salvage pathway in M. tuberculosis. MtIAGU-NH accepts inosine, adenosine, guanosine, and uridine as substrates, which may point to a pivotal metabolic role. OBJECTIVES Our aim was to construct a M. tuberculosis knockout strain for iunH gene, to evaluate in vitro growth and the effect of iunH deletion in M. tuberculosis in non-activated and activated macrophages models of infection. METHODS A M. tuberculosis knockout strain for iunH gene was obtained by allelic replacement, using pPR27xylE plasmid. The complemented strain was constructed by the transformation of the knockout strain with pNIP40::iunH. MtIAGU-NH expression was analysed by Western blot and LC-MS/MS. In vitro growth was evaluated in Sauton’s medium. Bacterial load of non-activated and interferon-γ activated RAW 264.7 cells infected with knockout strain was compared with wild-type and complemented strains. FINDINGS Western blot and LC-MS/MS validated iunH deletion at protein level. The iunH knockout led to a delay in M. tuberculosis growth kinetics in Sauton’s medium during log phase, but did not affect bases and nucleosides pool in vitro. No significant difference in bacterial load of knockout strain was observed when compared with both wild-type and complemented strains after infection of non-activated and interferon-γ activated RAW 264.7 cells. MAIN CONCLUSION The disruption of iunH gene does not influence M. tuberculosis growth in both non-activated and activated RAW 264.7 cells, which show that iunH gene is not important for macrophage invasion and virulence. Our results indicated that MtIAGU-NH is not a target for drug

  12. Progestins alter photo-transduction cascade and circadian rhythm network in eyes of zebrafish (Danio rerio)

    NASA Astrophysics Data System (ADS)

    Zhao, Yanbin; Fent, Karl

    2016-02-01

    Environmental progestins are implicated in endocrine disruption in vertebrates. Additional targets that may be affected in organisms are poorly known. Here we report that progesterone (P4) and drospirenone (DRS) interfere with the photo-transduction cascade and circadian rhythm network in the eyes of zebrafish. Breeding pairs of adult zebrafish were exposed to P4 and DRS for 21 days with different measured concentrations of 7–742 ng/L and 99-13´650 ng/L, respectively. Of totally 10 key photo-transduction cascade genes analyzed, transcriptional levels of most were significantly up-regulated, or normal down-regulation was attenuated. Similarly, for some circadian rhythm genes, dose-dependent transcriptional alterations were also observed in the totally 33 genes analyzed. Significant alterations occurred even at environmental relevant levels of 7 ng/L P4. Different patterns were observed for these transcriptional alterations, of which, the nfil3 family displayed most significant changes. Furthermore, we demonstrate the importance of sampling time for the determination and interpretation of gene expression data, and put forward recommendations for sampling strategies to avoid false interpretations. Our results suggest that photo-transduction signals and circadian rhythm are potential targets for progestins. Further studies are required to assess alterations on the protein level, on physiology and behavior, as well as on implications in mammals.

  13. Progestins alter photo-transduction cascade and circadian rhythm network in eyes of zebrafish (Danio rerio)

    PubMed Central

    Zhao, Yanbin; Fent, Karl

    2016-01-01

    Environmental progestins are implicated in endocrine disruption in vertebrates. Additional targets that may be affected in organisms are poorly known. Here we report that progesterone (P4) and drospirenone (DRS) interfere with the photo-transduction cascade and circadian rhythm network in the eyes of zebrafish. Breeding pairs of adult zebrafish were exposed to P4 and DRS for 21 days with different measured concentrations of 7–742 ng/L and 99-13´650 ng/L, respectively. Of totally 10 key photo-transduction cascade genes analyzed, transcriptional levels of most were significantly up-regulated, or normal down-regulation was attenuated. Similarly, for some circadian rhythm genes, dose-dependent transcriptional alterations were also observed in the totally 33 genes analyzed. Significant alterations occurred even at environmental relevant levels of 7 ng/L P4. Different patterns were observed for these transcriptional alterations, of which, the nfil3 family displayed most significant changes. Furthermore, we demonstrate the importance of sampling time for the determination and interpretation of gene expression data, and put forward recommendations for sampling strategies to avoid false interpretations. Our results suggest that photo-transduction signals and circadian rhythm are potential targets for progestins. Further studies are required to assess alterations on the protein level, on physiology and behavior, as well as on implications in mammals. PMID:26899944

  14. Generating Targeted Gene Knockout Lines in Physcomitrella patens to Study Evolution of Stress-Responsive Mechanisms

    PubMed Central

    Maronova, Monika; Kalyna, Maria

    2016-01-01

    The moss Physcomitrella patens possesses highly efficient homologous recombination allowing targeted gene manipulations and displays many features of the early land plants including high tolerance to abiotic stresses. It is therefore an invaluable model organism for studies of gene functions and comparative studies of evolution of stress responses in plants. Here, we describe a method for generating targeted gene knockout lines in P. patens using a polyethylene glycol-mediated transformation of protoplasts including basic in vitro growth, propagation, and maintenance techniques. PMID:26867627

  15. Establishment of mitochondrial pyruvate carrier 1 (MPC1) gene knockout mice with preliminary gene function analyses

    PubMed Central

    Li, Xiaoli; Li, Yaqing; Han, Gaoyang; Li, Xiaoran; Ji, Yasai; Fan, Zhirui; Zhong, Yali; Cao, Jing; Zhao, Jing; Mariusz, Goscinski; Zhang, Mingzhi; Wen, Jianguo; Nesland, Jahn M.; Suo, Zhenhe

    2016-01-01

    Pyruvate plays a critical role in the mitochondrial tricarboxylic acid (TCA) cycle, and it is the center product for the synthesis of amino acids, carbohydrates and fatty acids. Pyruvate transported across the inner mitochondrial membrane appears to be essential in anabolic and catabolic intermediary metabolism. The mitochondrial pyruvate carrier (MPC) mounted in the inner membrane of mitochondria serves as the channel to facilitate pyruvate permeating. In mammals, the MPC is formed by two paralogous subunits, MPC1 and MPC2. It is known that complete ablation of MPC2 in mice causes death on the 11th or 12th day of the embryonic period. However, MPC1 deletion and the knowledge of gene function in vivo are lacking. Using the new technology of gene manipulation known as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9 (CRISPR/Cas9) systems, we gained stable MPC1 gene heterozygous mutation mice models, and the heterozygous mutations could be stably maintained in their offsprings. Only one line with homozygous 27 bases deletion in the first exon was established, but no offsprings could be obtained after four months of mating experiments, indicating infertility of the mice with such homozygous deletion. The other line of MPC1 knockout (KO) mice was only heterozygous, which mutated in the first exon with a terminator shortly afterwards. These two lines of MPC1 KO mice showed lower fertility and significantly higher bodyweight in the females. We concluded that heterozygous MPC1 KO weakens fertility and influences the metabolism of glucose and fatty acid and bodyweight in mice. PMID:27835892

  16. Generation of hypoxanthine phosphoribosyltransferase gene knockout rabbits by homologous recombination and gene trapping through somatic cell nuclear transfer

    PubMed Central

    Yin, Mingru; Jiang, Weihua; Fang, Zhenfu; Kong, Pengcheng; Xing, Fengying; Li, Yao; Chen, Xuejin; Li, Shangang

    2015-01-01

    The rabbit is a common animal model that has been employed in studies on various human disorders, and the generation of genetically modified rabbit lines is highly desirable. Female rabbits have been successfully cloned from cumulus cells, and the somatic cell nuclear transfer (SCNT) technology is well established. The present study generated hypoxanthine phosphoribosyltransferase (HPRT) gene knockout rabbits using recombinant adeno-associated virus-mediated homologous recombination and SCNT. Gene trap strategies were employed to enhance the gene targeting rates. The male and female gene knockout fibroblast cell lines were derived by different strategies. When male HPRT knockout cells were used for SCNT, no live rabbits were obtained. However, when female HPRT+/− cells were used for SCNT, live, healthy rabbits were generated. The cloned HPRT+/− rabbits were fertile at maturity. We demonstrate a new technique to produce gene-targeted rabbits. This approach may also be used in the genetic manipulation of different genes or in other species. PMID:26522387

  17. 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

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

    PubMed

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

    2012-04-01

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

  19. Global Gene Expression and Focused Knockout Analysis Reveals Genes Associated with Fungal Fruiting Body Development in Neurospora crassa

    PubMed Central

    Wang, Zheng; Lopez-Giraldez, Francesc; Lehr, Nina; Farré, Marta; Common, Ralph; Trail, Frances

    2014-01-01

    Fungi can serve as highly tractable models for understanding genetic basis of sexual development in multicellular organisms. Applying a reverse-genetic approach to advance such a model, we used random and multitargeted primers to assay gene expression across perithecial development in Neurospora crassa. We found that functionally unclassified proteins accounted for most upregulated genes, whereas downregulated genes were enriched for diverse functions. Moreover, genes associated with developmental traits exhibited stage-specific peaks of expression. Expression increased significantly across sexual development for mating type gene mat a-1 and for mat A-1 specific pheromone precursor ccg-4. In addition, expression of a gene encoding a protein similar to zinc finger, stc1, was highly upregulated early in perithecial development, and a strain with a knockout of this gene exhibited arrest at the same developmental stage. A similar expression pattern was observed for genes in RNA silencing and signaling pathways, and strains with knockouts of these genes were also arrested at stages of perithecial development that paralleled their peak in expression. The observed stage specificity allowed us to correlate expression upregulation and developmental progression and to identify regulators of sexual development. Bayesian networks inferred from our expression data revealed previously known and new putative interactions between RNA silencing genes and pathways. Overall, our analysis provides a fine-scale transcriptomic landscape and novel inferences regarding the control of the multistage development process of sexual crossing and fruiting body development in N. crassa. PMID:24243796

  20. CRISPR/Cas9-mediated gene knockout in the ascidian Ciona intestinalis.

    PubMed

    Sasaki, Haruka; Yoshida, Keita; Hozumi, Akiko; Sasakura, Yasunori

    2014-09-01

    Knockout of genes with CRISPR/Cas9 is a newly emerged approach to investigate functions of genes in various organisms. We demonstrate that CRISPR/Cas9 can mutate endogenous genes of the ascidian Ciona intestinalis, a splendid model for elucidating molecular mechanisms for constructing the chordate body plan. Short guide RNA (sgRNA) and Cas9 mRNA, when they are expressed in Ciona embryos by means of microinjection or electroporation of their expression vectors, introduced mutations in the target genes. The specificity of target choice by sgRNA is relatively high compared to the reports from some other organisms, and a single nucleotide mutation at the sgRNA dramatically reduced mutation efficiency at the on-target site. CRISPR/Cas9-mediated mutagenesis will be a powerful method to study gene functions in Ciona along with another genome editing approach using TALE nucleases.

  1. Dual sgRNA-directed gene knockout using CRISPR/Cas9 technology in Caenorhabditis elegans

    PubMed Central

    Chen, Xiangyang; Xu, Fei; Zhu, Chengming; Ji, Jiaojiao; Zhou, Xufei; Feng, Xuezhu; Guang, Shouhong

    2014-01-01

    The CRISPR RNA-guided Cas9 nuclease gene-targeting system has been successfully used for genome editing in a variety of organisms. Here, we report the use of dual sgRNA-guided Cas9 nuclease to generate knockout mutants of protein coding genes, noncoding genes, and repetitive sequences in C. elegans. Co-injection of C. elegans with dual sgRNAs results in the removal of the interval between two sgRNAs and the loss-of-function phenotype of targeted genes. We sought to determine how large an interval can be eliminated and found that at least a 24 kb chromosome segment can be deleted using this dual sgRNA/Cas9 strategy. The deletion of large chromosome segments facilitates mutant screening by PCR and agarose electrophoresis. Thus, the use of the CRISPR/Cas9 system in combination with dual sgRNAs provides a powerful platform with which to easily generate gene knockout mutants in C. elegans. Our data also suggest that encoding multiple sgRNA sequences into a single CRISPR array to simultaneously edit several sites within the genome may cause the off-target deletion of chromosome sequences. PMID:25531445

  2. Transgenic gene knock-outs: functional genomics and therapeutic target selection.

    PubMed

    Harris, S; Foord, S M

    2000-11-01

    The completion of the first draft of the human genome presents both a tremendous opportunity and enormous challenge to the pharmaceutical industry since the whole community, with few exceptions, will soon have access to the same pool of candidate gene sequences from which to select future therapeutic targets. The commercial imperative to select and pursue therapeutically relevant genes from within the overall content of the genome will be particularly intense for those gene families that currently represent the chemically tractable or 'drugable' gene targets. As a consequence the emphasis within exploratory research has shifted towards the evaluation and adoption of technology platforms that can add additional value to the gene selection process, either through functional studies or direct/indirect measures of disease alignment e.g., genetics, differential gene expression, proteomics, tissue distribution, comparative species data etc. The selection of biological targets for the development of potential new medicines relies, in part, on the quality of the in vivo biological data that correlates a particular molecular target with the underlying pathophysiology of a disease. Within the pharmaceutical industry, studies employing transgenic animals and, in particular, animals with specific gene deletions are playing an increasingly important role in the therapeutic target gene selection, drug candidate selection and product development phases of the overall drug discovery process. The potential of phenotypic information from gene knock-outs to contribute to a high-throughput target selection/validation strategy has hitherto been limited by the resources required to rapidly generate and characterise a large number of knock-out transgenics in a timely fashion. The offerings of several companies that provide an opportunity to overcome these hurdles, albeit at a cost, are assessed with respect to the strategic business needs of the pharmaceutical industry.

  3. The effect of PDIA3 gene knockout on the mucosal immune function in IBS rats

    PubMed Central

    Zhuang, Zhao-Meng; Wang, Xiao-Teng; Zhang, Lu; Tao, Li-Yuan; Lv, Bin

    2015-01-01

    Objective: To observe the changes of intestinal inflammation on PDIA3 gene knockout IBS rats and its effect on immune function. Methods: 36 SD rats were randomly divided into four groups: the control group (n = 8); IBS- empty virus group (IBS-GFP, which); IBS-PDIA3 knockout group (n = 12); IBS- the control group (n = 12). After modeling, colon and ileocecal tissue pathology in each group were observed separately. Changes of immune and inflammatory markers were measured. At the same time, ultrastructural changes in each group were observed by electron microscopy. Results: Compared with the IBS control group, inflammation was reduced significantly in IBS-PDIA3 knockout group. IgE, IL-4 and IL-9 and the level of intestinal trypsin type were decreased significantly. Furthermore, mast cell degranulation and PAR 2 receptor reduced significantly. Conclusion: PDIA3 may play an important role in the development of IBS by mediating through immune responses of mucosal abnormalities. However, the mechanism needs to be confirmed in further study. PMID:26221224

  4. Generation of RUNX3 knockout pigs using CRISPR/Cas9-mediated gene targeting.

    PubMed

    Kang, J-T; Ryu, J; Cho, B; Lee, E-J; Yun, Y-J; Ahn, S; Lee, J; Ji, D-Y; Lee, K; Park, K-W

    2016-12-01

    Pigs are an attractive animal model to study the progression of cancer because of their anatomical and physiological similarities to human. However, the use of pig models for cancer research has been limited by availability of genetically engineered pigs which can recapitulate human cancer progression. Utilizing genome editing technologies such as CRISPR/Cas9 system allows us to generate genetically engineered pigs at a higher efficiency. In this study, specific CRISPR/Cas9 systems were used to target RUNX3, a known tumour suppressor gene, to generate a pig model that can induce gastric cancer in human. First, RUNX3 knockout cell lines carrying genetic modification (monoallelic or biallelic) of RUNX3 were generated by introducing engineered CRISPR/Cas9 system specific to RUNX3 into foetal fibroblast cells. Then, the genetically modified foetal fibroblast cells were used as donor cells for somatic cell nuclear transfer, followed by embryo transfer. We successfully obtained four live RUNX3 knockout piglets from two surrogates. The piglets showed the lack of RUNX3 protein in their internal organ system. Our results demonstrate that the CRISPR/Cas9 system is effective in inducing mutations on a specific locus of genome and the RUNX3 knockout pigs can be useful resources for human cancer research and to develop novel cancer therapies.

  5. Mapping ecologically relevant social behaviours by gene knockout in wild mice.

    PubMed

    Chalfin, Lea; Dayan, Molly; Levy, Dana Rubi; Austad, Steven N; Miller, Richard A; Iraqi, Fuad A; Dulac, Catherine; Kimchi, Tali

    2014-08-05

    The laboratory mouse serves as an important model system for studying gene, brain and behavioural interactions. Powerful methods of gene targeting have helped to decipher gene-function associations in human diseases. Yet, the laboratory mouse, obtained after decades of human-driven artificial selection, inbreeding, and adaptation to captivity, is of limited use for the study of fitness-driven behavioural responses that characterize the ancestral wild house mouse. Here, we demonstrate that the backcrossing of wild mice with knockout mutant laboratory mice retrieves behavioural traits exhibited exclusively by the wild house mouse, thereby unmasking gene functions inaccessible in the domesticated mutant model. Furthermore, we show that domestication had a much greater impact on females than on males, erasing many behavioural traits of the ancestral wild female. Hence, compared with laboratory mice, wild-derived mutant mice constitute an improved model system to gain insights into neuronal mechanisms underlying normal and pathological sexually dimorphic social behaviours.

  6. What do we learn from the murine Jacob/Nsmf gene knockout for human disease?

    PubMed Central

    Spilker, Christina; Grochowska, Katarzyna M.; Kreutz, Michael R.

    2016-01-01

    ABSTRACT Mutations in the NSMF gene have been related to Kallmann syndrome. Conflicting results have been reported on the subcellular localization of Jacob/NELF, the protein encoded by the NSMF gene. Some reports indicate an extracellular localization and a function as a guidance molecule for migration of GnRH-positive neurons from the olfactory placode to the hypothalamus. Other studies have shown protein transport of Jacob from synapse-to-nucleus and indicate a role of the protein in neuronal activity-dependent gene expression. A recent publication casts doubts on a major role of Jacob/NELF in Kallmann syndrome and neuronal migration of GnRH-positive neurons during early development. Instead a murine NSMF gene knockout results in hippocampal dysplasia, impaired BDNF-signaling during dendritogenesis, and phenotypes related to the lack of BDNF-induced nuclear import of Jacob in early postnatal development. PMID:27803842

  7. [Physiology of the visual retinal signal: From phototransduction to the visual cycle].

    PubMed

    Salesse, C

    2017-03-01

    The retinal photoreceptors (rods and cones) are responsible for light absorption and transduction of the signal, which is transmitted to the other retinal nerve cells and then to the brain. The chromophore of visual pigments of rods and cones is a particular isomer of a vitamin A derivative. Light absorption by this chromophore leads to its isomerization and to a phototransduction cascade, which results in photoreceptor hyperpolarization and cessation of glutamate secretion at their synaptic terminals. Phototransduction of cones and rods differs in their signal amplification and inactivation, which is consistent with their respective functions. The rods serve for dim light vision, whereas color and detailed vision is provided by cones. The rods are thus much more sensitive than cones, but the time course of cones' photoresponse is ∼10 times faster than that of rods. The orientation of cone visual pigments in the retina is optimized to achieve their function. The isomerized chromophore of visual pigments is regenerated by a mechanism known as the visual cycle. This process takes place mainly in the retinal pigment epithelium for the rods and the glial Müller cells for the cones. Mutations of a large number of proteins involved in visual phototransduction and in the retinoid visual cycle are responsible for hereditary diseases leading to photoreceptor degeneration. However, gene therapy offers quite a bit of hope for treatment.

  8. Phenotypes Associated with Knockouts of Eight Dense Granule Gene Loci (GRA2-9) in Virulent Toxoplasma gondii.

    PubMed

    Rommereim, Leah M; Bellini, Valeria; Fox, Barbara A; Pètre, Graciane; Rak, Camille; Touquet, Bastien; Aldebert, Delphine; Dubremetz, Jean-François; Cesbron-Delauw, Marie-France; Mercier, Corinne; Bzik, David J

    2016-01-01

    Toxoplasma gondii actively invades host cells and establishes a parasitophorous vacuole (PV) that accumulates many proteins secreted by the dense granules (GRA proteins). To date, at least 23 GRA proteins have been reported, though the function(s) of most of these proteins still remains unknown. We targeted gene knockouts at ten GRA gene loci (GRA1-10) to investigate the cellular roles and essentiality of these classical GRA proteins during acute infection in the virulent type I RH strain. While eight of these genes (GRA2-9) were successfully knocked out, targeted knockouts at the GRA1 and GRA10 loci were not obtained, suggesting these GRA proteins may be essential. As expected, the Δgra2 and Δgra6 knockouts failed to form an intravacuolar network (IVN). Surprisingly, Δgra7 exhibited hyper-formation of the IVN in both normal and lipid-free growth conditions. No morphological alterations were identified in parasite or PV structures in the Δgra3, Δgra4, Δgra5, Δgra8, or Δgra9 knockouts. With the exception of the Δgra3 and Δgra8 knockouts, all of the GRA knockouts exhibited defects in their infection rate in vitro. While the single GRA knockouts did not exhibit reduced replication rates in vitro, replication rate defects were observed in three double GRA knockout strains (Δgra4Δgra6, Δgra3Δgra5 and Δgra3Δgra7). However, the virulence of single or double GRA knockout strains in CD1 mice was not affected. Collectively, our results suggest that while the eight individual GRA proteins investigated in this study (GRA2-9) are not essential, several GRA proteins may provide redundant and potentially important functions during acute infection.

  9. Phenotypes Associated with Knockouts of Eight Dense Granule Gene Loci (GRA2-9) in Virulent Toxoplasma gondii

    PubMed Central

    Fox, Barbara A.; Pètre, Graciane; Rak, Camille; Touquet, Bastien; Aldebert, Delphine; Dubremetz, Jean-François; Cesbron-Delauw, Marie-France; Mercier, Corinne; Bzik, David J.

    2016-01-01

    Toxoplasma gondii actively invades host cells and establishes a parasitophorous vacuole (PV) that accumulates many proteins secreted by the dense granules (GRA proteins). To date, at least 23 GRA proteins have been reported, though the function(s) of most of these proteins still remains unknown. We targeted gene knockouts at ten GRA gene loci (GRA1-10) to investigate the cellular roles and essentiality of these classical GRA proteins during acute infection in the virulent type I RH strain. While eight of these genes (GRA2-9) were successfully knocked out, targeted knockouts at the GRA1 and GRA10 loci were not obtained, suggesting these GRA proteins may be essential. As expected, the Δgra2 and Δgra6 knockouts failed to form an intravacuolar network (IVN). Surprisingly, Δgra7 exhibited hyper-formation of the IVN in both normal and lipid-free growth conditions. No morphological alterations were identified in parasite or PV structures in the Δgra3, Δgra4, Δgra5, Δgra8, or Δgra9 knockouts. With the exception of the Δgra3 and Δgra8 knockouts, all of the GRA knockouts exhibited defects in their infection rate in vitro. While the single GRA knockouts did not exhibit reduced replication rates in vitro, replication rate defects were observed in three double GRA knockout strains (Δgra4Δgra6, Δgra3Δgra5 and Δgra3Δgra7). However, the virulence of single or double GRA knockout strains in CD1 mice was not affected. Collectively, our results suggest that while the eight individual GRA proteins investigated in this study (GRA2-9) are not essential, several GRA proteins may provide redundant and potentially important functions during acute infection. PMID:27458822

  10. Gene Expression Profiles of Main Olfactory Epithelium in Adenylyl Cyclase 3 Knockout Mice

    PubMed Central

    Wang, Zhenshan; Zhou, Yanfen; Luo, Yingtao; Zhang, Jing; Zhai, Yunpeng; Yang, Dong; Zhang, Zhe; Li, Yongchao; Storm, Daniel R.; Ma, Runlin Z.

    2015-01-01

    Adenylyl Cyclase 3 (AC3) plays an important role in the olfactory sensation-signaling pathway in mice. AC3 deficiency leads to defects in olfaction. However, it is still unknown whether AC3 deficiency affects gene expression or olfactory signal transduction pathways within the main olfactory epithelium (MOE). In this study, gene microarrays were used to screen differentially expressed genes in MOE from AC3 knockout (AC3−/−) and wild-type (AC3+/+) mice. The differentially expressed genes identified were subjected to bioinformatic analysis and verified by qRT-PCR. Gene expression in the MOE from AC3−/− mice was significantly altered, compared to AC3+/+ mice. Of the 41266 gene probes, 3379 had greater than 2-fold fold change in expression levels between AC3−/− and AC3+/+ mice, accounting for 8% of the total gene probes. Of these genes, 1391 were up regulated, and 1988 were down regulated, including 425 olfactory receptor genes, 99 genes that are specifically expressed in the immature olfactory neurons, 305 genes that are specifically expressed in the mature olfactory neurons, and 155 genes that are involved in epigenetic regulation. Quantitative RT-PCR verification of the differentially expressed epigenetic regulation related genes, olfactory receptors, ion transporter related genes, neuron development and differentiation related genes, lipid metabolism and membrane protein transport etc. related genes showed that P75NTR, Hinfp, Gadd45b, and Tet3 were significantly up-regulated, while Olfr370, Olfr1414, Olfr1208, Golf, Faim2, Tsg101, Mapk10, Actl6b, H2BE, ATF5, Kirrrel2, OMP, Drd2 etc. were significantly down-regulated. In summary, AC3 may play a role in proximal olfactory signaling and play a role in the regulation of differentially expressed genes in mouse MOE. PMID:26633363

  11. 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.

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

    PubMed

    Mahata, Barun; Biswas, Kaushik

    2017-01-01

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

  13. Cloning and knockout of phytoene desaturase gene in Sphingomonas elodea ATCC 31461 for economic recovery of gellan gum.

    PubMed

    Zhu, Liang; Wu, Xuechang; Li, Ou; Chen, Yamin; Qian, Chaodong; Teng, Yi; Tao, Xianglin; Gao, Haichun

    2011-09-01

    A gene encoding phytoene desaturase (crtI) in the carotenoid biosynthetic pathway of Sphingomonas elodea ATCC 31461, an industrial gellan gum-producing strain, was cloned and identified. This gene is predicted to encode a 492-amino acid protein with significant homology to the phytoene desaturase of other carotenogenic organisms. Knockout of crtI gene blocked yellow carotenoid pigment synthesis and resulted in the accumulation of colorless phytoene, confirming that it encodes phytoene desaturase. Further research indicates that the yield of gellan gum production by crtI gene knockout mutants is almost the same as that by the wild-type strain. In addition, a recovery method based on the colorless fermentation broth of the crtI gene knockout mutant was investigated. Compared to the volume of alcohol for the parent strain, much less alcohol (30%) is required in this recovery process; thus, the costs of downstream purification of gellan gum can be substantially reduced.

  14. 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

  15. High-throughput screening of mouse gene knockouts identifies established and novel skeletal phenotypes

    PubMed Central

    Brommage, Robert; Liu, Jeff; Hansen, Gwenn M; Kirkpatrick, Laura L; Potter, David G; Sands, Arthur T; Zambrowicz, Brian; Powell, David R; Vogel, Peter

    2014-01-01

    Screening gene function in vivo is a powerful approach to discover novel drug targets. We present high-throughput screening (HTS) data for 3 762 distinct global gene knockout (KO) mouse lines with viable adult homozygous mice generated using either gene-trap or homologous recombination technologies. Bone mass was determined from DEXA scans of male and female mice at 14 weeks of age and by microCT analyses of bones from male mice at 16 weeks of age. Wild-type (WT) cagemates/littermates were examined for each gene KO. Lethality was observed in an additional 850 KO lines. Since primary HTS are susceptible to false positive findings, additional cohorts of mice from KO lines with intriguing HTS bone data were examined. Aging, ovariectomy, histomorphometry and bone strength studies were performed and possible non-skeletal phenotypes were explored. Together, these screens identified multiple genes affecting bone mass: 23 previously reported genes (Calcr, Cebpb, Crtap, Dcstamp, Dkk1, Duoxa2, Enpp1, Fgf23, Kiss1/Kiss1r, Kl (Klotho), Lrp5, Mstn, Neo1, Npr2, Ostm1, Postn, Sfrp4, Slc30a5, Slc39a13, Sost, Sumf1, Src, Wnt10b), five novel genes extensively characterized (Cldn18, Fam20c, Lrrk1, Sgpl1, Wnt16), five novel genes with preliminary characterization (Agpat2, Rassf5, Slc10a7, Slc26a7, Slc30a10) and three novel undisclosed genes coding for potential osteoporosis drug targets. PMID:26273529

  16. Testing the role of predicted gene knockouts in human anthropometric trait variation

    PubMed Central

    Lessard, Samuel; Manning, Alisa K.; Low-Kam, Cécile; Auer, Paul L.; Giri, Ayush; Graff, Mariaelisa; Schurmann, Claudia; Yaghootkar, Hanieh; Luan, Jian'an; Esko, Tonu; Karaderi, Tugce; Bottinger, Erwin P.; Lu, Yingchang; Carlson, Chris; Caulfield, Mark; Dubé, Marie-Pierre; Jackson, Rebecca D.; Kooperberg, Charles; McKnight, Barbara; Mongrain, Ian; Peters, Ulrike; Reiner, Alex P.; Rhainds, David; Sotoodehnia, Nona; Hirschhorn, Joel N.; Scott, Robert A.; Munroe, Patricia B.; Frayling, Timothy M.; Loos, Ruth J.F.; North, Kari E.; Edwards, Todd L.; Tardif, Jean-Claude; Lindgren, Cecilia M.; Lettre, Guillaume

    2016-01-01

    Although the role of complete gene inactivation by two loss-of-function mutations inherited in trans is well-established in recessive Mendelian diseases, we have not yet explored how such gene knockouts (KOs) could influence complex human phenotypes. Here, we developed a statistical framework to test the association between gene KOs and quantitative human traits. Our method is flexible, publicly available, and compatible with common genotype format files (e.g. PLINK and vcf). We characterized gene KOs in 4498 participants from the NHLBI Exome Sequence Project (ESP) sequenced at high coverage (>100×), 1976 French Canadians from the Montreal Heart Institute Biobank sequenced at low coverage (5.7×), and >100 000 participants from the Genetic Investigation of ANthropometric Traits (GIANT) Consortium genotyped on an exome array. We tested associations between gene KOs and three anthropometric traits: body mass index (BMI), height and BMI-adjusted waist-to-hip ratio (WHR). Despite our large sample size and multiple datasets available, we could not detect robust associations between specific gene KOs and quantitative anthropometric traits. Our results highlight several limitations and challenges for future gene KO studies in humans, in particular when there is no prior knowledge on the phenotypes that might be affected by the tested gene KOs. They also suggest that gene KOs identified with current DNA sequencing methodologies probably do not strongly influence normal variation in BMI, height, and WHR in the general human population. PMID:26908616

  17. Testing the role of predicted gene knockouts in human anthropometric trait variation.

    PubMed

    Lessard, Samuel; Manning, Alisa K; Low-Kam, Cécile; Auer, Paul L; Giri, Ayush; Graff, Mariaelisa; Schurmann, Claudia; Yaghootkar, Hanieh; Luan, Jian'an; Esko, Tonu; Karaderi, Tugce; Bottinger, Erwin P; Lu, Yingchang; Carlson, Chris; Caulfield, Mark; Dubé, Marie-Pierre; Jackson, Rebecca D; Kooperberg, Charles; McKnight, Barbara; Mongrain, Ian; Peters, Ulrike; Reiner, Alex P; Rhainds, David; Sotoodehnia, Nona; Hirschhorn, Joel N; Scott, Robert A; Munroe, Patricia B; Frayling, Timothy M; Loos, Ruth J F; North, Kari E; Edwards, Todd L; Tardif, Jean-Claude; Lindgren, Cecilia M; Lettre, Guillaume

    2016-05-15

    Although the role of complete gene inactivation by two loss-of-function mutations inherited in trans is well-established in recessive Mendelian diseases, we have not yet explored how such gene knockouts (KOs) could influence complex human phenotypes. Here, we developed a statistical framework to test the association between gene KOs and quantitative human traits. Our method is flexible, publicly available, and compatible with common genotype format files (e.g. PLINK and vcf). We characterized gene KOs in 4498 participants from the NHLBI Exome Sequence Project (ESP) sequenced at high coverage (>100×), 1976 French Canadians from the Montreal Heart Institute Biobank sequenced at low coverage (5.7×), and >100 000 participants from the Genetic Investigation of ANthropometric Traits (GIANT) Consortium genotyped on an exome array. We tested associations between gene KOs and three anthropometric traits: body mass index (BMI), height and BMI-adjusted waist-to-hip ratio (WHR). Despite our large sample size and multiple datasets available, we could not detect robust associations between specific gene KOs and quantitative anthropometric traits. Our results highlight several limitations and challenges for future gene KO studies in humans, in particular when there is no prior knowledge on the phenotypes that might be affected by the tested gene KOs. They also suggest that gene KOs identified with current DNA sequencing methodologies probably do not strongly influence normal variation in BMI, height, and WHR in the general human population.

  18. Improving cold storage and processing traits in potato through targeted gene knockout.

    PubMed

    Clasen, Benjamin M; Stoddard, Thomas J; Luo, Song; Demorest, Zachary L; Li, Jin; Cedrone, Frederic; Tibebu, Redeat; Davison, Shawn; Ray, Erin E; Daulhac, Aurelie; Coffman, Andrew; Yabandith, Ann; Retterath, Adam; Haun, William; Baltes, Nicholas J; Mathis, Luc; Voytas, Daniel F; Zhang, Feng

    2016-01-01

    Cold storage of potato tubers is commonly used to reduce sprouting and extend postharvest shelf life. However, cold temperature stimulates the accumulation of reducing sugars in potato tubers. Upon high-temperature processing, these reducing sugars react with free amino acids, resulting in brown, bitter-tasting products and elevated levels of acrylamide--a potential carcinogen. To minimize the accumulation of reducing sugars, RNA interference (RNAi) technology was used to silence the vacuolar invertase gene (VInv), which encodes a protein that breaks down sucrose to glucose and fructose. Because RNAi often results in incomplete gene silencing and requires the plant to be transgenic, here we used transcription activator-like effector nucleases (TALENs) to knockout VInv within the commercial potato variety, Ranger Russet. We isolated 18 plants containing mutations in at least one VInv allele, and five of these plants had mutations in all VInv alleles. Tubers from full VInv-knockout plants had undetectable levels of reducing sugars, and processed chips contained reduced levels of acrylamide and were lightly coloured. Furthermore, seven of the 18 modified plant lines appeared to contain no TALEN DNA insertions in the potato genome. These results provide a framework for using TALENs to quickly improve traits in commercially relevant autotetraploid potato lines.

  19. Enhanced antinociceptive effects of morphine in histamine H2 receptor gene knockout mice.

    PubMed

    Mobarakeh, Jalal Izadi; Takahashi, Kazuhiro; Sakurada, Shinobu; Kuramasu, Atsuo; Yanai, Kazuhiko

    2006-09-01

    We have previously shown that antinociceptive effects of morphine are enhanced in histamine H1 receptor gene knockout mice. In the present study, involvement of supraspinal histamine H2 receptor in antinociception by morphine was examined using histamine H2 receptor gene knockout (H2KO) mice and histamine H2 receptor antagonists. Antinociception was evaluated by assays for thermal (hot-plate, tail-flick and paw-withdrawal tests), mechanical (tail-pressure test) and chemical (formalin and capsaicin tests) stimuli. Thresholds for pain perception in H2KO mice were higher than wild-type mice. Antinociceptive effects of intracerebroventricularly administered morphine were enhanced in the H2KO mice compared to wild-type mice. Intracerebroventricular co-administration of morphine and cimetidine produced significant antinociceptive effects in the wild-type mice when compared to morphine or cimetidine alone. Furthermore, zolantidine, a selective and hydrophobic H2 receptor antagonist, enhanced the effects of morphine in all nociceptive assays examined. These results suggest that histamine exerts inhibitory effects on morphine-induced antinociception through H2 receptors at the supraspinal level. Our present and previous studies suggest that H1 and H2 receptors cooperatively function to modulate pain perception in the central nervous system.

  20. Effect of chronic valproic Acid treatment on hepatic gene expression profile in wfs1 knockout mouse.

    PubMed

    Punapart, Marite; Eltermaa, Mall; Oflijan, Julia; Sütt, Silva; Must, Anne; Kõks, Sulev; Schalkwyk, Leonard C; Fernandes, Catherine; Vasar, Eero; Soomets, Ursel; Terasmaa, Anton

    2014-01-01

    Valproic acid (VPA) is a widely used anticonvulsant and mood-stabilizing drug whose use is often associated with drug-induced weight gain. Treatment with VPA has been shown to upregulate Wfs1 expression in vitro. Aim of the present study was to compare the effect of chronic VPA treatment in wild type (WT) and Wfs1 knockout (KO) mice on hepatic gene expression profile. Wild type, Wfs1 heterozygous, and homozygous mice were treated with VPA for three months (300 mg/kg i.p. daily) and gene expression profiles in liver were evaluated using Affymetrix Mouse GeneChip 1.0 ST array. We identified 42 genes affected by Wfs1 genotype, 10 genes regulated by VPA treatment, and 9 genes whose regulation by VPA was dependent on genotype. Among the genes that were regulated differentially by VPA depending on genotype was peroxisome proliferator-activated receptor delta (Ppard), whose expression was upregulated in response to VPA treatment in WT, but not in Wfs1 KO mice. Thus, regulation of Ppard by VPA is dependent on Wfs1 genotype.

  1. Engineering Human Stem Cell Lines with Inducible Gene Knockout using CRISPR/Cas9.

    PubMed

    Chen, Yuejun; Cao, Jingyuan; Xiong, Man; Petersen, Andrew J; Dong, Yi; Tao, Yunlong; Huang, Cindy Tzu-Ling; Du, Zhongwei; Zhang, Su-Chun

    2015-08-06

    Precise temporal control of gene expression or deletion is critical for elucidating gene function in biological systems. However, the establishment of human pluripotent stem cell (hPSC) lines with inducible gene knockout (iKO) remains challenging. We explored building iKO hPSC lines by combining CRISPR/Cas9-mediated genome editing with the Flp/FRT and Cre/LoxP system. We found that "dual-sgRNA targeting" is essential for biallelic knockin of FRT sequences to flank the exon. We further developed a strategy to simultaneously insert an activity-controllable recombinase-expressing cassette and remove the drug-resistance gene, thus speeding up the generation of iKO hPSC lines. This two-step strategy was used to establish human embryonic stem cell (hESC) and induced pluripotent stem cell (iPSC) lines with iKO of SOX2, PAX6, OTX2, and AGO2, genes that exhibit diverse structural layout and temporal expression patterns. The availability of iKO hPSC lines will substantially transform the way we examine gene function in human cells.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  3. CD38 gene knockout juvenile mice: a model of oxytocin signal defects in autism.

    PubMed

    Higashida, Haruhiro; Yokoyama, Shigeru; Munesue, Toshio; Kikuchi, Mitsuru; Minabe, Yoshio; Lopatina, Olga

    2011-01-01

    Oxytocin (OXT) in the hypothalamus is the biological basis of social recognition, trust, and bonding. We showed that CD38, a leukaemia cell marker, plays an important role in the hypothalamus in the process of OXT release in adult mice. Disruption of Cd38 (Cd38(-/-)) produced impairment of maternal behavior and male social recognition in mice, similar to the behavior observed in Oxt and OXT receptor (Oxtr) gene knockout (Oxt(-/-) and Oxtr(-/-), respectively) mice. Locomotor activity induced by separation from the dam was higher and the number of ultrasonic vocalization (USV) calls was lower in Cd38(-/-) than Cd38(+/+) pups. These phenotypes seemed to be caused by the high plasma OXT levels during development from neonates to 3-week-old juvenile mice. ADP-ribosyl cyclase activity was markedly lower in the knockout mice from birth, suggesting that weaning for mice is a critical time window of differentiating plasma OXT. Contribution by breastfeeding was an important exogenous source for regulating plasma OXT before weaning by the presence of OXT in milk and the dam's mammary glands. The dissimilarity of Cd38(-/-) infant behaviour to Oxt(-/-) or Oxtr(-/-) mice can be explained partly by this exogenous source of OXT. These results suggest that secretion of OXT into the brain in a CD38-dependent manner may play an important role in the development of social behavior, and mice with OXT signalling deficiency, including Cd38(-/-), Oxt(-/-) and Oxtr(-/-) mice are good animal models for developmental disorders, such as autism.

  4. Beta-oxidation in hepatocyte cultures from mice with peroxisomal gene knockouts.

    PubMed

    Dirkx, Ruud; Meyhi, Els; Asselberghs, Stanny; Reddy, Janardan; Baes, Myriam; Van Veldhoven, Paul P

    2007-06-08

    Beta-oxidation of carboxylates takes place both in mitochondria and peroxisomes and in each pathway parallel enzymes exist for each conversion step. In order to better define the substrate specificities of these enzymes and in particular the elusive role of peroxisomal MFP-1, hepatocyte cultures from mice with peroxisomal gene knockouts were used to assess the consequences on substrate degradation. Hepatocytes from mice with liver selective elimination of peroxisomes displayed severely impaired oxidation of 2-methylhexadecanoic acid, the bile acid intermediate trihydroxycholestanoic acid (THCA), and tetradecanedioic acid. In contrast, mitochondrial beta-oxidation rates of palmitate were doubled, despite the severely affected inner mitochondrial membrane. As expected, beta-oxidation of the branched chain compounds 2-methylhexadecanoic acid and THCA was reduced in hepatocytes from mice with inactivation of MFP-2. More surprisingly, dicarboxylic fatty acid oxidation was impaired in MFP-1 but not in MFP-2 knockout hepatocytes, indicating that MFP-1 might play more than an obsolete role in peroxisomal beta-oxidation.

  5. Efficient gene knock-out and knock-in with transgenic Cas9 in Drosophila.

    PubMed

    Xue, Zhaoyu; Ren, Mengda; Wu, Menghua; Dai, Junbiao; Rong, Yikang S; Gao, Guanjun

    2014-03-21

    Bacterial Cas9 nuclease induces site-specific DNA breaks using small gRNA as guides. Cas9 has been successfully introduced into Drosophila for genome editing. Here, we improve the versatility of this method by developing a transgenic system that expresses Cas9 in the Drosophila germline. Using this system, we induced inheritable knock-out mutations by injecting only the gRNA into embryos, achieved highly efficient mutagenesis by expressing gRNA from the promoter of a novel non-coding RNA gene, and recovered homologous recombination-based knock-in of a fluorescent marker at a rate of 4.5% by co-injecting gRNA with a circular DNA donor.

  6. DNA-free two-gene knockout in Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins

    PubMed Central

    Baek, Kwangryul; Kim, Duk Hyoung; Jeong, Jooyeon; Sim, Sang Jun; Melis, Anastasios; Kim, Jin-Soo; Jin, EonSeon; Bae, Sangsu

    2016-01-01

    Microalgae are versatile organisms capable of converting CO2, H2O, and sunlight into fuel and chemicals for domestic and industrial consumption. Thus, genetic modifications of microalgae for enhancing photosynthetic productivity, and biomass and bio-products generation are crucial for both academic and industrial applications. However, targeted mutagenesis in microalgae with CRISPR-Cas9 is limited. Here we report, a one-step transformation of Chlamydomonas reinhardtii by the DNA-free CRISPR-Cas9 method rather than plasmids that encode Cas9 and guide RNAs. Outcome was the sequential CpFTSY and ZEP two-gene knockout and the generation of a strain constitutively producing zeaxanthin and showing improved photosynthetic productivity. PMID:27466170

  7. Norepinephrine transporter knock-out alters expression of the genes connected with antidepressant drugs action.

    PubMed

    Solich, Joanna; Kolasa, Magdalena; Kusmider, Maciej; Faron-Gorecka, Agata; Pabian, Paulina; Zurawek, Dariusz; Szafran-Pilch, Kinga; Dziedzicka-Wasylewska, Marta

    2015-01-12

    Norepinephrine transporter knock-out mice (NET-KO) exhibit depression-resistant phenotypes. They manifest significantly shorter immobility times in both the forced swim test and the tail suspension test. Moreover, biochemical studies have revealed the up-regulation of other monoamine transporters (dopamine and serotonin) in the brains of NET-KO mice, similar to the phenomenon observed after the chronic pharmacological blockade of norepinephrine transporter by desipramine in wild-type (WT) animals. NET-KO mice are also resistant to stress, as we demonstrated previously by measuring plasma corticosterone concentration. In the present study, we used a microdissection technique to separate target brain regions and the TaqMan Low Density Array approach to test the expression of a group of genes in the NET-KO mice compared with WT animals. A group of genes with altered expression were identified in four brain structures (frontal and cingulate cortices, dentate gyrus of hippocampus and basal-lateral amygdala) of NET-KO mice compared with WT mice. These genes are known to be altered by antidepressant drugs administration. The most interesting gene is Crh-bp, which modulates the activity of corticotrophin--releasing hormone (CRH) and several CRH-family members. Generally, genetic disturbances within noradrenergic neurons result in biological changes, such as in signal transduction and intercellular communication, and may be linked to changes in noradrenaline levels in the brains of NET-KO mice.

  8. EFFECTS OF HEAT AND BROMOCHLOROACETIC ACID ON MALE REPRODUCTION IN HEAT SHOCK FACTOR-1 GENE KNOCKOUT MICE

    EPA Science Inventory

    Effects of heat and bromochloroacetic acid on male reproduction in heat shock factor-1 gene knockout mice.
    Luft JC1, IJ Benjamin2, JB Garges1 and DJ Dix1. 1Reproductive Toxicology Division, USEPA, RTP, NC, 27711 and 2Dept of Internal Medicine, Univ.of Texas Southwestern Med C...

  9. Opioid Analgesia in P450 Gene Cluster Knockout Mice: A Search for Analgesia-Relevant Isoforms

    PubMed Central

    Nalwalk, Julia W.; Ding, Xinxin; Scheer, Nico

    2015-01-01

    Cytochrome P450 monooxygenases (P450s), which are well-known drug-metabolizing enzymes, are thought to play a signal transduction role in µ opioid analgesia and may serve as high-affinity 3H-cimetidine (3HCIM) binding sites in the brain. 3HCIM binding sites may also be related to opioid or nonopioid analgesia. However, of the more than 100 murine P450 enzymes, the specific isoform(s) responsible for either function have not been identified. Presently, three lines of constitutive P450 gene cluster knockout (KO) mice with full-length deletions of 14 Cyp2c, 9 Cyp2d, and 7 Cyp3a genes were studied for deficiencies in 3HCIM binding and for opioid analgesia. Liver and brain homogenates from all three genotypes showed normal 3HCIM binding values, indicating that gene products of Cyp2d, Cyp3a, and Cyp2c are not 3HCIM-binding proteins. Cyp2d KO and Cyp3a KO mice showed normal antinociceptive responses to a moderate systemic dose of morphine (20 mg/kg, s.c.), thereby excluding 16 P450 isoforms as mediators of opioid analgesia. In contrast, Cyp2c KO mice showed a 41% reduction in analgesic responses following systemically (s.c.) administered morphine. However, the significance of brain Cyp2c gene products in opioid analgesia is uncertain because little or no analgesic deficits were noted in Cyp2c KO mice following intracerebroventricular or intrathecalmorphine administration, respectively. These results show that the gene products of Cyp2d and Cyp3a do not contribute to µ opioid analgesia in the central nervous system. A possible role for Cyp2c gene products in opioid analgesia requires further consideration. PMID:26109562

  10. GRK5-Knockout Mice Generated by TALEN-Mediated Gene Targeting.

    PubMed

    Nanjidsuren, Tsevelmaa; Park, Chae-Won; Sim, Bo-Woong; Kim, Sun-Uk; Chang, Kyu-Tae; Kang, Myung-Hwa; Min, Kwan-Sik

    2016-10-01

    Transcription activator-like effector nucleases (TALENs) are a new type of engineered nuclease that is very effective for directed gene disruption in any genome sequence. We investigated the generation of mice with genetic knockout (KO) of the G protein-coupled receptor kinase (GRK) 5 gene by microinjection of TALEN mRNA. TALEN vectors were designed to target exons 1, 3, and 5 of the mouse GRK5 gene. Flow cytometry showed that the activity of the TALEN mRNAs targeted to exons 1, 3, and 5 was 8.7%, 9.7%, and 12.7%, respectively. The TALEN mRNA for exon 5 was injected into the cytoplasm of 180 one-cell embryos. Of the 53 newborns, three (5.6%) were mutant founders (F0) with mutations. Two clones from F028 showed a 45-bp deletion and F039 showed the same biallelic non-frame-shifting 3-bp deletions. Three clones from F041 were shown to possess a combination of frame-shifting 2-bp deletions. All of the mutations were transmitted through the germline but not to all progenies (37.5%, 37.5%, and 57.1% for the F028, F039, and F041 lines, respectively). The homozygote GRK5-KO mice for 28 and 41 lines created on F3 progenies and the homozygous genotype was confirmed by PCR, T7E1 assay and sequencing.

  11. Dopamine neurons from transgenic mice with a knockout of the p53 gene resist MPTP neurotoxicity.

    PubMed

    Trimmer, P A; Smith, T S; Jung, A B; Bennett, J P

    1996-09-01

    We have examined MPTP toxicity to dopamine neurons of mice homozygous for a transgenic knockout of the p53 growth control gene (p53-/-). MPTP at a total dose of 96 mg/kg administered in four doses over two days produced a non-homogeneous loss of striatal dopamine transport sites and quantitatively reduced 3H-mazindol binding to similar degrees in p53-/- and wild type controls 2 and 3 weeks after starting MPTP. Nigral DA neurons stained immunohistochemically for tyrosine hydroxylase were counted using both manual and automated methods and found to be reduced 29-34% in wild type controls but were not reduced in p53-/-. Mean DA neuronal surface areas were reduced 63-68% by MPTP in controls and 35-50% in p53-/-. We conclude that p53 protein appears necessary for complete expression of MPTP neurotoxicity to dopamine neurons. Our findings suggest that the p53 gene and other growth control genes may regulate dopamine neuronal death in PD.

  12. Phototransduction Influences Metabolic Flux and Nucleotide Metabolism in Mouse Retina*

    PubMed Central

    Du, Jianhai; Rountree, Austin; Cleghorn, Whitney M.; Contreras, Laura; Lindsay, Ken J.; Sadilek, Martin; Gu, Haiwei; Djukovic, Danijel; Raftery, Dan; Satrústegui, Jorgina; Kanow, Mark; Chan, Lawrence; Tsang, Stephen H.; Sweet, Ian R.; Hurley, James B.

    2016-01-01

    Production of energy in a cell must keep pace with demand. Photoreceptors use ATP to maintain ion gradients in darkness, whereas in light they use it to support phototransduction. Matching production with consumption can be accomplished by coupling production directly to consumption. Alternatively, production can be set by a signal that anticipates demand. In this report we investigate the hypothesis that signaling through phototransduction controls production of energy in mouse retinas. We found that respiration in mouse retinas is not coupled tightly to ATP consumption. By analyzing metabolic flux in mouse retinas, we also found that phototransduction slows metabolic flux through glycolysis and through intermediates of the citric acid cycle. We also evaluated the relative contributions of regulation of the activities of α-ketoglutarate dehydrogenase and the aspartate-glutamate carrier 1. In addition, a comprehensive analysis of the retinal metabolome showed that phototransduction also influences steady-state concentrations of 5′-GMP, ribose-5-phosphate, ketone bodies, and purines. PMID:26677218

  13. A Jacob/Nsmf Gene Knockout Results in Hippocampal Dysplasia and Impaired BDNF Signaling in Dendritogenesis.

    PubMed

    Spilker, Christina; Nullmeier, Sven; Grochowska, Katarzyna M; Schumacher, Anne; Butnaru, Ioana; Macharadze, Tamar; Gomes, Guilherme M; Yuanxiang, PingAn; Bayraktar, Gonca; Rodenstein, Carolin; Geiseler, Carolin; Kolodziej, Angela; Lopez-Rojas, Jeffrey; Montag, Dirk; Angenstein, Frank; Bär, Julia; D'Hanis, Wolfgang; Roskoden, Thomas; Mikhaylova, Marina; Budinger, Eike; Ohl, Frank W; Stork, Oliver; Zenclussen, Ana C; Karpova, Anna; Schwegler, Herbert; Kreutz, Michael R

    2016-03-01

    Jacob, the protein encoded by the Nsmf gene, is involved in synapto-nuclear signaling and docks an N-Methyl-D-Aspartate receptor (NMDAR)-derived signalosome to nuclear target sites like the transcription factor cAMP-response-element-binding protein (CREB). Several reports indicate that mutations in NSMF are related to Kallmann syndrome (KS), a neurodevelopmental disorder characterized by idiopathic hypogonadotropic hypogonadism (IHH) associated with anosmia or hyposmia. It has also been reported that a protein knockdown results in migration deficits of Gonadotropin-releasing hormone (GnRH) positive neurons from the olfactory bulb to the hypothalamus during early neuronal development. Here we show that mice that are constitutively deficient for the Nsmf gene do not present phenotypic characteristics related to KS. Instead, these mice exhibit hippocampal dysplasia with a reduced number of synapses and simplification of dendrites, reduced hippocampal long-term potentiation (LTP) at CA1 synapses and deficits in hippocampus-dependent learning. Brain-derived neurotrophic factor (BDNF) activation of CREB-activated gene expression plays a documented role in hippocampal CA1 synapse and dendrite formation. We found that BDNF induces the nuclear translocation of Jacob in an NMDAR-dependent manner in early development, which results in increased phosphorylation of CREB and enhanced CREB-dependent Bdnf gene transcription. Nsmf knockout (ko) mice show reduced hippocampal Bdnf mRNA and protein levels as well as reduced pCREB levels during dendritogenesis. Moreover, BDNF application can rescue the morphological deficits in hippocampal pyramidal neurons devoid of Jacob. Taken together, the data suggest that the absence of Jacob in early development interrupts a positive feedback loop between BDNF signaling, subsequent nuclear import of Jacob, activation of CREB and enhanced Bdnf gene transcription, ultimately leading to hippocampal dysplasia.

  14. A Jacob/Nsmf Gene Knockout Results in Hippocampal Dysplasia and Impaired BDNF Signaling in Dendritogenesis

    PubMed Central

    Schumacher, Anne; Butnaru, Ioana; Macharadze, Tamar; Gomes, Guilherme M.; Yuanxiang, PingAn; Bayraktar, Gonca; Rodenstein, Carolin; Geiseler, Carolin; Kolodziej, Angela; Lopez-Rojas, Jeffrey; Montag, Dirk; Angenstein, Frank; Bär, Julia; D’Hanis, Wolfgang; Roskoden, Thomas; Mikhaylova, Marina; Budinger, Eike; Ohl, Frank W.; Stork, Oliver; Zenclussen, Ana C.; Karpova, Anna; Schwegler, Herbert; Kreutz, Michael R.

    2016-01-01

    Jacob, the protein encoded by the Nsmf gene, is involved in synapto-nuclear signaling and docks an N-Methyl-D-Aspartate receptor (NMDAR)-derived signalosome to nuclear target sites like the transcription factor cAMP-response-element-binding protein (CREB). Several reports indicate that mutations in NSMF are related to Kallmann syndrome (KS), a neurodevelopmental disorder characterized by idiopathic hypogonadotropic hypogonadism (IHH) associated with anosmia or hyposmia. It has also been reported that a protein knockdown results in migration deficits of Gonadotropin-releasing hormone (GnRH) positive neurons from the olfactory bulb to the hypothalamus during early neuronal development. Here we show that mice that are constitutively deficient for the Nsmf gene do not present phenotypic characteristics related to KS. Instead, these mice exhibit hippocampal dysplasia with a reduced number of synapses and simplification of dendrites, reduced hippocampal long-term potentiation (LTP) at CA1 synapses and deficits in hippocampus-dependent learning. Brain-derived neurotrophic factor (BDNF) activation of CREB-activated gene expression plays a documented role in hippocampal CA1 synapse and dendrite formation. We found that BDNF induces the nuclear translocation of Jacob in an NMDAR-dependent manner in early development, which results in increased phosphorylation of CREB and enhanced CREB-dependent Bdnf gene transcription. Nsmf knockout (ko) mice show reduced hippocampal Bdnf mRNA and protein levels as well as reduced pCREB levels during dendritogenesis. Moreover, BDNF application can rescue the morphological deficits in hippocampal pyramidal neurons devoid of Jacob. Taken together, the data suggest that the absence of Jacob in early development interrupts a positive feedback loop between BDNF signaling, subsequent nuclear import of Jacob, activation of CREB and enhanced Bdnf gene transcription, ultimately leading to hippocampal dysplasia. PMID:26977770

  15. BK Knockout by TALEN-Mediated Gene Targeting in Osteoblasts: KCNMA1 Determines the Proliferation and Differentiation of Osteoblasts

    PubMed Central

    Hei, Hongya; Gao, Jianjun; Dong, Jibin; Tao, Jie; Tian, Lulu; Pan, Wanma; Wang, Hongyu; Zhang, Xuemei

    2016-01-01

    Large conductance calcium-activated potassium (BK) channels participate in many important physiological functions in excitable tissues such as neurons, cardiac and smooth muscles, whereas the knowledge of BK channels in bone tissues and osteoblasts remains elusive. To investigate the role of BK channels in osteoblasts, we used transcription activator-like effector nuclease (TALEN) to establish a BK knockout cell line on rat ROS17/2.8 osteoblast, and detected the proliferation and mineralization of the BK-knockout cells. Our study found that the BK-knockout cells significantly decreased the ability of proliferation and mineralization as osteoblasts, compared to the wild type cells. The overall expression of osteoblast differentiation marker genes in the BK-knockout cells was significantly lower than that in wild type osteoblast cells. The BK-knockout osteoblast cell line in our study displays a phenotype decrease in osteoblast function which can mimic the pathological state of osteoblast and thus provide a working cell line as a tool for study of osteoblast function and bone related diseases. PMID:27329042

  16. Characterization of Frog Virus 3 knockout mutants lacking putative virulence genes.

    PubMed

    Andino, Francisco De Jesús; Grayfer, Leon; Chen, Guangchun; Chinchar, V Gregory; Edholm, Eva-Stina; Robert, Jacques

    2015-11-01

    To identify ranavirus virulence genes, we engineered Frog Virus 3 (FV3) knockout (KO) mutants defective for a putative viral caspase activation and recruitment domain-containing (CARD) protein (Δ64R-FV3) and a β-hydroxysteroid dehydrogenase homolog (Δ52L-FV3). Compared to wild type (WT) FV3, infection of Xenopus tadpoles with Δ64R- or Δ52L-FV3 resulted in significantly lower levels of mortality and viral replication. We further characterized these and two earlier KO mutants lacking the immediate-early18kDa protein (FV3-Δ18K) or the truncated viral homolog of eIF-2α (FV3-ΔvIF-2α). All KO mutants replicated as well as WT-FV3 in non-amphibian cell lines, whereas in Xenopus A6 kidney cells replication of ΔvCARD-, ΔvβHSD- and ΔvIF-2α-FV3 was markedly reduced. Furthermore, Δ64R- and ΔvIF-2α-FV3 were more sensitive to interferon than WT and Δ18-FV3. Notably, Δ64R-, Δ18K- and ΔvIF-2α- but not Δ52L-FV3 triggered more apoptosis than WT FV3. These data suggest that vCARD (64R) and vβ-HSD (52L) genes contribute to viral pathogenesis.

  17. TGFbeta inducible early gene-1 knockout mice display defects in bone strength and microarchitecture.

    PubMed

    Bensamoun, Sabine F; Hawse, John R; Subramaniam, Malayannan; Ilharreborde, Brice; Bassillais, Armelle; Benhamou, Claude L; Fraser, Daniel G; Oursler, Merry J; Amadio, Peter C; An, Kai-Nan; Spelsberg, Thomas C

    2006-12-01

    TGFbeta inducible early gene-1 (TIEG) is a member of the Sp/Krüppel-like transcription factor family originally cloned from human osteoblasts. We have previously demonstrated that TIEG plays a role in the expression of important osteoblast marker genes and in the maturation/differentiation of osteoblasts. To elucidate the function of TIEG in skeletal development and maintenance, we have generated a TIEG knockout (KO) mouse. Three-point bending tests demonstrated that the femurs of TIEG KO mice are significantly weaker than those of wild-type animals. pQCT analysis of tibias revealed significant decreases in bone content, density and size in KO animals compared to wild-type mice. Micro-CT analysis of the femoral head and vertebrae revealed increases in femoral head trabecular separation and decreases in cortical bone thickness and vertebral bone volume in KO mice relative to wild-type controls. In addition, electron microscopy indicated a significant decrease in osteocyte number in the femurs of KO mice. Taken together, these data demonstrate that the bones of TIEG KO mice display an osteopenic phenotype with significantly weaker bones and reduced amounts of cortical and trabecular bone. In summary, an important role for TIEG in skeletal development and/or homeostasis is indicated.

  18. Selective Photoreceptor Gene Knock-out Reveals a Regulatory Role for the Growth Behavior of Pseudomonas syringae.

    PubMed

    Shah, Rashmi; Pathak, Gopal; Drepper, Thomas; Gärtner, Wolfgang

    2016-07-01

    The plant pathogen Pseudomonas syringae (Ps) is a well-established model organism for bacterial infection of plants. The genome sequences of two pathovars, pv. syringae and pv. tomato, revealed one gene encoding a blue and two genes encoding red/far red light-sensing photoreceptors. Continuing former molecular characterization of the photoreceptor proteins, we here report selective photoreceptor gene disruption for pv. tomato aiming at identification of potentially regulatory functions of these photoreceptors. Transformation of Ps cells with linear DNA constructs yielded interposon mutations of the corresponding genes. Cell growth studies of the generated photoreceptor knock-out mutants revealed their role in light-dependent regulation of cell growth and motility. Disruption of the blue-light (BL) receptor gene caused a growth deregulation, in line with an observed increased virulence of this mutant (Moriconi et al., Plant J., 2013, 76, 322). Bacterial phytochrome-1 (BphP1) deletion mutant caused unaltered cell growth, but a stronger swarming capacity. Inactivation of its ortholog, BphP2, however, caused reduced growth and remarkably altered dendritic swarming behavior. Combined knock-out of both bacteriophytochromes reproduced the swarming pattern observed for the BphP2 mutant alone. A triple knock-out mutant showed a growth rate between that of the BL (deregulation) and the phytochrome-2 mutant (growth reduction).

  19. PDHA1 gene knockout in prostate cancer cells results in metabolic reprogramming towards greater glutamine dependence

    PubMed Central

    Li, Yaqing; Li, Xiaoran; Li, Xiaoli; Zhong, Yali; Ji, Yasai; Yu, Dandan; Zhang, Mingzhi; Wen, Jian-Guo; Zhang, Hongquan; Goscinski, Mariusz Adam; Nesland, Jahn M.; Suo, Zhenhe

    2016-01-01

    Alternative pathways of metabolism endowed cancer cells with metabolic stress. Inhibiting the related compensatory pathways might achieve synergistic anticancer results. This study demonstrated that pyruvate dehydrogenase E1α gene knockout (PDHA1 KO) resulted in alterations in tumor cell metabolism by rendering the cells with increased expression of glutaminase1 (GLS1) and glutamate dehydrogenase1 (GLUD1), leading to an increase in glutamine-dependent cell survival. Deprivation of glutamine induced cell growth inhibition, increased reactive oxygen species and decreased ATP production. Pharmacological blockade of the glutaminolysis pathway resulted in massive tumor cells apoptosis and dysfunction of ROS scavenge in the LNCaP PDHA1 KO cells. Further examination of the key glutaminolysis enzymes in human prostate cancer samples also revealed that higher levels of GLS1 and GLUD1 expression were significantly associated with aggressive clinicopathological features and poor clinical outcome. These insights supply evidence that glutaminolysis plays a compensatory role for cell survival upon alternative energy metabolism and targeting the glutamine anaplerosis of energy metabolism via GLS1 and GLUD1 in cancer cells may offer a potential novel therapeutic strategy. PMID:27462778

  20. Time-point and dosage of gene inactivation determine the tumor spectrum in conditional Ptch knockouts.

    PubMed

    Zibat, Arne; Uhmann, Anja; Nitzki, Frauke; Wijgerde, Mark; Frommhold, Anke; Heller, Tanja; Armstrong, Victor; Wojnowski, Leszek; Quintanilla-Martinez, Leticia; Reifenberger, Julia; Schulz-Schaeffer, Walter; Hahn, Heidi

    2009-06-01

    Mutations in Patched (PTCH) have been associated with tumors characteristic both for children [medulloblastoma (MB) and rhabdomyosarcoma (RMS)] and for elderly [basal cell carcinoma (BCC)]. The determinants of the variability in tumor onset and histology are unknown. We investigated the effects of the time-point and dosage of Ptch inactivation on tumor spectrum using conditional Ptch-knockout mice. Ptch heterozygosity induced prenatally resulted in the formation of RMS, which was accompanied by the silencing of the remaining wild-type Ptch allele. In contrast, RMS was observed neither after mono- nor biallelic postnatal deletion of Ptch. Postnatal biallelic deletion of Ptch led to BCC precancerous lesions of the gastrointestinal epithelium and mesenteric tumors. Hamartomatous gastrointestinal cystic tumors were induced by monoallelic, but not biallelic Ptch mutations, independently of the time-point of mutation induction. These data suggest that the expressivity of Ptch deficiency is largely determined by the time-point, the gene dose and mode of Ptch inactivation. Furthermore, they point to key differences in the tumorigenic mechanisms underlying adult and childhood tumors. The latter ones are unique among all tumors since their occurrence decreases rather than increases with age. A better understanding of mechanisms underlying this ontological restriction is of potential therapeutic value.

  1. Conditional knockout of the Slc5a6 gene in mouse intestine impairs biotin absorption.

    PubMed

    Ghosal, Abhisek; Lambrecht, Nils; Subramanya, Sandeep B; Kapadia, Rubina; Said, Hamid M

    2013-01-01

    The Slc5a6 gene expresses a plasma membrane protein involved in the transport of the water-soluble vitamin biotin; the transporter is commonly referred to as the sodium-dependent multivitamin transporter (SMVT) because it also transports pantothenic acid and lipoic acid. The relative contribution of the SMVT system toward carrier-mediated biotin uptake in the native intestine in vivo has not been established. We used a Cre/lox technology to generate an intestine-specific (conditional) SMVT knockout (KO) mouse model to address this issue. The KO mice exhibited absence of expression of SMVT in the intestine compared with sex-matched littermates as well as the expected normal SMVT expression in other tissues. About two-thirds of the KO mice died prematurely between the age of 6 and 10 wk. Growth retardation, decreased bone density, decreased bone length, and decreased biotin status were observed in the KO mice. Microscopic analysis showed histological abnormalities in the small bowel (shortened villi, dysplasia) and cecum (chronic active inflammation, dysplasia) of the KO mice. In vivo (and in vitro) transport studies showed complete inhibition in carrier-mediated biotin uptake in the intestine of the KO mice compared with their control littermates. These studies provide the first in vivo confirmation in native intestine that SMVT is solely responsible for intestinal biotin uptake. These studies also provide evidence for a casual association between SMVT function and normal intestinal health.

  2. Melanopsin Phototransduction Contributes to Light-Evoked Choroidal Expansion and Rod L-Type Calcium Channel Function In Vivo

    PubMed Central

    Berkowitz, Bruce A.; Schmidt, Tiffany; Podolsky, Robert H.; Roberts, Robin

    2016-01-01

    Purpose In humans, rodents, and pigeons, the dark → light transition signals nonretinal brain tissue to increase choroidal thickness, a major control element of choroidal blood flow, and thus of photoreceptor and retinal pigment epithelium function. However, it is unclear which photopigments in the retina relay the light signal to the brain. Here, we test the hypothesis that melanopsin (Opn4)-regulated phototransduction modulates light-evoked choroidal thickness expansion in mice. Methods Two-month-old C57Bl/6 wild-type (B6), 4- to 5-month-old C57Bl/6/129S6 wild-type (B6 + S6), and 2-month-old melanopsin knockout (Opn4−/−) on a B6 + S6 background were studied. Retinal anatomy was evaluated in vivo by optical coherence tomography and MRI. Choroidal thickness in dark and light were measured by diffusion-weighted MRI. Rod cell L-type calcium channel (LTCC) function in dark and light (manganese-enhanced MRI [MEMRI]) was also measured. Results Opn4−/− mice did not show the light-evoked expansion of choroidal thickness observed in B6 and B6 + S6 controls. Additionally, Opn4−/− mice had lower than normal rod cell and inner retinal LTCC function in the dark but not in the light. These deficits were not due to structural abnormalities because retinal laminar architecture and thickness, and choroidal thickness in the Opn4−/− mice were similar to controls. Conclusions First time evidence is provided that melanopsin phototransduction contributes to dark → light control of murine choroidal thickness. The data also highlight a contribution in vivo of melanopsin phototransduction to rod cell and inner retinal depolarization in the dark. PMID:27727394

  3. Kidney development and gene expression in the HIF2alpha knockout mouse.

    PubMed

    Steenhard, Brooke M; Freeburg, Paul B; Isom, Kathryn; Stroganova, Larysa; Borza, Dorin-Bogdan; Hudson, Billy G; St John, Patricia L; Zelenchuk, Adrian; Abrahamson, Dale R

    2007-04-01

    The hypoxia-inducible transcription factor-2 (HIF2), a heterodimer composed of HIF2alpha and HIF1beta subunits, drives expression of genes essential for vascularization, including vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR-2, Flk-1). Here, we used a HIF2alpha/LacZ transgenic mouse to define patterns of HIF2alpha transcription during kidney development and maturation. Our results from embryonic heterozygotes showed HIF2alpha/LacZ expression by apparently all renal endothelial cells. At 4 weeks of age, glomerular mesangial and vascular smooth muscle cells were also positive together with endothelial cells. These expression patterns were confirmed by electron microscopy using Bluo-gal as a beta-galactosidase substrate. Small numbers of glomerular and tubular epithelial cells were also positive at all stages examined. Light and electron microscopic examination of kidneys from HIF2alpha null embryos showed no defects in renal vascular development or nephrogenesis. Similarly, the same amounts of Flk-1 protein were seen on Western blots of kidney extracts from homozygous and heterozygous HIF2alpha mutants. To examine responsiveness of HIF2alpha null kidneys to hypoxia, embryonic day 13.5 metanephroi were cultured in room air or in mild (5% O(2)) hypoxia. For both heterozygous and null samples, VEGF mRNA levels doubled when metanephroi were cultured in mild hypoxia. Anterior chamber grafts of embryonic HIF2alpha knockouts were morphologically indistinguishable from heterozygous grafts. Endothelial markers, platelet endothelial cell adhesion molecule and BsLB4, as well as glomerular epithelial markers, GLEPP1 and WT-1, were all expressed appropriately. Finally, we undertook quantitative real-time polymerase chain reaction of kidneys from HIF2alpha null embryos and wild-type siblings and found no compensatory up-regulation of HIF1alpha or -3alpha. Our results show that, although HIF2alpha was widely transcribed by kidney endothelium and vascular

  4. CRISPR Knockout of the HuR Gene Causes a Xenograft Lethal Phenotype.

    PubMed

    Lal, Shruti; Cheung, Edwin C; Zarei, Mahsa; Preet, Ranjan; Chand, Saswati N; Mambelli-Lisboa, Nicole C; Romeo, Carmella; Stout, Matthew C; Londin, Eric; Goetz, Austin; Lowder, Cinthya Y; Nevler, Avinoam; Yeo, Charles J; Campbell, Paul M; Winter, Jordan M; Dixon, Dan A; Brody, Jonathan R

    2017-02-27

    Pancreatic ductal adenocarcinoma (PDA) is the third leading cause of cancer related deaths in the U.S., while colorectal cancer (CRC) is the third most common cancer. The RNA binding protein HuR (ELAVL1), supports a pro-oncogenic network in gastrointestinal (GI) cancer cells through enhanced HuR expression. Using a publically available database, HuR expression levels were determined to be increased in primary PDA and CRC tumor cohorts as compared to normal pancreas and colon tissues, respectively. CRISPR/Cas9 technology was successfully used to delete the HuR gene in both PDA (MIA PaCa-2 and Hs 766T) and CRC (HCT116) cell lines. HuR deficiency has a mild phenotype, in vitro, as HuR-deficient MIA PaCa-2 (MIA.HuR-KO(-/-)) cells had increased apoptosis when compared to isogenic wild-type (MIA.HuR-WT(+/+)) cells. Using this isogenic system, mRNAs were identified that specifically bound to HuR and were required for transforming a 2D culture into 3D (i.e., organoids). Importantly, HuR-deficient MIA PaCa-2 and Hs 766T cells were unable to engraft tumors in vivo compared to control HuR-proficient cells, demonstrating a unique xenograft lethal phenotype. While not as a dramatic phenotype, CRISPR knockout HuR HCT116 colon cancer cells (HCT.HuR-KO(-/-)) showed significantly reduced in vivo tumor growth compared to controls (HCT.HuR-WT(+/+)). Finally, HuR deletion affects KRAS activity and controls a subset of pro-oncogenic genes.

  5. Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes.

    PubMed

    Li, Ting; Huang, Sheng; Zhao, Xuefeng; Wright, David A; Carpenter, Susan; Spalding, Martin H; Weeks, Donald P; Yang, Bing

    2011-08-01

    Recent studies indicate that the DNA recognition domain of transcription activator-like (TAL) effectors can be combined with the nuclease domain of FokI restriction enzyme to produce TAL effector nucleases (TALENs) that, in pairs, bind adjacent DNA target sites and produce double-strand breaks between the target sequences, stimulating non-homologous end-joining and homologous recombination. Here, we exploit the four prevalent TAL repeats and their DNA recognition cipher to develop a 'modular assembly' method for rapid production of designer TALENs (dTALENs) that recognize unique DNA sequence up to 23 bases in any gene. We have used this approach to engineer 10 dTALENs to target specific loci in native yeast chromosomal genes. All dTALENs produced high rates of site-specific gene disruptions and created strains with expected mutant phenotypes. Moreover, dTALENs stimulated high rates (up to 34%) of gene replacement by homologous recombination. Finally, dTALENs caused no detectable cytotoxicity and minimal levels of undesired genetic mutations in the treated yeast strains. These studies expand the realm of verified TALEN activity from cultured human cells to an intact eukaryotic organism and suggest that low-cost, highly dependable dTALENs can assume a significant role for gene modifications of value in human and animal health, agriculture and industry.

  6. Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes

    SciTech Connect

    Li, T; Huang, S; Zhao, XF; Wright, DA; Carpenter, S; Spalding, MH; Weeks, DP; Yang, B

    2011-08-08

    Recent studies indicate that the DNA recognition domain of transcription activator-like (TAL) effectors can be combined with the nuclease domain of FokI restriction enzyme to produce TAL effector nucleases (TALENs) that, in pairs, bind adjacent DNA target sites and produce double-strand breaks between the target sequences, stimulating non-homologous end-joining and homologous recombination. Here, we exploit the four prevalent TAL repeats and their DNA recognition cipher to develop a 'modular assembly' method for rapid production of designer TALENs (dTALENs) that recognize unique DNA sequence up to 23 bases in any gene. We have used this approach to engineer 10 dTALENs to target specific loci in native yeast chromosomal genes. All dTALENs produced high rates of site-specific gene disruptions and created strains with expected mutant phenotypes. Moreover, dTALENs stimulated high rates (up to 34%) of gene replacement by homologous recombination. Finally, dTALENs caused no detectable cytotoxicity and minimal levels of undesired genetic mutations in the treated yeast strains. These studies expand the realm of verified TALEN activity from cultured human cells to an intact eukaryotic organism and suggest that low-cost, highly dependable dTALENs can assume a significant role for gene modifications of value in human and animal health, agriculture and industry.

  7. A Calcium-Relay Mechanism in Vertebrate Phototransduction

    PubMed Central

    2013-01-01

    Calcium-signaling in cells requires a fine-tuned system of calcium-transport proteins involving ion channels, exchangers, and ion-pumps but also calcium-sensor proteins and their targets. Thus, control of physiological responses very often depends on incremental changes of the cytoplasmic calcium concentration, which are sensed by calcium-binding proteins and are further transmitted to specific target proteins. This Review will focus on calcium-signaling in vertebrate photoreceptor cells, where recent physiological and biochemical data indicate that a subset of neuronal calcium sensor proteins named guanylate cyclase-activating proteins (GCAPs) operate in a calcium-relay system, namely, to make gradual responses to small changes in calcium. We will further integrate this mechanism in an existing computational model of phototransduction showing that it is consistent and compatible with the dynamics that are characteristic for the precise operation of the phototransduction pathways. PMID:23472635

  8. Individual Differences in Sleep Timing Relate to Melanopsin-Based Phototransduction in Healthy Adolescents and Young Adults

    PubMed Central

    van der Meijden, Wisse P.; Van Someren, Jamie L.; te Lindert, Bart H.W.; Bruijel, Jessica; van Oosterhout, Floor; Coppens, Joris E.; Kalsbeek, Andries; Cajochen, Christian; Bourgin, Patrice; Van Someren, Eus J.W.

    2016-01-01

    Study Objectives: Individual differences in sleep timing have been widely recognized and are of particular relevance in adolescents and young adults who often show mild to severely delayed sleep. The biological mechanisms underlying the between-subject variance remain to be determined. Recent human genetics studies showed an association between sleep timing and melanopsin gene variation, but support for functional effects on downstream pathways and behavior was not demonstrated before. We therefore investigated the association between the autonomic (i.e., pupil diameter) and behavioral (i.e., sleep timing) readouts of two different downstream brain areas, both affected by the same melanopsin-dependent retinal phototransduction: the olivary pretectal nucleus (OPN) and the suprachiasmatic nucleus (SCN). Methods: Our study population included 71 healthy individuals within an age range with known vulnerability to a delayed sleep phase (16.8–35.7 y, 37 males, 34 females). Pupillometry was performed to estimate functionality of the intrinsic melanopsin-signaling circuitry based on the OPN-mediated post-illumination pupil response (PIPR) to blue light. Sleep timing was quantified by estimating the SCN-mediated mid-sleep timing in three different ways in parallel: using a chronotype questionnaire, a sleep diary, and actigraphy. Results: All three measures consistently showed that those individuals with a later mid-sleep timing had a more pronounced PIPR (0.03 < P < 0.05), indicating a stronger blue-light responsiveness of the intrinsic melanopsin-based phototransduction circuitry. Conclusions: Trait-like individual differences in the melanopsin phototransduction circuitry contribute to individual differences in sleep timing. Blue light-sensitive young individuals are more prone to delayed sleep. Citation: van der Meijden WP, Van Someren JL; te Lindert BH, Bruijel J, van Oosterhout F, Coppens JE, Kalsbeek A, Cajochen C, Bourgin P, Van Someren EJ. Individual differences in

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

    PubMed

    Watanabe, Takahito; Noji, Sumihare; Mito, Taro

    2014-08-15

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

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

    PubMed

    Watanabe, Takahito; Noji, Sumihare; Mito, Taro

    2016-01-01

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

  11. Knockout targeting of the Drosophila nap1 gene and examination of DNA repair tracts in the recombination products.

    PubMed Central

    Lankenau, Susanne; Barnickel, Thorsten; Marhold, Joachim; Lyko, Frank; Mechler, Bernard M; Lankenau, Dirk-Henner

    2003-01-01

    We used ends-in gene targeting to generate knockout mutations of the nucleosome assembly protein 1 (Nap1) gene in Drosophila melanogaster. Three independent targeted null-knockout mutations were produced. No wild-type NAP1 protein could be detected in protein extracts. Homozygous Nap1(KO) knockout flies were either embryonic lethal or poorly viable adult escapers. Three additional targeted recombination products were viable. To gain insight into the underlying molecular processes we examined conversion tracts in the recombination products. In nearly all cases the I-SceI endonuclease site of the donor vector was replaced by the wild-type Nap1 sequence. This indicated exonuclease processing at the site of the double-strand break (DSB), followed by replicative repair at donor-target junctions. The targeting products are best interpreted either by the classical DSB repair model or by the break-induced recombination (BIR) model. Synthesis-dependent strand annealing (SDSA), which is another important recombinational repair pathway in the germline, does not explain ends-in targeting products. We conclude that this example of gene targeting at the Nap1 locus provides added support for the efficiency of this method and its usefulness in targeting any arbitrary locus in the Drosophila genome. PMID:12618400

  12. Pleiotropic consequences of gene knockouts in the phthiocerol dimycocerosate and phenolic glycolipid biosynthetic gene cluster of the opportunistic human pathogen Mycobacterium marinum.

    PubMed

    Mohandas, Poornima; Budell, William C; Mueller, Emily; Au, Andrew; Bythrow, Glennon V; Quadri, Luis E N

    2016-03-01

    Phthiocerol dimycocerosates (PDIMs) and phenolic glycolipids (PGLs) contribute to the pathogenicity of several mycobacteria. Biosynthesis of these virulence factors requires polyketide synthases and other enzymes that represent potential targets for the development of adjuvant antivirulence drugs. We used six isogenic Mycobacterium marinum mutants, each with a different gene knockout in the PDIM/PGL biosynthetic pathway, to probe the pleiotropy of mutations leading to PDIM(-) PGL(-), PDIM(+) PGL(-) or PDIM(-) PGL(+) phenotypes. We evaluated the M. marinum mutants for changes in antibiotic susceptibility, cell envelope permeability, biofilm formation, surface properties, sliding motility and virulence in an amoeba model. The analysis also permitted us to begin exploring the hypothesis that different gene knockouts rendering the same PDIM and/or PGL deficiency phenotypes lead to M. marinum mutants with equivalent pleiotropic profiles. Overall, the results of our study revealed a complex picture of pleiotropic patterns emerging from different gene knockouts, uncovered unexpected phenotypic inequalities between mutants, and provided new insight into the phenotypic consequences of gene knockouts in the PDIM/PGL biosynthetic pathway.

  13. Host knockout of E-prostanoid 2 receptors reduces tumor growth and causes major alterations of gene expression in prostaglandin E2-producing tumors

    PubMed Central

    Asting, Annika Gustafsson; Iresjö, Britt-Marie; Nilsberth, Camilla; Smedh, Ulrika; Lundholm, Kent

    2017-01-01

    Prostaglandin E2 (PGE2) is elevated in a variety of malignant tumors and has been shown to affect several hallmarks of cancer. Accordingly, the PGE2 receptor, E-prostanoid 2 (EP2), has been reported to be associated with patient survival and reduced tumor growth in EP2-knockout mice. Thus, the aim of the present study was to screen for major gene expression alterations in tumor tissue growing in EP2-knockout mice. EP2-knockout mice were bred and implanted with EP2 receptor-expressing and PGE2-producing epithelial-like tumors. Tumor tissue and plasma were collected and used for analyses with gene expression microarrays and multiplex enzyme-linked immunosorbent assays. Tumor growth, acute phase reactions/systemic inflammation and the expression of interleukin-6 were reduced in EP2-knockout tumor-bearing mice. Several hundreds of genes displayed major changes of expression in the tumor tissue when grown in EP2-knockout mice. Such gene alterations involved several different cellular functions, including stemness, migration and cell signaling. Besides gene expression, several long non-coding RNAs were downregulated in the tumors from the EP2-knockout mice. Overall, PGE2 signaling via host EP2 receptors affected a large number of different genes involved in tumor progression based on signaling between host stroma and tumor cells, which caused reduced tumor growth. PMID:28123585

  14. CRISPR/Cas9 mediated chicken Stra8 gene knockout and inhibition of male germ cell differentiation

    PubMed Central

    Zuo, Qisheng; Li, Dong; Zhang, Wenhui; Wang, Fei; Ji, Yanqin; Jin, Jing; Lu, Zhenyu; Wang, Man; Zhang, Chen; Li, Bichun

    2017-01-01

    An efficient genome editing approach had been established to construct the stable transgenic cell lines in the domestic chicken (Gallus gallus domesticus) at present. Our objectives were to investigate gene function in the differentiation process of chicken embryonic stem cells (ESCs) into spermatogonial stem cells(SSCs). Three guides RNA (gRNAs) were designed to knockout the Stra8 gene, and knockout efficiency was evaluated in domestic chicken cells using cleavage activity of in vitro transcription of gRNA, Luciferase-SSA assay, T7 endonuclease I assay(T7E1) and TA clone sequence. In addition, the Cas9/gRNA plasmid was transfected into ESCs to confirm the function of Stra8. SSA assay results showed that luciferase activity of the vector expressing gRNA-1 and gRNA- 2 was higher than that of gRNA-3. TA clone sequencing showed that the knockdown efficiency was 25% (10/40) in DF-1 cells, the knockdown efficiency was 23% (9/40) in chicken ESCs. T7E1 assay indicated that there were cleavage activity for three individuals, and the knockdown efficiency was 12% (3/25). Cell morphology, qRT-PCR, immunostaining and FCS indicated that Cas9/gRNA not only resulted in the knockout of Stra8 gene, but also suggested that the generation of SSCs was blocked by the Stra8 gene knockdown in vitro. Taken together, our results indicate that the CRISPR/Cas9 system could mediate stable Stra8 gene knockdown in domestic chicken’s cells and inhibit ECSs differentiation into SSCs. PMID:28234938

  15. CRISPR/Cas9 mediated chicken Stra8 gene knockout and inhibition of male germ cell differentiation.

    PubMed

    Zhang, Yani; Wang, Yingjie; Zuo, Qisheng; Li, Dong; Zhang, Wenhui; Wang, Fei; Ji, Yanqin; Jin, Jing; Lu, Zhenyu; Wang, Man; Zhang, Chen; Li, Bichun

    2017-01-01

    An efficient genome editing approach had been established to construct the stable transgenic cell lines in the domestic chicken (Gallus gallus domesticus) at present. Our objectives were to investigate gene function in the differentiation process of chicken embryonic stem cells (ESCs) into spermatogonial stem cells(SSCs). Three guides RNA (gRNAs) were designed to knockout the Stra8 gene, and knockout efficiency was evaluated in domestic chicken cells using cleavage activity of in vitro transcription of gRNA, Luciferase-SSA assay, T7 endonuclease I assay(T7E1) and TA clone sequence. In addition, the Cas9/gRNA plasmid was transfected into ESCs to confirm the function of Stra8. SSA assay results showed that luciferase activity of the vector expressing gRNA-1 and gRNA- 2 was higher than that of gRNA-3. TA clone sequencing showed that the knockdown efficiency was 25% (10/40) in DF-1 cells, the knockdown efficiency was 23% (9/40) in chicken ESCs. T7E1 assay indicated that there were cleavage activity for three individuals, and the knockdown efficiency was 12% (3/25). Cell morphology, qRT-PCR, immunostaining and FCS indicated that Cas9/gRNA not only resulted in the knockout of Stra8 gene, but also suggested that the generation of SSCs was blocked by the Stra8 gene knockdown in vitro. Taken together, our results indicate that the CRISPR/Cas9 system could mediate stable Stra8 gene knockdown in domestic chicken's cells and inhibit ECSs differentiation into SSCs.

  16. Cytokine knockouts in reproduction: the use of gene ablation to dissect roles of cytokines in reproductive biology.

    PubMed

    Ingman, Wendy V; Jones, Rebecca L

    2008-01-01

    Cytokines play many diverse and important roles in reproductive biology, and dissecting the complex interactions between these proteins and the different reproductive organs is a difficult task. One approach is to use gene ablation, or 'knockout', to analyse the effect of deletion of a single cytokine on mouse reproductive function. This review summarizes the essential roles of cytokines in reproductive biology that have been revealed by gene knockout studies, including development and regulation of the hypothalamo-pituitary-gondal axis, ovarian folliculogenesis, implantation and immune system modulation during pregnancy. However, successful utilization of this approach must consider the caveats associated with gene ablation studies, e.g. embryonic lethality, systemic effects of cytokine ablation on local reproductive processes and the limited exposure to pathogens in mice housed in laboratory conditions. New sophisticated technology that temporally or spatially regulates gene ablation can overcome some of these limitations. Discoveries on the roles of cytokines in reproductive function uncovered by gene ablation studies can now be applied to improve in vitro fertilization for infertile couples and in the development of contraceptive therapies.

  17. 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.

  18. 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.

  19. The sensitivity of light-evoked responses of retinal ganglion cells is decreased in nitric oxide synthase gene knockout mice.

    PubMed

    Wang, Guo-Yong; van der List, Deborah A; Nemargut, Joseph P; Coombs, Julie L; Chalupa, Leo M

    2007-11-30

    We have shown previously that increasing the production of nitric oxide (NO) results in a dampening of visual responses of retinal ganglion cells (G. Y. Wang, L. C. Liets, & L. M. Chalupa, 2003). To gain further insights into the role of NO in retinal function, we made whole-cell patch clamp recordings from ganglion cells of neural type nitric oxide synthase (nNOS) gene knockout mice. Here we show that in the dark-adapted state, the sensitivity of retinal ganglion cell to light stimulation is decreased in nNOS knockout animals. The lowest light intensities required to evoke optimal responses and the average intensities that evoked half-maximal responses were significantly higher in nNOS knockouts than in normal mice. Retinal histology and other features of light-evoked responses of ganglion cells in nNOS mice appeared to be indistinguishable from those of normal mice. Collectively, these results, in conjunction with our previous work, provide evidence that increasing levels of NO dampen visual responses of ganglion cells, while a lack of nNOS decreases the sensitivity of these neurons to light. Thus, NO levels in the retina are capable of modulating the information that ganglion cells convey to the visual centers of the brain.

  20. Circadian rhythms of clock gene expression in the cerebellum of serotonin-deficient Pet-1 knockout mice.

    PubMed

    Paulus, Erin V; Mintz, Eric M

    2016-01-01

    Serotonin plays an important role in the central regulation of circadian clock function. Serotonin levels are generally higher in the brain during periods of high activity, and these periods are in turn heavily regulated by the circadian clock located in the suprachiasmatic nucleus. However, the role of serotonin as a regulator of circadian rhythms elsewhere in the brain has not been extensively examined. In this study, we examined circadian rhythms of clock gene expression in the cerebellum in mice lacking the Pet-1 transcription factor, which results in a developed brain that is deficient in serotonin neurons. If serotonin helps to synchronize rhythms in brain regions other than the suprachiasmatic nucleus, we would expect to see differences in clock gene expression in these serotonin deficient mice. We found minor differences in the expression of Per1 and Per2 in the knockout mice as compared to wild type, but these differences were small and of questionable functional importance. We also measured the response of cerebellar clocks to injections of the serotonin agonist 8-OH-DPAT during the early part of the night. No effect on clock genes was observed, though the immediate-early gene Fos showed increased expression in wild type mice but not the knockouts. These results suggest that serotonin is not an important mediator of circadian rhythms in the cerebellum in a way that parallels its regulation of the circadian clock in the suprachiasmatic nucleus.

  1. [Gene knockout and knockin on the Escherichia coli lac operon loci using pBR322-red system].

    PubMed

    Chen, Wei; Yu, Mei; Li, Shan-Hu; Wang, Ming-Gang; Zhou, Jian-Guang

    2005-03-01

    pBR322-Red is a newly constructed recombineering plasmid, which contains a part of the pBR322 vector, a series of regulatory elements of lambda-prophage and Red recombination genes. In the beginning, we studied the best working conditions of pBR322-Red, and then modified lac operon in E. coli W3110 chromosome using the plasmid as follow: Firstly, we knockout the lacI gene using Red-mediated recombineering with overlapping single stranded DNA oligonucleotides. Secondly, we substituded the lacA and lacY genes with lacZ, a report gene, by Red-mediated linearized double strands DNA homologous recombination. Finally, we detected the expression of lacZ on these loci for the first time. The results suggested that pBR322-Red system is suitable for modifying W3110 chromosome with various recombination strategies.

  2. Improved generation of rat gene knockouts by target-selected mutagenesis in mismatch repair-deficient animals

    PubMed Central

    van Boxtel, Ruben; Toonen, Pim W; Verheul, Mark; van Roekel, Henk S; Nijman, Isaac J; Guryev, Victor; Cuppen, Edwin

    2008-01-01

    Background The laboratory rat (Rattus norvegicus) is one of the preferred model organisms in physiological and pharmacological research, although the availability of specific genetic models, especially gene knockouts, is limited. N-ethyl-N-nitrosourea (ENU)-driven target-selected mutagenesis is currently the most successful method in rats, although it is still very laborious and expensive. Results As ENU-induced DNA damage is normally recognized by the mismatch repair (MMR) system, we hypothesized that the effectiveness of the target-selected mutagenesis approach could be improved by using a MMR-deficient genetic background. Indeed, Msh6 knockout rats were found to be more sensitive to ENU treatment and the germ line mutation rate was boosted more than two-fold to 1 mutation per 585 kb. In addition, the molecular mutation spectrum was found to be changed in favor of generating knockout-type alleles by ~20%, resulting in an overall increase in efficiency of ~2.5 fold. The improved effectiveness was demonstrated by high throughput mutation discovery in 70 Mb of sequence in a set of only 310 mutant F1 rats. This resulted in the identification of 89 mutations of which four introduced a premature stopcodon and 64 resulted in amino acid changes. Conclusion Taken together, we show that the use of a MMR-deficient background considerably improves ENU-driven target-selected mutagenesis in the rat, thereby reducing animal use as well as screening costs. The use of a mismatch repair-deficient genetic background for improving mutagenesis and target-selected knockout efficiency is in principle applicable to any organism of interest. PMID:18840264

  3. Transcriptome analysis and RNA interference of cockroach phototransduction indicate three opsins and suggest a major role for TRPL channels

    PubMed Central

    French, Andrew S.; Meisner, Shannon; Liu, Hongxia; Weckström, Matti; Torkkeli, Päivi H.

    2015-01-01

    Our current understanding of insect phototransduction is based on a small number of species, but insects occupy many different visual environments. We created the retinal transcriptome of a nocturnal insect, the cockroach, Periplaneta americana to identify proteins involved in the earliest stages of compound eye phototransduction, and test the hypothesis that different visual environments are reflected in different molecular contributions to function. We assembled five novel mRNAs: two green opsins, one UV opsin, and one each TRP and TRPL ion channel homologs. One green opsin mRNA (pGO1) was 100–1000 times more abundant than the other opsins (pGO2 and pUVO), while pTRPL mRNA was 10 times more abundant than pTRP, estimated by transcriptome analysis or quantitative PCR (qPCR). Electroretinograms were used to record photoreceptor responses. Gene-specific in vivo RNA interference (RNAi) was achieved by injecting long (596–708 bp) double-stranded RNA into head hemolymph, and verified by qPCR. RNAi of the most abundant green opsin reduced both green opsins by more than 97% without affecting UV opsin, and gave a maximal reduction of 75% in ERG amplitude 7 days after injection that persisted for at least 19 days. RNAi of pTRP and pTRPL genes each specifically reduced the corresponding mRNA by 90%. Electroretinogram (ERG) reduction by pTRPL RNAi was slower than for opsin, reaching 75% attenuation by 21 days, without recovery at 29 days. pTRP RNAi attenuated ERG much less; only 30% after 21 days. Combined pTRP plus pTRPL RNAi gave only weak evidence of any cooperative interactions. We conclude that silencing retinal genes by in vivo RNAi using long dsRNA is effective, that visible light transduction in Periplaneta is dominated by pGO1, and that pTRPL plays a major role in cockroach phototransduction. PMID:26257659

  4. UVA phototransduction drives early melanin synthesis in human melanocytes.

    PubMed

    Wicks, Nadine L; Chan, Jason W; Najera, Julia A; Ciriello, Jonathan M; Oancea, Elena

    2011-11-22

    Exposure of human skin to solar ultraviolet radiation (UVR), a powerful carcinogen [1] comprising ~95% ultraviolet A (UVA) and ~5% ultraviolet B (UVB) at the Earth's surface, promotes melanin synthesis in epidermal melanocytes [2, 3], which protects skin from DNA damage [4, 5]. UVB causes DNA lesions [6] that lead to transcriptional activation of melanin-producing enzymes, resulting in delayed skin pigmentation within days [7]. In contrast, UVA causes primarily oxidative damage [8] and leads to immediate pigment darkening (IPD) within minutes, via an unknown mechanism [9, 10]. No receptor protein directly mediating phototransduction in skin has been identified. Here we demonstrate that exposure of primary human epidermal melanocytes (HEMs) to UVA causes calcium mobilization and early melanin synthesis. Calcium responses were abolished by treatment with G protein or phospholipase C (PLC) inhibitors or by depletion of intracellular calcium stores. We show that the visual photopigment rhodopsin [11] is expressed in HEMs and contributes to UVR phototransduction. Upon UVR exposure, significant melanin production was measured within one hour; cellular melanin continued to increase in a retinal- and calcium-dependent manner up to 5-fold after 24 hr. Our findings identify a novel UVA-sensitive signaling pathway in melanocytes that leads to calcium mobilization and melanin synthesis and may underlie the mechanism of IPD in human skin.

  5. The phototransduction cascade in the isolated chick pineal gland revisited.

    PubMed

    Holthues, Heike; Vollrath, Lutz

    2004-03-05

    It is well established that the isolated chick pineal gland is directly light sensitive and that melatonin synthesis of the gland can be inhibited by exposing the gland to light during scotophase. Since not all the steps of the phototransduction cascade have been clarified to the same extent as in the retina, we have treated isolated chick pineal glands with 90 min of light during scotophase and with drugs that affect key-components of vertebrate phototransduction, i.e., cyclic guanosine monophosphate (cGMP) phosphodiesterase 6 (PDE6), cGMP levels and cGMP-gated calcium channels. The endpoint measured was the activity of the rate-limiting enzyme of melatonin synthesis, arylalkylamine N-acetyltransferase (AA-NAT), which is inhibited by light. The effects on AA-NAT activity of light were negated by addition of dipyridamol and zaprinast, either of which inhibits the light-induced activation of PDE6. The effect of light was also counteracted by the nitric oxide donor sodium nitroprusside and C-type natriuretic peptide, both of which increase cGMP levels, and by the calcium channel agonist Bay K 8644, which prevents the cGMP-decrease-induced closure of cGMP-gated calcium channels. Inhibition of nitric oxide synthase (NOS) by N(G)-nitro-l-arginine did not influence the inhibitory effect of light, suggesting that the NOS pathway does not play a role. Since the light effect on AA-NAT activity involves both cGMP and cyclic adenosine monophosphate (cAMP) hydrolysis, we have also studied whether the cGMP-inhibited cAMP phosphodiesterase 3 (PDE3) is involved. As the specific PDE3 inhibitor cilostamide is without effect, we assume that the light-induced decrease of cAMP levels does not involve PDE3. These results taken together strongly suggest that the investigated steps of the phototransduction cascade in the isolated chick pineal gland are basically similar to those in the retina.

  6. Enhanced morphine-induced antinociception in histamine H3 receptor gene knockout mice.

    PubMed

    Mobarakeh, Jalal Izadi; Takahashi, Kazuhiro; Yanai, Kazuhiko

    2009-09-01

    Previous studies have implicated a potential role for histamine H3 receptor in pain processing. There have been conflicting data, however, on the roles of H3 receptors in pain perception, and little information is available about the role of spinal histamine H3 receptors in morphine-induced antinociception. In the present study we examined the role of histamine H3 receptor in morphine-induced antinociception using histamine H3 receptor knockout mice and a histamine H3 receptor antagonist. Anitinociception was evaluated by assays for four nociceptive stimuli: hot-plate, tail-flick, paw-withdrawal, and formalin tests. Antinociception induced by morphine (0.125 nmol/5 microl, i.t.) was significantly augmented in histamine H3 receptor knockout (-/-) mice compared to the wild-type (+/+) mice in all four assays of pain. Furthermore, the effect of intrathecally administered morphine with thioperamide, a histamine H3 antagonist, was examined in C57BL/6J mice. A low dose of i.t. administered thioperamide (0.125 nmol/5 microl) alone had no significant effect on the nociceptive response. In contrast, the combination of morphine (0.125 nmol/5 microl, i.t.) with the same dose of thioperamide resulted in a significant reduction in the pain-related behaviors in all four nociceptive tests. These results suggest that histamine exerts inhibitory effects on morphine-induced antinociception through H3 receptors at the spinal level.

  7. [Effect of different carbon sources on pyruvic acid production by using lpdA gene knockout Escherichia coli].

    PubMed

    Shen, Dongqian; Feng, Xiaoyu; Lin, Dongqiang; Yao, Shanjing

    2009-09-01

    We studied the ability of lpdA gene knockout Escherichia coli to ferment different sugars in mineral salts medium for the production of pyruvate. The sugars studied were glucose, fructose, xylose and mannose at a concentration of 10 g/L. At the same time, effect of inoculum size on lpdA fermentation with glucose was studied. The strain was able to use all sugars for biomass generation and pyruvate production. The lpdA knockout mutant converted glucose, fructose, xylose and mannose to pyruvate with yields of 0.884 g/g, 0.802 g/g, 0.817 g/g and 0.808 g/L, respectively. The pyruvate accumulation curve coupled with cell growth except for mannose as carbon source. When the inoculation size increased, the rate of glucose consumption, pyruvate accumulation and cell growth increased but lower pyruvate concentration. This study demonstrates that E. coli lpdA mutant has the potential to produce pyruvic acid from xylose and mannose.

  8. Highly efficient gene knockout by injection of TALEN mRNAs into oocytes and host transfer in Xenopus laevis.

    PubMed

    Nakajima, Keisuke; Yaoita, Yoshio

    2015-01-16

    Zinc-finger nucleases, transcription activator-like effector nucleases (TALENs) and the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system are potentially powerful tools for producing tailor-made knockout animals. However, their mutagenic activity is not high enough to induce mutations at all loci of a target gene throughout an entire tadpole. In this study, we present a highly efficient method for introducing gene modifications at almost all target sequences in randomly selected embryos. The gene modification activity of TALEN is enhanced by adopting the host-transfer technique. In our method, the efficiency is further improved by injecting TALEN mRNAs fused to the 3'UTR of the Xenopus DEADSouth gene into oocytes, which are then transferred into a host female frog, where they are ovulated and fertilized. The addition of the 3'UTR of the DEADSouth gene promotes mRNA translation in the oocytes and increases the expression of TALEN proteins to near-maximal levels three hours post fertilization (hpf). In contrast, TALEN mRNAs without this 3'UTR are translated infrequently in oocytes. Our data suggest that genomic DNA is more sensitive to TALEN proteins from fertilization to the midblastula (MBT) stage. Our method works by increasing the levels of TALEN proteins during the pre-MBT stages.

  9. Targeted Disruption of the Meprin β Gene in Mice Leads to Underrepresentation of Knockout Mice and Changes in Renal Gene Expression Profiles

    PubMed Central

    Norman, Lourdes P.; Jiang, Weiping; Han, Xiaoli; Saunders, Thomas L.; Bond, Judith S.

    2003-01-01

    Meprins are multidomain zinc metalloproteases that are highly expressed in mammalian kidney and intestinal brush border membranes and in leukocytes and certain cancer cells. Mature meprins are oligomers of evolutionarily related, separately encoded α and/or β subunits. Homooligomers of meprin α are secreted; oligomers containing meprin β are plasma membrane associated. Meprin substrates include bioactive peptides and extracellular matrix proteins. Meprins have been implicated in cancer and intestinal inflammation. Additionally, meprin β is a candidate gene for diabetic nephropathy. To elucidate in vivo functions of these metalloproteases, meprin β null mice were generated by targeted disruption of the meprin β gene on mouse chromosome 18q12. Analyses of meprin β knockout mice indicated that (i) 50% fewer null mice are born than the Mendelian distribution predicts, (ii) null mice that survive develop normally and are viable and fertile, (iii) meprin β knockout mice lack membrane-associated meprin α in kidney and intestine, and (iv) null mice have changes in renal gene expression profiles compared to wild-type mice as assessed by microarray analyses. Thus, disruption of the meprin β allele in mice affects embryonic viability, birth weight, renal gene expression profiles, and the distribution of meprin α in kidney and intestine. PMID:12556482

  10. Reconstructing gene regulatory networks from knock-out data using Gaussian Noise Model and Pearson Correlation Coefficient.

    PubMed

    Mohamed Salleh, Faridah Hani; Arif, Shereena Mohd; Zainudin, Suhaila; Firdaus-Raih, Mohd

    2015-12-01

    A gene regulatory network (GRN) is a large and complex network consisting of interacting elements that, over time, affect each other's state. The dynamics of complex gene regulatory processes are difficult to understand using intuitive approaches alone. To overcome this problem, we propose an algorithm for inferring the regulatory interactions from knock-out data using a Gaussian model combines with Pearson Correlation Coefficient (PCC). There are several problems relating to GRN construction that have been outlined in this paper. We demonstrated the ability of our proposed method to (1) predict the presence of regulatory interactions between genes, (2) their directionality and (3) their states (activation or suppression). The algorithm was applied to network sizes of 10 and 50 genes from DREAM3 datasets and network sizes of 10 from DREAM4 datasets. The predicted networks were evaluated based on AUROC and AUPR. We discovered that high false positive values were generated by our GRN prediction methods because the indirect regulations have been wrongly predicted as true relationships. We achieved satisfactory results as the majority of sub-networks achieved AUROC values above 0.5.

  11. Rapid-throughput skeletal phenotyping of 100 knockout mice identifies 9 new genes that determine bone strength.

    PubMed

    Bassett, J H Duncan; Gogakos, Apostolos; White, Jacqueline K; Evans, Holly; Jacques, Richard M; van der Spek, Anne H; Ramirez-Solis, Ramiro; Ryder, Edward; Sunter, David; Boyde, Alan; Campbell, Michael J; Croucher, Peter I; Williams, Graham R

    2012-01-01

    Osteoporosis is a common polygenic disease and global healthcare priority but its genetic basis remains largely unknown. We report a high-throughput multi-parameter phenotype screen to identify functionally significant skeletal phenotypes in mice generated by the Wellcome Trust Sanger Institute Mouse Genetics Project and discover novel genes that may be involved in the pathogenesis of osteoporosis. The integrated use of primary phenotype data with quantitative x-ray microradiography, micro-computed tomography, statistical approaches and biomechanical testing in 100 unselected knockout mouse strains identified nine new genetic determinants of bone mass and strength. These nine new genes include five whose deletion results in low bone mass and four whose deletion results in high bone mass. None of the nine genes have been implicated previously in skeletal disorders and detailed analysis of the biomechanical consequences of their deletion revealed a novel functional classification of bone structure and strength. The organ-specific and disease-focused strategy described in this study can be applied to any biological system or tractable polygenic disease, thus providing a general basis to define gene function in a system-specific manner. Application of the approach to diseases affecting other physiological systems will help to realize the full potential of the International Mouse Phenotyping Consortium.

  12. Efficient generation of FVII gene knockout mice using CRISPR/Cas9 nuclease and truncated guided RNAs

    PubMed Central

    An, Liyou; Hu, Yeshu; Chang, Shiwei; Zhu, Xiumei; Ling, Pingping; Zhang, Fenli; Liu, Jiao; Liu, Yanhong; Chen, Yexiang; Yang, Lan; Presicce, Giorgio Antonio; Du, Fuliang

    2016-01-01

    We investigated the effects of 5′-end truncated CRISPR RNA-guided Cas9 nuclease (tru-RGN, 17/18 nucleotides) on genome editing capability in NIH/3T3 cells, and its efficiencies on generating Factor VII (FVII) gene-knockout (KO) mice. In cultured cells, RGNs on-target editing activity had been varied when gRNAs was truncated, higher at Site Two (tF7–2 vs. F7–2, 49.5 vs. 30.1%) while lower in other two sites (Site One, tF7–1 vs.F7–1, 12.1 vs. 23.6%; Site Three, tF7–3 vs.F7–3, 7.7 vs 10.9%) (P < 0.05). Out of 15 predicated off–target sites, tru-RGNs showed significantly decreased frequencies at 5 sites. By microinjecting tru-RGN RNAs into zygotes, FVII KO mice were generated with higher efficiency at Site Two (80.1 vs. 35.8%) and Site One (55.0 vs 3.7%) (P < 0.05), but not at Site three (39.4 vs 27.8%) (P > 0.05) when compared with standard RGN controls. Knockout FVII mice demonstrated a delayed prothrombin time and decreased plasma FVII expression. Our study first demonstrates that truncated gRNAs to 18 complementary nucleotides and Cas9 nucleases, can effectively generate FVII gene KO mice with a significantly higher efficiency in a site-dependent manner. In addition, the off-target frequency was much lower in KO mice than in cell lines via RGN expression vector-mediated genome editing. PMID:27139777

  13. Using CRISPR/Cas9-Mediated GLA Gene Knockout as an In Vitro Drug Screening Model for Fabry Disease

    PubMed Central

    Song, Hui-Yung; Chiang, Huai-Chih; Tseng, Wei-Lien; Wu, Ping; Chien, Chian-Shiu; Leu, Hsin-Bang; Yang, Yi-Ping; Wang, Mong-Lien; Jong, Yuh-Jyh; Chen, Chung-Hsuan; Yu, Wen-Chung; Chiou, Shih-Hwa

    2016-01-01

    The CRISPR/Cas9 Genome-editing system has revealed promising potential for generating gene mutation, deletion, and correction in human cells. Application of this powerful tool in Fabry disease (FD), however, still needs to be explored. Enzyme replacement therapy (ERT), a regular administration of recombinant human α Gal A (rhα-GLA), is a currently available and effective treatment to clear the accumulated Gb3 in FD patients. However, the short half-life of rhα-GLA in human body limits its application. Moreover, lack of an appropriate in vitro disease model restricted the high-throughput screening of drugs for improving ERT efficacy. Therefore, it is worth establishing a large-expanded in vitro FD model for screening potential candidates, which can enhance and prolong ERT potency. Using CRISPR/Cas9-mediated gene knockout of GLA in HEK-293T cells, we generated GLA-null cells to investigate rhα-GLA cellular pharmacokinetics. The half-life of administrated rhα-GLA was around 24 h in GLA-null cells; co-administration of proteasome inhibitor MG132 and rhα-GLA significantly restored the GLA enzyme activity by two-fold compared with rhα-GLA alone. Furthermore, co-treatment of rhα-GLA/MG132 in patient-derived fibroblasts increased Gb3 clearance by 30%, compared with rhα-GLA treatment alone. Collectively, the CRISPR/Cas9-mediated GLA-knockout HEK-293T cells provide an in vitro FD model for evaluating the intracellular pharmacokinetics of the rhα-GLA as well as for screening candidates to prolong rhα-GLA potency. Using this model, we demonstrated that MG132 prolongs rhα-GLA half-life and enhanced Gb3 clearance, shedding light on the direction of enhancing ERT efficacy in FD treatment. PMID:27983599

  14. Using CRISPR/Cas9-Mediated GLA Gene Knockout as an In Vitro Drug Screening Model for Fabry Disease.

    PubMed

    Song, Hui-Yung; Chiang, Huai-Chih; Tseng, Wei-Lien; Wu, Ping; Chien, Chian-Shiu; Leu, Hsin-Bang; Yang, Yi-Ping; Wang, Mong-Lien; Jong, Yuh-Jyh; Chen, Chung-Hsuan; Yu, Wen-Chung; Chiou, Shih-Hwa

    2016-12-13

    The CRISPR/Cas9 Genome-editing system has revealed promising potential for generating gene mutation, deletion, and correction in human cells. Application of this powerful tool in Fabry disease (FD), however, still needs to be explored. Enzyme replacement therapy (ERT), a regular administration of recombinant human α Gal A (rhα-GLA), is a currently available and effective treatment to clear the accumulated Gb3 in FD patients. However, the short half-life of rhα-GLA in human body limits its application. Moreover, lack of an appropriate in vitro disease model restricted the high-throughput screening of drugs for improving ERT efficacy. Therefore, it is worth establishing a large-expanded in vitro FD model for screening potential candidates, which can enhance and prolong ERT potency. Using CRISPR/Cas9-mediated gene knockout of GLA in HEK-293T cells, we generated GLA-null cells to investigate rhα-GLA cellular pharmacokinetics. The half-life of administrated rhα-GLA was around 24 h in GLA-null cells; co-administration of proteasome inhibitor MG132 and rhα-GLA significantly restored the GLA enzyme activity by two-fold compared with rhα-GLA alone. Furthermore, co-treatment of rhα-GLA/MG132 in patient-derived fibroblasts increased Gb3 clearance by 30%, compared with rhα-GLA treatment alone. Collectively, the CRISPR/Cas9-mediated GLA-knockout HEK-293T cells provide an in vitro FD model for evaluating the intracellular pharmacokinetics of the rhα-GLA as well as for screening candidates to prolong rhα-GLA potency. Using this model, we demonstrated that MG132 prolongs rhα-GLA half-life and enhanced Gb3 clearance, shedding light on the direction of enhancing ERT efficacy in FD treatment.

  15. Orexin gene transfer into the amygdala suppresses both spontaneous and emotion-induced cataplexy in orexin-knockout mice.

    PubMed

    Liu, Meng; Blanco-Centurion, Carlos; Konadhode, Roda Rani; Luan, Liju; Shiromani, Priyattam J

    2016-03-01

    Narcolepsy is a chronic sleep disorder linked to the loss of orexin-producing neurons in the hypothalamus. Cataplexy, a sudden loss of muscle tone during waking, is an important distinguishing symptom of narcolepsy and it is often triggered by strong emotions. The neural circuit underlying cataplexy attacks is not known, but is likely to involve the amygdala, a region implicated in regulating emotions. In mice models of narcolepsy, transfer of the orexin gene into surrogate neurons has been successful in ameliorating narcoleptic symptoms. However, it is not known whether this method also blocks cataplexy triggered by strong emotions. To examine this possibility, the gene encoding mouse prepro-orexin was transferred into amygdala neurons of orexin-knockout (KO) mice (rAAV-orexin; n = 8). Orexin-KO mice that did not receive gene transfer (no-rAAV; n = 7) or received only the reporter gene (rAAV-GFP; n = 7) served as controls. Three weeks later, the animal's sleep and behaviour were recorded at night (no-odour control night), followed by another recording at night in the presence of predator odour (odour night). Orexin-KO mice given the orexin gene transfer into surrogate amygdala neurons had significantly less spontaneous bouts of cataplexy, and predator odour did not induce cataplexy compared with control mice. Moreover, the mice with orexin gene transfer were awake more during the odour night. These results demonstrate that orexin gene transfer into amygdala neurons can suppress both spontaneous and emotion-induced cataplexy attacks in narcoleptic mice. It suggests that manipulating amygdala pathways is a potential strategy for treating cataplexy in narcolepsy.

  16. [Roles of histamine receptors in pain perception: a study using receptors gene knockout mice].

    PubMed

    Yanai, Kazuhiko; Mobarakeh, Jalal Izadi; Kuramasu, Atsuo; Sakurada, Shinobu

    2003-11-01

    To study the participation of histamine H1- and H2-receptors in pain perception, H1 and H2 receptor knockout (KO) mice were examined for pain threshold by means of three kinds of nociceptive tasks. These included assays for thermal, mechanical, and chemical nociception. H1KO mice showed significantly fewer nociceptive responses to the hot-plate, tail-flick, tail-pressure, paw-withdrawal, formalin, capsaicin, and abdominal constriction tests. Sensitivity to noxious stimuli in H1KO mice was significantly decreased when compared to wild-type mice. The antinociceptive phenotypes of H2KO were relatively less prominent when compared to H1KO mice. We also examined the antinociceptive effects of intrathecally-, intracerebroventricularly-, and subcutaneously-administered morphine in H1KO and H2KO mice. In these nociceptive assays, the antinociceptive effects produced by morphine were more enhanced in both H1KO and H2KO mice. The effects of histamine H1- and H2-receptor antagonists on morphine-induced antinociception were studied in ICR mice. The intrathecal, intracerebroventricular and subcutaneous co-administrations of d-chlorpheniramine enhanced the effects of morphine in all nociceptive assays examined. In addition, intrathecal co-administrations of cimetidine enhanced the antinociception of morphine in the hot plate tests. These results suggest that existing H1 and H2 receptors play an inhibitory role in morphine-induced antinociception in the spinal and supra-spinal levels.

  17. Knockout of the abetalipoproteinemia gene in mice: reduced lipoprotein secretion in heterozygotes and embryonic lethality in homozygotes.

    PubMed

    Raabe, M; Flynn, L M; Zlot, C H; Wong, J S; Véniant, M M; Hamilton, R L; Young, S G

    1998-07-21

    Abetalipoproteinemia, an inherited human disease characterized by a near-complete absence of the apolipoprotein (apo) B-containing lipoproteins in the plasma, is caused by mutations in the gene for microsomal triglyceride transfer protein (MTP). We used gene targeting to knock out the mouse MTP gene (Mttp). In heterozygous knockout mice (Mttp+/- ), the MTP mRNA, protein, and activity levels were reduced by 50%, in both liver and intestine. Compared with control mice (Mttp+/+), chow-fed Mttp+/- mice had reduced plasma levels of low-density lipoprotein cholesterol and had a 28% reduction in plasma apoB100 levels. On a high-fat diet, the Mttp+/- mice exhibited a marked reduction in total plasma cholesterol levels, compared with those in Mttp+/+ mice. Both the livers of adult Mttp+/- mice and the visceral endoderm of the yolk sacs from Mttp+/- embryos manifested an accumulation of cytosolic fat. All homozygous embryos (Mttp-/-) died during embryonic development. In the visceral endoderm of Mttp-/- yolk sacs, lipoprotein synthesis was virtually absent, and there was a marked accumulation of cytosolic fat droplets. In summary, half-normal MTP levels do not support normal levels of lipoprotein synthesis and secretion, and a complete deficiency of MTP causes lethal developmental abnormalities, perhaps because of an impaired capacity of the yolk sac to export lipids to the developing embryo.

  18. Efficient bi-allelic gene knockout and site-specific knock-in mediated by TALENs in pigs.

    PubMed

    Yao, Jing; Huang, Jiaojiao; Hai, Tang; Wang, Xianlong; Qin, Guosong; Zhang, Hongyong; Wu, Rong; Cao, Chunwei; Xi, Jianzhong Jeff; Yuan, Zengqiang; Zhao, Jianguo

    2014-11-05

    Pigs are ideal organ donors for xenotransplantation and an excellent model for studying human diseases, such as neurodegenerative disease. Transcription activator-like effector nucleases (TALENs) are used widely for gene targeting in various model animals. Here, we developed a strategy using TALENs to target the GGTA1, Parkin and DJ-1 genes in the porcine genome using Large White porcine fibroblast cells without any foreign gene integration. In total, 5% (2/40), 2.5% (2/80), and 22% (11/50) of the obtained colonies of fibroblast cells were mutated for GGTA1, Parkin, and DJ-1, respectively. Among these mutant colonies, over 1/3 were bi-allelic knockouts (KO), and no off-target cleavage was detected. We also successfully used single-strand oligodeoxynucleotides to introduce a short sequence into the DJ-1 locus. Mixed DJ-1 mutant colonies were used as donor cells for somatic cell nuclear transfer (SCNT), and three female piglets were obtained (two were bi-allelically mutated, and one was mono-allelically mutated). Western blot analysis showed that the expression of the DJ-1 protein was disrupted in KO piglets. These results imply that a combination of TALENs technology with SCNT can efficiently generate bi-allelic KO pigs without the integration of exogenous DNA. These DJ-1 KO pigs will provide valuable information for studying Parkinson's disease.

  19. Effect of Cyp27A1 gene dosage on atherosclerosis development in ApoE-knockout mice.

    PubMed

    Zurkinden, Line; Solcà, Curzio; Vögeli, Isabelle A; Vogt, Bruno; Ackermann, Daniel; Erickson, Sandra K; Frey, Felix J; Sviridov, Dmitri; Escher, Geneviève

    2014-03-01

    In humans, sterol 27-hydroxylase (CYP27A1) deficiency leads to cholesterol deposition in tendons and vasculature. Thus, in addition to its role in bile acid synthesis, where it converts cholesterol to 27-hydroxycholesterol (27-OHC), CYP27A1 may also be atheroprotective. Cyp27A1-deficient (Cyp27A1(-/-)) mice were crossed with apolipoprotein E (apoE)-deficient mice. Cyp27A1(+/+)/apoE(-/-) [ApoE-knockout (KO)], Cyp27A1(+/-)/apoE(-/-) heterozygous (het), and Cyp27A1(-/-)/apoE(-/-) [double-knockout (DKO)] mice were challenged with a Western diet (WD) for 3 and 6 mo. ApoE-KO mice fed a chow diet or a WD were used as the control. The severity of atherosclerosis in DKO mice was reduced 10-fold. Compared with the control, the DKO mice had no 27-OHC, total plasma cholesterol and low-density lipoprotein and very low density lipoprotein (LDL/VLDL) concentrations were reduced 2-fold, and HDL was elevated 2-fold. Expression of hepatic CYP7A1, CYP3A, and CYP8B1 were 5- to 10-fold higher. 3-Hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) activity increased 4-fold. Fecal cholesterol was increased. In contrast, het mice fed a WD developed accelerated atherosclerosis and severe skin lesions, possibly because of reduced reverse cholesterol transport due to diminished 27-OHC production. CYP27A1 activity is involved in the control of cholesterol homeostasis and development of atherosclerosis with a distinct gene dose-dependent effect.

  20. Microarray analysis of active cardiac remodeling genes in a familial hypertrophic cardiomyopathy mouse model rescued by a phospholamban knockout

    PubMed Central

    Rajan, Sudarsan; Pena, James R.; Jegga, Anil G.; Aronow, Bruce J.; Wolska, Beata M.

    2013-01-01

    Familial hypertrophic cardiomyopathy (FHC) is a disease characterized by ventricular hypertrophy, fibrosis, and aberrant systolic and/or diastolic function. Our laboratories have previously developed two mouse models that affect cardiac performance. One mouse model encodes an FHC-associated mutation in α-tropomyosin: Glu → Gly at amino acid 180, designated as Tm180. These mice display a phenotype that is characteristic of FHC, including severe cardiac hypertrophy with fibrosis and impaired physiological performance. The other model was a gene knockout of phospholamban (PLN KO), a regulator of calcium uptake in the sarcoplasmic reticulum of cardiomyocytes; these hearts exhibit hypercontractility with no pathological abnormalities. Previous work in our laboratories shows that when mice were genetically crossed between the PLN KO and Tm180, the progeny (PLN KO/Tm180) display a rescued hypertrophic phenotype with improved morphology and cardiac function. To understand the changes in gene expression that occur in these models undergoing cardiac remodeling (Tm180, PLN KO, PLN KO/Tm180, and nontransgenic control mice), we conducted microarray analyses of left ventricular tissue at 4 and 12 mo of age. Expression profiling reveals that 1,187 genes changed expression in direct response to the three genetic models. With these 1,187 genes, 11 clusters emerged showing normalization of transcript expression in the PLN KO/Tm180 hearts. In addition, 62 transcripts are highly involved in suppression of the hypertrophic phenotype. Confirmation of the microarray analysis was conducted by quantitative RT-PCR. These results provide insight into genes that alter expression during cardiac remodeling and are active during modulation of the cardiomyopathic phenotype. PMID:23800848

  1. Morphological and genetic characterization of group I Clostridium botulinum type B strain 111 and the transcriptional regulator spoIIID gene knockout mutant in sporulation.

    PubMed

    Hosomi, Koji; Kuwana, Ritsuko; Takamatsu, Hiromu; Kohda, Tomoko; Kozaki, Shunji; Mukamoto, Masafumi

    2015-06-01

    Clostridium botulinum is a heat-resistant spore-forming bacterium that causes the serious paralytic illness botulism. Heat-resistant spores may cause food sanitation hazards and sporulation plays a central role in the survival of C. botulinum. We observed morphological changes and investigated the role of the transcriptional regulator SpoIIID in the sporulation of C. botulinum type B strain 111 in order to elucidate the molecular mechanism in C. botulinum. C. botulinum type B formed heat-resistant spores through successive morphological changes corresponding to those of Bacillus subtilis, a spore-forming model organism. An analysis of the spoIIID gene knockout mutant revealed that the transcriptional regulator SpoIIID contributed to heat-resistant spore formation by C. botulinum type B and activated the transcription of the sigK gene later during sporulation. Transcription of the spoIIID gene, which differed from that in B. subtilis and Clostridium difficile, was observed in the sigE gene knockout mutant of C. botulinum type B. An analysis of the sigF gene knockout mutant showed that the sporulation-specific sigma factor SigF was essential for transcription of the spoIIID gene in C. botulinum type B. These results suggest that the regulation of sporulation in C. botulinum is not similar to that in B. subtilis and other clostridia.

  2. TNF-α Gene Knockout in Triple Negative Breast Cancer Cell Line Induces Apoptosis

    PubMed Central

    Pileczki, Valentina; Braicu, Cornelia; Gherman, Claudia D.; Berindan-Neagoe, Ioana

    2013-01-01

    Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory cytokine involved in the promotion and progression of cancer, including triple negative breast cancer cells. Thus, there is significant interest in understanding the molecular signaling pathways that connect TNF-α with the survival of tumor cells. In our experiments, we used as an in vitro model for triple negative breast cancer the cell line Hs578T. The purpose of this study is to determine the gene expression profiling of apoptotic signaling networks after blocking TNF-α formation by using specially designed siRNA molecules to target TNF-α messenger RNA. Knockdown of TNF-α gene was associated with cell proliferation inhibition and apoptosis, as observed by monitoring the cell index using the xCELLigence RTCA System and flow cytometry. PCR array technology was used to examine the transcript levels of 84 genes involved in apoptosis. 15 genes were found to be relevant after comparing the treated group with the untreated one of which 3 were down-regulated and 12 up-regulated. The down-regulated genes are all involved in cell survival, whereas the up-regulated ones are involved in and interact with pro-apoptotic pathways. The results described here indicate that the direct target of TNF-α in the Hs578T breast cancer cell line increases the level of certain pro-apoptotic factors that modulate different cellular networks that direct the cells towards death. PMID:23263670

  3. Mammalian Axoneme Central Pair Complex Proteins: Broader Roles Revealed by Gene Knockout Phenotypes

    PubMed Central

    Teves, Maria E.; Nagarkatti-Gude, David R.; Zhang, Zhibing; Strauss, Jerome F.

    2016-01-01

    The axoneme genes, their encoded proteins, their functions and the structures they form are largely conserved across species. Much of our knowledge of the function and structure of axoneme proteins in cilia and flagella is derived from studies on model organisms like the green algae, Chlamydomonas reinhardtii. The core structure of cilia and flagella is the axoneme, which in most motile cilia and flagella contains a 9 + 2 configuration of microtubules. The two central microtubules are the scaffold of the central pair complex (CPC). Mutations that disrupt CPC genes in Chlamydomonas and other model organisms result in defects in assembly, stability and function of the axoneme, leading to flagellar motility defects. However, targeted mutations generated in mice in the orthologous CPC genes have revealed significant differences in phenotypes of mutants compared to Chlamydomonas. Here we review observations that support the concept of cell-type specific roles for the CPC genes in mice, and an expanded repertoire of functions for the products of these genes in cilia, including non-motile cilia, and other microtubule-associated cellular functions. PMID:26785425

  4. Long-term rescue of cone photoreceptor degeneration in retinitis pigmentosa 2 (RP2)-knockout mice by gene replacement therapy

    PubMed Central

    Mookherjee, Suddhasil; Hiriyanna, Suja; Kaneshiro, Kayleigh; Li, Linjing; Li, Yichao; Li, Wei; Qian, Haohua; Li, Tiansen; Khanna, Hemant; Colosi, Peter; Swaroop, Anand; Wu, Zhijian

    2015-01-01

    Retinal neurodegenerative diseases are especially attractive targets for gene replacement therapy, which appears to be clinically effective for several monogenic diseases. X-linked forms of retinitis pigmentosa (XLRP) are relatively severe blinding disorders, resulting from progressive photoreceptor dysfunction primarily caused by mutations in RPGR or RP2 gene. With a goal to develop gene therapy for the XLRP-RP2 disease, we first performed detailed characterization of the Rp2-knockout (Rp2-KO) mice and observed early-onset cone dysfunction, which was followed by progressive cone degeneration, mimicking cone vision impairment in XLRP patients. The mice also exhibited distinct and significantly delayed falling phase of photopic b-wave of electroretinogram (ERG). Concurrently, we generated a self-complementary adeno-associated viral (AAV) vector carrying human RP2-coding sequence and demonstrated its ability to mediate stable RP2 protein expression in mouse photoreceptors. A long-term efficacy study was then conducted in Rp2-KO mice following AAV-RP2 vector administration. Preservation of cone function was achieved with a wide dose range over 18-month duration, as evidenced by photopic ERG and optomotor tests. The slower b-wave kinetics was also completely restored. Morphologically, the treatment preserved cone viability, corrected mis-trafficking of M-cone opsin and restored cone PDE6 expression. The therapeutic effect was achieved even in mice that received treatment at an advanced disease stage. The highest AAV-RP2 dose group demonstrated retinal toxicity, highlighting the importance of careful vector dosing in designing future human trials. The wide range of effective dose, a broad treatment window and long-lasting therapeutic effects should make the RP2 gene therapy attractive for clinical development. PMID:26358772

  5. Generation and characterization of NV gene-knockout recombinant viral hemorrhagic septicemia virus (VHSV) genotype IVa.

    PubMed

    Kim, Min Sun; Kim, Dong Soo; Kim, Ki Hong

    2011-11-03

    A recombinant viral hemorrhagic septicemia virus (rVHSV-deltaNV-EGFP) containing the enhanced green fluorescent protein (EGFP) gene instead of the NV gene was produced using the reverse-genetics method. For use as a positive control, another recombinant virus (rVHSV-wild) was also generated, which had an identical nucleotide sequence to the wild-type VHSV genome except for a few artificially replaced nucleotides. The rVHSVs were rescued using a system controlled by T7 RNA polymerase supplied by a retroviral vector. Generation of rVHSV-deltaNV-EGFP and rVHSV-wild was confirmed by sequencing of RT-PCR products, and rescue of infectious rVHSVs was confirmed by observation of plaque formation. Replication efficiency of rVHSV-wild was distinctly lower than that of wild-type VHSV, suggesting that the artificially replaced nucleotides, especially when immediately preceding the G or NV gene start codons, might affect the replication of the virus. Replication of rVHSV-deltaNV-EGFP was slightly lower than that of rVHSV-wild when epithelioma papulosum cyprini cells were infected with multiplicity of infection (MOI) 1.0, but much lower when cells were infected with MOI 0.00001. These results suggest that the NV gene plays an important role in VHSV replication through interactions with host-cell responses, and the lower replication ability of rVHSV-wild compared to wild-type VHSV might be caused by replaced nucleotides just before the NV gene open reading frame (ORF) rather than the G gene ORF. In olive flounder Paralichthys olivaceus, rVHSV-wild produced slower-progressing mortalities than wild-type VHSV, whereas rVHSV-deltaNV-EGFP pathogenesis was highly attenuated. These results suggest that the NV protein of VHSV may play an important role not only in viral replication but also in viral pathogenesis.

  6. Knockout of G protein β5 impairs brain development and causes multiple neurologic abnormalities in mice

    PubMed Central

    Zhang, Jian-Hua; Pandey, Mritunjay; Seigneur, Erica M.; Panicker, Leelamma M.; Koo, Lily; Schwartz, Owen M.; Chen, Weiping; Chen, Ching-Kang; Simonds, William F.

    2011-01-01

    Gβ5 is a divergent member of the signal-transducing G protein β subunit family encoded by GNB5 and expressed principally in brain and neuronal tissue. Among heterotrimeric Gβ isoforms, Gβ5 is unique in its ability to heterodimerize with members of the R7 subfamily of the regulator of G protein signaling (RGS) proteins that contain G protein-γ like domains. Previous studies employing Gnb5 knockout (KO) mice have shown that Gβ5 is an essential stabilizer of such RGS proteins and regulates the deactivation of retinal phototransduction and the proper functioning of retinal bipolar cells. However, little is known of the function of Gβ5 in the brain outside the visual system. We show here that mice lacking Gβ5 have a markedly abnormal neurologic phenotype that includes impaired development, tiptoe-walking, motor learning and coordination deficiencies, and hyperactivity. We further show that Gβ5-deficient mice have abnormalities of neuronal development in cerebellum and hippocampus. We find that the expression of both mRNA and protein from multiple neuronal genes is dysregulated in Gnb5 KO mice. Taken together with previous observations from Gnb5 KO mice, our findings suggest a model in which Gβ5 regulates dendritic arborization and/or synapse formation during development, in part by effects on gene expression. PMID:21883221

  7. Phenotypic characterization of recessive gene knockout rat models of Parkinson's disease.

    PubMed

    Dave, Kuldip D; De Silva, Shehan; Sheth, Niketa P; Ramboz, Sylvie; Beck, Melissa J; Quang, Changyu; Switzer, Robert C; Ahmad, Syed O; Sunkin, Susan M; Walker, Dan; Cui, Xiaoxia; Fisher, Daniel A; McCoy, Aaron M; Gamber, Kevin; Ding, Xiaodong; Goldberg, Matthew S; Benkovic, Stanley A; Haupt, Meredith; Baptista, Marco A S; Fiske, Brian K; Sherer, Todd B; Frasier, Mark A

    2014-10-01

    Recessively inherited loss-of-function mutations in the PTEN-induced putative kinase 1(Pink1), DJ-1 (Park7) and Parkin (Park2) genes are linked to familial cases of early-onset Parkinson's disease (PD). As part of its strategy to provide more tools for the research community, The Michael J. Fox Foundation for Parkinson's Research (MJFF) funded the generation of novel rat models with targeted disruption ofPink1, DJ-1 or Parkin genes and determined if the loss of these proteins would result in a progressive PD-like phenotype. Pathological, neurochemical and behavioral outcome measures were collected at 4, 6 and 8months of age in homozygous KO rats and compared to wild-type (WT) rats. Both Pink1 and DJ-1 KO rats showed progressive nigral neurodegeneration with about 50% dopaminergic cell loss observed at 8 months of age. ThePink1 KO and DJ-1 KO rats also showed a two to three fold increase in striatal dopamine and serotonin content at 8 months of age. Both Pink1 KO and DJ-1 KO rats exhibited significant motor deficits starting at 4months of age. However, Parkin KO rats displayed normal behaviors with no neurochemical or pathological changes. These results demonstrate that inactivation of the Pink1 or DJ-1 genes in the rat produces progressive neurodegeneration and early behavioral deficits, suggesting that these recessive genes may be essential for the survival of dopaminergic neurons in the substantia nigra (SN). These MJFF-generated novel rat models will assist the research community to elucidate the mechanisms by which these recessive genes produce PD pathology and potentially aid in therapeutic development.

  8. Lost in phototransduction: a few facts and hypotheses on cephalopod photoresponse.

    PubMed

    Moccia, Francesco; Cristo, Carlo Di; Di Cosmo, Anna

    2009-06-01

    Cephalopods are endowed with the most sophisticated nervous system among invertebrates and exhibit a repertoire of complex behaviors, such as spatial and observational learning. Cephalopod eyes supply a wide range of information which are utilized for these learning behaviors. Although our understanding of vertebrate physiology greatly benefited from the sub-cellular analysis of cephalopod nervous system, as shown by the discovery of the ionic bases of action potentials and of the Ca2+ requirement for neurotransmitter release Surprisingly, the cellular basis by which the visual system drives the sophisticated repertoire of cephalopod behaviors are still poorly understood. In this review, we will describe the current knowledge about cephalopod phototransduction. Light excites cephalopod photoreceptors by either inducing Ca2+ release from intracellular stores or activating cation-permeable channels by an as yet unknown mechanism. A 92 kDa protein, which is homologous to the Drosophila transient receptor potential (TRP) gene, is the most likely mediator of light-induced currents in cephalopods. A number of models which explain the mechanism whereby cephalopod TRP channel is gated by light will be discussed.

  9. Knockout of the adp gene related with colonization in Bacillus nematocida B16 using customized transcription activator-like effectors nucleases

    PubMed Central

    Niu, Qiuhong; Zheng, Haoying; Zhang, Lin; Qin, Fujun; Facemire, Loryn; Zhang, Guo; Cao, Feng; Zhang, Ke-qin; Huang, Xiaowei; Yang, Jianwei; He, Lei; Liu, Chanjuan

    2015-01-01

    Bacillus nematocida B16 is able to dominate in the intestines of the worm Caenorhabditis elegans in ‘Trojan horse’ pathogenic mechanism. The adp is one candidate gene which potentially play a vital role in the colonization from our previous random mutagenesis screening results. To analyse the functional role of this gene, we constructed the adp knockout mutant through customized transcription activator-like effectors nucleases (TALEN), which has been successfully used in yeasts, nematodes, zebrafish and human pluripotent cells. Here, we first time report this knockout method in bacteria on this paper. Bioassay experiments demonstrated that the adp knockout mutant of B16 showed considerably lower colonization activity, reduced numbers of intestines and less than 80% nematocidal activity compared with the wild-type strain when infected for 48 h. However, no obvious change on proteolytic activity was observed in the mutant. Conversely, the complementation of adp gene restored most of the above deficient phenotypes. These results indicated that the adp gene was involved in surface adhesion and played a comparatively important role in colonizing host nematodes. Moreover, TALENs successfully disrupt target genes in bacteria. PMID:25912819

  10. Restoration of hearing in the VGLUT3 knockout mouse using virally mediated gene therapy.

    PubMed

    Akil, Omar; Seal, Rebecca P; Burke, Kevin; Wang, Chuansong; Alemi, Aurash; During, Matthew; Edwards, Robert H; Lustig, Lawrence R

    2012-07-26

    Mice lacking the vesicular glutamate transporter-3 (VGLUT3) are congenitally deaf due to loss of glutamate release at the inner hair cell afferent synapse. Cochlear delivery of VGLUT3 using adeno-associated virus type 1 (AAV1) leads to transgene expression in only inner hair cells (IHCs), despite broader viral uptake. Within 2 weeks of AAV1-VGLUT3 delivery, auditory brainstem response (ABR) thresholds normalize, along with partial rescue of the startle response. Lastly, we demonstrate partial reversal of the morphologic changes seen within the afferent IHC ribbon synapse. These findings represent a successful restoration of hearing by gene replacement in mice, which is a significant advance toward gene therapy of human deafness.

  11. Knockout of Lysosomal Enzyme-Targeting Gene Causes Abnormalities in Mouse Pup Isolation Calls

    PubMed Central

    Barnes, Terra D.; Holy, Timothy E.

    2017-01-01

    Humans lacking a working copy of the GNPTAB gene suffer from the metabolic disease Mucolipidosis type II (MLII). MLII symptoms include mental retardation, skeletal deformities and cartilage defects as well as a speech delay with most subjects unable to utter single words (Otomo et al., 2009; Cathey et al., 2010; Leroy et al., 2012). Here we asked whether mice lacking a copy of Gnptab gene exhibited vocal abnormities. We recorded ultrasonic vocalizations from 5 to 8 day old mice separated from their mother and littermates. Although Gnptab−/− pups emitted a similar number of calls, several features of the calls were different from their wild type littermates. Gnptab−/− mice showed a decrease in the length of calls, an increase in the intra-bout pause duration, significantly fewer pitch jumps with smaller mean size, and an increase in the number of isolated calls. In addition, Gnptab−/− mice vocalizations had less power, particularly in the higher frequencies. Gnptab+/− mouse vocalizations did not appear to be affected. We then attempted to classify these recordings using these features to determine the genotype of the animal. We were able to correctly identify 87% of the recordings as either Gnptab−/− or Gnptab+/+ pup, significantly better than chance, demonstrating that genotype is a strong predictor of vocalization phenotype. These data show that deletion of genes in the lysosomal enzyme targeting pathway affect mouse pup isolation calls. PMID:28101008

  12. Generation of biallelic knock-out sheep via gene-editing and somatic cell nuclear transfer

    PubMed Central

    Li, Honghui; Wang, Gui; Hao, Zhiqiang; Zhang, Guozhong; Qing, Yubo; Liu, Shuanghui; Qing, Lili; Pan, Weirong; Chen, Lei; Liu, Guichun; Zhao, Ruoping; Jia, Baoyu; Zeng, Luyao; Guo, Jianxiong; Zhao, Lixiao; Zhao, Heng; Lv, Chaoxiang; Xu, Kaixiang; Cheng, Wenmin; Li, Hushan; Zhao, Hong-Ye; Wang, Wen; Wei, Hong-Jiang

    2016-01-01

    Transgenic sheep can be used to achieve genetic improvements in breeds and as an important large-animal model for biomedical research. In this study, we generated a TALEN plasmid specific for ovine MSTN and transfected it into fetal fibroblast cells of STH sheep. MSTN biallelic-KO somatic cells were selected as nuclear donor cells for SCNT. In total, cloned embryos were transferred into 37 recipient gilts, 28 (75.7%) becoming pregnant and 15 delivering, resulting in 23 lambs, 12 of which were alive. Mutations in the lambs were verified via sequencing and T7EI assay, and the gene mutation site was consistent with that in the donor cells. Off-target analysis was performed, and no off-target mutations were detected. MSTN KO affected the mRNA expression of MSTN relative genes. The growth curve for the resulting sheep suggested that MSTN KO caused a remarkable increase in body weight compared with those of wild-type sheep. Histological analyses revealed that MSTN KO resulted in muscle fiber hypertrophy. These findings demonstrate the successful generation of MSTN biallelic-KO STH sheep via gene editing in somatic cells using TALEN technology and SCNT. These MSTN mutant sheep developed and grew normally, and exhibited increased body weight and muscle growth. PMID:27654750

  13. Gene knockouts reveal separate functions for two cytoplasmic dyneins in Tetrahymena thermophila.

    PubMed

    Lee, S; Wisniewski, J C; Dentler, W L; Asai, D J

    1999-03-01

    In many organisms, there are multiple isoforms of cytoplasmic dynein heavy chains, and division of labor among the isoforms would provide a mechanism to regulate dynein function. The targeted disruption of somatic genes in Tetrahymena thermophila presents the opportunity to determine the contributions of individual dynein isoforms in a single cell that expresses multiple dynein heavy chain genes. Substantial portions of two Tetrahymena cytoplasmic dynein heavy chain genes were cloned, and their motor domains were sequenced. Tetrahymena DYH1 encodes the ubiquitous cytoplasmic dynein Dyh1, and DYH2 encodes a second cytoplasmic dynein isoform, Dyh2. The disruption of DYH1, but not DYH2, resulted in cells with two detectable defects: 1) phagocytic activity was inhibited, and 2) the cells failed to distribute their chromosomes correctly during micronuclear mitosis. In contrast, the disruption of DYH2 resulted in a loss of regulation of cell size and cell shape and in the apparent inability of the cells to repair their cortical cytoskeletons. We conclude that the two dyneins perform separate tasks in Tetrahymena.

  14. Generation of biallelic knock-out sheep via gene-editing and somatic cell nuclear transfer.

    PubMed

    Li, Honghui; Wang, Gui; Hao, Zhiqiang; Zhang, Guozhong; Qing, Yubo; Liu, Shuanghui; Qing, Lili; Pan, Weirong; Chen, Lei; Liu, Guichun; Zhao, Ruoping; Jia, Baoyu; Zeng, Luyao; Guo, Jianxiong; Zhao, Lixiao; Zhao, Heng; Lv, Chaoxiang; Xu, Kaixiang; Cheng, Wenmin; Li, Hushan; Zhao, Hong-Ye; Wang, Wen; Wei, Hong-Jiang

    2016-09-22

    Transgenic sheep can be used to achieve genetic improvements in breeds and as an important large-animal model for biomedical research. In this study, we generated a TALEN plasmid specific for ovine MSTN and transfected it into fetal fibroblast cells of STH sheep. MSTN biallelic-KO somatic cells were selected as nuclear donor cells for SCNT. In total, cloned embryos were transferred into 37 recipient gilts, 28 (75.7%) becoming pregnant and 15 delivering, resulting in 23 lambs, 12 of which were alive. Mutations in the lambs were verified via sequencing and T7EI assay, and the gene mutation site was consistent with that in the donor cells. Off-target analysis was performed, and no off-target mutations were detected. MSTN KO affected the mRNA expression of MSTN relative genes. The growth curve for the resulting sheep suggested that MSTN KO caused a remarkable increase in body weight compared with those of wild-type sheep. Histological analyses revealed that MSTN KO resulted in muscle fiber hypertrophy. These findings demonstrate the successful generation of MSTN biallelic-KO STH sheep via gene editing in somatic cells using TALEN technology and SCNT. These MSTN mutant sheep developed and grew normally, and exhibited increased body weight and muscle growth.

  15. [Effects of knockout ECM25/YJL201W gene in brewing yeast on beer flavor stability].

    PubMed

    Zhang, Yixin; Li, Qi; Shen, Wei; Xie, Yan; Gu, Guoxian

    2008-08-01

    The ECM25 deletion mutant of industrial brewing yeast, G03/a, was constructed by replacing the ECM25 gene with the kanMX gene. The transformant was verified to be genetically stable. The PCR analysis showed that ECM25 gene in the G-03/a was deleted. Under aerobic conditions of ll degrees C and 28 degrees C, compared with the host strain G-03, the excretive glutathione concentration of G-03/a increased by 21.4% and 14.7%, respectively. Strains G-03 and G-03/a were inoculated in flasks and cultivated continuously for 4 generations. Compared with the host strain G-03, the glutathione concentration in the main fermentation broth and final beer of strain G-03/a increased by 32.1% and 13.8%, the stability index (SI) increased by 7.7% and 5.3%, respectively, and the flavor resistance staling value (RSV value) in final beer increased by 45.0%. During EBC fermentation, the glutathione concentration in the main fermentation broth of strain G-03/a increased by 34.0%, compared with the host strain G-03. Furthermore, no significant difference in routine fermentation parameters was found. The strain G-03/a is proved to be an excellent anti-staling brewing yeast to improve beer flavor stability.

  16. The Construction of Transgenic and Gene Knockout/Knockin Mouse Models of Human Disease

    PubMed Central

    Doyle, Alfred; McGarry, Michael P.; Lee, Nancy A.; Lee, James J.

    2012-01-01

    The genetic and physiological similarities between mice and humans have focused considerable attention on rodents as potential models of human health and disease. Together with the wealth of resources, knowledge, and technologies surrounding the mouse as a model system, these similarities have propelled this species to the forefront of biomedical research. The advent of genomic manipulation has quickly led to the creation and use of genetically engineered mice as powerful tools for cutting edge studies of human disease research, including the discovery, refinement, and utility of many currently available therapeutic regimes. In particular, the creation of genetically modified mice as models of human disease has remarkably changed our ability to understand the molecular mechanisms and cellular pathways underlying disease states. Moreover, the mouse models resulting from gene transfer technologies have been important components correlating an individual’s gene expression profile to the development of disease pathologies. The objective of this review is to provide physician-scientists with an expansive historical and logistical overview of the creation of mouse models of human disease through gene transfer technologies. Our expectation is that this will facilitate on-going disease research studies and may initiate new areas of translational research leading to enhanced patient care. PMID:21800101

  17. 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 (Cnr2tm1Zim) 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 (Cnr2Dgen) receptor knockout mice developed augmented severity of disease consistent with the genetically and pharmacologically-distinct, Cnr2tm1Zim 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

  18. 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

  19. Knockout of the alanine racemase gene in Aeromonas hydrophila HBNUAh01 results in cell wall damage and enhanced membrane permeability.

    PubMed

    Liu, Dong; Zhang, Lu; Xue, Wen; Wang, Yaping; Ju, Jiansong; Zhao, Baohua

    2015-07-01

    This study focused on the alanine racemase gene (alr-2), which is involved in the synthesis of d-alanine that forms the backbone of the cell wall. A stable alr-2 knockout mutant of Aeromonas hydrophila HBNUAh01 was constructed. When the mutant was supplemented with d-alanine, growth was unaffected; deprivation of d-alanine caused the growth arrest of the starved mutant cells, but not cell lysis. No alanine racemase activity was detected in the culture of the mutant. Additionally, a membrane permeability assay showed increasing damage to the cell wall during d-alanine starvation. No such damage was observed in the wild type during culture. Scanning and transmission electron microscopy analyses revealed deficiencies of the cell envelope and perforation of the cell wall. Leakage of UV-absorbing substances from the mutants was also observed. Thus, the partial viability of the mutants and their independence of d-alanine for growth indicated that inactivation of alr-2 does not impose an auxotrophic requirement for d-alanine.

  20. Adenoviral gene therapy of the Tay-Sachs disease in hexosaminidase A-deficient knock-out mice.

    PubMed

    Guidotti, J E; Mignon, A; Haase, G; Caillaud, C; McDonell, N; Kahn, A; Poenaru, L

    1999-05-01

    The severe neurodegenerative disorder, Tays-Sachs disease, is caused by a beta-hexosaminidase alpha-subunit deficiency which prevents the formation of lysosomal heterodimeric alpha-beta enzyme, hexosaminidase A (HexA). No treatment is available for this fatal disease; however, gene therapy could represent a therapeutic approach. We previously have constructed and characterized, in vitro, adenoviral and retroviral vectors coding for alpha- and beta-subunits of the human beta-hexosaminidases. Here, we have determined the in vivo strategy which leads to the highest HexA activity in the maximum number of tissues in hexA -deficient knock-out mice. We demonstrated that intravenous co-administration of adenoviral vectors coding for both alpha- and beta-subunits, resulting in preferential liver transduction, was essential to obtain the most successful results. Only the supply of both subunits allowed for HexA overexpression leading to massive secretion of the enzyme in serum, and full or partial enzymatic activity restoration in all peripheral tissues tested. The enzymatic correction was likely to be due to direct cellular transduction by adenoviral vectors and/or uptake of secreted HexA by different organs. These results confirmed that the liver was the preferential target organ to deliver a large amount of secreted proteins. In addition, the need to overexpress both subunits of heterodimeric proteins in order to obtain a high level of secretion in animals defective in only one subunit is emphasized. The endogenous non-defective subunit is otherwise limiting.

  1. The kiss/kissr systems are dispensable for zebrafish reproduction: evidence from gene knockout studies.

    PubMed

    Tang, Haipei; Liu, Yun; Luo, Daji; Ogawa, Satoshi; Yin, Yike; Li, Shuisheng; Zhang, Yong; Hu, Wei; Parhar, Ishwar S; Lin, Haoran; Liu, Xiaochun; Cheng, Christopher H K

    2015-02-01

    The kiss1/gpr54 signaling system is considered to be a critical regulator of reproduction in most vertebrates. However, this presumption has not been tested vigorously in nonmammalian vertebrates. Distinct from mammals, multiple kiss1/gpr54 paralogous genes (kiss/kissr) have been identified in nonmammalian vertebrates, raising the possibility of functional redundancy among these genes. In this study, we have systematically generated the zebrafish kiss1(-/-), kiss2(-/-), and kiss1(-/-);kiss2(-/-) mutant lines as well as the kissr1(-/-), kissr2(-/-), and kissr1(-/-);kissr2(-/-) mutant lines using transcription activator-like effector nucleases. We have demonstrated that spermatogenesis and folliculogenesis as well as reproductive capability are not impaired in all of these 6 mutant lines. Collectively, our results indicate that kiss/kissr signaling is not absolutely required for zebrafish reproduction, suggesting that the kiss/kissr systems play nonessential roles for reproduction in certain nonmammalian vertebrates. These findings also demonstrated that fish and mammals have evolved different strategies for neuroendocrine control of reproduction.

  2. Triple immunoglobulin gene knockout transchromosomic cattle: bovine lambda cluster deletion and its effect on fully human polyclonal antibody production.

    PubMed

    Matsushita, Hiroaki; Sano, Akiko; Wu, Hua; Jiao, Jin-An; Kasinathan, Poothappillai; Sullivan, Eddie J; Wang, Zhongde; Kuroiwa, Yoshimi

    2014-01-01

    Towards the goal of producing fully human polyclonal antibodies (hpAbs or hIgGs) in transchromosomic (Tc) cattle, we previously reported that Tc cattle carrying a human artificial chromosome (HAC) comprising the entire unrearranged human immunoglobulin (Ig) heavy-chain (hIGH), kappa-chain (hIGK), and lambda-chain (hIGL) germline loci produced physiological levels of hIgGs when both of the bovine immunoglobulin mu heavy-chains, bIGHM and bIGHML1, were homozygously inactivated (bIGHM-/-, bIGHML1-/-; double knockouts or DKO). However, because endogenous bovine immunoglobulin light chain loci are still intact, the light chains are produced both from the hIGK and hIGL genomic loci on the HAC and from the endogenous bovine kappa-chain (bIGK) and lambda-chain (bIGL) genomic loci, resulting in the production of fully hIgGs (both Ig heavy-chains and light-chains are of human origin: hIgG/hIgκ or hIgG/hIgλ) and chimeric hIgGs (Ig heavy-chains are of human origin while the Ig light-chains are of bovine origin: hIgG/bIgκ or hIgG/bIgλ). To improve fully hIgG production in Tc cattle, we here report the deletion of the entire bIGL joining (J) and constant (C) gene cluster (bIGLJ1-IGLC1 to bIGLJ5-IGLC5) by employing Cre/loxP mediated site-specific chromosome recombination and the production of triple knockout (bIGHM-/-, bIGHML1-/- and bIGL-/-; TKO) Tc cattle. We further demonstrate that bIGL cluster deletion greatly improves fully hIgGs production in the sera of TKO Tc cattle, with 51.3% fully hIgGs (hIgG/hIgκ plus hIgG/hIgλ).

  3. Fatty Liver and Insulin Resistance in the Liver-Specific Knockout Mice of Mitogen Inducible Gene-6

    PubMed Central

    Park, Byung Kil; Kim, Hee-Youn; Lee, Jun Choul; Kim, Koon Soon; Jeong, Won Hoon; Kim, Ki Young

    2016-01-01

    Mitogen inducible gene-6 (Mig-6) is a feedback inhibitor of epidermal growth factor receptor (EGFR) signaling pathway. The liver-specific knockout mice of the Mig-6 gene (Mig-6d/d) showed hepatomegaly and increased hypercholesterolemia. In this study, the biomarkers of insulin resistance and the effects of high-fat diets in the wild (Mig-6f/f) and Mig-6d/d mice were analyzed. The fasting plasma concentrations of glucose, triglyceride, cholesterols, free fatty acids, and HOMA-IR were measured and the glucose tolerance and insulin resistance tests were performed in the 25-week-old Mig-6f/f and the Mig-6d/d mice. The protein levels of active insulin receptor, glucose 6-phosphatase, and phosphoenolpyruvate carboxykinase were analyzed in the liver and fat. The fasting plasma cholesterol and glucose concentration were higher in the Mig-6d/d mice than the Mig-6f/f mice with increased fat deposition in the liver. But the Mig-6d/d mice had the improved glucose intolerance and insulin resistance without increased amount of phosphoinsulin receptor after insulin infusion in the liver. The hepatic concentration of phosphoenolpyruvate carboxykinase was increased in fasting Mig-6d/d mice. The feeding of high-fat diet accelerated the plasma lipids profiles and HOMA-IR in the Mig-6d/d mice but had no differential effects in oral glucose tolerance test and insulin tolerance test in both genotypes. These results suggest that the activated EGFR signaling might increase the fasting plasma glucose concentration through inducing the hepatic steatosis and the improved whole-body insulin resistance in the KO mice be caused by decreased adipogenesis in fat tissues. PMID:28053990

  4. Conditional knockout of Foxc2 gene in kidney: efficient generation of conditional alleles of single-exon gene by double-selection system.

    PubMed

    Motojima, Masaru; Ogiwara, Sanae; Matsusaka, Taiji; Kim, Sang Yong; Sagawa, Nobuho; Abe, Koichiro; Ohtsuka, Masato

    2016-02-01

    Foxc2 is a single-exon gene and a key regulator in development of multiple organs, including kidney. To avoid embryonic lethality of conventional Foxc2 knockout mice, we conditionally deleted Foxc2 in kidneys. Conditional targeting of a single-exon gene involves the large floxed gene segment spanning from promoter region to coding region to avoid functional disruption of the gene by the insertion of a loxP site. Therefore, in ES cell clones surviving a conventional single-selection, e.g., neomycin-resistant gene (neo) alone, homologous recombination between the long floxed segment and target genome results in a high incidence of having only one loxP site adjacent to the selection marker. To avoid this limitation, we employed a double-selection system. We generated a Foxc2 targeting construct in which a floxed segment contained 4.6 kb mouse genome and two different selection marker genes, zeocin-resistant gene and neo, that were placed adjacent to each loxP site. After double-selection by zeocin and neomycin, 72 surviving clones were screened that yielded three correctly targeted clones. After floxed Foxc2 mice were generated by tetraploid complementation, we removed the two selection marker genes by a simultaneous-single microinjection of expression vectors for Dre and Flp recombinases into in vitro-fertilized eggs. To delete Foxc2 in mouse kidneys, floxed Foxc2 mice were mated with Pax2-Cre mice. Newborn Pax2-Cre; Foxc2(loxP/loxP) mice showed kidney hypoplasia and glomerular cysts. These results indicate the feasibility of generating floxed Foxc2 mice by double-selection system and simultaneous removal of selection markers with a single microinjection.

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

    PubMed

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

    2014-12-01

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

  6. A T-DNA Insertion Knockout of the Bifunctional Lysine-Ketoglutarate Reductase/Saccharopine Dehydrogenase Gene Elevates Lysine Levels in Arabidopsis Seeds1

    PubMed Central

    Zhu, Xiaohong; Tang, Guiliang; Granier, Fabienne; Bouchez, David; Galili, Gad

    2001-01-01

    Plants possess both anabolic and catabolic pathways for the essential amino acid lysine (Lys). However, although the biosynthetic pathway was clearly shown to regulate Lys accumulation in plants, the functional significance of Lys catabolism has not been experimentally elucidated. To address this issue, we have isolated an Arabidopsis knockout mutant with a T-DNA inserted into exon 13 of the gene encoding Lys ketoglutarate reductase/saccharopine dehydrogenase. This bifunctional enzyme controls the first two steps of Lys catabolism. The phenotype of the LKR/SDH knockout was indistinguishable from wild-type plants under normal growth conditions, suggesting that Lys catabolism is not an essential pathway under standard growth conditions. However, mature seeds of the knockout mutant over-accumulated Lys compared with wild-type plants. This report provides the first direct evidence for the functional significance of Lys catabolism in regulating Lys accumulation in seeds. Such a knockout mutant may also provide new perspectives to improve the level of the essential amino acid Lys in plant seeds. PMID:11500552

  7. Complete knockout of the lactate dehydrogenase A gene is lethal in pyruvate dehydrogenase kinase 1, 2, 3 down-regulated CHO cells.

    PubMed

    Yip, Shirley S M; Zhou, Meixia; Joly, John; Snedecor, Bradley; Shen, Amy; Crawford, Yongping

    2014-09-01

    Accumulation of high level of lactate can negatively impact cell growth during fed-batch culture process. In this study, we attempted to knockout the lactate dehydrogenase A (LDHA) gene in CHO cells in order to attenuate the lactate level. To prevent the potential deleterious effect of pyruvate accumulation, consequent to LDHA knockout, on cell culture, we chose a pyruvate dehydrogenase kinase 1, 2, and 3 (PDHK1, 2, and 3) knockdown cell line in which to knock out LDHA alleles. Around 3,000 clones were screened to obtain 152 mutants. Only heterozygous mutants were identified. An attempt to knockout the remaining wild-type allele from one such heterozygote yielded only two mutants after screening 567 clones. One had an extra valine. Another evidenced a duplication event, possessing at lease one wild-type and two different frameshifted alleles. Both mutants still retained LDH activity. Together, our data strongly suggest that a complete knockout of LDHA is lethal in CHO cells, despite simultaneous down-regulation of PDHK1, 2, and 3.

  8. Use of RDA analysis of knockout mice to identify myeloid genes regulated in vivo by PU.1 and C/EBPalpha.

    PubMed Central

    Iwama, A; Zhang, P; Darlington, G J; McKercher, S R; Maki, R; Tenen, D G

    1998-01-01

    PU.1 and C/EBPalpha are transcription factors essential for normal myeloid development. Loss-of-function mutation of PU.1 leads to an absolute block in monocyte/macrophage development and abnormal granulocytic development while that of C/EBPalpha causes a selective block in neutrophilic differentiation. In order to understand these phenotypes, we studied the role of PU.1 and C/EBPalpha in the regulation of myeloid target genes in vivo . Northern blot analysis revealed that mRNAs encoding receptors for M-CSF, G-CSF and GM-CSF, were expressed at low levels in PU.1(-/-) fetal liver compared with wild type. To identify additional myeloid genes regulated by PU.1 and C/EBPalpha, we performed representational difference analysis (RDA), a PCR-based subtractive hybridization using fetal livers from wild type and PU.1 or C/EBPalpha knockout mice. By introducing a new modification of RDA, that of tissue-specific gene suppression, we could selectively identify a set of differentially expressed genes specific to myeloid cells. Differentially expressed genes included both primary and secondary granule protein genes. In addition, eight novel genes were identified that were upregulated in expression during myeloid differentiation. These methods provide a general strategy for elucidating the genes affected in murine knockout models. PMID:9611252

  9. The probabilistic cell: implementation of a probabilistic inference by the biochemical mechanisms of phototransduction.

    PubMed

    Houillon, Audrey; Bessière, Pierre; Droulez, Jacques

    2010-09-01

    When we perceive the external world, our brain has to deal with the incompleteness and uncertainty associated with sensory inputs, memory and prior knowledge. In theoretical neuroscience probabilistic approaches have received a growing interest recently, as they account for the ability to reason with incomplete knowledge and to efficiently describe perceptive and behavioral tasks. How can the probability distributions that need to be estimated in these models be represented and processed in the brain, in particular at the single cell level? We consider the basic function carried out by photoreceptor cells which consists in detecting the presence or absence of light. We give a system-level understanding of the process of phototransduction based on a bayesian formalism: we show that the process of phototransduction is equivalent to a temporal probabilistic inference in a Hidden Markov Model (HMM), for estimating the presence or absence of light. Thus, the biochemical mechanisms of phototransduction underlie the estimation of the current state probability distribution of the presence of light. A classical descriptive model describes the interactions between the different molecular messengers, ions, enzymes and channel proteins occurring within the photoreceptor by a set of nonlinear coupled differential equations. In contrast, the probabilistic HMM model is described by a discrete recurrence equation. It appears that the binary HMM has a general solution in the case of constant input. This allows a detailed analysis of the dynamics of the system. The biochemical system and the HMM behave similarly under steady-state conditions. Consequently a formal equivalence can be found between the biochemical system and the HMM. Numerical simulations further extend the results to the dynamic case and to noisy input. All in all, we have derived a probabilistic model equivalent to a classical descriptive model of phototransduction, which has the additional advantage of assigning a

  10. Protective effects of sunscreening agents on photocarcinogenesis, photoaging, and DNA damage in XPA gene knockout mice.

    PubMed

    Horiki, S; Miyauchi-Hashimoto, H; Tanaka, K; Nikaido, O; Horio, T

    2000-10-01

    We investigated the protective effects of commercial sunscreening agents against UVB-induced photoresponses in group A xeroderma pigmentosum (XPA) model mice. XPA gene-deficient mice are defective in nucleotide excision repair and show a high incidence of skin tumors and severe acute inflammation in response to UVB irradiation, in a similar manner to XP patients. SPF 10 and SPF 60 sunscreens protected partially and almost completely, respectively, ear swelling responses produced by UVB up to 200 mJ/cm2 in (-/-) mice. XPA (-/-) mice were irradiated three times a week to a cumulative dose of 2.6 J/cm2 UVB for a period of 24 weeks with or without SPF 10 or SPF 60 sunscreen. UV-induced skin tumors had developed in all unprotected (-/-) mice (13.3 tumors per mouse) at the completion of UVB irradiation. The SPF 60 sunscreen afforded stronger protection against photocarcinogenesis (1.0 tumors per mouse) than the SPF 10 sunscreen (4.4 tumors per mouse). Regarding photoaging, SPF 60 sunscreen also protected against mast cell infiltration (79% inhibition), elastic fiber accumulation, and dermal cyst proliferation in XPA (-/-) mice compared with unprotected (-/-) mice. In (-/-) mice, the SPF 60 sunscreen provided stronger protection against cyclobutane pyrimidine dimer formation shown immunohistologically following irradiation with 200 mJ/cm2 UVB than the SPF 10 sunscreen. The XPA model mouse is a useful animal for the evaluation of the photoprotective ability of sunscreens because photoresponses, even chronic changes, can be easily and quickly induced experimentally.

  11. Aggravated renal inflammatory responses in TRPV1 gene knockout mice subjected to DOCA-salt hypertension.

    PubMed

    Wang, Youping; Wang, Donna H

    2009-12-01

    To test the hypothesis that deletion of the transient receptor potential vanilloid type 1 (TRPV1) channel exaggerates hypertension-induced renal inflammatory response, wild-type (WT) or TRPV1-null mutant (TRPV1(-/-)) mice were subjected to uninephrectomy and deoxycorticosterone acetate (DOCA)-salt treatment for 4 wk. Mean arterial pressure (MAP) determined by radiotelemetry increased in DOCA-salt-treated WT or TRPV1(-/-) mice, whereas there was no difference in MAP between two strains at the baseline or after DOCA-salt treatment. DOCA-salt treatment increased urinary excretion of albumin and 8-isoprostane in both WT and TRPV1(-/-) mice, and the increases were greater in magnitude in the latter strain. Periodic acid-Schiff and Mason's trichrome staining showed that kidneys of DOCA-salt-treated TRPV1(-/-) mice exhibited more severe glomerulosclerosis and tubulointerstitial injury compared with DOCA-salt-treated WT mice. NF-kappaB assay showed that DOCA-salt treatment increased renal activated NF-kappaB concentrations in TRPV1(-/-) mice compared with WT mice. Immunostaining and ELISA assay revealed that DOCA-salt-treated TRPV1(-/-) mice had enhanced renal infiltration of monocyte/macrophage and lymphocyte, as well as increased renal levels of proinflammatory cytokine (TNF-alpha, IL-6) and chemokine (MCP-1) compared with DOCA-salt-treated WT mice. Renal ICAM-1 but not VCAM-1 expression was also greater in DOCA-salt-treated TRPV1(-/-) than WT mice. Dexamethasone (DEXA), an immunosuppressive drug, conveyed a renoprotective effect that was greater in DOCA-salt-treated TRPV1(-/-) compared with WT mice. These data show that renal inflammation is exacerbated in DOCA-salt hypertension when TRPV1 gene is deleted and that the deterioration is ameliorated by DEXA treatment, indicating that TRPV1 may act as a potential regulator of the inflammatory process to lessen renal injury in DOCA-salt hypertension.

  12. A large-scale zebrafish gene knockout resource for the genome-wide study of gene function.

    PubMed

    Varshney, Gaurav K; Lu, Jing; Gildea, Derek E; Huang, Haigen; Pei, Wuhong; Yang, Zhongan; Huang, Sunny C; Schoenfeld, David; Pho, Nam H; Casero, David; Hirase, Takashi; Mosbrook-Davis, Deborah; Zhang, Suiyuan; Jao, Li-En; Zhang, Bo; Woods, Ian G; Zimmerman, Steven; Schier, Alexander F; Wolfsberg, Tyra G; Pellegrini, Matteo; Burgess, Shawn M; Lin, Shuo

    2013-04-01

    With the completion of the zebrafish genome sequencing project, it becomes possible to analyze the function of zebrafish genes in a systematic way. The first step in such an analysis is to inactivate each protein-coding gene by targeted or random mutation. Here we describe a streamlined pipeline using proviral insertions coupled with high-throughput sequencing and mapping technologies to widely mutagenize genes in the zebrafish genome. We also report the first 6144 mutagenized and archived F1's predicted to carry up to 3776 mutations in annotated genes. Using in vitro fertilization, we have rescued and characterized ~0.5% of the predicted mutations, showing mutation efficacy and a variety of phenotypes relevant to both developmental processes and human genetic diseases. Mutagenized fish lines are being made freely available to the public through the Zebrafish International Resource Center. These fish lines establish an important milestone for zebrafish genetics research and should greatly facilitate systematic functional studies of the vertebrate genome.

  13. Histone Deacetylase Inhibition Rescues Gene Knockout Levels Achieved with Integrase-Defective Lentiviral Vectors Encoding Zinc-Finger Nucleases

    PubMed Central

    Pelascini, Laetitia P.L.; Maggio, Ignazio; Liu, Jin; Holkers, Maarten; Cathomen, Toni

    2013-01-01

    Abstract Zinc-finger nucleases (ZFNs) work as dimers to induce double-stranded DNA breaks (DSBs) at predefined chromosomal positions. In doing so, they constitute powerful triggers to edit and to interrogate the function of genomic sequences in higher eukaryotes. A preferred route to introduce ZFNs into somatic cells relies on their cotransduction with two integrase-defective lentiviral vectors (IDLVs) each encoding a monomer of a functional heterodimeric pair. The episomal nature of IDLVs diminishes the risk of genotoxicity and ensures the strict transient expression profile necessary to minimize deleterious effects associated with long-term ZFN activity. However, by deploying IDLVs and conventional lentiviral vectors encoding HPRT1- or eGFP-specific ZFNs, we report that DSB formation at target alleles is limited after IDLV-mediated ZFN transfer. This IDLV-specific underperformance stems, to a great extent, from the activity of chromatin-remodeling histone deacetylases (HDACs). Importantly, the prototypic and U.S. Food and Drug Administration–approved inhibitors of metal-dependent HDACs, trichostatin A and vorinostat, respectively, did not hinder illegitimate recombination-mediated repair of targeted chromosomal DSBs. This allowed rescuing IDLV-mediated site-directed mutagenesis to levels approaching those achieved by using their isogenic chromosomally integrating counterparts. Hence, HDAC inhibition constitutes an efficacious expedient to incorporate in genome-editing strategies based on transient IDLV-mediated ZFN expression. Finally, we compared two of the most commonly used readout systems to measure targeted gene knockout activities based on restriction and mismatch-sensitive endonucleases. These experiments indicate that these enzymatic assays display a similar performance. PMID:24059449

  14. [BLG gene knockout and hLF gene knock-in at BLG locus in goat by TALENs].

    PubMed

    Song, Shaozheng; Zhu, Mengmin; Yuan, Yuguo; Rong, Yao; Xu, Sheng; Chen, Si; Mei, Junyan; Cheng, Yong

    2016-03-01

    To knock out β-lactoglobulin (BLG) gene and insert human lactoferrin (hLF) coding sequence at BLG locus of goat, the transcription activator-like effector nucleases (TALEN) mediated recombination was used to edit the BLG gene of goat fetal fibroblast, then as donor cells for somatic cell nuclear transfer. We designed a pair of specific plasmid TALEN-3-L/R for goat BLG exon III recognition sites, and BLC14-TK vector containing a negative selection gene HSV-TK, was used for the knock in of hLF gene. TALENs plasmids were transfected into the goat fetal fibroblast cells, and the cells were screened three days by 2 μg/mL puromycin. DNA cleavage activities of cells were verified by PCR amplification and DNA production sequencing. Then, targeting vector BLC14-TK and plasmids TALEN-3-L/R were co-transfected into goat fetal fibroblasts, both 700 μg/mL G418 and 2 μg/mL GCV were simultaneously used to screen G418-resistant cells. Detections of integration and recombination were implemented to obtain cells with hLF gene site-specific integration. We chose targeting cells as donor cells for somatic cell nuclear transfer. The mutagenicity of TALEN-3-L/R was between 25% and 30%. A total of 335 reconstructed embryos with 6 BLG-/hLF+ targeting cell lines were transferred into 16 recipient goats. There were 9 pregnancies confirmed by ultrasound on day 30 to 35 (pregnancy rate of 39.1%), and one of 50-day-old fetus with BLG-/hLF+ was achieved. These results provide the basis for hLF gene knock-in at BLG locus of goat and cultivating transgenic goat of low allergens and rich hLF in the milk.

  15. A large-scale zebrafish gene knockout resource for the genome-wide study of gene function

    PubMed Central

    Varshney, Gaurav K.; Lu, Jing; Gildea, Derek E.; Huang, Haigen; Pei, Wuhong; Yang, Zhongan; Huang, Sunny C.; Schoenfeld, David; Pho, Nam H.; Casero, David; Hirase, Takashi; Mosbrook-Davis, Deborah; Zhang, Suiyuan; Jao, Li-En; Zhang, Bo; Woods, Ian G.; Zimmerman, Steven; Schier, Alexander F.; Wolfsberg, Tyra G.; Pellegrini, Matteo; Burgess, Shawn M.; Lin, Shuo

    2013-01-01

    With the completion of the zebrafish genome sequencing project, it becomes possible to analyze the function of zebrafish genes in a systematic way. The first step in such an analysis is to inactivate each protein-coding gene by targeted or random mutation. Here we describe a streamlined pipeline using proviral insertions coupled with high-throughput sequencing and mapping technologies to widely mutagenize genes in the zebrafish genome. We also report the first 6144 mutagenized and archived F1's predicted to carry up to 3776 mutations in annotated genes. Using in vitro fertilization, we have rescued and characterized ∼0.5% of the predicted mutations, showing mutation efficacy and a variety of phenotypes relevant to both developmental processes and human genetic diseases. Mutagenized fish lines are being made freely available to the public through the Zebrafish International Resource Center. These fish lines establish an important milestone for zebrafish genetics research and should greatly facilitate systematic functional studies of the vertebrate genome. PMID:23382537

  16. Tissue distribution of products of the mouse decay-accelerating factor (DAF) genes. Exploitation of a Daf1 knock-out mouse and site-specific monoclonal antibodies.

    PubMed

    Lin, F; Fukuoka, Y; Spicer, A; Ohta, R; Okada, N; Harris, C L; Emancipator, S N; Medof, M E

    2001-10-01

    Decay-accelerating factor (DAF) is a membrane regulator of C3 activation that protects self cells from autologous complement attack. In humans, DAF is uniformly expressed as a glycosylphosphatidylinositol (GPI)-anchored molecule. In mice, both GPI-anchored and transmembrane-anchored DAF proteins are produced, each of which can be derived from two different genes (Daf1 and Daf2). In this report, we describe a Daf1 gene knock-out mouse arising as the first product of a strategy for targeting one or both Daf genes. As part of the work, we characterize recently described monoclonal antibodies against murine DAF protein using deletion mutants synthesized in yeast, and then employ the monoclonal antibodies in conjunction with wild-type and the Daf1 knock-out mice to determine the tissue distribution of the mouse Daf1 and Daf2 gene products. To enhance the immunohistochemical detection of murine DAF protein, we utilized the sensitive tyramide fluorescence method. In wild-type mice, we found strong DAF labelling of glomeruli, airway and gut epithelium, the spleen, vascular endothelium throughout all tissues, and seminiferous tubules of the testis. In Daf1 knock-out mice, DAF labelling was ablated in most tissues, but strong labelling of the testis and splenic dendritic cells remained. In both sites, reverse transcription-polymerase chain reaction analyses identified both GPI and transmembrane forms of Daf2 gene-derived protein. The results have relevance for studies of in vivo murine DAF function and of murine DAF structure.

  17. Liver-specific inactivation of the abetalipoproteinemia gene completely abrogates very low density lipoprotein/low density lipoprotein production in a viable conditional knockout mouse.

    PubMed

    Chang, B H; Liao, W; Li, L; Nakamuta, M; Mack, D; Chan, L

    1999-03-05

    Conventional knockout of the microsomal triglyceride transfer protein large subunit (lMTP) gene is embryonic lethal in the homozygous state in mice. We have produced a conditional lMTP knockout mouse by inserting loxP sequences flanking exons 5 and 6 by gene targeting. Homozygous floxed mice were born live with normal plasma lipids. Intravenous injection of an adenovirus harboring Cre recombinase (AdCre1) produced deletion of exons 5 and 6 and disappearance of lMTP mRNA and immunoreactive protein in a liver-specific manner. There was also disappearance of plasma apolipoprotein (apo) B-100 and marked reduction in apoB-48 levels. Wild-type mice showed no response, and heterozygous mice, an intermediate response, to AdCre1. Wild-type mice doubled their plasma cholesterol level following a high cholesterol diet. This hypercholesterolemia was abolished in AdCre1-treated lMTP-/- mice, the result of a complete absence of very low/intermediate/low density lipoproteins and a slight reduction in high density lipoprotein. Heterozygous mice showed an intermediate lipoprotein phenotype. The rate of accumulation of plasma triglyceride following Triton WR1339 treatment in lMTP-/- mice was <10% that in wild-type animals, indicating a failure of triglyceride-rich lipoprotein production. Pulse-chase experiments using hepatocytes isolated from wild-type and lMTP-/- mice revealed a failure of apoB secretion in lMTP-/- animals. Therefore, the liver-specific inactivation of the lMTP gene completely abrogates apoB-100 and very low/intermediate/low density lipoprotein production. These conditional knockout mice are a useful in vivo model for studying the role of MTP in apoB biosynthesis and the biogenesis of apoB-containing lipoproteins.

  18. Random Splicing of Several Exons Caused by a Single Base Change in the Target Exon of CRISPR/Cas9 Mediated Gene Knockout

    PubMed Central

    Kapahnke, Marcel; Banning, Antje; Tikkanen, Ritva

    2016-01-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)-associated sequence 9 (CRISPR/Cas9) system is widely used for genome editing purposes as it facilitates an efficient knockout of a specific gene in, e.g. cultured cells. Targeted double-strand breaks are introduced to the target sequence of the guide RNAs, which activates the cellular DNA repair mechanism for non-homologous-end-joining, resulting in unprecise repair and introduction of small deletions or insertions. Due to this, sequence alterations in the coding region of the target gene frequently cause frame-shift mutations, facilitating degradation of the mRNA. We here show that such CRISPR/Cas9-mediated alterations in the target exon may also result in altered splicing of the respective pre-mRNA, most likely due to mutations of splice-regulatory sequences. Using the human FLOT-1 gene as an example, we demonstrate that such altered splicing products also give rise to aberrant protein products. These may potentially function as dominant-negative proteins and thus interfere with the interpretation of the data generated with these cell lines. Since most researchers only control the consequences of CRISPR knockout at genomic and protein level, our data should encourage to also check the alterations at the mRNA level. PMID:27983621

  19. Deep Brain Photoreceptor (val-opsin) Gene Knockout Using CRISPR/Cas Affects Chorion Formation and Embryonic Hatching in the Zebrafish

    PubMed Central

    Hang, Chong Yee; Moriya, Shogo; Ogawa, Satoshi; Parhar, Ishwar S.

    2016-01-01

    Non-rod non-cone photopigments in the eyes and the brain can directly mediate non-visual functions of light in non-mammals. This was supported by our recent findings on vertebrate ancient long (VAL)-opsin photopigments encoded by the val-opsinA (valopa) and val-opsinB (valopb) genes in zebrafish. However, the physiological functions of valop isoforms remain unknown. Here, we generated valop-mutant zebrafish using CRISPR/Cas genome editing, and examined the phenotypes of loss-of-function mutants. F0 mosaic mutations and germline transmission were confirmed via targeted insertions and/or deletions in the valopa or valopb gene in F1 mutants. Based on in silico analysis, frameshift mutations converted VAL-opsin proteins to non-functional truncated forms with pre-mature stop codons. Most F1 eggs or embryos from F0 female valopa/b mutants showed either no or only partial chorion elevation, and the eggs or embryos died within 26 hour-post-fertilization. However, most F1 embryos from F0 male valopa mutant developed but hatched late compared to wild-type embryos, which hatched at 4 day-post-fertilization. Late-hatched F1 offspring included wild-type and mutants, indicating the parental effects of valop knockout. This study shows valop gene knockout affects chorion formation and embryonic hatching in the zebrafish. PMID:27792783

  20. DECREASED EXPRESSION LEVEL OF APOPTOSIS-RELATED GENES AND/OR PROTEINS IN SKELETAL MUSCLES, BUT NOT IN HEARTS, OF GROWTH HORMONE RECEPTOR KNOCKOUT MICE

    PubMed Central

    Gesing, Adam; Masternak, Michal M.; Wang, Feiya; Lewinski, Andrzej; Karbownik-Lewinska, Malgorzata; Bartke, Andrzej

    2013-01-01

    The long-lived growth hormone (GH) receptor knockout (GHRKO; KO) mice are GH resistant due to targeted disruption of the GH receptor (Ghr) gene. Apoptosis is a physiological process in which cells play an active role in their own death and is a normal component of the development and health of multicellular organisms. Aging is associated with the progressive loss of strength of skeletal and heart muscles. Calorie restriction (CR) is a well known experimental model to delay aging and increase lifespan. The aim of the study was to examine the expression of the following apoptosis-related genes: caspase-3, caspase-9, caspase-8, bax, bcl-2, Smac/DIABLO, p53 and cytochrome c1 (cyc1) in the skeletal muscles and hearts of female normal and GHRKO mice, fed ad libitum or subjected to 40% CR for 6 months, starting at 2 months of age. Moreover, skeletal muscle caspase-3, caspase-9, caspase-8, bax, bcl-2, Smac/DIABLO, Apaf-1, bad, phospho-bad (pbad), phospho-p53 (pp53) and cytochrome c (cyc) protein expression levels were assessed. Results Expression of caspase-3, caspase-9, bax and Smac/DIABLO genes and proteins was decreased in GHRKO’s skeletal muscles. The Apaf-1 protein expression also was diminished in this tissue. In contrast, bcl-2 and pbad protein levels were increased in skeletal muscles in knockouts. No changes were demonstrated for the examined genes expression in GHRKO’s hearts except for the increased level of cyc1 mRNA. CR did not alter the expression of the examined genes and proteins in skeletal muscles of knockouts vs. normal (N) mice. In heart homogenates, CR increased caspase-3 mRNA level as compared to ad libitum (AL) mice. Conclusion decreased expression of certain pro-apoptotic genes and/or proteins may constitute the potential mechanism of prolonged longevity in GHRKO mice, protecting these animals from aging; this potential beneficial mechanism is not affected by calorie restriction. PMID:21321312

  1. The evolution of phototransduction from an ancestral cyclic nucleotide gated pathway

    PubMed Central

    Plachetzki, David C.; Fong, Caitlin R.; Oakley, Todd H.

    2010-01-01

    The evolutionary histories of complex traits are complicated because such traits are comprised of multiple integrated and interacting components, which may have different individual histories. Phylogenetic studies of complex trait evolution often do not take this into account, instead focusing only on the history of whole, integrated traits; for example, mapping eyes as simply present or absent through history. Using the biochemistry of animal vision as a model, we demonstrate how investigating the individual components of complex systems can aid in elucidating both the origins and diversification of such systems. Opsin-based phototransduction underlies all visual phenotypes in animals, using complex protein cascades that translate light information into changes in cyclic nucleotide gated (CNG) or canonical transient receptor potential (TRPC) ion-channel activity. Here we show that CNG ion channels play a role in cnidarian phototransduction. Transcripts of a CNG ion channel co-localize with opsin in specific cell types of the eyeless cnidarian Hydra magnipapillata. Further, the CNG inhibitor cis-diltiazem ablates a stereotypical photoresponse in the hydra. Our findings in the Cnidaria, the only non-bilaterian lineage to possess functional opsins, allow us to trace the history of CNG-based photosensitivity to the very origin of animal phototransduction. Our general analytical approach, based on explicit phylogenetic analysis of individual components, contrasts the deep evolutionary history of CNG-based phototransduction, today used in vertebrate vision, with the more recent assembly of TRPC-based systems that are common to protostome (e.g. fly and mollusc) vision. PMID:20219739

  2. UV light phototransduction activates transient receptor potential A1 ion channels in human melanocytes.

    PubMed

    Bellono, Nicholas W; Kammel, Laura G; Zimmerman, Anita L; Oancea, Elena

    2013-02-05

    Human skin is constantly exposed to solar ultraviolet radiation (UVR), the most prevalent environmental carcinogen. Humans have the unique ability among mammals to respond to UVR by increasing their skin pigmentation, a protective process driven by melanin synthesis in epidermal melanocytes. The molecular mechanisms used by melanocytes to detect and respond to long-wavelength UVR (UVA) are not well understood. We recently identified a UVA phototransduction pathway in melanocytes that is mediated by G protein-coupled receptors and leads to rapid calcium mobilization. Here we report that in human epidermal melanocytes physiological doses of UVR activate a retinal-dependent current mediated by transient receptor potential A1 (TRPA1) ion channels. The TRPA1 photocurrent is UVA-specific and requires G protein and phospholipase C signaling, thus contributing to UVA-induced calcium responses to mediate downstream cellular effects and providing evidence for TRPA1 function in mammalian phototransduction. Remarkably, TRPA1 activation is required for the UVR-induced and retinal-dependent early increase in cellular melanin. Our results show that TRPA1 is essential for a unique extraocular phototransduction pathway in human melanocytes that is activated by physiological doses of UVR and results in early melanin synthesis.

  3. The Role of Mislocalized Phototransduction in Photoreceptor Cell Death of Retinitis Pigmentosa

    PubMed Central

    Nakao, Takeshi; Tsujikawa, Motokazu; Notomi, Shoji; Ikeda, Yasuhiro; Nishida, Kohji

    2012-01-01

    Most of inherited retinal diseases such as retinitis pigmentosa (RP) cause photoreceptor cell death resulting in blindness. RP is a large family of diseases in which the photoreceptor cell death can be caused by a number of pathways. Among them, light exposure has been reported to induce photoreceptor cell death. However, the detailed mechanism by which photoreceptor cell death is caused by light exposure is unclear. In this study, we have shown that even a mild light exposure can induce ectopic phototransduction and result in the acceleration of rod photoreceptor cell death in some vertebrate models. In ovl, a zebrafish model of outer segment deficiency, photoreceptor cell death is associated with light exposure. The ovl larvae show ectopic accumulation of rhodopsin and knockdown of ectopic rhodopsin and transducin rescue rod photoreceptor cell death. However, knockdown of phosphodiesterase, the enzyme that mediates the next step of phototransduction, does not. So, ectopic phototransduction activated by light exposure, which leads to rod photoreceptor cell death, is through the action of transducin. Furthermore, we have demonstrated that forced activation of adenylyl cyclase in the inner segment leads to rod photoreceptor cell death. For further confirmation, we have also generated a transgenic fish which possesses a human rhodopsin mutation, Q344X. This fish and rd10 model mice show photoreceptor cell death caused by adenylyl cyclase. In short, our study indicates that in some RP, adenylyl cyclase is involved in photoreceptor cell death pathway; its inhibition is potentially a logical approach for a novel RP therapy. PMID:22485131

  4. Phototransduction early steps model based on Beer-Lambert optical law.

    PubMed

    Salido, Ezequiel M; Servalli, Leonardo N; Gomez, Juan Carlos; Verrastro, Claudio

    2017-02-01

    The amount of available rhodopsin on the photoreceptor outer segment and its change over time is not considered in classic models of phototransduction. Thus, those models do not take into account the absorptance variation of the outer segment under different brightness conditions. The relationship between the light absorbed by a medium and its absorptance is well described by the Beer-Lambert law. This newly proposed model implements the absorptance variation phenomenon in a set of equations that admit photons per second as input and results in active rhodopsins per second as output. This study compares the classic model of phototransduction developed by Forti et al. (1989) to this new model by using different light stimuli to measure active rhodopsin and photocurrent. The results show a linear relationship between light stimulus and active rhodopsin in the Forti model and an exponential saturation in the new model. Further, photocurrent values have shown that the new model behaves equivalently to the experimental and theoretical data as published by Forti in dark-adapted rods, but fits significantly better under light-adapted conditions. The new model successfully introduced a physics optical law to the standard model of phototransduction adding a new processing layer that had not been mathematically implemented before. In addition, it describes the physiological concept of saturation and delivers outputs in concordance to input magnitudes.

  5. Knockout of GH3 genes in the moss Physcomitrella patens leads to increased IAA levels at elevated temperature and in darkness.

    PubMed

    Mittag, Jennifer; Gabrielyan, Anastasia; Ludwig-Müller, Jutta

    2015-12-01

    Two proteins of the GRETCHEN HAGEN3 (GH3) family of acyl acid amido synthetases from the moss Physcomitrella patens conjugate indole-3-acetic acid (IAA) to a series of amino acids. The possible function of altered auxin levels in the moss in response to two different growth perturbations, elevated temperatures and darkness, was analyzed using a) the recently described double knockout lines in both P. patens GH3 genes (GH3-doKO) and b) a previously characterized line harboring an auxin-inducible soybean GH3 promoter::reporter fused to β-glucuronidase (G1-GUS). The GUS activity as marker of the auxin response increased at higher temperatures and after cultivation in the darkness for a period of up to four weeks. Generally, the double knockout plants grew more slowly than the wild type (WT). The altered growth conditions influenced the phenotypes of the double knockout lines differently from that of WT moss. Higher temperatures negatively affected GH3-doKO plants compared to WT which was shown by stronger loss of chlorophyll. On the other hand, a positive effect was found on the concentrations of free IAA which increased at 28 °C in the GH3-doKO lines compared to WT plants. A different factor, namely darkness vs. a light/dark cycle caused the adverse phenotype concerning chlorophyll concentrations. Mutant moss plants showed higher chlorophyll concentrations than WT and these correlated with higher free IAA in the plant population that was classified as green. Our data show that growth perturbations result in higher free IAA levels in the GH3-doKO mutants, but in one case - growth in darkness - the mutants could cope better with the condition, whereas at elevated temperatures the mutants were more sensitive than WT. Thus, GH3 function in P. patens WT could lie in the regulation of IAA concentrations under unfavorable environmental conditions.

  6. Colony-live —a high-throughput method for measuring microbial colony growth kinetics— reveals diverse growth effects of gene knockouts in Escherichia coli

    PubMed Central

    2014-01-01

    Background Precise quantitative growth measurements and detection of small growth changes in high-throughput manner is essential for fundamental studies of bacterial cell. However, an inherent tradeoff for measurement quality in high-throughput methods sacrifices some measurement quality. A key challenge has been how to enhance measurement quality without sacrificing throughput. Results We developed a new high-throughput measurement system, termed Colony-live. Here we show that Colony-live provides accurate measurement of three growth values (lag time of growth (LTG), maximum growth rate (MGR), and saturation point growth (SPG)) by visualizing colony growth over time. By using a new normalization method for colony growth, Colony-live gives more precise and accurate growth values than the conventional method. We demonstrated the utility of Colony-live by measuring growth values for the entire Keio collection of Escherichia coli single-gene knockout mutants. By using Colony-live, we were able to identify subtle growth defects of single-gene knockout mutants that were undetectable by the conventional method quantified by fixed time-point camera imaging. Further, Colony-live can reveal genes that influence the length of the lag-phase and the saturation point of growth. Conclusions Measurement quality is critical to achieving the resolution required to identify unique phenotypes among a diverse range of phenotypes. Sharing high-quality genome-wide datasets should benefit many researchers who are interested in specific gene functions or the architecture of cellular systems. Our Colony-live system provides a new powerful tool to accelerate accumulation of knowledge of microbial growth phenotypes. PMID:24964927

  7. Effects of SIRT1 gene knock-out via activation of SREBP2 protein-mediated PI3K/AKT signaling on osteoarthritis in mice.

    PubMed

    Yu, Fei; Zeng, Hui; Lei, Ming; Xiao, De-Ming; Li, Wei; Yuan, Hao; Lin, Jian-Jing

    2016-10-01

    This study investigated the effects of SIRT1 gene knock-out on osteoarthritis in mice, and the possible roles of SREBP2 protein and the PI3K/AKT signaling pathway in the effects. Mice were randomly divided into a normal group and a SIRT1 gene knock-out group (6 mice in each group). In these groups, one side of the knee anterior cruciate ligament was traversed, and the ipsilateral medial meniscus was cut to establish an osteoarthritis model of knee joint. The countralateral synovial bursa was cut out, serving as controls. The knee joint specimens were then divided into four groups: SIRT1(+/+) control group (group A, n=6); SIRT1(+/+) osteoarthritis group (group B, n=6); SIRT1(-/-) control group (group C, n=6); SIRT1(-/-) osteoarthritis group (group D, n=6). HE staining, Masson staining, Safranin O-Fast Green staining and Van Gieson staining were used to observe the morphological changes in the articular cartilage of the knee. Immunohistochemical staining was employed to detect the expression of SIRT1, SREBP2, VEGF, AKT, HMGCR and type II collagen proteins. SA-β-gal staining was utilized to evaluate chondrocyte aging. The results showed clear knee joint cartilage destruction and degeneration in the SIRT1(-/-) osteoarthritis group. The tidal line was twisted and displaced anteriorly. Type II collagen was destroyed and distributed unevenly. Compared with the SIRT1(+/+) osteoarthritis group and SIRT1(-/-) control group, SIRT1 protein expression was not obviously changed in the SIRT1(-/-) osteoarthritis group (P>0.05), while the expression levels of the SREBP2, VEGF and HMGCR proteins were significantly increased (P<0.05) and the levels of AKT and type II collagen proteins were significantly decreased (P<0.05). SIRT1 gene knock-out may aggravate cartilage degeneration in osteoarthritis by activating the SREBP2 protein-mediated PI3K/AKT signalling pathway, suggesting that SIRT1 gene may play a protective role against osteoarthritis.

  8. Production of a mouse strain with impaired glucose tolerance by systemic heterozygous knockout of the glucokinase gene and its feasibility as a prediabetes model

    PubMed Central

    SAITO, Mikako; KANEDA, Asako; SUGIYAMA, Tae; IIDA, Ryousuke; OTOKUNI, Keiko; KABURAGI, Misako; MATSUOKA, Hideaki

    2015-01-01

    Exon II of glucokinase (Gk) was deleted to produce a systemic heterozygous Gk knockout (Gk+/−) mouse. The relative expression levels of Gk in the heart, lung, liver, stomach, and pancreas in Gk+/− mice ranged from 0.41–0.68 versus that in wild (Gk+/+) mice. On the other hand, its expression levels in the brain, adipose tissue, and muscle ranged from 0.95–1.03, and its expression levels in the spleen and kidney were nearly zero. Gk knockout caused no remarkable off-target effect on the expression of 7 diabetes causing genes (Shp, Hnf1a, Hnf1b, Irs1, Irs2, Kir6.2, and Pdx1) in 10 organs. The glucose tolerance test was conducted to determine the blood glucose concentrations just after fasting for 24 h (FBG) and at 2 h after high-glucose application (GTT2h). The FBG-GTT2h plots obtained with the wild strain fed the control diet (CD), Gk+/− strain fed the CD, and Gk+/− strain fed the HFD were distributed in separate areas in the FBG-GTT2h diagram. The respective areas could be defined as the normal state, prediabetes state, and diabetes state, respectively. Based on the results, the criteria for prediabetes could be defined for the Gk+/− strain developed in this study. PMID:25765873

  9. Neutral Lipids and Peroxisome Proliferator-Activated Receptor-γ Control Pulmonary Gene Expression and Inflammation-Triggered Pathogenesis in Lysosomal Acid Lipase Knockout Mice

    PubMed Central

    Lian, Xuemei; Yan, Cong; Qin, Yulin; Knox, Lana; Li, Tingyu; Du, Hong

    2005-01-01

    The functional roles of neutral lipids in the lung are poorly understood. However, blocking cholesteryl ester and triglyceride metabolism in lysosomal acid lipase gene knockout mice (lal−/−) results in severe pathogenic phenotypes in the lung, including massive neutrophil infiltration, foamy macrophage accumulation, unwanted cell growth, and emphysema. To elucidate the mechanism underlining these pathologies, we performed Affymetrix GeneChip microarray analysis of 1-, 3-, and 6-month-old mice and identified aberrant gene expression that progressed with age. Among changed genes, matrix metalloproteinase (MMP)-12, apoptosis inhibitor 6 (Api-6), erythroblast transformation-specific domain (Ets) transcription factor family member Spi-C, and oncogene MafB were increased 100-, 70-, 40-, and 10-fold, respectively, in lal−/− lungs versus the wild-type lungs. The pathogenic increases of these molecules occurred primarily in alveolar type II epithelial cells. Transcriptional activities of the MMP-12 and Api-6 promoters were stimulated by Spi-C or MafB in respiratory epithelial cells. Treatment with 9-hydroxyoctadecanoic acids and ciglitazone significantly rescued lal−/− pulmonary inflammation and aberrant gene expression. In addition, both compounds as well as peroxisome proliferator-activated receptor gamma inhibited MMP-12 and Api-6 promoter activities. These data suggest that inflammation-triggered cell growth and emphysema during lysosomal acid lipase deficiency are partially caused by peroxisome proliferator-activated receptor-γ inactivation. PMID:16127159

  10. The sub-optimal phenotypes of double-knockout mutants of Escherichia coli depend on the order of gene deletions.†

    PubMed Central

    Gawand, Pratish; Abukar, Fatumina Said; Venayak, Naveen; Partow, Siavash; Motter, Adilson E.; Mahadevan, Radhakrishnan

    2016-01-01

    Metabolic networks are characterized by multiple redundant reactions that do not have a clear biological function. The redundancies in the metabolic networks are implicated in adaptation to random mutations and survival under different environmental conditions. Reactions that are not active under wild-type growth conditions, but get transiently activated after a mutation event such as gene deletion are known as latent reactions. Characterization of multiple-gene knockout mutants can identify the physiological roles of latent reactions. In this study, we characterized double-gene deletion mutants of E. coli with an aim to investigate the sub-optimal physiology of the mutants and the plausible roles of latent reactions. Specifically, we investigated the effects of deletion of the glyoxylate-shunt gene aceA (encoding a latent reaction enzyme, isocitrate lyase) on the growth characteristics of the mutant E. coli Δpgi. The deletion of aceA reduced the growth rate of E. coli Δpgi, indicating that the activation of the glyoxylate shunt plays an important role in adaptation of the mutant E. coli Δpgi. We also investigated the effect of the order of the gene deletions on the growth rates and substrate uptake rates of the double-gene deletion mutants. The results indicate that the order in which genes are deleted determines the phenotype of the mutants during the sub-optimal growth phase. To elucidate the mechanism behind the difference between the observed phenotypes, we carried out transcriptomic analysis and constraint-based modeling of the mutants. Transcriptomic analysis showed differential expression of the gene aceK (encoding the protein isocitrate dehydrogenase kinase) involved in controlling the isocitrate flux through the TCA cycle and the glyoxylate shunt. Higher acetate production in the E. coli ΔaceA1 Δpgi2 mutant was consistent with the increased aceK expression, which limits the TCA cycle flux and causes acetate production via overflow metabolism. PMID

  11. Construction of a Gene Knockout System for Application in Paenibacillus alvei CCM 2051T, Exemplified by the S-Layer Glycan Biosynthesis Initiation Enzyme WsfP▿

    PubMed Central

    Zarschler, Kristof; Janesch, Bettina; Zayni, Sonja; Schäffer, Christina; Messner, Paul

    2009-01-01

    The gram-positive bacterium Paenibacillus alvei CCM 2051T is covered by an oblique surface layer (S-layer) composed of glycoprotein subunits. The S-layer O-glycan is a polymer of [→3)-β-d-Galp-(1[α-d-Glcp-(1→6)]→4)-β-d-ManpNAc-(1→] repeating units that is linked by an adaptor of -[GroA-2→OPO2→4-β-d-ManpNAc-(1→4)]→3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-β-d-Galp-(1→ to specific tyrosine residues of the S-layer protein. For elucidation of the mechanism governing S-layer glycan biosynthesis, a gene knockout system using bacterial mobile group II intron-mediated gene disruption was developed. The system is further based on the sgsE S-layer gene promoter of Geobacillus stearothermophilus NRS 2004/3a and on the Geobacillus-Bacillus-Escherichia coli shuttle vector pNW33N. As a target gene, wsfP, encoding a putative UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase, representing the predicted initiation enzyme of S-layer glycan biosynthesis, was disrupted. S-layer protein glycosylation was completely abolished in the insertional P. alvei CCM 2051T wsfP mutant, according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis evidence and carbohydrate analysis. Glycosylation was fully restored by plasmid-based expression of wsfP in the glycan-deficient P. alvei mutant, confirming that WsfP initiates S-layer protein glycosylation. This is the first report on the successful genetic manipulation of bacterial S-layer protein glycosylation in vivo, including transformation of and heterologous gene expression and gene disruption in the model organism P. alvei CCM 2051T. PMID:19304819

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

    PubMed

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

    2015-07-01

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

  13. Facilitation of Direct Conditional Knockout of Essential Genes in Bacillus licheniformis DSM13 by Comparative Genetic Analysis and Manipulation of Genetic Competence▿ †

    PubMed Central

    Hoffmann, Kerstin; Wollherr, Antje; Larsen, Michael; Rachinger, Michael; Liesegang, Heiko; Ehrenreich, Armin; Meinhardt, Friedhelm

    2010-01-01

    The genetic manageability of the biotechnologically important Bacillus licheniformis is hampered due to its poor transformability, whereas Bacillus subtilis efficiently takes up DNA during genetic competence, a quorum-sensing-dependent process. Since the sensor histidine kinase ComP, encoded by a gene of the quorum-sensing module comQXPA of B. licheniformis DSM13, was found to be inactive due to an insertion element within comP, the coding region was exchanged with a functional copy. Quorum sensing was restored, but the already-poor genetic competence dropped further. The inducible expression of the key regulator for the transcription of competence genes, ComK, in trans resulted in highly competent strains and facilitated the direct disruption of genes, as well as the conditional knockout of an essential operon. As ComK is inhibited at low cell densities by a proteolytic complex in which MecA binds ComK and such inhibition is antagonized by the interaction of MecA with ComS (the expression of the latter is controlled by cell density in B. subtilis), we performed an in silico analysis of MecA and the hitherto unidentified ComS, which revealed differences for competent and noncompetent strains, indicating that the reduced competence possibly is due to a nonfunctional coupling of the comQXPA-encoded quorum module and ComK. The obtained increased genetic tractability of this industrial workhorse should improve a wide array of scientific investigations. PMID:20543043

  14. Model-assisted formate dehydrogenase-O (fdoH) gene knockout for enhanced succinate production in Escherichia coli from glucose and glycerol carbon sources.

    PubMed

    Mienda, Bashir Sajo; Shamsir, Mohd Shahir; Md Illias, Rosli

    2016-11-01

    Succinic acid is an important platform chemical that has broad applications and is been listed as one of the top twelve bio-based chemicals produced from biomass by the US Department of Energy. The metabolic role of Escherichia coli formate dehydrogenase-O (fdoH) under anaerobic conditions in relation to succinic acid production remained largely unspecified. Herein we report, what are to our knowledge, the first metabolic fdoH gene knockout that have enhanced succinate production using glucose and glycerol substrates in E. coli. Using the most recent E. coli reconstruction iJO1366, we engineered its host metabolism to enhance the anaerobic succinate production by deleting the fdoH gene, which blocked H(+) conduction across the mutant cell membrane for the enhanced succinate production. The engineered mutant strain BMS4 showed succinate production of 2.05 g l(-1) (41.2-fold in 7 days) from glycerol and .39 g l(-1) (6.2-fold in 1 day) from glucose. This work revealed that a single deletion of the fdoH gene is sufficient to increase succinate production in E. coli from both glucose and glycerol substrates.

  15. Cas-Database: web-based genome-wide guide RNA library design for gene knockout screens using CRISPR-Cas9

    PubMed Central

    Park, Jeongbin; Kim, Jin-Soo; Bae, Sangsu

    2016-01-01

    Motivation: CRISPR-derived RNA guided endonucleases (RGENs) have been widely used for both gene knockout and knock-in at the level of single or multiple genes. RGENs are now available for forward genetic screens at genome scale, but single guide RNA (sgRNA) selection at this scale is difficult. Results: We develop an online tool, Cas-Database, a genome-wide gRNA library design tool for Cas9 nucleases from Streptococcus pyogenes (SpCas9). With an easy-to-use web interface, Cas-Database allows users to select optimal target sequences simply by changing the filtering conditions. Furthermore, it provides a powerful way to select multiple optimal target sequences from thousands of genes at once for the creation of a genome-wide library. Cas-Database also provides a web application programming interface (web API) for advanced bioinformatics users. Availability and implementation: Free access at http://www.rgenome.net/cas-database/. Contact: sangsubae@hanyang.ac.kr or jskim01@snu.ac.kr Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27153724

  16. The FKBP5 Gene Affects Alcohol Drinking in Knockout Mice and Is Implicated in Alcohol Drinking in Humans.

    PubMed

    Qiu, Bin; Luczak, Susan E; Wall, Tamara L; Kirchhoff, Aaron M; Xu, Yuxue; Eng, Mimy Y; Stewart, Robert B; Shou, Weinian; Boehm, Stephen L; Chester, Julia A; Yong, Weidong; Liang, Tiebing

    2016-08-05

    FKBP5 encodes FK506-binding protein 5, a glucocorticoid receptor (GR)-binding protein implicated in various psychiatric disorders and alcohol withdrawal severity. The purpose of this study is to characterize alcohol preference and related phenotypes in Fkbp5 knockout (KO) mice and to examine the role of FKBP5 in human alcohol consumption. The following experiments were performed to characterize Fkpb5 KO mice. (1) Fkbp5 KO and wild-type (WT) EtOH consumption was tested using a two-bottle choice paradigm; (2) The EtOH elimination rate was measured after intraperitoneal (IP) injection of 2.0 g/kg EtOH; (3) Blood alcohol concentration (BAC) was measured after 3 h limited access of alcohol; (4) Brain region expression of Fkbp5 was identified using LacZ staining; (5) Baseline corticosterone (CORT) was assessed. Additionally, two SNPs, rs1360780 (C/T) and rs3800373 (T/G), were selected to study the association of FKBP5 with alcohol consumption in humans. Participants were college students (n = 1162) from 21-26 years of age with Chinese, Korean or Caucasian ethnicity. The results, compared to WT mice, for KO mice exhibited an increase in alcohol consumption that was not due to differences in taste sensitivity or alcohol metabolism. Higher BAC was found in KO mice after 3 h of EtOH access. Fkbp5 was highly expressed in brain regions involved in the regulation of the stress response, such as the hippocampus, amygdala, dorsal raphe and locus coeruleus. Both genotypes exhibited similar basal levels of plasma corticosterone (CORT). Finally, single nucleotide polymorphisms (SNPs) in FKBP5 were found to be associated with alcohol drinking in humans. These results suggest that the association between FKBP5 and alcohol consumption is conserved in both mice and humans.

  17. The FKBP5 Gene Affects Alcohol Drinking in Knockout Mice and Is Implicated in Alcohol Drinking in Humans

    PubMed Central

    Qiu, Bin; Luczak, Susan E.; Wall, Tamara L.; Kirchhoff, Aaron M.; Xu, Yuxue; Eng, Mimy Y.; Stewart, Robert B.; Shou, Weinian; Boehm, Stephen L.; Chester, Julia A.; Yong, Weidong; Liang, Tiebing

    2016-01-01

    FKBP5 encodes FK506-binding protein 5, a glucocorticoid receptor (GR)-binding protein implicated in various psychiatric disorders and alcohol withdrawal severity. The purpose of this study is to characterize alcohol preference and related phenotypes in Fkbp5 knockout (KO) mice and to examine the role of FKBP5 in human alcohol consumption. The following experiments were performed to characterize Fkpb5 KO mice. (1) Fkbp5 KO and wild-type (WT) EtOH consumption was tested using a two-bottle choice paradigm; (2) The EtOH elimination rate was measured after intraperitoneal (IP) injection of 2.0 g/kg EtOH; (3) Blood alcohol concentration (BAC) was measured after 3 h limited access of alcohol; (4) Brain region expression of Fkbp5 was identified using LacZ staining; (5) Baseline corticosterone (CORT) was assessed. Additionally, two SNPs, rs1360780 (C/T) and rs3800373 (T/G), were selected to study the association of FKBP5 with alcohol consumption in humans. Participants were college students (n = 1162) from 21–26 years of age with Chinese, Korean or Caucasian ethnicity. The results, compared to WT mice, for KO mice exhibited an increase in alcohol consumption that was not due to differences in taste sensitivity or alcohol metabolism. Higher BAC was found in KO mice after 3 h of EtOH access. Fkbp5 was highly expressed in brain regions involved in the regulation of the stress response, such as the hippocampus, amygdala, dorsal raphe and locus coeruleus. Both genotypes exhibited similar basal levels of plasma corticosterone (CORT). Finally, single nucleotide polymorphisms (SNPs) in FKBP5 were found to be associated with alcohol drinking in humans. These results suggest that the association between FKBP5 and alcohol consumption is conserved in both mice and humans. PMID:27527158

  18. Application of the Saccharomyces cerevisiae FLP/FRT recombination system in filamentous fungi for marker recycling and construction of knockout strains devoid of heterologous genes.

    PubMed

    Kopke, Katarina; Hoff, Birgit; Kück, Ulrich

    2010-07-01

    To overcome the limited availability of antibiotic resistance markers in filamentous fungi, we adapted the FLP/FRT recombination system from the yeast Saccharomyces cerevisiae for marker recycling. We tested this system in the penicillin producer Penicillium chrysogenum using different experimental approaches. In a two-step application, we first integrated ectopically a nourseothricin resistance cassette flanked by the FRT sequences in direct repeat orientation (FRT-nat1 cassette) into a P. chrysogenum recipient. In the second step, the gene for the native yeast FLP recombinase, and in parallel, a codon-optimized P. chrysogenum flp (Pcflp) recombinase gene, were transferred into the P. chrysogenum strain carrying the FRT-nat1 cassette. The corresponding transformants were analyzed by PCR, growth tests, and sequencing to verify successful recombination events. Our analysis of several single- and multicopy transformants showed that only when the codon-optimized recombinase was present could a fully functional recombination system be generated in P. chrysogenum. As a proof of application of this system, we constructed a DeltaPcku70 knockout strain devoid of any heterologous genes. To further improve the FLP/FRT system, we produced a flipper cassette carrying the FRT sites as well as the Pcflp gene together with a resistance marker. This cassette allows the controlled expression of the recombinase gene for one-step marker excision. Moreover, the applicability of the optimized FLP/FRT recombination system in other fungi was further demonstrated by marker recycling in the ascomycete Sordaria macrospora. Here, we discuss the application of the optimized FLP/FRT recombination system as a molecular tool for the genetic manipulation of filamentous fungi.

  19. Generation of Interleukin-2 Receptor Gamma Gene Knockout Pigs from Somatic Cells Genetically Modified by Zinc Finger Nuclease-Encoding mRNA

    PubMed Central

    Watanabe, Masahito; Nakano, Kazuaki; Matsunari, Hitomi; Matsuda, Taisuke; Maehara, Miki; Kanai, Takahiro; Kobayashi, Mirina; Matsumura, Yukina; Sakai, Rieko; Kuramoto, Momoko; Hayashida, Gota; Asano, Yoshinori; Takayanagi, Shuko; Arai, Yoshikazu; Umeyama, Kazuhiro; Nagaya, Masaki; Hanazono, Yutaka; Nagashima, Hiroshi

    2013-01-01

    Zinc finger nuclease (ZFN) is a powerful tool for genome editing. ZFN-encoding plasmid DNA expression systems have been recently employed for the generation of gene knockout (KO) pigs, although one major limitation of this technology is the use of potentially harmful genome-integrating plasmid DNAs. Here we describe a simple, non-integrating strategy for generating KO pigs using ZFN-encoding mRNA. The interleukin-2 receptor gamma (IL2RG) gene was knocked out in porcine fetal fibroblasts using ZFN-encoding mRNAs, and IL2RG KO pigs were subsequently generated using these KO cells through somatic cell nuclear transfer (SCNT). The resulting IL2RG KO pigs completely lacked a thymus and were deficient in T and NK cells, similar to human X-linked SCID patients. Our findings demonstrate that the combination of ZFN-encoding mRNAs and SCNT provides a simple robust method for producing KO pigs without genomic integration. PMID:24130776

  20. AAV-mediated RLBP1 gene therapy improves the rate of dark adaptation in Rlbp1 knockout mice

    PubMed Central

    Choi, Vivian W; Bigelow, Chad E; McGee, Terri L; Gujar, Akshata N; Li, Hui; Hanks, Shawn M; Vrouvlianis, Joanna; Maker, Michael; Leehy, Barrett; Zhang, Yiqin; Aranda, Jorge; Bounoutas, George; Demirs, John T; Yang, Junzheng; Ornberg, Richard; Wang, Yu; Martin, Wendy; Stout, Kelly R; Argentieri, Gregory; Grosenstein, Paul; Diaz, Danielle; Turner, Oliver; Jaffee, Bruce D; Police, Seshidhar R; Dryja, Thaddeus P

    2015-01-01

    Recessive mutations in RLBP1 cause a form of retinitis pigmentosa in which the retina, before its degeneration leads to blindness, abnormally slowly recovers sensitivity after exposure to light. To develop a potential gene therapy for this condition, we tested multiple recombinant adeno-associated vectors (rAAVs) composed of different promoters, capsid serotypes, and genome conformations. We generated rAAVs in which sequences from the promoters of the human RLBP1, RPE65, or BEST1 genes drove the expression of a reporter gene (green fluorescent protein). A promoter derived from the RLBP1 gene mediated expression in the retinal pigment epithelium and Müller cells (the intended target cell types) at qualitatively higher levels than in other retinal cell types in wild-type mice and monkeys. With this promoter upstream of the coding sequence of the human RLBP1 gene, we compared the potencies of vectors with an AAV2 versus an AAV8 capsid in transducing mouse retinas, and we compared vectors with a self-complementary versus a single-stranded genome. The optimal vector (scAAV8-pRLBP1-hRLBP1) had serotype 8 capsid and a self-complementary genome. Subretinal injection of scAAV8-pRLBP1-hRLBP1 in Rlbp1 nullizygous mice improved the rate of dark adaptation based on scotopic (rod-plus-cone) and photopic (cone) electroretinograms (ERGs). The effect was still present after 1 year. PMID:26199951

  1. Analysis of differential gene expression profiles in Caenorhabditis elegans knockouts for the v-SNARE master protein 1.

    PubMed

    Rodriguez, Ashley; McKay, Kody; Graham, Melanie; Dittrich, Josiah; Holgado, Andrea M

    2014-06-01

    At chemical synapses, neurons communicate information to other cells by secreting neurotransmitters or neuropeptides into the synaptic cleft, which then bind to receptors on the target cell. Preliminary work performed in our laboratory has shown that mutant nematodes lacking a protein called VSM-1 have increased synaptic density compared with the wild type. Consequently, we hypothesized that genes expressed in vsm-1 mutants mediate enhanced synaptogenesis. To identify these genes of interest, we utilized microarray technology and quantitative PCR. To this end, first we isolated the total RNA from young-adult wild-type and vsm-1 mutant Caenorhabditis elegans. Next, we synthesized cDNA from reverse transcription of the isolated RNA. Hybridization of the cDNA to a microarray was performed to facilitate gene expression profiling. Finally, fluorescently labeled microarrays were analyzed, and the identities of induced and repressed genes were uncovered in the open-source software Magic Tool. Analyses of microarray experiments performed using three independent biological samples per strain and three technical replicas and dye swaps showed induction of genes coding for major sperm proteins and repression of SPP-2 in vsm-1 mutants. Microarray results were also validated and quantified by using quantitative PCR.

  2. Effects of glucose-dependent insulinotropic polypeptide receptor knockout and a high-fat diet on cognitive function and hippocampal gene expression in mice.

    PubMed

    Lennox, Rachel; Lennox, Rachael R; Moffett, R Charlotte; Moffett, Charlotte; Porter, David W; Irwin, Nigel; Gault, Victor A; Flatt, Peter R

    2015-07-01

    It has been previously demonstrated that compromise of glucose-dependent insulinotropic polypeptide receptor (GIPR) action and chronic consumption of a high-fat diet can independently impair memory and learning ability, however, the underlying pathology remain to be elucidated. The present study investigated the effects of GIPR knockout (KO), alone and in combination with a high-fat diet, on aspects of cognitive function and hippocampal gene expression in mice. In object recognition tests, normal mice exhibited effective memory, preferring to investigate the novel over the familiar object. However, wild-type (WT) mice fed a high-fat diet and GIPR KO mice fed a standard or high-fat diet demonstrated no such discrimination, suggesting the impairment of memory function. This decline in cognitive function was associated with marked changes in the expression levels of hippocampal genes involved in memory and learning. The chronic consumption of a high-fat diet decreased the hippocampal gene expression levels of mammalian target of rapamycin (mTOR), neurotrophic tyrosine kinase receptor type 2 (NTRK2) and synaptophysin. Notably, the GIPR KO mice fed a high-fat diet exhibited no reduction in the hippocampal expression of synaptophysin expression, however, the GIPR KO mice fed a standard rodent maintenance diet exhibited reduced hippocampal expression of mTOR compared with the WT controls. These data highlighted the importance of intact GIPR signalling and dietary composition in modulating memory and learning, and hippocampal pathways involved in the maintenance of synaptic plasticity, including mTOR and NTRK2, appear to be key in this regard.

  3. Creation of fragrant rice by targeted knockout of the OsBADH2 gene using TALEN technology.

    PubMed

    Shan, Qiwei; Zhang, Yi; Chen, Kunling; Zhang, Kang; Gao, Caixia

    2015-08-01

    Fragrant rice is favoured worldwide because of its agreeable scent. The presence of a defective badh2 allele encoding betaine aldehyde dehydrogenase (BADH2) results in the synthesis of 2-acetyl-1-pyrroline (2AP), which is a major fragrance compound. Here, transcription activator-like effector nucleases (TALENs) were engineered to target and disrupt the OsBADH2 gene. Six heterozygous mutants (30%) were recovered from 20 transgenic hygromycin-resistant lines. Sanger sequencing confirmed that these lines had various indel mutations at the TALEN target site. All six transmitted the BADH2 mutations to the T1 generation; and four T1 mutant lines tested also efficiently transmitted the mutations to the T2 generation. Mutant plants carrying only the desired DNA sequence change but not the TALEN transgene were obtained by segregation in the T1 and T2 generations. The 2AP content of rice grains of the T1 lines with homozygous mutations increased from 0 to 0.35-0.75 mg/kg, which was similar to the content of a positive control variety harbouring the badh2-E7 mutation. We also simultaneously introduced three different pairs of TALENs targeting three separate rice genes into rice cells by bombardment and obtained lines with mutations in one, two and all three genes. These results indicate that targeted mutagenesis using TALENs is a useful approach to creating important agronomic traits.

  4. Expression, purification and characterization of zinc-finger nuclease to knockout the goat beta-lactoglobulin gene.

    PubMed

    Song, Yujie; Cui, Chenchen; Zhu, Hongmei; Li, Qian; Zhao, Fan; Jin, Yaping

    2015-08-01

    Engineered zinc-finger nucleases (ZFNs) have been widely used for precise genome editing. ZFNs can induce DNA double-strand breaks at specific genomic locations and drive the introduction of an insertion or deletion of base pairs at the targeted region, consequently resulting in a loss-of-function mutation. In this study, we investigated the cloning, expression and purification of ZFN fusion proteins targeting the goat beta-lactoglobulin (BLG) gene and detected the cleavage activities of these ZFN proteins in vitro and in cells, respectively. The results showed that the pET-BLG-LFN and pET-BLG-RFN prokaryotic expression plasmids can be constructed correctly and expressed efficiently in Escherichia coli BL21 (DE3) cells to produce the 6× His-tagged ZFN proteins that can be purified by Ni-IDA-Sefinose Column. The predetermined sequence of BLG can be recognized and excised both in vitro and in goat fibroblasts by the purified ZFN fusion proteins, which demonstrated that the purified ZFN fusion proteins can be used as gene modification tools to knock out the BLG gene. Furthermore, these results lay the foundation for eliminating allergen BLG from goat milk and improving the quality of goat milk products.

  5. The mu-opioid receptor gene-dose dependent reductions in G-protein activation in the pons/medulla and antinociception induced by endomorphins in mu-opioid receptor knockout mice.

    PubMed

    Mizoguchi, H; Narita, M; Oji, D E; Suganuma, C; Nagase, H; Sora, I; Uhl, G R; Cheng, E Y; Tseng, L F

    1999-01-01

    There appear to be different relationships between mu-opioid receptor densities and the acute and neuroadaptive mu-opioid agonist-induced responses of the multiple opioid neuronal systems, including important pons/medulla circuits. The recent success in creating mu-opioid receptor knockout mice allows studies of mu-opioid agonist-induced pharmacological and physiological effects in animals that express no, one or two copies of the mu-opioid receptor gene. We now report that the binding of mu-opioid receptor ligand, [3H][D-Ala2,NHPhe4,Gly-ol]enkephalin to membrane preparations of the pons/medulla was reduced by half in heterozygous mu-opioid receptor knockout mice and eliminated in homozygous mu-opioid receptor knockout mice. The endogenous mu-opioid agonist peptides endomorphin-1 and -2 activate G-proteins in the pons/medulla from wild-type mice in a concentration-dependent fashion, as assessed using [35S]guanosine-5'-o-(3-thio)triphosphate binding. This stimulation was reduced to half of the wild-type levels in heterozygous mice and eliminated in homozygous knockout mice. The intracerebroventricular injection of either endomorphin-1 or endomorphin-2 produced marked antinociception in the hot-plate and tail-flick tests in wild-type mice. These antinociceptive actions were significantly reduced in heterozygous mu-opioid receptor knockout mice, and virtually abolished in homozygous knockout mice. The mu-opioid receptors are the principal molecular targets for endomorphin-induced G-protein activation in the pons/medulla and the antinociception caused by the intracerebroventricular administration of mu-opioid agonists. These data support the notion that there are limited physiological mu-opioid receptor reserves for inducing G-protein activation in the pons/medulla and for the nociceptive modulation induced by the central administration of endomorphin-1 and -2.

  6. AAV8(Y733F)-mediated gene therapy in a Spata7 knockout mouse model of Leber congenital amaurosis and retinitis pigmentosa

    PubMed Central

    Zhong, Hua; Eblimit, Aiden; Moayedi, Yalda; Boye, Sanford L; Chiodo, Vince A; Chen, Yiyun; Li, Yumei; Nichols, Ralph M; Hauswirth, William W; Chen, Rui; Mardon, Graeme

    2016-01-01

    Loss of SPATA7 function causes the pathogenesis of Leber congenital amaurosis and retinitis pigmentosa. Spata7 knockout mice mimic human SPATA7–related retinal disease with apparent photoreceptor degeneration observed as early as postnatal day 15 (P15). To test the efficacy of adeno-associated virus (AAV)-mediated gene therapy for rescue of photoreceptor survival and function in Spata7 mutant mice, we employed the AAV8(Y733F) vector carrying hGRK1-driven full-length FLAG-tagged Spata7 cDNA to target both rod and cone photoreceptors. Following subretinal injection of this vector, FLAG-tagged SPATA7 was found to co-localize with endogenous SPATA7 in wild-type mice. In Spata7 mutant mice initially treated at P15, we observed improvement of photoresponse, photoreceptor ultrastructure, and significant alleviation of photoreceptor degeneration. Furthermore we performed treatments at P28 and P56 and found that all treatments (P15-P56) can ameliorate rod and cone loss in the long term (1 year); however, none efficiently protect photoreceptors from degeneration by 86 weeks of age since only a small amount of treated photoreceptors can survive to this time. This study demonstrates long-term improvement of photoreceptor function by AAV8(Y733F)-introduced Spata7 expression in a mouse model as potential treatment of the human disease but also suggests that treated mutant photoreceptors still undergo progressive degeneration. PMID:25965394

  7. AAV8(Y733F)-mediated gene therapy in a Spata7 knockout mouse model of Leber congenital amaurosis and retinitis pigmentosa.

    PubMed

    Zhong, H; Eblimit, A; Moayedi, Y; Boye, S L; Chiodo, V A; Chen, Y; Li, Y; Nichols, R M; Hauswirth, W W; Chen, R; Mardon, G

    2015-08-01

    Loss of SPATA7 function causes the pathogenesis of Leber congenital amaurosis and retinitis pigmentosa. Spata7 knockout mice mimic human SPATA7-related retinal disease with apparent photoreceptor degeneration observed as early as postnatal day 15 (P15). To test the efficacy of adeno-associated virus (AAV)-mediated gene therapy for rescue of photoreceptor survival and function in Spata7 mutant mice, we employed the AAV8(Y733F) vector carrying hGRK1-driven full-length FLAG-tagged Spata7 cDNA to target both rod and cone photoreceptors. Following subretinal injection of this vector, FLAG-tagged SPATA7 was found to colocalize with endogenous SPATA7 in wild-type mice. In Spata7 mutant mice initially treated at P15, we observed improvement of photoresponse, photoreceptor ultrastructure and significant alleviation of photoreceptor degeneration. Furthermore, we performed treatments at P28 and P56 and found that all treatments (P15-P56) can ameliorate rod and cone loss in the long term (1 year); however, none efficiently protect photoreceptors from degeneration by 86 weeks of age as only a small amount of treated photoreceptors can survive to this time. This study demonstrates long-term improvement of photoreceptor function by AAV8(Y733F)-introduced Spata7 expression in a mouse model as potential treatment of the human disease, but also suggests that treated mutant photoreceptors still undergo progressive degeneration.

  8. Comparative N-linked glycan analysis of wild-type and α1,3-galactosyltransferase gene knock-out pig fibroblasts using mass spectrometry approaches.

    PubMed

    Park, Hae-Min; Kim, Yoon-Woo; Kim, Kyoung-Jin; Kim, Young June; Yang, Yung-Hun; Jin, Jang Mi; Kim, Young Hwan; Kim, Byung-Gee; Shim, Hosup; Kim, Yun-Gon

    2015-01-31

    Carbohydrate antigens expressed on pig cells are considered to be major barriers in pig-to-human xenotransplantation. Even after α1,3-galactosyltransferase gene knock-out (GalT-KO) pigs are generated, potential non-Gal antigens are still existed. However, to the best of our knowledge there is no extensive study analyzing N-glycans expressed on the GalT-KO pig tissues or cells. Here, we identified and quantified totally 47 N-glycans from wild-type (WT) and GalT-KO pig fibroblasts using mass spectrometry. First, our results confirmed the absence of galactose-alpha-1,3-galactose (α-Gal) residue in the GalT-KO pig cells. Interestingly, we showed that the level of overall fucosylated N-glycans from GalT-KO pig fibroblasts is much higher than from WT pig fibroblasts. Moreover, the relative quantity of the N-glycolylneuraminic acid (NeuGc) antigen is slightly higher in the GalT-KO pigs. Thus, this study will contribute to a better understanding of cellular glycan alterations on GalT-KO pigs for successful xenotransplantation.

  9. Bacillus subtilis Mutants with Knockouts of the Genes Encoding Ribonucleases RNase Y and RNase J1 Are Viable, with Major Defects in Cell Morphology, Sporulation, and Competence

    PubMed Central

    Figaro, Sabine; Durand, Sylvain; Gilet, Laetitia; Cayet, Nadège; Sachse, Martin

    2013-01-01

    The genes encoding the ribonucleases RNase J1 and RNase Y have long been considered essential for Bacillus subtilis cell viability, even before there was concrete knowledge of their function as two of the most important enzymes for RNA turnover in this organism. Here we show that this characterization is incorrect and that ΔrnjA and Δrny mutants are both viable. As expected, both strains grow relatively slowly, with doubling times in the hour range in rich medium. Knockout mutants have major defects in their sporulation and competence development programs. Both mutants are hypersensitive to a wide range of antibiotics and have dramatic alterations to their cell morphologies, suggestive of cell envelope defects. Indeed, RNase Y mutants are significantly smaller in diameter than wild-type strains and have a very disordered peptidoglycan layer. Strains lacking RNase J1 form long filaments in tight spirals, reminiscent of mutants of the actin-like proteins (Mre) involved in cell shape determination. Finally, we combined the rnjA and rny mutations with mutations in other components of the degradation machinery and show that many of these strains are also viable. The implications for the two known RNA degradation pathways of B. subtilis are discussed. PMID:23504012

  10. Disruption to social dyadic interactions but not emotional/anxiety-related behaviour in mice with heterozygous 'knockout' of the schizophrenia risk gene neuregulin-1.

    PubMed

    O'Tuathaigh, Colm M P; O'Connor, Anne-Marie; O'Sullivan, Gerard J; Lai, Donna; Harvey, Richard; Croke, David T; Waddington, John L

    2008-02-15

    Clinical genetic studies have implicated neuregulin-1 [NRG1] as a leading susceptibility gene for schizophrenia. NRG1 is known to play a significant role in the developing brain, which is consistent with the prevailing neurodevelopmental model of schizophrenia. Thus, the emotional and social phenotype of adult mice with heterozygous 'knockout' of transmembrane [TM]-domain NRG1 was examined further in both sexes. Emotional/anxiety-related behaviour was assessed using the elevated plus-maze and the light-dark test. Social behaviour was examined in terms of dyadic interactions between NRG1 mutants and an unfamiliar C57BL6 conspecific in a novel environment. There was no effect of NRG1 genotype on performance in either test of emotionality/anxiety. However, previous reports of hyperactivity in NRG1 mutants were confirmed in both paradigms. In the test of social interaction, aggressive following was increased in NRG1 mutants of both sexes, together with an increase in walkovers in female mutants. These findings elaborate the specificity of the NRG1 phenotype for the social rather than the emotional/anxiety-related domain. They indicate that NRG1 is involved in the regulation of reciprocal social interaction behaviour and thus suggest a putative role for NRG1 in a schizophrenia-related endophenotype.

  11. Knock-out of Arabidopsis metal transporter gene IRT1 results in iron deficiency accompanied by cell differentiation defects.

    PubMed

    Henriques, Rossana; Jásik, Ján; Klein, Markus; Martinoia, Enrico; Feller, Urs; Schell, Jeff; Pais, Maria S; Koncz, Csaba

    2002-11-01

    IRT1 and IRT2 are members of the Arabidopsis ZIP metal transporter family that are specifically induced by iron deprivation in roots and act as heterologous suppressors of yeast mutations inhibiting iron and zinc uptake. Although IRT1 and IRT2 are thought to perform redundant functions as root-specific metal transporters, insertional inactivation of the IRT1 gene alone results in typical symptoms of iron deficiency causing severe leaf chlorosis and lethality in soil. The irt1 mutation is characterized by specific developmental defects, including a drastic reduction of chloroplast thylakoid stacking into grana and lack of palisade parenchyma differentiation in leaves, reduced number of vascular bundles in stems, and irregular patterns of enlarged endodermal and cortex cells in roots. Pulse labeling with 59Fe through the root system shows that the irt1 mutation reduces iron accumulation in the shoots. Short-term labeling with 65Zn reveals no alteration in spatial distribution of zinc, but indicates a lower level of zinc accumulation. In comparison to wild-type, the irt1 mutant responds to iron and zinc deprivation by altered expression of certain zinc and iron transporter genes, which results in the activation of ZIP1 in shoots, reduction of ZIP2 transcript levels in roots, and enhanced expression of IRT2 in roots. These data support the conclusion that IRT1 is an essential metal transporter required for proper development and regulation of iron and zinc homeostasis in Arabidopsis.

  12. The low density lipoprotein receptor-related protein 1: Unique tissue-specific functions revealed by selective gene knockout studies

    PubMed Central

    Lillis, Anna P.; Van Duyn, Lauren B.; Murphy-Ullrich, Joanne E.; Strickland, Dudley K.

    2008-01-01

    The low-density lipoprotein (LDL) receptor-related protein (originally called LRP, but now referred to as LRP1) is a large endocytic receptor that is widely expressed in several tissues. LRP1 is a member of the LDL receptor family that plays diverse roles in various biological processes including lipoprotein metabolism, degradation of proteases, activation of lysosomal enzymes and cellular entry of bacterial toxins and viruses. Deletion of the LRP1 gene leads to lethality in mice, revealing a critical, but as of yet, undefined role in development. Tissue-specific gene deletion studies reveal an important contribution of LRP1 in the vasculature, central nervous system, in macrophages and in adipocytes. Three important properties of LRP1 dictate its diverse role in physiology: first, its ability to recognize more than thirty distinct ligands; second, its ability to bind a large number of cytoplasmic adaptor proteins via determinants located on its cytoplasmic domain in a phosphorylation-specific manner; and third, its ability to associate with and modulate the activity of other transmembrane receptors such as integrins and receptor tyrosine kinases. PMID:18626063

  13. Metabolic flux screening of Saccharomyces cerevisiae single knockout strains on glucose and galactose supports elucidation of gene function.

    PubMed

    Velagapudi, Vidya R; Wittmann, Christoph; Schneider, Konstantin; Heinzle, Elmar

    2007-12-01

    New methods for an extended physiological characterization of yeast at a microtiter plate scale were applied to 27 deletion mutants of Saccharomyces cerevisiae cultivated on glucose and galactose as sole carbon sources. In this way, specific growth rates, specific rates of glucose consumption and ethanol production were determined. Flux distribution, particularly concerning branching into the pentose phosphate pathway was determined using a new (13)C-labelling method using MALDI-ToF-mass spectrometry. On glucose, the growth was predominantly fermentative whereas on galactose respiration was more active with correspondingly lower ethanol production. Some deletion strains showed unexpected behavior providing very informative data about the function of the corresponding gene. Deletion of malic enzyme gene, MAE1, did not show any significant phenotype when grown on glucose but a drastically increased branching from glucose 6-phosphate into the pentose phosphate pathway when grown on galactose. This allows the conclusion that MAE1 is important for the supply of NADPH during aerobic growth on galactose.

  14. Deletion of the UT receptor gene results in the selective loss of urotensin-II contractile activity in aortae isolated from UT receptor knockout mice

    PubMed Central

    Behm, David J; Harrison, Stephen M; Ao, Zhaohui; Maniscalco, Kristeen; Pickering, Susan J; Grau, Evelyn V; Woods, Tina N; Coatney, Robert W; Doe, Christopher P A; Willette, Robert N; Johns, Douglas G; Douglas, Stephen A

    2003-01-01

    Urotensin-II (U-II) is among the most potent mammalian vasoconstrictors identified and may play a role in the aetiology of essential hypertension. Currently, only one mouse U-II receptor (UT) gene has been cloned. It is postulated that this protein is solely responsible for mediating U-II-induced vasoconstriction. This hypothesis has been investigated in the present study, which assessed basal haemodynamics and vascular reactivity to hU-II in wild-type (UT(+/+)) and UT receptor knockout (UT(−/−)) mice. Basal left ventricular end-diastolic and end-systolic volumes/pressures, stroke volumes, mean arterial blood pressures, heart rates, cardiac outputs and ejection fractions in UT(+/+) mice and in UT(−/−) mice were similar. Relative to UT(+/+) mouse isolated thoracic aorta, where hU-II was a potent spasmogen (pEC50=8.26±0.08) that evoked relatively little vasoconstriction (17±2% 60 mM KCl), vessels isolated from UT(−/−) mice did not respond to hU-II. However, in contrast, the superior mesenteric artery isolated from both the genotypes did not contract in the presence of hU-II. Reactivity to unrelated vasoconstrictors (phenylephrine, endothelin-1, KCl) and endothelium-dependent/independent vasodilator agents (carbachol, sodium nitroprusside) was similar in the aorta and superior mesenteric arteries isolated from both the genotypes. The present study is the first to directly link hU-II-induced vasoconstriction with the UT receptor. Deletion of the UT receptor gene results in loss of hU-II contractile action with no ‘nonspecific' alterations in vascular reactivity. However, as might be predicted based on the limited contractile efficacy recorded in vitro, the contribution that hU-II and its receptor make to basal systemic haemodynamics appears to be negligible in this species. PMID:12770952

  15. CRISPR-mediated knockout of Cybb in NSG mice establishes a model of chronic granulomatous disease for human stem cell gene therapy transplants.

    PubMed

    Sweeney, Colin L; Choi, Uimook; Liu, Chengyu; Koontz, Sherry M; Ha, Seung-Kwon; Malech, Harry L

    2017-03-06

    Chronic granulomatous disease (CGD) is characterized by defects in production of microbicidal reactive oxygen species (ROS) by phagocytes. Testing of gene and cell therapies for treatment of CGD in human hematopoietic cells requires pre-clinical transplant models. The use of the lymphocyte-deficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mouse strain for human hematopoietic cell xenografts to test CGD therapies is complicated by the presence of functional mouse granulocytes capable of producing ROS for subsequent bacterial and fungal killing. To establish a phagocyte-defective mouse model of X-linked CGD (X-CGD) in NSG mice, we utilized clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 for targeted knock-out of mouse Cybb on the X-chromosome by microinjection of NSG mouse zygotes with Cas9 mRNA and CRISPR single-guide RNA targeting Cybb exon 1 or exon 3. This resulted in a high incidence of indel formation at the CRISPR target site, with all mice exhibiting deletions in at least one Cybb allele based on sequence analysis of tail snip DNA. A female mouse heterozygous for a 235-bp deletion in Cybb exon 1 was bred to an NSG male to establish the X-CGD NSG mouse strain, NSG.Cybb[KO]. Resulting male offspring with the 235-bp deletion were found to be defective for production of ROS by neutrophils and other phagocytes, and demonstrated increased susceptibility to spontaneous bacterial and fungal infections with granulomatous inflammation. The establishment of the phagocyte-defective NSG.Cybb[KO] mouse model enables the in vivo assessment of gene and cell therapy strategies for treating CGD in human hematopoietic cell transplants without obfuscation by functional mouse phagocytes, and may also be useful for modeling of other phagocyte disorders in humanized NSG mouse xenografts.

  16. Echium oil reduces plasma lipids and hepatic lipogenic gene expression in apoB100-only LDL receptor knockout mice.

    PubMed

    Zhang, Ping; Boudyguina, Elena; Wilson, Martha D; Gebre, Abraham K; Parks, John S

    2008-10-01

    We tested the hypothesis that dietary supplementation with echium oil (EO), which is enriched in stearidonic acid (SDA; 18:4 n-3), the product of Delta-6 desaturation of 18:3 n-3, will decrease plasma triglyceride (TG) concentrations and result in conversion of SDA to eicosapentaenoic acid (EPA) in the liver. Mildly hypertriglyceridemic mice (apoB100-only LDLrKO) were fed a basal diet containing 10% calories as palm oil (PO) and 0.2% cholesterol for 4 weeks, after which they were randomly assigned to experimental diets consisting of the basal diet plus supplementation of 10% of calories as PO, EO or fish oil (FO) for 8 weeks. The EO and FO experimental diets decreased plasma TG and VLDL lipid concentration, and hepatic TG content compared to PO, and there was a significant correlation between hepatic TG content and plasma TG concentration among diet groups. EO fed mice had plasma and liver lipid EPA enrichment that was greater than PO-fed mice but less than FO-fed mice. Down-regulation of several genes involved in hepatic TG biosynthesis was similar for mice fed EO and FO and significantly lower compared to those fed PO. In conclusion, EO may provide a botanical alternative to FO for reduction of plasma TG concentrations.

  17. Gene knockout demonstrates that vip3A contributes to the pathogenesis of Bacillus thuringiensis toward Agrotis ipsilon and Spodoptera exigua.

    PubMed

    Donovan, W P; Donovan, J C; Engleman, J T

    2001-07-01

    Vip3A is an 89-kDa protein secreted by Bacillus thuringiensis during vegetative growth. To determine the importance of Vip3A for the insect pathogenicity of B. thuringiensis the vip3A gene was deleted from strain HD1, yielding strain HD1Deltavip3A. Compared with HD1, strain HD1Deltavip3A was one-fourth as toxic to Agrotis ipsilon larvae and less than one-tenth as toxic to Spodoptera exigua larvae. When streptomycin was included in the S. exigua diet the toxicity of HD1Deltavip3A was approximately half that of HD1. Addition of HD1 spores increased the toxicity of purified Cry1 protein more than 600-fold against S. exigua, whereas addition of HD1Deltavip3A spores increased toxicity of Cry1 protein approximately 10-fold. These results demonstrate that an important component of B. thuringiensis insecticidal activity against S. exigua is the synthesis of Vip3A protein by B. thuringiensis cells after ingestion of spores and crystal proteins by insect larvae.

  18. Hypercholesterolemia in low density lipoprotein receptor knockout mice and its reversal by adenovirus-mediated gene delivery.

    PubMed Central

    Ishibashi, S; Brown, M S; Goldstein, J L; Gerard, R D; Hammer, R E; Herz, J

    1993-01-01

    We employed homologous recombination in embryonic stem cells to produce mice lacking functional LDL receptor genes. Homozygous male and female mice lacking LDL receptors (LDLR-/- mice) were viable and fertile. Total plasma cholesterol levels were twofold higher than those of wild-type litter-mates, owing to a seven- to ninefold increase in intermediate density lipoproteins (IDL) and LDL without a significant change in HDL. Plasma triglyceride levels were normal. The half-lives for intravenously administered 125I-VLDL and 125I-LDL were prolonged by 30-fold and 2.5-fold, respectively, but the clearance of 125I-HDL was normal in the LDLR-/- mice. Unlike wild-type mice, LDLR-/- mice responded to moderate amounts of dietary cholesterol (0.2% cholesterol/10% coconut oil) with a major increase in the cholesterol content of IDL and LDL particles. The elevated IDL/LDL level of LDLR-/- mice was reduced to normal 4 d after the intravenous injection of a recombinant replication-defective adenovirus encoding the human LDL receptor driven by the cytomegalovirus promoter. The virus restored expression of LDL receptor protein in the liver and increased the clearance of 125I-VLDL. We conclude that the LDL receptor is responsible in part for the low levels of VLDL, IDL, and LDL in wild-type mice and that adenovirus-encoded LDL receptors can acutely reverse the hypercholesterolemic effects of LDL receptor deficiency. Images PMID:8349823

  19. 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

  20. Relaxin-3 receptor (Rxfp3) gene knockout mice display reduced running wheel activity: implications for role of relaxin-3/RXFP3 signalling in sustained arousal.

    PubMed

    Hosken, Ihaia T; Sutton, Steven W; Smith, Craig M; Gundlach, Andrew L

    2015-02-01

    Anatomical and pharmacological evidence suggests the neuropeptide, relaxin-3, is the preferred endogenous ligand for the relaxin family peptide-3 receptor (RXFP3) and suggests a number of putative stress- and arousal-related roles for RXFP3 signalling. However, in vitro and in vivo evidence demonstrates exogenous relaxin-3 can activate other relaxin peptide family receptors, and the role of relaxin-3/RXFP3 signalling in specific brain circuits and associated behaviours in mice is not well described. In this study, we characterised the behaviour of cohorts of male and female Rxfp3 gene knockout (KO) mice (C57/B6J(RXFP3TM1/DGen)), relative to wild-type (WT) littermates to determine if this receptor KO strain has a similar phenotype to its ligand KO equivalent. Rxfp3 KO mice displayed similar performance to WT littermates in several acute behavioural paradigms designed to gauge motor coordination (rotarod test), spatial memory (Y-maze), depressive-like behaviour (repeat forced-swim test) and sensorimotor gating (prepulse inhibition of acoustic startle). Notably however, male and female Rxfp3 KO mice displayed robust and consistent (dark phase) hypoactivity on voluntary home-cage running wheels (∼20-60% less activity/h), and a small but significant decrease in anxiety-like behavioural traits in the elevated plus maze and light/dark box paradigms. Importantly, this phenotype is near identical to that observed in two independent lines of relaxin-3 KO mice, suggesting these phenotypes are due to the elimination of ligand or receptor and RXFP3-linked signalling. Furthermore, this behavioural characterisation of Rxfp3 KO mice identifies them as a useful experimental model for studying RXFP3-linked signalling and assessing the selectivity and/or potential off-target actions of RXFP3 agonists and antagonists, which could lead to an improved understanding of dysfunctional arousal in mental health disorders, including depression, anxiety, insomnia and neurodegenerative

  1. Human knockout research: new horizons and opportunities.

    PubMed

    Alkuraya, Fowzan S

    2015-02-01

    Although numerous approaches have been pursued to understand the function of human genes, Mendelian genetics has by far provided the most compelling and medically actionable dataset. Biallelic loss-of-function (LOF) mutations are observed in the majority of autosomal recessive Mendelian disorders, representing natural human knockouts and offering a unique opportunity to study the physiological and developmental context of these genes. The restriction of such context to 'disease' states is artificial, however, and the recent ability to survey entire human genomes for biallelic LOF mutations has revealed a surprising landscape of knockout events in 'healthy' individuals, sparking interest in their role in phenotypic diversity beyond disease causation. As I discuss in this review, the potentially wide implications of human knockout research warrant increased investment and multidisciplinary collaborations to overcome existing challenges and reap its benefits.

  2. Effects of NV gene knock-out recombinant viral hemorrhagic septicemia virus (VHSV) on Mx gene expression in Epithelioma papulosum cyprini (EPC) cells and olive flounder (Paralichthys olivaceus).

    PubMed

    Kim, Min Sun; Kim, Ki Hong

    2012-03-01

    To determine whether the NV gene of viral hemorrhagic septicemia virus (VHSV) is related to the type I interferon response of hosts, expression of Mx gene in Epithelioma papulosum cyprini (EPC) cells and in olive flounder (Paralichthys olivaceus) in response to infection with either wild-type VHSV or recombinant VHSVs (rVHSV-ΔNV-EGFP and rVHSV-wild) was investigated. A reporter vector was constructed for measuring Mx gene expression using olive flounder Mx promoter, in which the reporter Metridia luciferase was designed to be excreted to culture medium to facilitate measurement. The highest increase of luciferase activity was detected from supernatant of cells infected with rVHSV-ΔNV-EGFP. In contrast cells infected with wild-type VHSV showed a slight increase of the luciferase activity. Interestingly, cells infected with rVHSV-wild that has artificially changed nucleotides just before and after the NV gene ORF, also showed highly increased luciferase activity, but the increased amplitude was lower than that by rVHSV-ΔNV-EGFP. These results strongly suggest that the NV protein of VHSV plays an important role in suppressing interferon response in host cells, which provides a condition for the viruses to efficiently proliferate in host cells. In an in vivo experiment, the Mx gene expression in olive flounder challenged with the rVHSV-ΔNV-EGFP was clearly higher than fish challenged with rVHSV-wild or wild-type VHSV, suggesting that lacking of the NV gene in the genome of rVHSV-ΔNV-EGFP brought to strong interferon response that subsequently inhibit viral replication in fish.

  3. Human Knockout Carriers: Dead, Diseased, Healthy, or Improved?

    PubMed Central

    Narasimhan, Vagheesh M.; Xue, Yali; Tyler-Smith, Chris

    2016-01-01

    Whole-genome and whole-exome sequence data from large numbers of individuals reveal that we all carry many variants predicted to inactivate genes (knockouts). This discovery raises questions about the phenotypic consequences of these knockouts and potentially allows us to study human gene function through the investigation of homozygous loss-of-function carriers. Here, we discuss strategies, recent results, and future prospects for large-scale human knockout studies. We examine their relevance to studying gene function, population genetics, and importantly, the implications for accurate clinical interpretations. PMID:26988438

  4. Profound induction of hepatic cholesteryl ester transfer protein transgene expression in apolipoprotein E and low density lipoprotein receptor gene knockout mice. A novel mechanism signals changes in plasma cholesterol levels.

    PubMed Central

    Masucci-Magoulas, L; Plump, A; Jiang, X C; Walsh, A; Breslow, J L; Tall, A R

    1996-01-01

    The plasma cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl esters from HDL to other lipoproteins and is a key regulated component of reverse cholesterol transport. Dietary hypercholesterolemia results in increased hepatic CETP gene transcription and higher plasma CETP levels. To investigate the mechanisms by which the liver senses hypercholesterolemia, mice containing a natural flanking region CETP transgene (NFR-CETP transgene) were bred with apo E or LDL receptor gene knockout mice (E0 or LDLr0 mice). Compared to NFR-CETP transgenic (Tg) mice with intact apo E genes, in NFR-CETP Tg/E0 mice there was an eightfold induction of plasma CETP levels and a parallel increase in hepatic CETP mRNA levels. Other sterol-responsive genes (LDL receptor and hydroxymethyl glutaryl CoA reductase) also showed evidence of altered regulation with decreased abundance of their mRNAs in the E0 background. A similar induction of plasma CETP and hepatic CETP mRNA levels resulted from breeding the NFR-CETP transgene into the LDL receptor gene knockout background. When placed on a high cholesterol diet, there was a further increase in CETP levels in both E0 and LDLr0 backgrounds. In CETP Tg, CETP Tg/E0, and CETP Tg/LDLr0 mice on different diets, plasma CETP and CETP mRNA levels were highly correlated with plasma cholesterol levels. The results indicate that hepatic CETP gene expression is driven by a mechanism which senses changes in plasma cholesterol levels independent of apo E and LDL receptors. Hepatic sterol-sensitive genes have mechanisms to sense hypercholesterolemia that do not require classical receptor-mediated lipoprotein uptake. PMID:8550828

  5. Guwiyang Wurra--'Fire Mouse': a global gene knockout model for TSPO/PBR drug development, loss-of-function and mechanisms of compensation studies.

    PubMed

    Middleton, Ryan J; Liu, Guo-Jun; Banati, Richard B

    2015-08-01

    The highly conserved 18-kDa translocator protein (TSPO) or peripheral benzodiazepine receptor (PBR), is being investigated as a diagnostic and therapeutic target for disease conditions ranging from inflammation to neurodegeneration and behavioural illnesses. Many functions have been attributed to TSPO/PBR including a role in the mitochondrial permeability transition pore (MPTP), steroidogenesis and energy metabolism. In this review, we detail the recent developments in determining the physiological role of TSPO/PBR, specifically based on data obtained from the recently generated Tspo knockout mouse models. In addition to defining the role of TSPO/PBR, we also describe the value of Tspo knockout mice in determining the selectivity, specificity and presence of any off-target effects of TSPO/PBR ligands.

  6. Residual Cajal bodies in coilin knockout mice fail to recruit Sm snRNPs and SMN, the spinal muscular atrophy gene product

    PubMed Central

    Tucker, Karen E.; Berciano, Maria Teresa; Jacobs, Erica Y.; LePage, David F.; Shpargel, Karl B.; Rossire, Jennifer J.; Chan, Edward K.L.; Lafarga, Miguel; Conlon, Ronald A.; Matera, A. Gregory

    2001-01-01

    Cajal bodies (CBs) are nuclear suborganelles involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs). In addition to snRNPs, they are highly enriched in basal transcription and cell cycle factors, the nucleolar proteins fibrillarin (Fb) and Nopp140 (Nopp), the survival motor neuron (SMN) protein complex, and the CB marker protein, p80 coilin. We report the generation of knockout mice lacking the COOH-terminal 487 amino acids of coilin. Northern and Western blot analyses demonstrate that we have successfully removed the full-length coilin protein from the knockout animals. Some homozygous mutant animals are viable, but their numbers are reduced significantly when crossed to inbred backgrounds. Analysis of tissues and cell lines from mutant animals reveals the presence of extranucleolar foci that contain Fb and Nopp but not other typical nucleolar markers. These so-called “residual” CBs neither condense Sm proteins nor recruit members of the SMN protein complex. Transient expression of wild-type mouse coilin in knockout cells results in formation of CBs and restores these missing epitopes. Our data demonstrate that full-length coilin is essential for proper formation and/or maintenance of CBs and that recruitment of snRNP and SMN complex proteins to these nuclear subdomains requires sequences within the coilin COOH terminus. PMID:11470819

  7. Development of an efficient agrobacterium-mediated gene targeting system for rice and analysis of rice knockouts lacking granule-bound starch synthase (Waxy) and β1,2-xylosyltransferase.

    PubMed

    Ozawa, Kenjirou; Wakasa, Yuhya; Ogo, Yuko; Matsuo, Kouki; Kawahigashi, Hiroyuki; Takaiwa, Fumio

    2012-04-01

    We have developed a high-frequency method for Agrobacterium-mediated gene targeting by combining an efficient transformation system using rice suspension-cultured calli and a positive/negative selection system. Compared with the conventional transformation system using calli on solid medium, transformation using suspension-cultured calli resulted in a 5- to 10-fold increase in the number of resistant calli per weight of starting material after positive/negative selection. Homologous recombination occurred in about 1.5% of the positive/negative selected calli. To evaluate the efficacy of our method, we show in this report that knockout rice plants containing either a disrupted Waxy (granule-bound starch synthase) or a disrupted Xyl (β1,2-xylosyltransferase) gene can be easily obtained by homologous recombination. Study of gene function using homologous recombination in higher plants can now be considered routine work as a direct result of this technical advance.

  8. Knockout mouse production assisted by Blm knockdown

    PubMed Central

    FUKUDA, Mikiko; INOUE, Mayuko; MURAMATSU, Daisuke; MIYACHI, Hitoshi; SHINKAI, Yoichi

    2015-01-01

    Production of knockout mice using targeted embryonic stem cells (ESCs) is a powerful approach for investigating the function of specific genes in vivo. Although the protocol for gene targeting via homologous recombination (HR) in ESCs is already well established, the targeting efficiency varies at different target loci and is sometimes too low. It is known that knockdown of the Bloom syndrome gene, BLM, enhances HR-mediated gene targeting efficiencies in various cell lines. However, it has not yet been investigated whether this approach in ESCs is applicable for successful knockout mouse production. Therefore, we attempted to answer this question. Consistent with previous reports, Blm knockdown enhanced gene targeting efficiencies for three gene loci that we examined by 2.3–4.1-fold. Furthermore, the targeted ESC clones generated good chimeras and were successful in germline transmission. These data suggest that Blm knockdown provides a general benefit for efficient ESC-based and HR-mediated knockout mouse production. PMID:26598326

  9. Manipulation of Mouse Embryonic Stem Cells for Knockout Mouse Production

    PubMed Central

    Limaye, Advait; Hall, Bradford; Kulkarni, Ashok B

    2009-01-01

    The establishment of mouse embryonic stem (ES) cell liness has allowed for the generation of the knockout mouse. ES cells that are genetically altered in culture can then be manipulated to derive a whole mouse containing the desired mutation. To successfully generate a knockout mouse, however, the ES cells must be carefully cultivated in a pluripotent state throughout the gene targeting experiment. This unit describes detailed step-by-step protocols, reagents, equipment, and strategies needed for the successful generation of gene knockout embryonic stem cells using homologous recombination technologies. PMID:19731225

  10. The kinetics of inactivation of the rod phototransduction cascade with constant Ca2+i

    PubMed Central

    1996-01-01

    A rich variety of mechanisms govern the inactivation of the rod phototransduction cascade. These include rhodopsin phosphorylation and subsequent binding of arrestin; modulation of rhodopsin kinase by S- modulin (recoverin); regulation of G-protein and phosphodiesterase inactivation by GTPase-activating factors; and modulation of guanylyl cyclase by a high-affinity Ca(2+)-binding protein. The dependence of several of the inactivation mechanisms on Ca2+i makes it difficult to assess the contributions of these mechanisms to the recovery kinetics in situ, where Ca2+i is dynamically modulated during the photoresponse. We recorded the circulating currents of salamander rods, the inner segments of which are held in suction electrodes in Ringer's solution. We characterized the response kinetics to flashes under two conditions: when the outer segments are in Ringer's solution, and when they are in low-Ca2+ choline solutions, which we show clamp Ca2+i very near its resting level. At T = 20-22 degrees C, the recovery phases of responses to saturating flashes producing 10(2.5)-10(4.5) photoisomerizations under both conditions are characterized by a dominant time constant, tau c = 2.4 +/- 0.4 s, the value of which is not dependent on the solution bathing the outer segment and therefore not dependent on Ca2+i. We extended a successful model of activation by incorporating into it a first-order inactivation of R*, and a first-order, simultaneous inactivation of G-protein (G*) and phosphodiesterase (PDE*). We demonstrated that the inactivation kinetics of families of responses obtained with Ca2+i clamped to rest are well characterized by this model, having one of the two inactivation time constants (tau r* or tau PDE*) equal to tau c, and the other time constant equal to 0.4 +/- 0.06 s. PMID:8741728

  11. Gene knockout using transcription activator-like effector nucleases (TALENs) reveals that human NDUFA9 protein is essential for stabilizing the junction between membrane and matrix arms of complex I.

    PubMed

    Stroud, David A; Formosa, Luke E; Wijeyeratne, Xiaonan W; Nguyen, Thanh N; Ryan, Michael T

    2013-01-18

    Transcription activator-like effector nucleases (TALENs) represent a promising approach for targeted knock-out of genes in cultured human cells. We used TALEN-technology to knock out the nuclear gene encoding NDUFA9, a subunit of mitochondrial respiratory chain complex I in HEK293T cells. Screening for the knock-out revealed a mixture of NDUFA9 cell clones that harbored partial deletions of the mitochondrial N-terminal targeting signal but were still capable of import. A cell line lacking functional copies of both NDUFA9 alleles resulted in a loss of NDUFA9 protein expression, impaired assembly of complex I, and cells incapable of growth in galactose medium. Cells lacking NDUFA9 contained a complex I subcomplex consisting of membrane arm subunits but not marker subunits of the matrix arm. Re-expression of NDUFA9 restored the defects in complex I assembly. We conclude that NDUFA9 is involved in stabilizing the junction between membrane and matrix arms of complex I, a late assembly step critical for complex I biogenesis and activity.

  12. 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.

  13. Heat shock response: lessons from mouse knockouts.

    PubMed

    Christians, E S; Benjamin, I J

    2006-01-01

    Organisms are endowed with integrated regulatory networks that transduce and amplify incoming signals into effective responses, ultimately imparting cell death and/or survival pathways. As a conserved cytoprotective mechanism from bacteria to humans, the heat shock response has been established as a paradigm for inducible gene expression, stimulating the interests of biologists and clinicians alike to tackle fundamental questions related to the molecular switches, lineage-specific requirements, unique and/or redundant roles, and even efforts to harness the response therapeutically. Gene targeting studies in mice confirm HSF1 as a master regulator required for cell growth, embryonic development, and reproduction. For example, sterility of Hsf1-null female but not null male mice established strict requirements for maternal HSF1 expression in the oocyte. Yet Hsf2 knockouts by three independent laboratories have not fully clarified the role of mammalian HSF2 for normal development, fertility, and postnatal neuronal function. In contrast, Hsf4 knockouts have provided a consistent demonstration for HSF4's critical role during lens formation. In the future, molecular analysis of HSF knockout mice will bring new insights to HSF interactions, foster better understanding of gene regulation at the genome level, lead to a better integration of the HSF pathway in life beyond heat shock, the classical laboratory challenge.

  14. Altered gene expression and functional activity of opioid receptors in the cerebellum of CB1 cannabinoid receptor knockout mice after acute treatments with cannabinoids.

    PubMed

    Páldyová, Estera; Bereczki, E; Sántha, M; Wenger, T; Borsodi, Anna; Benyhe, S

    2007-01-01

    Numerous studies have shown functional links between the cannabinoid and opioid systems. The goal of this study was to evaluate whether acute treatments by endogenous cannabinoid agonist, selective CB1 or CB2 receptor antagonists modulate the expression of mu- (MOR) and delta- (DOR) opioid receptor mRNA levels and functional activity in the cerebellum of transgenic mice deficient in the CB1 type of cannabis receptors. We examined the effect of noladin ether (endogenous cannabinoid agonist) pretreatment on MOR and DOR mRNA expression by using reverse transcription and real-time polimerase chain reaction (PCR) and the ability of subsequent application of the opioid agonists to activate G-proteins, as measured by [35S]GTPgammaS binding, in wild-type (CB1+/+) and CB1 cannabinoid receptor deficient (CB1-/-, 'knockout', K.O.) mice. The acute administration of noladin ether markedly reduced MOR-mediated G-protein activation and caused a significant increase in the level of MOR mRNAs in the cerebella of wildtype, but not in the CB1-/- mice. No significant differences were observed in DOR functional activity and mRNA expression in wild-type animals. In CB1-/- mice the expression of DOR mRNA increased after noladin ether treatment, but no changes were found in DOR functional activity. In addition, Rimonabant (selective central cannabinoid CB1 receptor antagonist) and SR144528 (selective peripheral cannabinoid CB2 receptor antagonist) caused significant potentiation in MOR functional activity in the wild-type animals, whereas DOR mediated G-protein activation was increased in the CB1-/- mice. In contrast, Rimonabant and SR144528 decreased the MOR and DOR mRNA expressions in both CB1+/+ and CB1-/- mice. Taken together, these results indicate that acute treatment with cannabinoids causes alterations in MOR and DOR mRNA expression and functional activity in the cerebella of wild-type and CB1 knockout mice indicating indirect interactions between these two signaling systems.

  15. Post-illumination pupil response after blue light: Reliability of optimized melanopsin-based phototransduction assessment.

    PubMed

    van der Meijden, Wisse P; te Lindert, Bart H W; Bijlenga, Denise; Coppens, Joris E; Gómez-Herrero, Germán; Bruijel, Jessica; Kooij, J J Sandra; Cajochen, Christian; Bourgin, Patrice; Van Someren, Eus J W

    2015-10-01

    ± 3.6 yr) we examined the potential confounding effects of dark adaptation, time of the day (morning vs. afternoon), body posture (upright vs. supine position), and 24-h environmental light history on the PIPR assessment. Mixed effect regression models were used to analyze these possible confounders. A supine position caused larger PIPR-mm (β = 0.29 mm, SE = 0.10, p = 0.01) and PIPR-% (β = 4.34%, SE = 1.69, p = 0.02), which was due to an increase in baseline dark pupil diameter; this finding is of relevance for studies requiring a supine posture, as in functional Magnetic Resonance Imaging, constant routine protocols, and bed-ridden patients. There were no effects of dark adaptation, time of day, and light history. In conclusion, the presented method provides a reliable and robust assessment of the PIPR to allow for studies on individual differences in melanopsin-based phototransduction and effects of interventions.

  16. The broken mouse: the role of development, plasticity and environment in the interpretation of phenotypic changes in knockout mice.

    PubMed

    Gingrich, J A; Hen, R

    2000-02-01

    With the advent of gene knockout technology has arisen the problem of how to interpret the resulting phenotypic changes in mice lacking specific genes. This problem is especially relevant when applied to behavioral phenotypes of knockout mice, which are difficult to interpret. Of particular interest are the roles of development and compensatory changes, as well as other factors, such as the influence of the gene knockout on nearby genes, the effect of the genetic background strain, maternal behavioral influences, and pleiotrophy.

  17. Construction of an alpha toxin gene knockout mutant of Clostridium perfringens type A by use of a mobile group II intron.

    PubMed

    Chen, Yue; McClane, Bruce A; Fisher, Derek J; Rood, Julian I; Gupta, Phalguni

    2005-11-01

    In developing Clostridium perfringens as a safe vaccine vector, the alpha toxin gene (plc) in the bacterial chromosome must be permanently inactivated. Disrupting genes in C. perfringens by traditional mutagenesis methods is very difficult. Therefore, we developed a new strategy using group II intron-based Target-Tron technology to inactivate the plc gene in C. perfringens ATCC 3624. Western blot analysis showed no production of alpha toxin protein in the culture supernatant of the plc mutant. Advantages of this technology, such as site specificity, relatively high frequency of insertion, and introduction of no antibiotic resistance genes into the chromosome, could facilitate construction of other C. perfringens mutants.

  18. Gene expression profiling studies in regenerating nerves in a mouse model for CMT1X: uninjured Cx32-knockout peripheral nerves display expression profile of injured wild type nerves.

    PubMed

    Freidin, Mona; Asche-Godin, Samantha; Abrams, Charles K

    2015-01-01

    X-linked Charcot-Marie-Tooth disease (CMT1X) is an inherited peripheral neuropathy caused by mutations in GJB1, the human gene for Connexin32 (Cx32). This present study uses Ilumina Ref8-v2 BeadArray to examine the expression profiles of injured and uninjured sciatic nerves at 5, 7, and 14 days post-crush injury (dpi) from Wild Type (WT) and Cx32-knockout (Cx32KO) mice to identify the genes and signaling pathways that are dysregulated in the absence of Schwann cell Cx32. Given the assumption that loss of Schwann cell Cx32 disrupts the regeneration and maintenance of myelinated nerve leading to a demyelinating neuropathy in CMT1X, we initially hypothesized that nerve crush injury would result in significant increases in differential gene expression in Cx32KO mice relative to WT nerves. However, microarray analysis revealed a striking collapse in the number of differentially expressed genes at 5 and 7 dpi in Cx32KO nerves relative to WT, while uninjured and 14 dpi time points showed large numbers of differentially regulated genes. Further comparisons within each genotype showed limited changes in Cx32KO gene expression following crush injury when compared to uninjured Cx32KO nerves. By contrast, WT nerves exhibited robust changes in gene expression at 5 and 7 dpi with no significant differences in gene expression by 14dpi relative to uninjured WT nerve samples. Taken together, these data suggest that the gene expression profile in uninjured Cx32KO sciatic nerve strongly resembles that of a WT nerve following injury and that loss of Schwann cell Cx32 leads to a basal state of gene expression similar to that of an injured WT nerve. These findings support a role for Cx32 in non-myelinating and regenerating populations of Schwann cells in normal axonal maintenance in re-myelination, and regeneration of peripheral nerve following injury. Disruption of Schwann cell-axonal communication in CMT1X may cause dysregulation of signaling pathways that are essential for the

  19. [An efficient genetic knockout system based on linear DNA fragment homologous recombination for halophilic archaea].

    PubMed

    Xiaoli, Wang; Chuang, Jiang; Jianhua, Liu; Xipeng, Liu

    2015-04-01

    With the development of functional genomics, gene-knockout is becoming an important tool to elucidate gene functions in vivo. As a good model strain for archaeal genetics, Haloferax volcanii has received more attention. Although several genetic manipulation systems have been developed for some halophilic archaea, it is time-consuming because of the low percentage of positive clones during the second-recombination selection. These classical gene knockout methods are based on DNA recombination between the genomic homologous sequence and the circular suicide plasmid, which carries a pyrE selection marker and two DNA fragments homologous to the upstream and downstream fragments of the target gene. Many wild-type clones are obtained through a reverse recombination between the plasmid and genome in the classic gene knockout method. Therefore, it is necessary to develop an efficient gene knockout system to increase the positive clone percentage. Here we report an improved gene knockout method using a linear DNA cassette consisting of upstream and downstream homologous fragments, and the pyrE marker. Gene deletions were subsequently detected by colony PCR analysis. We determined the efficiency of our knockout method by deleting the xpb2 gene from the H. volcanii genome, with the percentage of positive clones higher than 50%. Our method provides an efficient gene knockout strategy for halophilic archaea.

  20. Generation and characterization of novel cytochrome P450 Cyp2c gene cluster knockout and CYP2C9 humanized mouse lines.

    PubMed

    Scheer, Nico; Kapelyukh, Yury; Chatham, Lynsey; Rode, Anja; Buechel, Sandra; Wolf, C Roland

    2012-12-01

    Compared with rodents and many other animal species, the human cytochrome P450 (P450) Cyp2c gene cluster varies significantly in the multiplicity of functional genes and in the substrate specificity of its enzymes. As a consequence, the use of wild-type animal models to predict the role of human CYP2C enzymes in drug metabolism and drug-drug interactions is limited. Within the human CYP2C cluster CYP2C9 is of particular importance, because it is one of the most abundant P450 enzymes in human liver, and it is involved in the metabolism of a wide variety of important drugs and environmental chemicals. To investigate the in vivo functions of cytochrome P450 Cyp2c genes and to establish a model for studying the functions of CYP2C9 in vivo, we have generated a mouse model with a deletion of the murine Cyp2c gene cluster and a corresponding humanized model expressing CYP2C9 specifically in the liver. Despite the high number of functional genes in the mouse Cyp2c cluster and the reported roles of some of these proteins in different biological processes, mice deleted for Cyp2c genes were viable and fertile but showed certain phenotypic alterations in the liver. The expression of CYP2C9 in the liver also resulted in viable animals active in the metabolism and disposition of a number of CYP2C9 substrates. These mouse lines provide a powerful tool for studying the role of Cyp2c genes and of CYP2C9 in particular in drug disposition and as a factor in drug-drug interaction.

  1. The knock-out of ARP3a gene affects F-actin cytoskeleton organization altering cellular tip growth, morphology and development in moss Physcomitrella patens.

    PubMed

    Finka, Andrija; Saidi, Younousse; Goloubinoff, Pierre; Neuhaus, Jean-Marc; Zrÿd, Jean-Pierre; Schaefer, Didier G

    2008-10-01

    The seven subunit Arp2/3 complex is a highly conserved nucleation factor of actin microfilaments. We have isolated the genomic sequence encoding a putative Arp3a protein of the moss Physcomitrella patens. The disruption of this ARP3A gene by allele replacement has generated loss-of-function mutants displaying a complex developmental phenotype. The loss-of function of ARP3A gene results in shortened, almost cubic chloronemal cells displaying affected tip growth and lacking differentiation to caulonemal cells. In moss arp3a mutants, buds differentiate directly from chloronemata to form stunted leafy shoots having differentiated leaves similar to wild type. Yet, rhizoids never differentiate from stem epidermal cells. To characterize the F-actin organization in the arp3a-mutated cells, we disrupted ARP3A gene in the previously described HGT1 strain expressing conditionally the GFP-talin marker. In vivo observation of the F-actin cytoskeleton during P. patens development demonstrated that loss-of-function of Arp3a is associated with the disappearance of specific F-actin cortical structures associated with the establishment of localized cellular growth domains. Finally, we show that constitutive expression of the P. patens Arp3a and its Arabidopsis thaliana orthologs efficiently complement the mutated phenotype indicating a high degree of evolutionary conservation of the Arp3 function in land plants.

  2. CRISPR/Cas9 mediated knockout of the abdominal-A homeotic gene in the global pest, diamondback moth (Plutella xylostella).

    PubMed

    Huang, Yuping; Chen, Yazhou; Zeng, Baosheng; Wang, Yajun; James, Anthony A; Gurr, Geoff M; Yang, Guang; Lin, Xijian; Huang, Yongping; You, Minsheng

    2016-08-01

    The diamondback moth, Plutella xylostella (L.), is a worldwide agricultural pest that has developed resistance to multiple classes of insecticides. Genetics-based approaches show promise as alternative pest management approaches but require functional studies to identify suitable gene targets. Here we use the CRISPR/Cas9 system to target a gene, abdominal-A, which has an important role in determining the identity and functionality of abdominal segments. We report that P. xylostella abdominal-A (Pxabd-A) has two structurally-similar splice isoforms (A and B) that differ only in the length of exon II, with 15 additional nucleotides in isoform A. Pxabd-A transcripts were detected in all developmental stages, and particularly in pupae and adults. CRISPR/Cas9-based mutagenesis of Pxabd-A exon I produced 91% chimeric mutants following injection of 448 eggs. Phenotypes with abnormal prolegs and malformed segments were visible in hatched larvae and unhatched embryos, and various defects were inherited by the next generation (G1). Genotyping of mutants demonstrated several mutations at the Pxabd-A genomic locus. The results indicate that a series of insertions and deletions were induced in the Pxabd-A locus, not only in G0 survivors but also in G1 individuals, and this provides a foundation for genome editing. Our study demonstrates the utility of the CRISPR/Cas9 system for targeting genes in an agricultural pest and therefore provides a foundation the development of novel pest management tools.

  3. Knockout of crtB or crtI gene blocks the carotenoid biosynthetic pathway in Deinococcus radiodurans R1 and influences its resistance to oxidative DNA-damaging agents due to change of free radicals scavenging ability.

    PubMed

    Zhang, Lei; Yang, Qiao; Luo, Xuesong; Fang, Chengxiang; Zhang, Qiuju; Tang, Yali

    2007-10-01

    Deinococcus radiodurans R1, a red-pigmented strain of the extremely radioresistant genus Deinococcus, contains a major carotenoid namely deinoxanthin. The high resistance of this organism against the lethal actions of DNA-damaging agents including ionizing radiation and ultraviolet light (UV) has been widely reported. However, the possible antioxidant role of carotenoids in this strain has not been completely elucidated. In this study, we constructed two colorless mutants by knockout of crtB and crtI genes, respectively. Comparative analysis of the two colorless mutants and the wild type showed that the two colorless mutants were more sensitive to ionizing radiation, UV, and hydrogen peroxide, but not to mitomycin-C (MMC). With electron spin resonance (ESR) and spin trapping techniques, we observed that hydroxyl radical signals occurred in the suspensions of UV irradiated Deinococcus radiodurans cells and the intensity of signals was influenced by carotenoids levels. We further showed that the carotenoid extract from the wild type could obviously scavenge superoxide anions generated by the irradiated riboflavin/EDTA system. These results suggest that carotenoids in D. radiodurans R1 function as free radical scavengers to protect this organism against the deleterious effects of oxidative DNA-damaging agents.

  4. Sarcocystis neurona infection in gamma interferon gene knockout (KO) mice: comparative infectivity of sporocysts in two strains of KO mice, effect of trypsin digestion on merozoite viability, and infectivity of bradyzoites to KO mice and cell culture.

    PubMed

    Dubey, J P; Sundar, N; Kwok, O C H; Saville, W J A

    2013-09-01

    The protozoan Sarcocystis neurona is the primary cause of Equine Protozoal Myeloencephalitis (EPM). EPM or EPM-like illness has been reported in horses, sea otters, and several other mammals. The gamma interferon gene knockout (KO) mouse is often used as a model to study biology and discovery of new therapies against S. neurona because it is difficult to induce clinical EPM in other hosts, including horses. In the present study, infectivity of three life cycle stages (merozoites, bradyzoites, sporozoites) to KO mice and cell culture was studied. Two strains of KO mice (C57-black, and BALB/c-derived, referred here as black or white) were inoculated orally graded doses of S. neurona sporocysts; 12 sporocysts were infective to both strains of mice and all infected mice died or became ill within 70 days post-inoculation. Although there was no difference in infectivity of sporocysts to the two strains of KO mice, the disease was more severe in black mice. S. neurona bradyzoites were not infectious to KO mice and cell culture. S. neurona merozoites survived 120 min incubation in 0.25% trypsin, indicating that trypsin digestion can be used to recover S. neurona from tissues of acutely infected animals.

  5. Deficiency in pulmonary surfactant proteins in mice with fatty acid binding protein 4-Cre-mediated knockout of the tuberous sclerosis complex 1 gene.

    PubMed

    Xiang, Xinxin; Yuan, Fang; Zhao, Jing; Li, Ziru; Wang, Xian; Guan, Youfei; Tang, Chaoshu; Sun, Guang; Li, Yin; Zhang, Weizhen

    2013-03-01

    Tuberous sclerosis complex 1 (TSC1) forms a heterodimmer with tuberous sclerosis complex 2, to inhibit signalling by the mammalian target of rapamycin (mTOR) complex 1 (mTORC1). The mTORC1 stimulates cell growth by promoting anabolic cellular processes, such as gene transcription and protein translation, in response to growth factors and nutrient signals. Originally designed to test the role of TSC1 in adipocyte function, mice in which the gene for TSC1 was specifically deleted by the fatty acid binding protein 4 (FABP4)-Cre (Fabp4-Tsc1cKO mice) died prematurely within 48 h after birth. The Fabp4-Tsc1cKO mouse revealed a much smaller phenotype relative to the wild-type littermates. Maternal administration of rapamycin, a classical mTOR inhibitor, significantly increased the survival time of Fabp4-Tsc1cKO mice for up to 23 days. Both macroscopic and microscopic haemorrhages were observed in the lungs of Fabp4-Tsc1cKO mice, while other tissues showed no significant changes. Levels of surfactant proteins A and B demonstrated a significant decrease in the Fabp4-Tsc1cKO mice, which was rescued by maternal injection of rapamycin. Co-localization of FABP4 or TSC1 with surfactant protein B was also detected in neonatal pulmonary tissues. Our study suggests that TSC1-mTORC1 may be critical for the synthesis of surfactant proteins A and B.

  6. Activation of IKK/NF-κB provokes renal inflammatory responses in guanylyl cyclase/natriuretic peptide receptor-A gene-knockout mice

    PubMed Central

    Das, Subhankar; Periyasamy, Ramu

    2012-01-01

    The present study was aimed at determining the consequences of the disruption of guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) gene (Npr1) on proinflammatory responses of nuclear factor kappa B, inhibitory kappa B kinase, and inhibitory kappa B alpha (NF-κB, IKK, IκBα) in the kidneys of mutant mice. The results showed that the disruption of Npr1 enhanced the renal NF-κB binding activity by 3.8-fold in 0-copy (−/−) mice compared with 2-copy (+/+) mice. In parallel, IKK activity and IκBα protein phosphorylation were increased by 8- and 11-fold, respectively, in the kidneys of 0-copy mice compared with wild-type mice. Interestingly, IκBα was reduced by 80% and the expression of proinflammatory cytokines and renal fibrosis were significantly enhanced in 0-copy mice than 2-copy mice. Treatment of 0-copy mice with NF-κB inhibitors andrographolide, pyrrolidine dithiocarbamate, and etanercept showed a substantial reduction in renal fibrosis, attenuation of proinflammatory cytokines gene expression, and significantly reduced IKK activity and IkBα phosphorylation. These findings indicate that the systemic disruption of Npr1 activates the renal NF-κB pathways in 0-copy mice, which transactivates the expression of various proinflammatory cytokines to initiate renal remodeling; however, inhibition of NF-κB pathway repairs the abnormal renal pathology in mutant mice. PMID:22318993

  7. Single and Compound Knock-outs of MicroRNA (miRNA)-155 and Its Angiogenic Gene Target CCN1 in Mice Alter Vascular and Neovascular Growth in the Retina via Resident Microglia.

    PubMed

    Yan, Lulu; Lee, Sangmi; Lazzaro, Douglas R; Aranda, Jacob; Grant, Maria B; Chaqour, Brahim

    2015-09-18

    The response of the retina to ischemic insult typically leads to aberrant retinal neovascularization, a major cause of blindness. The epigenetic regulation of angiogenic gene expression by miRNAs provides new prospects for their therapeutic utility in retinal neovascularization. Here, we focus on miR-155, a microRNA functionally important in inflammation, which is of paramount importance in the pathogenesis of retinal neovascularization. Whereas constitutive miR-155-deficiency in mice results in mild vascular defects, forced expression of miR-155 causes endothelial hyperplasia and increases microglia count and activation. The mouse model of oxygen-induced retinopathy, which recapitulates ischemia-induced aberrant neovessel growth, is characterized by increased expression of miR-155 and localized areas of microglia activation. Interestingly, miR-155 deficiency in mice reduces microglial activation, curtails abnormal vessel growth, and allows for rapid normalization of the retinal vasculature following ischemic insult. miR-155 binds to the 3'-UTR and represses the expression of the CCN1 gene, which encodes an extracellular matrix-associated integrin-binding protein that both promotes physiological angiogenesis and harnesses growth factor-induced abnormal angiogenic responses. Single CCN1 deficiency or double CCN1 and miR-155 knock-out in mice causes retinal vascular malformations typical of faulty maturation, mimicking the vascular alterations of miR-155 gain of function. During development, the miR-155/CCN1 regulatory axis balances the proangiogenic and proinflammatory activities of microglia to allow for their function as guideposts for sprout fusion and anastomosis. Under ischemic conditions, dysregulated miR-155 and CCN1 expression increases the inflammatory load and microglial activation, prompting aberrant angiogenic responses. Thus, miR-155 functions in tandem with CCN1 to modulate inflammation-induced vascular homeostasis and repair.

  8. Fusion of SpCas9 to E. coli Rec A protein enhances CRISPR-Cas9 mediated gene knockout in mammalian cells.

    PubMed

    Lin, Lin; Petersen, Trine Skov; Jensen, Kristopher Torp; Bolund, Lars; Kühn, Ralf; Luo, Yonglun

    2017-03-01

    Mammalian cells repair double-strand DNA breaks (DSB) by a range of different pathways following DSB induction by the engineered clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein Cas9. While CRISPR-Cas9 thus enables predesigned modifications of the genome, applications of CRISPR-Cas9-mediated genome-editing are frequently hampered by the unpredictable and varying pathways for DSB repair in mammalian cells. Here we present a strategy of fusing Cas9 to recombinant proteins for fine-tuning of the DSB repair preferences in mammalian cells. By fusing Streptococcus Pyogenes Cas9 (SpCas9) to the recombinant protein A (Rec A, NP_417179.1) from Escherichia coli, we create a recombinant Cas9 protein (rSpCas9) which enhances the generation of indel mutations at DSB sites in mammalian cells, increases the frequency of DSB repair by homology-directed single-strand annealing (SSA), and represses homology-directed gene conversion by approximately 33%. Our study thus proves for the first time that fusing SpCas9 to recombinant proteins can influence the balance between DSB repair pathways in mammalian cells. This approach may form the basis for further investigations of the applications of recombinant Cas9 proteins to fine-tuning DSB repair pathways in eukaryotic cells.

  9. Evidence for early and progressive ultrasonic vocalization and oromotor deficits in a PINK1 gene knockout rat model of Parkinson's disease.

    PubMed

    Grant, Laura M; Kelm-Nelson, Cynthia A; Hilby, Breanna L; Blue, Katherine V; Paul Rajamanickam, Eunice S; Pultorak, Joshua D; Fleming, Shelia M; Ciucci, Michelle R

    2015-11-01

    Parkinson's disease (PD) is a progressive neurodegenerative disease that leads to a wide range of motor and nonmotor deficits. Specifically, voice and swallow deficits manifest early, are devastating to quality of life, and are difficult to treat with standard medical therapies. The pathological hallmarks of PD include accumulation of the presynaptic protein α-synuclein (αSyn) as well as degeneration of substantia nigra dopaminergic neurons. However, there is no clear understanding of how or when this pathology contributes to voice and swallow dysfunction in PD. The present study evaluates the effect of loss of function of the phosphatase and tensin homolog-induced putative kinase 1 gene in rats (PINK1(-/-) ), a model of autosomal recessive PD in humans, on vocalization, oromotor and limb function, and neurodegenerative pathologies. Behavioral measures include ultrasonic vocalizations, tongue force, biting, and gross motor performance that are assayed at 2, 4, 6, and 8 months of age. Aggregated αSyn and tyrosine hydroxylase immunoreactivity (TH-ir) were measured at 8 months. We show that, compared with wild-type controls, PINK1(-/-) rats develop (1) early and progressive vocalization and oromotor deficits, (2) reduced TH-ir in the locus coeruleus that correlates with vocal loudness and tongue force, and (3) αSyn neuropathology in brain regions important for cranial sensorimotor control. This novel approach of characterizing a PINK1(-/-) genetic model of PD provides the foundational work required to define behavioral biomarkers for the development of disease-modifying therapeutics for PD patients.

  10. Essential role of chicken ovalbumin upstream promoter-transcription factor II in insulin secretion and insulin sensitivity revealed by conditional gene knockout.

    PubMed

    Bardoux, Pascale; Zhang, Pili; Flamez, Daisy; Perilhou, Anaïs; Lavin, Tiphaine Aguirre; Tanti, Jean-François; Hellemans, Karine; Gomas, Emmanuel; Godard, Cécile; Andreelli, Fabrizio; Buccheri, Maria Antonietta; Kahn, Axel; Le Marchand-Brustel, Yannick; Burcelin, Rémy; Schuit, Frans; Vasseur-Cognet, Mireille

    2005-05-01

    Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) has been implicated in the control of blood glucose by its potent effect on expression and signaling of various nuclear receptors. To understand the role of COUP-TFII in glucose homeostasis, conditional COUP-TFII-deficient mice were generated and crossed with mice expressing Cre under the control of rat insulin II gene promoter, resulting in deletion of COUP-TFII in pancreatic beta-cells. Homozygous mutants died before birth for yet undetermined reasons. Heterozygous mice appeared healthy at birth and showed normal growth and fertility. When challenged intraperitoneally, the animals had glucose intolerance associated with reduced glucose-stimulated insulin secretion. Moreover, these heterozygous mice presented a mild increase in fasting and random-fed circulating insulin levels. In accordance, islets isolated from these animals exhibited higher insulin secretion in low glucose conditions and markedly decreased glucose-stimulated insulin secretion. Their pancreata presented normal microscopic architecture and insulin content up to 16 weeks of study. Altered insulin secretion was associated with peripheral insulin resistance in whole animals. It can be concluded that COUP-TFII is a new, important regulator of glucose homeostasis and insulin sensitivity.

  11. A knockout mutation in the lignin biosynthesis gene CCR1 explains a major QTL for acid detergent lignin content in Brassica napus seeds.

    PubMed

    Liu, Liezhao; Stein, Anna; Wittkop, Benjamin; Sarvari, Pouya; Li, Jiana; Yan, Xingying; Dreyer, Felix; Frauen, Martin; Friedt, Wolfgang; Snowdon, Rod J

    2012-05-01

    Seed coat phenolic compounds represent important antinutritive fibre components that cause a considerable reduction in value of seed meals from oilseed rape (Brassica napus). The nutritionally most important fibre compound is acid detergent lignin (ADL), to which a significant contribution is made by phenylpropanoid-derived lignin precursors. In this study, we used bulked-segregant analysis in a population of recombinant inbred lines (RILs) from a cross of the Chinese oilseed rape lines GH06 (yellow seed, low ADL) and P174 (black seed, high ADL) to identify markers with tight linkage to a major quantitative trait locus (QTL) for seed ADL content. Fine mapping of the QTL was performed in a backcross population comprising 872 BC(1)F(2) plants from a cross of an F(7) RIL from the above-mentioned population, which was heterozygous for this major QTL and P174. A 3:1 phenotypic segregation for seed ADL content indicated that a single, dominant, major locus causes a substantial reduction in ADL. This locus was successively narrowed to 0.75 cM using in silico markers derived from a homologous Brassica rapa sequence contig spanning the QTL. Subsequently, we located a B. rapa orthologue of the key lignin biosynthesis gene CINNAMOYL CO-A REDUCTASE 1 (CCR1) only 600 kbp (0.75 cM) upstream of the nearest linked marker. Sequencing of PCR amplicons, covering the full-length coding sequences of Bna.CCR1 homologues, revealed a locus in P174 whose sequence corresponds to the Brassica oleracea wild-type allele from chromosome C8. In GH06, however, this allele is replaced by a homologue derived from chromosome A9 that contains a loss-of-function frameshift mutation in exon 1. Genetic and physical map data infer that this loss-of-function allele has replaced a functional Bna.CCR1 locus on chromosome C8 in GH06 by homoeologous non-reciprocal translocation.

  12. Generation of conditional knockout alleles for PRL-3.

    PubMed

    Yan, Hong; Kong, Dong; Ge, Xiaomei; Gao, Xiang; Han, Xiao

    2011-11-01

    Phosphatase of regenerating liver-3 (PRL-3) is a member of the protein tyrosine phosphatase (PTP) superfamily and is highly expressed in cancer metastases. For better understanding of the role of PRL-3 in tumor metastasis, we applied a rapid and efficient method for generating PRL-3 floxed mice and investigated its phenotypes. A BAC retrieval strategy was applied to construct the PRL-3 conditional gene-targeting vector. Exon 4 was selected for deletion to generate a nonfunctional prematurely terminated short peptide as it will cause a frame-shift mutation. Conditional knockout PRL-3 mice were generated by using the Cre-loxP system and were validated by Southern blot and RT-PCR analysis. Further analysis revealed the phenotype characteristics of PRL-3 knockout mice and wildtype mice. In this study, we successfully constructed the PRL-3 conditional knockout mice, which will be helpful to clarify the roles of PRL-3 and the mechanisms in tumor metastasis.

  13. Quantitative modeling of the molecular steps underlying shut-off of rhodopsin activity in rod phototransduction

    PubMed Central

    Kraft, Timothy W.

    2016-01-01

    biochemical expectations. However, for the arrestin knockout (Arr−/−) phenotype, the predictions deviated from experimental findings and led us to invoke a low-activity state that R* enters before arrestin binding. Our simulations of this three-state R* shut-off model are very similar to those of the binary model in the WT case but are preferred because they appear to accurately predict the mean SPRs for four mutant phenotypes, Arr+/−, Arr−/−, GRK1+/−, and GRK1−/−, in addition to the WT phenotype. When we additionally treated the formation and shut-off of activated phosphodiesterase (E*) as stochastic, the simulated SPRs appeared even more similar to real SPRs, and there was very little change in the ensemble mean and standard deviation or in the amplitude distribution. Conclusions We conclude that the conventional model of graded reduction in R* activity through successive phosphorylation steps appears to be inconsistent with experimental results. Instead, we find that two variants of a model in which R* activity initially remains high and then declines abruptly after several phosphorylation steps appears capable of providing a better description of experimentally measured SPRs. PMID:27375353

  14. INDUCTION OF MAMMARY GLAND DEVELOPMENT IN ESTROGEN RECEPTOR-ALPHA KNOCKOUT MICE

    EPA Science Inventory

    Mammary glands from the estrogen receptor knockout ( ERKO) mouse do not undergo ductal morphogenesis or alveolar development. Disrupted Er signaling may result in reduced estrogen-responsive gene products in the mammary gland or reduced mammotropic hormones that contribute t...

  15. Knockout, Transfer and Spectroscopic Factors

    NASA Astrophysics Data System (ADS)

    Kemper, Kirby; Keeley, Nicholas; Rusek, Krzysztof

    2011-10-01

    As derived quantities rather than observables, spectroscopic factors extracted from fits to data are model dependent. The main source of uncertainty is the choice of binding potential, but other factors such as adequate modeling of the reaction mechanism, the Perey effect, choice of distorting nuclear potentials etc. can also play a significant role. Recently, there has been some discussion of apparent discrepancies in spectroscopic factors derived from knockout reactions compared to those obtained from low-energy direct reactions. It should be possible to reconcile these discrepancies and we explore this prospect by attempting to describe the 10Be(d,t)9Be data of Nucl. Phys. A157, 305 (1970) using the 10Be/9Be form factors from a recent knockout study, Phys. Rev. Lett. 106, 162502 (2011). The influence of such factors as choice of distorting potentials and multi-step reactions paths will be explored.

  16. RDGBα, a PtdIns-PtdOH transfer protein, regulates G-protein-coupled PtdIns(4,5)P2 signalling during Drosophila phototransduction

    PubMed Central

    Yadav, Shweta; Garner, Kathryn; Georgiev, Plamen; Li, Michelle; Gomez-Espinosa, Evelyn; Panda, Aniruddha; Mathre, Swarna; Okkenhaug, Hanneke; Cockcroft, Shamshad; Raghu, Padinjat

    2015-01-01

    ABSTRACT Many membrane receptors activate phospholipase C (PLC) during signalling, triggering changes in the levels of several plasma membrane lipids including phosphatidylinositol (PtdIns), phosphatidic acid (PtdOH) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. It is widely believed that exchange of lipids between the plasma membrane and endoplasmic reticulum (ER) is required to restore lipid homeostasis during PLC signalling, yet the mechanism remains unresolved. RDGBα (hereafter RDGB) is a multi-domain protein with a PtdIns transfer protein (PITP) domain (RDGB-PITPd). We find that, in vitro, the RDGB-PITPd binds and transfers both PtdOH and PtdIns. In Drosophila photoreceptors, which experience high rates of PLC activity, RDGB function is essential for phototransduction. We show that binding of PtdIns to RDGB-PITPd is essential for normal phototransduction; however, this property is insufficient to explain the in vivo function because another Drosophila PITP (encoded by vib) that also binds PtdIns cannot rescue the phenotypes of RDGB deletion. In RDGB mutants, PtdIns(4,5)P2 resynthesis at the plasma membrane following PLC activation is delayed and PtdOH levels elevate. Thus RDGB couples the turnover of both PtdIns and PtdOH, key lipid intermediates during G-protein-coupled PtdIns(4,5)P2 turnover. PMID:26203165

  17. Deconstructing mammalian reproduction: using knockouts to define fertility pathways.

    PubMed

    Roy, Angshumoy; Matzuk, Martin M

    2006-02-01

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

  18. Central nervous system-specific knockout of steroidogenic factor 1.

    PubMed

    Kim, Ki Woo; Zhao, Liping; Parker, Keith L

    2009-03-05

    Steroidogenic factor 1 (SF-1) is a nuclear receptor that plays important roles in the hypothalamus-pituitary-steroidogenic organ axis. Global knockout studies in mice revealed the essential in vivo roles of SF-1 in the ventromedial hypothalamic (VMH) nucleus, adrenal glands, and gonads. One limitation of global SF-1 knockout mice is their early postnatal death from adrenocortical insufficiency. To overcome limitations of the global knockout mice and to delineate the roles of SF-1 in the brain, we used Cre/loxP recombination technology to genetically ablate SF-1 specifically in the central nervous system (CNS). Mice with CNS-specific knockout of SF-1 mediated by nestin-Cre showed increased anxiety-like behavior, revealing a crucial role of SF-1 in a complex behavioral phenotype. Our studies with CNS-specific SF-1 KO mice also defined roles of SF-1 in regulating the VMH expression of target genes implicated in anxiety and energy homeostasis. Therefore, this review will focus on our recent studies defining the functional roles of SF-1 in the VMH linked to anxiety and energy homeostasis.

  19. Pleiotropic effects in Eya3 knockout mice

    PubMed Central

    Söker, Torben; Dalke, Claudia; Puk, Oliver; Floss, Thomas; Becker, Lore; Bolle, Ines; Favor, Jack; Hans, Wolfgang; Hölter, Sabine M; Horsch, Marion; Kallnik, Magdalena; Kling, Eva; Moerth, Corinna; Schrewe, Anja; Stigloher, Christian; Topp, Stefanie; Gailus-Durner, Valerie; Naton, Beatrix; Beckers, Johannes; Fuchs, Helmut; Ivandic, Boris; Klopstock, Thomas; Schulz, Holger; Wolf, Eckhard; Wurst, Wolfgang; Bally-Cuif, Laure; de Angelis, Martin Hrabé; Graw, Jochen

    2008-01-01

    Background In Drosophila, mutations in the gene eyes absent (eya) lead to severe defects in eye development. The functions of its mammalian orthologs Eya1-4 are only partially understood and no mouse model exists for Eya3. Therefore, we characterized the phenotype of a new Eya3 knockout mouse mutant. Results Expression analysis of Eya3 by in-situ hybridizations and β-Gal-staining of Eya3 mutant mice revealed abundant expression of the gene throughout development, e.g. in brain, eyes, heart, somites and limbs suggesting pleiotropic effects of the mutated gene. A similar complex expression pattern was observed also in zebrafish embryos. The phenotype of young adult Eya3 mouse mutants was systematically analyzed within the German Mouse Clinic. There was no obvious defect in the eyes, ears and kidneys of Eya3 mutant mice. Homozygous mutants displayed decreased bone mineral content and shorter body length. In the lung, the tidal volume at rest was decreased, and electrocardiography showed increased JT- and PQ intervals as well as decreased QRS amplitude. Behavioral analysis of the mutants demonstrated a mild increase in exploratory behavior, but decreased locomotor activity and reduced muscle strength. Analysis of differential gene expression revealed 110 regulated genes in heart and brain. Using real-time PCR, we confirmed Nup155 being down regulated in both organs. Conclusion The loss of Eya3 in the mouse has no apparent effect on eye development. The wide-spread expression of Eya3 in mouse and zebrafish embryos is in contrast to the restricted expression pattern in Xenopus embryos. The loss of Eya3 in mice leads to a broad spectrum of minor physiological changes. Among them, the mutant mice move less than the wild-type mice and, together with the effects on respiratory, muscle and heart function, the mutation might lead to more severe effects when the mice become older. Therefore, future investigations of Eya3 function should focus on aging mice. PMID:19102749

  20. Dcdc2 knockout mice display exacerbated developmental disruptions following knockdown of doublecortin.

    PubMed

    Wang, Y; Yin, X; Rosen, G; Gabel, L; Guadiana, S M; Sarkisian, M R; Galaburda, A M; Loturco, J J

    2011-09-08

    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 (wt) 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.

  1. Simultaneous paralogue knockout using a CRISPR-concatemer in mouse small intestinal organoids.

    PubMed

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

    2016-10-27

    Approaches based on genetic modification have been invaluable for investigating a wide array of biological processes, with gain- and loss-of-function approaches frequently used to investigate gene function. However, the presence of paralogues, and hence possible genetic compensation, for many genes necessitates the knockout (KO) of all paralogous genes in order to observe clear phenotypic change. CRISPR technology, the most recently described tool for gene editing, can generate KOs with unprecedented ease and speed and has been used in adult stem cell-derived organoids for single gene knockout, gene knock-in and gene correction. However, the simultaneous targeting of multiple genes in organoids by CRISPR technology has not previously been described. Here we describe a rapid, scalable and cost effective method for generating double knockouts in organoids. By concatemerizing multiple gRNA expression cassettes, we generated a 'gRNA concatemer vector'. Our method allows the rapid assembly of annealed synthetic DNA oligos into the final vector in a single step. This approach facilitates simultaneous delivery of multiple gRNAs to allow up to 4 gene KO in one step, or potentially to increase the efficiency of gene knockout by providing multiple gRNAs targeting one gene. As a proof of concept, we knocked out negative regulators of the Wnt pathway in small intestinal organoids, thereby removing their growth dependence on the exogenous Wnt enhancer, R-spondin1.

  2. The evolution of thymic lymphomas in p53 knockout mice

    PubMed Central

    Dudgeon, Crissy; Chan, Chang; Kang, Wenfeng; Sun, Yvonne; Emerson, Ryan; Robins, Harlan

    2014-01-01

    Germline deletion of the p53 gene in mice gives rise to spontaneous thymic (T-cell) lymphomas. In this study, the p53 knockout mouse was employed as a model to study the mutational evolution of tumorigenesis. The clonality of the T-cell repertoire from p53 knockout and wild-type thymic cells was analyzed at various ages employing TCRβ sequencing. These data demonstrate that p53 knockout thymic lymphomas arose in an oligoclonal fashion, with tumors evolving dominant clones over time. Exon sequencing of tumor DNA revealed that all of the independently derived oligoclonal mouse tumors had a deletion in the Pten gene prior to the formation of the TCRβ rearrangement, produced early in development. This was followed in each independent clone of the thymic lymphoma by the amplification or overexpression of cyclin Ds and Cdk6. Alterations in the expression of Ikaros were common and blocked further development of CD-4/CD-8 T cells. While the frequency of point mutations in the genome of these lymphomas was one per megabase, there were a tremendous number of copy number variations producing the tumors’ driver mutations. The initial inherited loss of p53 functions appeared to delineate an order of genetic alterations selected for during the evolution of these thymic lymphomas. PMID:25452272

  3. GARP2 accelerates retinal degeneration in rod cGMP-gated cation channel β-subunit knockout mice

    PubMed Central

    DeRamus, Marci L.; Stacks, Delores A.; Zhang, Youwen; Huisingh, Carrie E.; McGwin, Gerald; Pittler, Steven J.

    2017-01-01

    The Cngb1 locus-encoded β-subunit of rod cGMP-gated cation channel and associated glutamic acid rich proteins (GARPs) are required for phototransduction, disk morphogenesis, and rod structural integrity. To probe individual protein structure/function of the GARPs, we have characterized several transgenic mouse lines selectively restoring GARPs on a Cngb1 knockout (X1−/−) mouse background. Optical coherence tomography (OCT), light and transmission electron microscopy (TEM), and electroretinography (ERG) were used to analyze 6 genotypes including WT at three and ten weeks postnatal. Comparison of aligned histology/OCT images demonstrated that GARP2 accelerates the rate of degeneration. ERG results are consistent with the structural analyses showing the greatest attenuation of function when GARP2 is present. Even 100-fold or more overexpression of GARP1 could not accelerate degeneration as rapidly as GARP2, and when co-expressed GARP1 attenuated the structural and functional deficits elicited by GARP2. These results indicate that the GARPs are not fully interchangeable and thus, likely have separate and distinct functions in the photoreceptor. We also present a uniform murine OCT layer naming nomenclature system that is consistent with human retina layer designations to standardize murine OCT, which will facilitate data evaluation across different laboratories. PMID:28198469

  4. Human Genetic Disorders and Knockout Mice Deficient in Glycosaminoglycan

    PubMed Central

    2014-01-01

    Glycosaminoglycans (GAGs) are constructed through the stepwise addition of respective monosaccharides by various glycosyltransferases and maturated by epimerases and sulfotransferases. The structural diversity of GAG polysaccharides, including their sulfation patterns and sequential arrangements, is essential for a wide range of biological activities such as cell signaling, cell proliferation, tissue morphogenesis, and interactions with various growth factors. Studies using knockout mice of enzymes responsible for the biosynthesis of the GAG side chains of proteoglycans have revealed their physiological functions. Furthermore, mutations in the human genes encoding glycosyltransferases, sulfotransferases, and related enzymes responsible for the biosynthesis of GAGs cause a number of genetic disorders including chondrodysplasia, spondyloepiphyseal dysplasia, and Ehlers-Danlos syndromes. This review focused on the increasing number of glycobiological studies on knockout mice and genetic diseases caused by disturbances in the biosynthetic enzymes for GAGs. PMID:25126564

  5. Iron regulatory protein-2 knockout increases perihematomal ferritin expression and cell viability after intracerebral hemorrhage.

    PubMed

    Chen, Mai; Awe, Olatilewa O; Chen-Roetling, Jing; Regan, Raymond F

    2010-06-14

    Iron is deposited in perihematomal tissue after an intracerebral hemorrhage (ICH), and may contribute to oxidative injury. Cell culture studies have demonstrated that enhancing ferritin expression by targeting iron regulatory protein (IRP) binding activity reduces cellular vulnerability to iron and hemoglobin. In order to assess the therapeutic potential of this approach after striatal ICH, the effect of IRP1 or IRP2 gene knockout on ferritin expression and injury was quantified. Striatal ferritin in IRP1 knockout mice was similar to that in wild-type controls 3 days after stereotactic injection of artificial CSF or autologous blood. Corresponding levels in IRP2 knockouts were increased by 11-fold and 8.4-fold, respectively, compared with wild-type. Protein carbonylation, a sensitive marker of hemoglobin neurotoxicity, was increased by 2.4-fold in blood-injected wild-type striata, was not altered by IRP1 knockout, but was reduced by approximately 60% by IRP2 knockout. Perihematomal cell viability in wild-type mice, assessed by MTT assay, was approximately half of that in contralateral striata at 3 days, and was significantly increased in IRP2 knockouts but not in IRP1 knockouts. Protection was also observed when hemorrhage was induced by collagenase injection. These results suggest that IRP2 binding activity reduces ferritin expression in the striatum after ICH, preventing an optimal response to elevated local iron concentrations. IRP2 binding activity may be a novel therapeutic target after hemorrhagic CNS injuries.

  6. Iron Regulatory Protein-2 Knockout Increases Perihematomal Ferritin Expression and Cell Viability after Intracerebral Hemorrhage

    PubMed Central

    Chen, Mai; Awe, Olatilewa O.; Chen-Roetling, Jing; Regan, Raymond F.

    2010-01-01

    Iron is deposited in perihematomal tissue after an intracerebral hemorrhage (ICH), and may contribute to oxidative injury. Cell culture studies have demonstrated that enhancing ferritin expression by targeting iron regulatory protein (IRP) binding activity reduces cellular vulnerability to iron and hemoglobin. In order to assess the therapeutic potential of this approach after striatal ICH, the effect of IRP1 or IRP2 gene knockout on ferritin expression and injury was quantified. Striatal ferritin in IRP1 knockout mice was similar to that in wild-type controls three days after stereotactic injection of artificial CSF or autologous blood. Corresponding levels in IRP2 knockouts were increased by 11-fold and 8.4-fold, respectively, compared with wild-type. Protein carbonylation, a sensitive marker of hemoglobin neurotoxicity, was increased by 2.4-fold in blood-injected wild-type striata, was not altered by IRP1 knockout, but was reduced by approximately 60% by IRP2 knockout. Perihematomal cell viability in wild-type mice, assessed by MTT assay, was approximately half of that in contralateral striata at three days, and was significantly increased in IRP2 knockouts but not in IRP1 knockouts. Protection was also observed when hemorrhage was induced by collagenase injection. These results suggest that IRP2 binding activity reduces ferritin expression in the striatum after ICH, preventing an optimal response to elevated local iron concentrations. IRP2 binding activity may be a novel therapeutic target after hemorrhagic CNS injuries. PMID:20399759

  7. Phosphorylation of the Drosophila transient receptor potential ion channel is regulated by the phototransduction cascade and involves several protein kinases and phosphatases.

    PubMed

    Voolstra, Olaf; Bartels, Jonas-Peter; Oberegelsbacher, Claudia; Pfannstiel, Jens; Huber, Armin

    2013-01-01

    Protein phosphorylation plays a cardinal role in regulating cellular processes in eukaryotes. Phosphorylation of proteins is controlled by protein kinases and phosphatases. We previously reported the light-dependent phosphorylation of the Drosophila transient receptor potential (TRP) ion channel at multiple sites. TRP generates the receptor potential upon stimulation of the photoreceptor cell by light. An eye-enriched protein kinase C (eye-PKC) has been implicated in the phosphorylation of TRP by in vitro studies. Other kinases and phosphatases of TRP are elusive. Using phosphospecific antibodies and mass spectrometry, we here show that phosphorylation of most TRP sites depends on the phototransduction cascade and the activity of the TRP ion channel. A candidate screen to identify kinases and phosphatases provided in vivo evidence for an involvement of eye-PKC as well as other kinases and phosphatases in TRP phosphorylation.

  8. AMPK: Lessons from transgenic and knockout animals

    PubMed Central

    Viollet, Benoit; Athea, Yoni; Mounier, Remi; Guigas, Bruno; Zarrinpashneh, Elham; Horman, Sandrine; Lantier, Louise; Hebrard, Sophie; Devin-Leclerc, Jocelyne; Beauloye, Christophe; Foretz, Marc; Andreelli, Fabrizio; Ventura-Clapier, Renee; Bertrand, Luc

    2009-01-01

    AMP-activated protein kinase (AMPK), a phylogenetically conserved serine/threonine protein kinase, has been proposed to function as a ‘fuel gauge’ to monitor cellular energy status in response to nutritional environmental variations. AMPK system is a regulator of energy balance that, once activated by low energy status, switches on ATP-producing catabolic pathways (such as fatty acid oxidation and glycolysis), and switches off ATP-consuming anabolic pathways (such as lipogenesis), both by short-term effect on phosphorylation of regulatory proteins and by long-term effect on gene expression. Numerous observations obtained with pharmacological activators and agents that deplete intracellular ATP have been supportive of AMPK playing a role in the control of energy metabolism but none of these studies have provided conclusive evidence. Relatively recent developments in our understanding of precisely how AMPK complexes might operate to control energy metabolism is due in part to the development of transgenic and knockout mouse models. Although there are inevitable caveats with genetic models, some important findings have emerged. In the present review, we discuss recent findings obtained from animal models with inhibition or activation of AMPK signaling pathway. PMID:19273052

  9. Kanamycin ototoxicity in glutamate transporter knockout mice.

    PubMed

    Shimizu, Yoshitaka; Hakuba, Nobuhiro; Hyodo, Jun; Taniguchi, Masafumi; Gyo, Kiyofumi

    2005-06-03

    Glutamate-aspartate transporter (GLAST), a powerful glutamate uptake system, removes released glutamate from the synaptic cleft and facilitates the re-use of glutamate as a neurotransmitter recycling system. Aminoglycoside-induced hearing loss is mediated via a glutamate excitotoxic process. We investigated the effect of aminoglycoside ototoxicity in GLAST knockout mice using the recorded auditory brainstem response (ABR) and number of hair cells in the cochlea. Kanamycin (100 mg/mL) was injected directly into the posterior semicircular canal of mice. Before the kanamycin treatment, there was no difference in the ABR threshold average between the wild-type and knockout mice. Kanamycin injection aggravated the ABR threshold in the GLAST knockout mice compared with the wild-type mice, and the IHC degeneration was more severe in the GLAST knockout mice. These findings suggest that GLAST plays an important role in preventing the degeneration of inner hair cells in aminoglycoside ototoxicity.

  10. Knock-Out Models Reveal New Aquaporin Functions

    PubMed Central

    Verkman, Alan S.

    2013-01-01

    Knockout mice have been informative in the discovery of unexpected biological functions of aquaporins. Knockout mice have confirmed the predicted roles of aquaporins in transepithelial fluid transport, as in the urinary concentrating mechanism and glandular fluid secretion. A less obvious, though predictable role of aquaporins is in tissue swelling under stress, as in the brain in stroke, tumor and infection. Phenotype analysis of aquaporin knockout mice has revealed several unexpected cellular roles of aquaporins whose mechanisms are being elucidated. Aquaporins facilitate cell migration, as seen in aquaporin-dependent tumor angiogenesis and tumor metastasis, by a mechanism that may involve facilitated water transport in lamellipodia of migrating cells. The ‘aquaglyceroporins’, aquaporins that transport both glycerol and water, regulate glycerol content in epidermis, fat and other tissues, and lead to a multiplicity of interesting consequences of gene disruption including dry skin, resistance to skin carcinogenesis, impaired cell proliferation and altered fat metabolism. An even more surprising role of a mammalian aquaporin is in neural signal transduction in the central nervous system. The many roles of aquaporins might be exploited for clinical benefit by modulation of aquaporin expression/function – as diuretics, and in the treatment of brain swelling, glaucoma, epilepsy, obesity and cancer. PMID:19096787

  11. Generation of TALEN-mediated FH knockout rat model.

    PubMed

    Yu, Dandan; Zhong, Yali; Li, Xiaoran; Li, Yaqing; Li, Xiaoli; Cao, Jing; Fan, Zhirui; Fan, Huijie; Yuan, Long; Xu, Benling; Yuan, Yuan; Zhang, Hongquan; Ji, Zhenyu; Wen, Jian-Guo; Zhang, Mingzhi; Nesland, Jahn M; Suo, Zhenhe

    2016-09-20

    Transcription activator-like effector nucleases (TALENs) are valuable tools for precise genome engineering of laboratory animals. Here we utilized this technique for efficient site-specific gene modification to create a fumarate hydratase (FH) gene knockout rat model, in which there was an 11 base-pair deletion in the first exon of the FH gene in 111 rats. 18 live-born targeted mutation offsprings were produced from 80 injected zygotes with 22.5% efficiency, indicating high TALEN knockout success in rat zygots. Only heterozygous deletion was observed in the offsprings. Sixteen pairs of heterozygous FH knockout (FH+/-) rats were arranged for mating experiments for six months without any homozygous KO rat identified. Sequencing from the pregnant rats embryo samples showed no homozygous FH KO, indicating that homozygous FH KO is embryonically lethal. Comparatively, the litter size was decreased in both male and female FH+/- KO rats. There was no behaviour difference between the FH+/- KO and the control rats except that the FH+/- KO male rats showed significantly higher body weight in the 16-week observation period. Clinical haematology and biochemical examinations showed hematopoietic and kidney dysfunction in the FH+/- KO rats. Small foci of anaplastic lesions of tubular epithelial cells around glomeruli were identified in the FH+/- kidney, and these anaplastic cells were comparatively positive for Ki67, p53 and Sox9, and such findings are most probably related to the kidney dysfunction reflected by the biochemical examinations of the rats. In conclusion, we have successfully established an FH+/- KO rat model, which will be useful for further functional FH studies.

  12. Generation of TALEN-mediated FH knockout rat model

    PubMed Central

    Yu, Dandan; Zhong, Yali; Li, Xiaoran; Li, Yaqing; Li, Xiaoli; Cao, Jing; Fan, Zhirui; Fan, Huijie; Yuan, Long; Xu, Benling; Yuan, Yuan; Zhang, Hongquan; Ji, Zhenyu; Wen, Jian-Guo; Zhang, Mingzhi; Nesland, Jahn M.; Suo, Zhenhe

    2016-01-01

    Transcription activator-like effector nucleases (TALENs) are valuable tools for precise genome engineering of laboratory animals. Here we utilized this technique for efficient site-specific gene modification to create a fumarate hydratase (FH) gene knockout rat model, in which there was an 11 base-pair deletion in the first exon of the FH gene in 111 rats. 18 live-born targeted mutation offsprings were produced from 80 injected zygotes with 22.5% efficiency, indicating high TALEN knockout success in rat zygots. Only heterozygous deletion was observed in the offsprings. Sixteen pairs of heterozygous FH knockout (FH+/−) rats were arranged for mating experiments for six months without any homozygous KO rat identified. Sequencing from the pregnant rats embryo samples showed no homozygous FH KO, indicating that homozygous FH KO is embryonically lethal. Comparatively, the litter size was decreased in both male and female FH+/− KO rats. There was no behaviour difference between the FH+/− KO and the control rats except that the FH+/− KO male rats showed significantly higher body weight in the 16-week observation period. Clinical haematology and biochemical examinations showed hematopoietic and kidney dysfunction in the FH+/− KO rats. Small foci of anaplastic lesions of tubular epithelial cells around glomeruli were identified in the FH+/− kidney, and these anaplastic cells were comparatively positive for Ki67, p53 and Sox9, and such findings are most probably related to the kidney dysfunction reflected by the biochemical examinations of the rats. In conclusion, we have successfully established an FH+/− KO rat model, which will be useful for further functional FH studies. PMID:27556703

  13. Molecular mechanisms of cocaine reward: Combined dopamine and serotonin transporter knockouts eliminate cocaine place preference

    PubMed Central

    Sora, Ichiro; Hall, F. Scott; Andrews, Anne M.; Itokawa, Masanari; Li, Xiao-Fei; Wei, Hong-Bing; Wichems, Christine; Lesch, Klaus-Peter; Murphy, Dennis L.; Uhl, George R.

    2001-01-01

    Cocaine blocks uptake by neuronal plasma membrane transporters for dopamine (DAT), serotonin (SERT), and norepinephrine (NET). Cocaine reward/reinforcement has been linked to actions at DAT or to blockade of SERT. However, knockouts of neither DAT, SERT, or NET reduce cocaine reward/reinforcement, leaving substantial uncertainty about cocaine's molecular mechanisms for reward. Conceivably, the molecular bases of cocaine reward might display sufficient redundancy that either DAT or SERT might be able to mediate cocaine reward in the other's absence. To test this hypothesis, we examined double knockout mice with deletions of one or both copies of both the DAT and SERT genes. These mice display viability, weight gain, histologic features, neurochemical parameters, and baseline behavioral features that allow tests of cocaine influences. Mice with even a single wild-type DAT gene copy and no SERT copies retain cocaine reward/reinforcement, as measured by conditioned place-preference testing. However, mice with no DAT and either no or one SERT gene copy display no preference for places where they have previously received cocaine. The serotonin dependence of cocaine reward in DAT knockout mice is thus confirmed by the elimination of cocaine place preference in DAT/SERT double knockout mice. These results provide insights into the brain molecular targets necessary for cocaine reward in knockout mice that develop in their absence and suggest novel strategies for anticocaine medication development. PMID:11320258

  14. Ultrastructural analysis of megakaryocytes in GPV knockout mice.

    PubMed

    Poujol, C; Ramakrishnan, V; DeGuzman, F; Nurden, A T; Phillips, D R; Nurden, P

    2000-08-01

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

  15. Knockout of leucine aminopeptidase in Toxoplasma gondii using CRISPR/Cas9.

    PubMed

    Zheng, Jun; Jia, Honglin; Zheng, Yonghui

    2015-02-01

    Leucine aminopeptidases of the M17 peptidase family represent ideal drug targets for therapies directed against the pathogens Plasmodium, Babesia and Trypanosoma. Previously, we characterised Toxoplasma gondii leucine aminopeptidase and demonstrated its role in regulating the levels of free amino acids. In this study, we evaluated the potential of T. gondii leucine aminopeptidase as a drug target in T. gondii by a knockout method. Existing knockout methods for T. gondii have many drawbacks; therefore, we developed a new technique that takes advantage of the CRISPR/Cas9 system. We first chose a Cas9 target site in the gene encoding T. gondii leucine aminopeptidase and then constructed a knockout vector containing Cas9 and the single guide RNA. After transfection, single tachyzoites were cloned in 96-well plates by limiting dilution. Two transfected strains derived from a single clone were cultured in Vero cells, and then subjected to expression analysis by western blotting. The phenotypic analysis revealed that knockout of T. gondii leucine aminopeptidase resulted in inhibition of attachment/invasion and replication; both the growth and attachment/invasion capacity of knockout parasites were restored by complementation with a synonymously substituted allele of T. gondii leucine aminopeptidase. Mouse experiments demonstrated that T. gondii leucine aminopeptidase knockout somewhat reduced the pathogenicity of T. gondii. An enzymatic activity assay showed that T. gondii leucine aminopeptidase knockout reduced the processing of a leucine aminopeptidase-specific substrate in T. gondii. The absence of leucine aminopeptidase activity could be slightly compensated for in T. gondii. Overall, T. gondii leucine aminopeptidase knockout influenced the growth of T. gondii, but did not completely block parasite development, virulence or enzymatic activity. Therefore, we conclude that leucine aminopeptidase would be useful only as an adjunctive drug target in T. gondii.

  16. Phenotype of the taurine transporter knockout mouse.

    PubMed

    Warskulat, Ulrich; Heller-Stilb, Birgit; Oermann, Evelyn; Zilles, Karl; Haas, Helmut; Lang, Florian; Häussinger, Dieter

    2007-01-01

    This chapter reports present knowledge on the properties of mice with disrupted gene coding for the taurine transporter (taut-/- mice). Study of those mice unraveled some of the roles of taurine and its membrane transport for the development and maintenance of normal organ functions and morphology. When compared with wild-type controls, taut-/- mice have decreased taurine levels in skeletal and heart muscle by about 98%, in brain, kidney, plasma, and retina by 80 to 90%, and in liver by about 70%. taut-/- mice exhibit a lower body mass as well as a strongly reduced exercise capacity compared with taut+/- and wild-type mice. Furthermore, taut-/- mice show a variety of pathological features, for example, subtle derangement of renal osmoregulation, changes in neuroreceptor expression, and loss of long-term potentiation in the striatum, and they develop clinically relevant age-dependent disorders, for example, visual, auditory, and olfactory dysfunctions, unspecific hepatitis, and liver fibrosis. Taurine-deficient animal models such as acutely dietary-manipulated foxes and cats, pharmacologically induced taurine-deficient rats, and taurine transporter knockout mouse are powerful tools allowing identification of the mechanisms and complexities of diseases mediated by impaired taurine transport and taurine depletion (Chapman et al., 1993; Heller-Stilb et al., 2002; Huxtable, 1992; Lake, 1993; Moise et al., 1991; Novotny et al., 1991; Pion et al., 1987; Timbrell et al., 1995; Warskulat et al., 2004, 2006b). Taurine, which is the most abundant amino acid in many tissues, is normally found in intracellular concentrations of 10 to 70 mmol/kg in mammalian heart, brain, skeletal muscle, liver, and retina (Chapman et al., 1993; Green et al., 1991; Huxable, 1992; Timbrell et al., 1995). These high taurine levels are maintained by an ubiquitous expression of Na(+)-dependent taurine transporter (TAUT) in the plasma membrane (Burg, 1995; Kwon and Handler, 1995; Lang et al., 1998

  17. Sensitivity of heterozygous α1,6-fucosyltransferase knock-out mice to cigarette smoke-induced emphysema: implication of aberrant transforming growth factor-β signaling and matrix metalloproteinase gene expression.

    PubMed

    Gao, Congxiao; Maeno, Toshitaka; Ota, Fumi; Ueno, Manabu; Korekane, Hiroaki; Takamatsu, Shinji; Shirato, Ken; Matsumoto, Akio; Kobayashi, Satoshi; Yoshida, Keiichi; Kitazume, Shinobu; Ohtsubo, Kazuaki; Betsuyaku, Tomoko; Taniguchi, Naoyuki

    2012-05-11

    We previously demonstrated that a deficiency in core fucosylation caused by the genetic disruption of α1,6-fucosyltransferase (Fut8) leads to lethal abnormalities and the development of emphysematous lesions in the lung by attenuation of TGF-β1 receptor signaling. Herein, we investigated the physiological relevance of core fucosylation in the pathogenesis of emphysema using viable heterozygous knock-out mice (Fut8(+/-)) that were exposed to cigarette smoke (CS). The Fut8(+/-) mice exhibited a marked decrease in FUT8 activity, and matrix metalloproteinase (MMP)-9 activities were elevated in the lung at an early stage of exposure. Emphysema developed after a 3-month CS exposure, accompanied by the recruitment of large numbers of macrophages to the lung. CS exposure substantially and persistently elevated the expression level of Smad7, resulting in a significant reduction of Smad2 phosphorylation (which controls MMP-9 expression) in Fut8(+/-) mice and Fut8-deficient embryonic fibroblast cells. These in vivo and in vitro studies show that impaired core fucosylation enhances the susceptibility to CS and constitutes at least part of the disease process of emphysema, in which TGF-β-Smad signaling is impaired and the MMP-mediated destruction of lung parenchyma is up-regulated.

  18. Proton Knock-Out in Hall A

    SciTech Connect

    Kees de Jager

    2002-06-01

    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.

  19. Brief Report: Altered Social Behavior in Isolation-Reared "Fmr1" Knockout Mice

    ERIC Educational Resources Information Center

    Heitzer, Andrew M.; Roth, Alexandra K.; Nawrocki, Lauren; Wrenn, Craige C.; Valdovinos, Maria G.

    2013-01-01

    Social behavior abnormalities in Fragile X syndrome (FXS) are characterized by social withdrawal, anxiety, and deficits in social cognition. To assess these deficits, a model of FXS, the "Fmr1" knockout mouse ("Fmr1" KO), has been utilized. This mouse model has a null mutation in the fragile X mental retardation 1 gene ("Fmr1") and displays…

  20. Loss of cone cyclic nucleotide-gated channel leads to alterations in light response modulating system and cellular stress response pathways: a gene expression profiling study

    PubMed Central

    Ma, Hongwei; Thapa, Arjun; Morris, Lynsie M.; Michalakis, Stylianos; Biel, Martin; Frank, Mark Barton; Bebak, Melissa; Ding, Xi-Qin

    2013-01-01

    The cone photoreceptor cyclic nucleotide-gated (CNG) channel is essential for central and color vision and visual acuity. Mutations in the channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophy. We investigated the gene expression profiles in mouse retina with CNG channel deficiency using whole genome expression microarrays. As cones comprise only 2 to 3% of the total photoreceptor population in the wild-type mouse retina, the mouse lines with CNG channel deficiency on a cone-dominant background, i.e. Cnga3−/−/Nrl−/− and Cngb3−/−/Nrl−/− mice, were used in our study. Comparative data analysis revealed a total of 105 genes altered in Cnga3−/−/Nrl−/− and 92 in Cngb3−/−/Nrl−/− retinas, relative to Nrl−/− retinas, with 27 genes changed in both genotypes. The differentially expressed genes primarily encode proteins associated with cell signaling, cellular function maintenance and gene expression. Ingenuity pathway analysis (IPA) identified 26 and 9 canonical pathways in Cnga3−/−/Nrl−/− and Cngb3−/−/Nrl−/− retinas, respectively, with 6 pathways being shared. The shared pathways include phototransduction, cAMP/PKA-mediated signaling, endothelin signaling, and EIF2/endoplasmic reticulum (ER) stress, whereas the IL-1, CREB, and purine metabolism signaling were found to specifically associate with Cnga3 deficiency. Thus, CNG channel deficiency differentially regulates genes that affect cell processes such as phototransduction, cellular survival and gene expression, and such regulations play a crucial role(s) in the retinal adaptation to impaired cone phototransduction. Though lack of Cnga3 and Cngb3 shares many common pathways, deficiency of Cnga3 causes more significant alterations in gene expression. This work provides insights into how cones respond to impaired phototransduction at the gene expression levels. PMID:23740940

  1. Generation of a New Model Rat: Nrf2 Knockout Rats Are Sensitive to Aflatoxin B1 Toxicity.

    PubMed

    Taguchi, Keiko; Takaku, Misaki; Egner, Patricia A; Morita, Masanobu; Kaneko, Takehito; Mashimo, Tomoji; Kensler, Thomas W; Yamamoto, Masayuki

    2016-07-01

    THE TRANSCRIPTION FACTOR NRF2: (NF-E2-related-factor 2) REGULATES A BATTERY OF ANTIOXIDATIVE STRESS-RESPONSE GENES AND DETOXICATION GENES, AND NRF2 KNOCKOUT LINES OF MICE HAVE BEEN CONTRIBUTING CRITICALLY TO THE CLARIFICATION OF ROLES THAT NRF2 PLAYS FOR CELL PROTECTION HOWEVER, THERE ARE APPARENT LIMITATIONS IN USE OF THE MOUSE MODELS FOR INSTANCE, RATS EXHIBIT MORE SUITABLE FEATURES FOR TOXICOLOGICAL OR PHYSIOLOGICAL EXAMINATIONS THAN MICE IN THIS STUDY, WE GENERATED 2 LINES OF NRF2 KNOCKOUT RATS BY USING A GENOME EDITING TECHNOLOGY; 1 LINE HARBORS A 7-BP DELETION Δ7 AND THE OTHER LINE HARBORS A 1-BP INSERTION +1 IN THE NRF2 GENE IN THE LIVERS OF RATS HOMOZYGOUSLY DELETING THE NRF2 GENE, AN ACTIVATOR OF NRF2 SIGNALING, CDDO-IM, COULD NOT INDUCE EXPRESSION OF REPRESENTATIVE NRF2 TARGET GENES TO EXAMINE ALTERED TOXICOLOGICAL RESPONSE, WE TREATED THE NRF2 KNOCKOUT RATS WITH AFLATOXIN B1 AFB1, A CARCINOGENIC MYCOTOXIN THAT ELICITS GENE MUTATIONS THROUGH BINDING OF ITS METABOLITES TO DNA AND FOR WHICH THE RAT HAS BEEN PROPOSED AS A REASONABLE SURROGATE FOR HUMAN TOXICITY INDEED, IN THE NRF2 KNOCKOUT RAT LIVERS THE ENZYMES OF THE AFB1 DETOXICATION PATHWAY WERE SIGNIFICANTLY DOWNREGULATED SINGLE DOSE ADMINISTRATION OF AFB1 INCREASED HEPATOTOXICITY AND BINDING OF AFB1-N7-GUANINE TO HEPATIC DNA IN NRF2 KNOCKOUT RATS COMPARED WITH WILD-TYPE NRF2 KNOCKOUT RATS REPEATEDLY TREATED WITH AFB1 WERE PRONE TO LETHALITY AND CDDO-IM WAS NO LONGER PROTECTIVE THESE RESULTS DEMONSTRATE THAT NRF2 KNOCKOUT RATS ARE QUITE SENSITIVE TO AFB1 TOXICITIES AND THIS RAT GENOTYPE EMERGES AS A NEW MODEL ANIMAL IN TOXICOLOGY.

  2. Mice expressing the human CYP7A1 gene in the mouse CYP7A1 knock-out background lack induction of CYP7A1 expression by cholesterol feeding and have increased hypercholesterolemia when fed a high fat diet.

    PubMed

    Chen, Jean Y; Levy-Wilson, Beatriz; Goodart, Sheryl; Cooper, Allen D

    2002-11-08

    Cholesterol 7alpha-hydroxylase (CYP7A1) catalyzes the rate-limiting step in the pathway responsible for the formation of the majority of bile acids. Transcription of the gene is regulated by the size of the bile acid pool and dietary and hormonal factors. The farnesoid X receptor and the liver X receptor (LXR) are responsible for regulation by bile acids and cholesterol, respectively. To study the effects of dietary cholesterol and fat upon expression of the human CYP7A1 gene, mice were generated by crossing transgenic mice carrying the human CYP7A1 gene with mice that were homozygous knock-outs (CYP7A1(-/-)). The mice (mCYP7A1(-/-)/hCYP7A1) expressed the human gene at much higher levels than did the transgenics bred in the wild-type background. A diet containing 1% cholic acid reduced the expression of the human gene in mCYP7A1(-/-)/hCYP7A1 mice to undetectable levels. Cholestyramine (5%) increased the level of expression of the human gene and the mouse gene. Thus, farnesoid X receptor-mediated regulation was preserved. A diet containing 2% cholesterol increased expression of the mouse gene in wild-type mice, but it did not affect expression of the human gene in mCYP7A1(-/-)/hCYP7A1 mice. None of the diets altered the serum cholesterol or triglyceride levels in these mice; 1% cholic acid caused a redistribution of cholesterol from the high density lipoprotein to the low density lipoprotein density in the humanized mice but not in wild-type mice. A diet containing 30% saturated fat and 2% cholesterol caused a decrease in CYP7A1 levels in mCYP7A1(-/-)/hCYP7A1 mice. The serum cholesterol levels rose in all mice fed this diet. The increase was greater in the mCYP7A1(-/-)/hCYP7A1 mice. Together, these data suggest that the lack of an LXR element in the region from -56 to -49 of the human CYP7A1 promoter may account for some of the differences in response to diets between humans and rodents.

  3. The knockout of secretin in cerebellar Purkinje cells impairs mouse motor coordination and motor learning.

    PubMed

    Zhang, Li; Chung, Sookja Kim; Chow, Billy Kwok Chong

    2014-05-01

    Secretin (SCT) was first considered to be a gut hormone regulating gastrointestinal functions when discovered. Recently, however, central actions of SCT have drawn intense research interest and are supported by the broad distribution of SCT in specific neuronal populations and by in vivo physiological studies regarding its role in water homeostasis and food intake. The direct action of SCT on a central neuron was first discovered in cerebellar Purkinje cells in which SCT from cerebellar Purkinje cells was found to potentiate GABAergic inhibitory transmission from presynaptic basket cells. Because Purkinje neurons have a major role in motor coordination and learning functions, we hypothesize a behavioral modulatory function for SCT. In this study, we successfully generated a mouse model in which the SCT gene was deleted specifically in Purkinje cells. This mouse line was tested together with SCT knockout and SCT receptor knockout mice in a full battery of behavioral tasks. We found that the knockout of SCT in Purkinje neurons did not affect general motor ability or the anxiety level in open field tests. However, knockout mice did exhibit impairments in neuromuscular strength, motor coordination, and motor learning abilities, as shown by wire hanging, vertical climbing, and rotarod tests. In addition, SCT knockout in Purkinje cells possibly led to the delayed development of motor neurons, as supported by the later occurrence of key neural reflexes. In summary, our data suggest a role in motor coordination and motor learning for SCT expressed in cerebellar Purkinje cells.

  4. Altered Expression of EPO Might Underlie Hepatic Hemangiomas in LRRK2 Knockout Mice

    PubMed Central

    Xiao, Kaifu; Zhang, Zhuohua

    2016-01-01

    Parkinson's disease (PD) is a severe neurodegenerative disorder caused by progressive loss of dopaminergic neurons in the substantia nigra pars compacta of the midbrain. The molecular mechanism of PD pathogenesis is unclear. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are a common genetic cause of familial and sporadic PD. However, studies on LRRK2 mutant mice revealed no visible dopaminergic neuronal loss in the midbrain. While surveying a LRRK2 knockout mouse strain, we found that old animals developed age-dependent hepatic vascular growths similar to cavernous hemangiomas. In livers of these hemangioma-positive LRRK2 knockout mice, we detected an increased expression of the HIF-2α protein and significant reactivation of the expression of the HIF-2α target gene erythropoietin (EPO), a finding consistent with a role of the HIF-2α pathway in blood vessel vascularization. We also found that the kidney EPO expression was reduced to 20% of the wild-type level in 18-month-old LRRK2 knockout mice. Unexpectedly, this reduction was restored to wild-type levels when the knockout mice were 22 months to 23 months old, implying a feedback mechanism regulating kidney EPO expression. Our findings reveal a novel function of LRRK2 in regulating EPO expression and imply a potentially novel relationship between PD genes and hematopoiesis. PMID:27872856

  5. Altered Expression of EPO Might Underlie Hepatic Hemangiomas in LRRK2 Knockout Mice.

    PubMed

    Wu, Ben; Xiao, Kaifu; Zhang, Zhuohua; Ma, Long

    2016-01-01

    Parkinson's disease (PD) is a severe neurodegenerative disorder caused by progressive loss of dopaminergic neurons in the substantia nigra pars compacta of the midbrain. The molecular mechanism of PD pathogenesis is unclear. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are a common genetic cause of familial and sporadic PD. However, studies on LRRK2 mutant mice revealed no visible dopaminergic neuronal loss in the midbrain. While surveying a LRRK2 knockout mouse strain, we found that old animals developed age-dependent hepatic vascular growths similar to cavernous hemangiomas. In livers of these hemangioma-positive LRRK2 knockout mice, we detected an increased expression of the HIF-2α protein and significant reactivation of the expression of the HIF-2α target gene erythropoietin (EPO), a finding consistent with a role of the HIF-2α pathway in blood vessel vascularization. We also found that the kidney EPO expression was reduced to 20% of the wild-type level in 18-month-old LRRK2 knockout mice. Unexpectedly, this reduction was restored to wild-type levels when the knockout mice were 22 months to 23 months old, implying a feedback mechanism regulating kidney EPO expression. Our findings reveal a novel function of LRRK2 in regulating EPO expression and imply a potentially novel relationship between PD genes and hematopoiesis.

  6. Hair-Cell Mechanotransduction Persists in TRP Channel Knockout Mice

    PubMed Central

    Niksch, Paul D.; Webber, Roxanna M.; Garcia-Gonzalez, Miguel; Watnick, Terry; Zhou, Jing; Vollrath, Melissa A.; Corey, David P.

    2016-01-01

    Members of the TRP superfamily of ion channels mediate mechanosensation in some organisms, and have been suggested as candidates for the mechanotransduction channel in vertebrate hair cells. Some TRP channels can be ruled out based on lack of an inner ear phenotype in knockout animals or pore properties not similar to the hair-cell channel. Such studies have excluded Trpv4, Trpa1, Trpml3, Trpm1, Trpm3, Trpc1, Trpc3, Trpc5, and Trpc6. However, others remain reasonable candidates. We used data from an RNA-seq analysis of gene expression in hair cells as well as data on TRP channel conductance to narrow the candidate group. We then characterized mice lacking functional Trpm2, Pkd2, Pkd2l1, Pkd2l2 and Pkd1l3, using scanning electron microscopy, auditory brainstem response, permeant dye accumulation, and single-cell electrophysiology. In all of these TRP-deficient mice, and in double and triple knockouts, mechanotransduction persisted. Together with published studies, these results argue against the participation of any of the 33 mouse TRP channels in hair cell transduction. PMID:27196058

  7. TALEN-based knockout library for human microRNAs.

    PubMed

    Kim, Young-Kook; Wee, Gabbine; Park, Joha; Kim, Jongkyu; Baek, Daehyun; Kim, Jin-Soo; Kim, V Narry

    2013-12-01

    Various technical tools have been developed to probe the functions of microRNAs (miRNAs), yet their application has been limited by low efficacy and specificity. To overcome the limitations, we used transcription activator-like effector nucleases (TALENs) to knock out human miRNA genes. We designed and produced a library of 540 pairs of TALENs for 274 miRNA loci, focusing on potentially important miRNAs. The knockout procedure takes only 2-4 weeks and can be applied to any cell type. As a case study, we generated knockout cells for two related miRNAs, miR-141 and miR-200c, which belong to the highly conserved miR-200 family. Interestingly, miR-141 and miR-200c, despite their overall similarity, suppress largely nonoverlapping groups of targets, thus suggesting that functional miRNA-target interaction requires strict seed-pairing. Our study illustrates the potency of TALEN technology and provides useful resources for miRNA research.

  8. Maize-targeted mutagenesis: A knockout resource for maize.

    PubMed

    May, Bruce P; Liu, Hong; Vollbrecht, Erik; Senior, Lynn; Rabinowicz, Pablo D; Roh, Donna; Pan, Xiaokang; Stein, Lincoln; Freeling, Mike; Alexander, Danny; Martienssen, Rob

    2003-09-30

    We describe an efficient system for site-selected transposon mutagenesis in maize. A total of 43,776 F1 plants were generated by using Robertson's Mutator (Mu) pollen parents and self-pollinated to establish a library of transposon-mutagenized seed. The frequency of new seed mutants was between 10-4 and 10-5 per F1 plant. As a service to the maize community, maize-targeted mutagenesis selects insertions in genes of interest from this library by using the PCR. Pedigree, knockout, sequence, phenotype, and other information is stored in a powerful interactive database (maize-targeted mutagenesis database) that enables analysis of the entire population and the handling of knockout requests. By inhibiting Mu activity in most F1 plants, we sought to reduce somatic insertions that may cause false positives selected from pooled tissue. By monitoring the remaining Mu activity in the F2, however, we demonstrate that seed phenotypes depend on it, and false positives occur in lines that appear to lack it. We conclude that more than half of all mutations arising in this population are suppressed on losing Mu activity. These results have implications for epigenetic models of inbreeding and for functional genomics.

  9. 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

  10. Rhamnolipid and poly (hydrozyalkanoate) biosynthesis in 3-hydrozyacyl-ACP:COA transacylase (phaG) - knockouts of pseudomonas chloroaphis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The 3-hydroxyacyl-ACP:CoA transacylase gene (phaG(Pc30761)) of P. chlororaphis NRRL B-30761 was cloned and analyzed. The nucleotide and translated amino-acid sequences of phaG(Pc30761) had 99% identities (at 100% query coverage) with the phaG gene of P. fluorescens O6. Two phaG-knockout strains of...

  11. A novel CYP17A1 deletion causes a functional knockout of the steroid enzyme 17-hydroxylase and 17,20-lyase in a Turkish family and illustrates the precise role of the CYP17A1 gene

    PubMed Central

    Camats, Núria; Üstyol, Ala; Atabek, Mehmet Emre; Dick, Bernhard; Flück, Christa E

    2015-01-01

    Key Clinical Message A novel homozygous long-range deletion of the CYP17A1 gene abolished protein expression and caused the severest form of 17-hydroxylase deficiency in one kindred of a Turkish family. The affected subjects presented with 46,XY sex reversal and 46,XX lack of pubertal development as well as severe hypertension. PMID:26509008

  12. Adenosine A2A Receptor Gene Knockout Prevents l-3,4-Dihydroxyphenylalanine-Induced Dyskinesia by Downregulation of Striatal GAD67 in 6-OHDA-Lesioned Parkinson’s Mice

    PubMed Central

    Yin, Su-bing; Zhang, Xiao-guang; Chen, Shuang; Yang, Wen-ting; Zheng, Xia-wei; Zheng, Guo-qing

    2017-01-01

    l-3,4-Dihydroxyphenylalanine (l-DOPA) remains the primary pharmacological agent for the symptomatic treatment of Parkinson’s disease (PD). However, the development of l-DOPA-induced dyskinesia (LID) limits the long-term use of l-DOPA for PD patients. Some data have reported that adenosine A2A receptor (A2AR) antagonists prevented LID in animal model of PD. However, the mechanism in which adenosine A2AR blockade alleviates the symptoms of LID has not been fully clarified. Here, we determined to knock out (KO) the gene of A2AR and explored the possible underlying mechanisms implicated in development of LID in a mouse model of PD. A2AR gene KO mice were unilaterally injected into the striatum with 6-hydroxydopamine (6-OHDA) in order to damage dopamine neurons on one side of the brain. 6-OHDA-lesioned mice were then injected once daily for 21 days with l-DOPA. Abnormal involuntary movements (AIMs) were evaluated on days 3, 8, 13, and 18 after l-DOPA administration, and real-time polymerase chain reaction and immunohistochemistry for glutamic acid decarboxylase (GAD) 65 and GAD67 were performed. We found that A2AR gene KO was effective in reducing AIM scores and accompanied with decrease of striatal GAD67, rather than GAD65. These results demonstrated that the possible mechanism involved in alleviation of AIM symptoms by A2AR gene KO might be through reducing the expression of striatal GAD67. PMID:28377741

  13. IdealKnock: A framework for efficiently identifying knockout strategies leading to targeted overproduction.

    PubMed

    Gu, Deqing; Zhang, Cheng; Zhou, Shengguo; Wei, Liujing; Hua, Qiang

    2016-04-01

    In recent years, computer aided redesigning methods based on genome-scale metabolic network models (GEMs) have played important roles in metabolic engineering studies; however, most of these methods are hindered by intractable computing times. In particular, methods that predict knockout strategies leading to overproduction of desired biochemical are generally unable to do high level prediction because the computational time will increase exponentially. In this study, we propose a new framework named IdealKnock, which is able to efficiently evaluate potentials of the production for different biochemical in a system by merely knocking out pathways. In addition, it is also capable of searching knockout strategies when combined with the OptKnock or OptGene framework. Furthermore, unlike other methods, IdealKnock suggests a series of mutants with targeted overproduction, which enables researchers to select the one of greatest interest for experimental validation. By testing the overproduction of a large number of native metabolites, IdealKnock showed its advantage in successfully breaking through the limitation of maximum knockout number in reasonable time and suggesting knockout strategies with better performance than other methods. In addition, gene-reaction relationship is well considered in the proposed framework.

  14. FMR1 Knockout mice: A model to study fragile X mental retardation

    SciTech Connect

    Oostra, B.A.; Bakker, C.E.; Reyniers, E.

    1994-09-01

    The fragile X syndrome is the most frequent form of inherited mental retardation in humans with an incidence of 1 in 1250 males and 1 in 2500 females. The clinical syndrome includes moderate to severe mental retardation, autistic behavior, macroorchidism, and facial features, such as long face with mandibular prognathism and large, everted ears. The molecular basis for this disease is a large expansion of a triplet repeat (CGG){sub n} in the 5{prime} untranslated region of the FMR1 gene. Due to this large expansion of the CGG repeat, the promoter region becomes methylated and the FMR1 gene is subsequently silenced. Hardly anything is known about the physiologic function of FMR1 and the pathologic mechanisms leading to these symptoms. Since the FMR1 gene is highly conserved in the mouse, we used the mouse to design a knockout model for the fragile X syndrome. These knockout mice lacking Fmrp have normal litter size suggesting that FMR1 is not essential in human gametogenesis and embryonic development. The knockout mice show the abnormalities also seen in the affected organs of human patients. Mutant mice show a gradual development through time of macroorchidism. In the knockout mice we observed cognitive defects in the form of deficits in learning (as shown by the hidden platform Morris water maze task) and behavioral abnormalities such as increased exploratory behavior and hyperactivity. Therefore this knockout mouse may serve as a valuable tool in studying the role of FMR1 in the fragile X syndrome and may serve as a model to elucidate the mechanisms involved in macroorchidism, abnormal behavior, and mental retardation.

  15. Modeling fragile X syndrome in the Fmr1 knockout mouse.

    PubMed

    Kazdoba, Tatiana M; Leach, Prescott T; Silverman, Jill L; Crawley, Jacqueline N

    2014-11-01

    Fragile X Syndrome (FXS) is a commonly inherited form of intellectual disability and one of the leading genetic causes for autism spectrum disorder. Clinical symptoms of FXS can include impaired cognition, anxiety, hyperactivity, social phobia, and repetitive behaviors. FXS is caused by a CGG repeat mutation which expands a region on the X chromosome containing the FMR1 gene. In FXS, a full mutation (> 200 repeats) leads to hypermethylation of FMR1, an epigenetic mechanism that effectively silences FMR1 gene expression and reduces levels of the FMR1 gene product, fragile X mental retardation protein (FMRP). FMRP is an RNA-binding protein that is important for the regulation of protein expression. In an effort to further understand how loss of FMR1 and FMRP contribute to FXS symptomology, several FXS animal models have been created. The most well characterized rodent model is the Fmr1 knockout (KO) mouse, which lacks FMRP protein due to a disruption in its Fmr1 gene. Here, we review the behavioral phenotyping of the Fmr1 KO mouse to date, and discuss the clinical relevance of this mouse model to the human FXS condition. While much remains to be learned about FXS, the Fmr1 KO mouse is a valuable tool for understanding the repercussions of functional loss of FMRP and assessing the efficacy of pharmacological compounds in ameliorating the molecular and behavioral phenotypes relevant to FXS.

  16. Minor compensatory changes in SAGE Mdr1a (P-gp), Bcrp, and Mrp2 knockout rats do not detract from their utility in the study of transporter-mediated pharmacokinetics.

    PubMed

    Zamek-Gliszczynski, Maciej J; Goldstein, Keith M; Paulman, April; Baker, Thomas K; Ryan, Timothy P

    2013-06-01

    Mdr1a-, Bcrp-, and Mrp2-knockout rats are a more practical species for absorption, distribution, metabolism, and excretion (ADME) studies than murine models and previously demonstrated expected alterations in the pharmacokinetics of various probe substrates. At present, gene expression and pathology changes were systematically studied in the small intestine, liver, kidney, and brain tissue from male SAGE Mdr1a, Bcrp, and Mrp2 knockout rats versus wild-type Sprague-Dawley controls. Gene expression data supported the relevant knockout genotype. As expected, Mrp2 knockout rats were hyperbilirubinemic and exhibited upregulation of hepatic Mrp3. Overall, few alterations were observed within 112 ADME-relevant genes. The two potentially most consequential changes were upregulation of intestinal carboxylesterase in Mdr1a knockouts and catechol-O-methyltransferase in all tissues of Bcrp knockout rats. Previously reported upregulation of hepatic Mdr1b P-glycoprotein in proprietary Wistar Mdr1a knockout rats was not observed in the SAGE counterpart investigated herein. Relative liver and kidney weights were 22-53% higher in all three knockouts, with microscopic increases in hepatocyte size in Mdr1a and Mrp2 knockout rats and glomerular size in Bcrp and Mrp2 knockouts. Increased relative weight of clearing organs is quantitatively consistent with reported increases in the clearance of drugs that are not substrates of the knocked-out transporter. Overall, SAGE knockout rats demonstrated modest compensatory changes, which do not preclude their general application to study transporter-mediated pharmacokinetics. However, until future studies elucidate the magnitude of functional change, caution is warranted in rare instances of extensive metabolism by catechol-O-methyltransferase in Bcrp knockouts and intestinal carboxylesterase in Mdr1a knockout rats, specifically for molecules with free catechol groups and esters subject to gut-wall hydrolysis.

  17. 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.

  18. Effect of knockout of α2δ-1 on action potentials in mouse sensory neurons

    PubMed Central

    Margas, Wojciech; Ferron, Laurent; Nieto-Rostro, Manuela; Schwartz, Arnold; Dolphin, Annette C.

    2016-01-01

    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 Ca2+ 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 Ca2+ 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 Ca2+-activation of large conductance calcium-activated potassium (BK) and small conductance calcium-activated potassium (SK) channels in these events. In conclusion, the reduced Ca2+ 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 Ca2+ and ATP together to control life and death’. PMID:27377724

  19. Knockout of Zebrafish Ovarian Aromatase Gene (cyp19a1a) by TALEN and CRISPR/Cas9 Leads to All-male Offspring Due to Failed Ovarian Differentiation

    PubMed Central

    Lau, Esther Shuk-Wa; Zhang, Zhiwei; Qin, Mingming; Ge, Wei

    2016-01-01

    Sexual or gonadal differentiation is a complex event and its mechanism remains elusive in teleosts. Despite its complexity and plasticity, the process of ovarian differentiation is believed to involve gonadal aromatase (cyp19a1a) in nearly all species studied. However, most data concerning the role of aromatase have come from gene expression analysis or studies involving pharmacological approaches. There has been a lack of genetic evidence for the importance of aromatase in gonadal differentiation, especially the timing when the enzyme starts to exert its effect. This is due to the lack of appropriate loss-of-function approaches in fish models for studying gene functions. This situation has changed recently with the development of genome editing technologies, namely TALEN and CRISPR/Cas9. Using both TALEN and CRISPR/Cas9, we successfully established three mutant zebrafish lines lacking the ovarian aromatase. As expected, all mutant fish were males, supporting the view that aromatase plays a critical role in directing ovarian differentiation and development. Further analysis showed that the ovarian aromatase did not seem to affect the formation of so-called juvenile ovary and oocyte-like germ cells; however, it was essential for further differentiation of the juvenile ovary into the true ovary. PMID:27876832

  20. Altered Sleep Homeostasis in Rev-erbα Knockout Mice

    PubMed Central

    Mang, Géraldine M.; La Spada, Francesco; Emmenegger, Yann; Chappuis, Sylvie; Ripperger, Jürgen A.; Albrecht, Urs; Franken, Paul

    2016-01-01

    Study Objectives: The nuclear receptor REV-ERBα is a potent, constitutive transcriptional repressor critical for the regulation of key circadian and metabolic genes. Recently, REV-ERBα's involvement in learning, neurogenesis, mood, and dopamine turnover was demonstrated suggesting a specific role in central nervous system functioning. We have previously shown that the brain expression of several core clock genes, including Rev-erbα, is modulated by sleep loss. We here test the consequences of a loss of REV-ERBα on the homeostatic regulation of sleep. Methods: EEG/EMG signals were recorded in Rev-erbα knockout (KO) mice and their wild type (WT) littermates during baseline, sleep deprivation, and recovery. Cortical gene expression measurements after sleep deprivation were contrasted to baseline. Results: Although baseline sleep/wake duration was remarkably similar, KO mice showed an advance of the sleep/wake distribution relative to the light-dark cycle. After sleep onset in baseline and after sleep deprivation, both EEG delta power (1–4 Hz) and sleep consolidation were reduced in KO mice indicating a slower increase of homeostatic sleep need during wakefulness. This slower increase might relate to the smaller increase in theta and gamma power observed in the waking EEG prior to sleep onset under both conditions. Indeed, the increased theta activity during wakefulness predicted delta power in subsequent NREM sleep. Lack of Rev-erbα increased Bmal1, Npas2, Clock, and Fabp7 expression, confirming the direct regulation of these genes by REV-ERBα also in the brain. Conclusions: Our results add further proof to the notion that clock genes are involved in sleep homeostasis. Because accumulating evidence directly links REV-ERBα to dopamine signaling the altered homeostatic regulation of sleep reported here are discussed in that context. Citation: Mang GM, La Spada F, Emmenegger Y, Chappuis S, Ripperger JA, Albrecht U, Franken P. Altered sleep homeostasis in Rev

  1. In vivo transduction by intravenous injection of a lentiviral vector expressing human ADA into neonatal ADA gene knockout mice: a novel form of enzyme replacement therapy for ADA deficiency.

    PubMed

    Carbonaro, Denise A; Jin, Xiangyang; Petersen, Denise; Wang, Xingchao; Dorey, Fred; Kil, Ki Soo; Aldrich, Melissa; Blackburn, Michael R; Kellems, Rodney E; Kohn, Donald B

    2006-06-01

    Using a mouse model of adenosine deaminase-deficient severe combined immune deficiency syndrome (ADA-deficient SCID), we have developed a noninvasive method of gene transfer for the sustained systemic expression of human ADA as enzyme replacement therapy. The method of delivery is a human immunodeficiency virus 1-based lentiviral vector given systemically by intravenous injection on day 1 to 2 of life. In this article we characterize the biodistribution of the integrated vector, the expression levels of ADA enzyme activity in various tissues, as well as the efficacy of systemic ADA expression to correct the ADA-deficient phenotype in this mouse model. The long-term expression of enzymatically active ADA achieved by this method, primarily from transduction of liver and lung, restored immunologic function and significantly extended survival. These studies illustrate the potential for sustained in vivo production of enzymatically active ADA, as an alternative to therapy by frequent injection of exogenous ADA protein.

  2. Universal statistics of the knockout tournament

    NASA Astrophysics Data System (ADS)

    Baek, Seung Ki; Yi, Il Gu; Park, Hye Jin; Kim, Beom Jun

    2013-11-01

    We study statistics of the knockout tournament, where only the winner of a fixture progresses to the next. We assign a real number called competitiveness to each contestant and find that the resulting distribution of prize money follows a power law with an exponent close to unity if the competitiveness is a stable quantity and a decisive factor to win a match. Otherwise, the distribution is found narrow. The existing observation of power law distributions in various kinds of real sports tournaments therefore suggests that the rules of those games are constructed in such a way that it is possible to understand the games in terms of the contestants' inherent characteristics of competitiveness.

  3. Universal statistics of the knockout tournament.

    PubMed

    Baek, Seung Ki; Yi, Il Gu; Park, Hye Jin; Kim, Beom Jun

    2013-11-12

    We study statistics of the knockout tournament, where only the winner of a fixture progresses to the next. We assign a real number called competitiveness to each contestant and find that the resulting distribution of prize money follows a power law with an exponent close to unity if the competitiveness is a stable quantity and a decisive factor to win a match. Otherwise, the distribution is found narrow. The existing observation of power law distributions in various kinds of real sports tournaments therefore suggests that the rules of those games are constructed in such a way that it is possible to understand the games in terms of the contestants' inherent characteristics of competitiveness.

  4. Knockout of Foxp2 disrupts vocal development in mice

    PubMed Central

    Castellucci, Gregg A.; McGinley, Matthew J.; McCormick, David A.

    2016-01-01

    The FOXP2 gene is important for the development of proper speech motor control in humans. However, the role of the gene in general vocal behavior in other mammals, including mice, is unclear. Here, we track the vocal development of Foxp2 heterozygous knockout (Foxp2+/−) mice and their wildtype (WT) littermates from juvenile to adult ages, and observe severe abnormalities in the courtship song of Foxp2+/− mice. In comparison to their WT littermates, Foxp2+/− mice vocalized less, produced shorter syllable sequences, and possessed an abnormal syllable inventory. In addition, Foxp2+/− song also exhibited irregular rhythmic structure, and its development did not follow the consistent trajectories observed in WT vocalizations. These results demonstrate that the Foxp2 gene is critical for normal vocal behavior in juvenile and adult mice, and that Foxp2 mutant mice may provide a tractable model system for the study of the gene’s role in general vocal motor control. PMID:26980647

  5. Knockout of Vasohibin-1 Gene in Mice Results in Healthy Longevity with Reduced Expression of Insulin Receptor, Insulin Receptor Substrate 1, and Insulin Receptor Substrate 2 in Their White Adipose Tissue

    PubMed Central

    Takeda, Eichi; Suzuki, Yasuhiro; Yamada, Tetsuya; Katagiri, Hideki

    2017-01-01

    Vasohibin-1 (Vash1), originally isolated as an endothelium-derived angiogenesis inhibitor, has a characteristic of promoting stress tolerance in endothelial cells (ECs). We therefore speculated that the lack of the vash1 gene would result in a short lifespan. However, to our surprise, vash1−/− mice lived significantly longer with a milder senescence phenotype than wild-type (WT) mice. We sought the cause of this healthy longevity and found that vash1−/− mice exhibited mild insulin resistance along with reduced expression of the insulin receptor (insr), insulin receptor substrate 1 (irs-1), and insulin receptor substrate 2 (irs-2) in their white adipose tissue (WAT) but not in their liver or skeletal muscle. The expression of vash1 dominated in the WAT among those 3 organs. Importantly, vash1−/− mice did not develop diabetes even when fed a high-fat diet. These results indicate that the expression of vash1 was required for the normal insulin sensitivity of the WAT and that the target molecules for this activity were insr, irs1, and irs2. The lack of vash1 caused mild insulin resistance without the outbreak of overt diabetes and might contribute to healthy longevity. PMID:28367331

  6. 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.

  7. Rapid phenotyping of knockout mice to identify genetic determinants of bone strength

    PubMed Central

    Freudenthal, Bernard; Logan, John; Croucher, Peter I

    2016-01-01

    The genetic determinants of osteoporosis remain poorly understood, and there is a large unmet need for new treatments in our ageing society. Thus, new approaches for gene discovery in skeletal disease are required to complement the current genome-wide association studies in human populations. The International Knockout Mouse Consortium (IKMC) and the International Mouse Phenotyping Consortium (IMPC) provide such an opportunity. The IKMC generates knockout mice representing each of the known protein-coding genes in C57BL/6 mice and, as part of the IMPC initiative, the Origins of Bone and Cartilage Disease project identifies mutants with significant outlier skeletal phenotypes. This initiative will add value to data from large human cohorts and provide a new understanding of bone and cartilage pathophysiology, ultimately leading to the identification of novel drug targets for the treatment of skeletal disease. PMID:27535945

  8. Increased Renal Proximal Convoluted Tubule Transport Contributes to Hypertension in Cyp4a14 Knockout Mice

    PubMed Central

    Quigley, Raymond; Chakravarty, Sumana; Zhao, Xueying; Imig, John D.; Capdevila, Jorge H.

    2009-01-01

    Background/Aims Disrupting the enzyme Cyp4a14 in mice leads to hypertension, which is more severe in the male mice and appears to be due to androgen excess. Because the Cyp4a14 enzyme is located in the proximal tubule of the kidney, we hypothesized that there could be dysregulation of transport in this segment that could contribute to the hypertension. Methods Wild-type (SV/129) mice and mice that had targeted disruption of the Cyp4a14 gene were studied. Proximal convoluted tubules (PCT) from knockout and wild-type mice were dissected and perfused in vitrofor measurement of volume absorption (JV). Expression of the sodium-hydrogen exchanger 3 (NHE3), the predominant transporter responsible for sodium transport in this segment, was measured by immunoblot. Renal vascular (afferent arteriole) responses to angiotensin and endothelin were also measured. Results PCT volume absorption was elevated in tubules from the Cyp4a14 knockout mice as compared to the wild-type mice. Brush border membrane NHE3 expression was almost 2-fold higher in Cyp4a14 knockout mice than in wild-type mice. No difference was found in the afferent arteriolar response. Conclusion Thus, hypertension in the Cyp4a14 knockout mice appears to be driven by excessive fluid reabsorption in the proximal tubule, which is secondary to overexpression of NHE3. PMID:19713718

  9. Glutamate transporter type 3 knockout reduces brain tolerance to focal brain ischemia in mice.

    PubMed

    Li, Liaoliao; Zuo, Zhiyi

    2011-05-01

    Excitatory amino-acid transporters (EAATs) transport glutamate into cells under physiologic conditions. Excitatory amino-acid transporter type 3 (EAAT3) is the major neuronal EAAT and also uptakes cysteine, the rate-limiting substrate for synthesis of glutathione. Thus, we hypothesize that EAAT3 contributes to providing brain ischemic tolerance. Male 8-week-old EAAT3 knockout mice on CD-1 mouse gene background and wild-type CD-1 mice were subjected to right middle cerebral artery occlusion for 90 minutes. Their brain infarct volumes, neurologic functions, and brain levels of glutathione, nitrotyrosine, and 4-hydroxy-2-nonenal (HNE) were evaluated. The EAAT3 knockout mice had bigger brain infarct volumes and worse neurologic deficit scores and motor coordination functions than did wild-type mice, no matter whether these neurologic outcome parameters were evaluated at 24 hours or at 4 weeks after brain ischemia. The EAAT3 knockout mice contained higher levels of HNE in the ischemic penumbral cortex and in the nonischemic cerebral cortex than did wild-type mice. Glutathione levels in the ischemic and nonischemic cortices of EAAT3 knockout mice tended to be lower than those of wild-type mice. Our results suggest that EAAT3 is important in limiting ischemic brain injury after focal brain ischemia. This effect may involve attenuating brain oxidative stress.

  10. Altered Reward Circuitry in the Norepinephrine Transporter Knockout Mouse

    PubMed Central

    Hall, F. Scott; Uhl, George R.; Bearer, Elaine L.; Jacobs, Russell E.

    2013-01-01

    Synaptic levels of the monoamine neurotransmitters dopamine, serotonin, and norepinephrine are modulated by their respective plasma membrane transporters, albeit with a few exceptions. Monoamine transporters remove monoamines from the synaptic cleft and thus influence the degree and duration of signaling. Abnormal concentrations of these neuronal transmitters are implicated in a number of neurological and psychiatric disorders, including addiction, depression, and attention deficit/hyperactivity disorder. This work concentrates on the norepinephrine transporter (NET), using a battery of in vivo magnetic resonance imaging techniques and histological correlates to probe the effects of genetic deletion of the norepinephrine transporter on brain metabolism, anatomy and functional connectivity. MRS recorded in the striatum of NET knockout mice indicated a lower concentration of NAA that correlates with histological observations of subtle dysmorphisms in the striatum and internal capsule. As with DAT and SERT knockout mice, we detected minimal structural alterations in NET knockout mice by tensor-based morphometric analysis. In contrast, longitudinal imaging after stereotaxic prefrontal cortical injection of manganese, an established neuronal circuitry tracer, revealed that the reward circuit in the NET knockout mouse is biased toward anterior portions of the brain. This is similar to previous results observed for the dopamine transporter (DAT) knockout mouse, but dissimilar from work with serotonin transporter (SERT) knockout mice where Mn2+ tracings extended to more posterior structures than in wildtype animals. These observations correlate with behavioral studies indicating that SERT knockout mice display anxiety-like phenotypes, while NET knockouts and to a lesser extent DAT knockout mice display antidepressant-like phenotypic features. Thus, the mainly anterior activity detected with manganese-enhanced MRI in the DAT and NET knockout mice is likely indicative of

  11. [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.

  12. apo B gene knockout in mice results in embryonic lethality in homozygotes and neural tube defects, male infertility, and reduced HDL cholesterol ester and apo A-I transport rates in heterozygotes.

    PubMed Central

    Huang, L S; Voyiaziakis, E; Markenson, D F; Sokol, K A; Hayek, T; Breslow, J L

    1995-01-01

    apo B is a structural constituent of several classes of lipoprotein particles, including chylomicrons, VLDL, and LDL. To better understand the role of apo B in the body, we have used gene targeting in embryonic stem cells to create a null apo B allele in the mouse. Homozygous apo B deficiency led to embryonic lethality, with resorption of all embryos by gestational day 9. Heterozygotes showed an increased tendency to intrauterine death with some fetuses having incomplete neural tube closure and some live-born heterozygotes developing hydrocephalus. The majority of male heterozygotes were sterile, although the genitourinary system and sperm were grossly normal. Viable heterozygotes had normal triglycerides, but total, LDL, and HDL cholesterol levels were decreased by 37, 37, and 39%, respectively. Hepatic and intestinal apo B mRNA levels were decreased in heterozygotes, presumably contributing to the decreased LDL levels through decreased synthesis of apo B-containing lipoproteins. Kinetic studies indicated that heterozygotes had decreased transport rates of HDL cholesterol ester and apo A-I. As liver and intestinal apo A-I mRNA levels were unchanged, the mechanism for decreased apo A-I transport must be posttranscriptional. Heterozygotes also had normal cholesterol absorption and a normal response of the plasma lipoprotein pattern to chronic consumption of a high fat, high cholesterol, Western-type diet. In summary, we report a mouse model for apo B deficiency with several phenotypic features that were unexpected based on clinical studies of apo B-deficient humans, such as embryonic lethality in homozygotes and neural tube closure defects, male infertility, and a major defect in HDL production in heterozygotes. This model presents an opportunity to study the mechanisms underlying these phenotypic changes. Images PMID:7593600

  13. Transgenic knockout mice with exclusively human sickle hemoglobinand sickle cell disease

    SciTech Connect

    Paszty, C.; Brion, C.; Manci, E.; Witkowska, E.; Stevens, M.; Narla, M.; Rubin, E.

    1997-06-13

    To create mice expressing exclusively human sicklehemoglobin (HbS), transgenic mice expressing human alpha-, gamma-, andbeta[S]-globin were generated and bred with knockout mice that haddeletions of the murine alpha- and beta-globin genes. These sickle cellmice have the major features (irreversibly sickled red cells, anemia,multiorgan pathology) found in humans with sickle cell disease and, assuch, represent a useful in vivo system to accelerate the development ofimproved therapies for this common genetic disease.

  14. Cytochrome P450 1b1 in polycyclic aromatic hydrocarbon (PAH)-induced skin carcinogenesis: Tumorigenicity of individual PAHs and coal-tar extract, DNA adduction and expression of select genes in the Cyp1b1 knockout mouse

    SciTech Connect

    Siddens, Lisbeth K.; Bunde, Kristi L.; Harper, Tod A.; McQuistan, Tammie J.; Löhr, Christiane V.; Bramer, Lisa M.; Waters, Katrina M.; Tilton, Susan C.; Krueger, Sharon K.; and others

    2015-09-01

    FVB/N mice wild-type, heterozygous or null for Cyp 1b1 were used in a two-stage skin tumor study comparing PAH, benzo[a]pyrene (BaP), dibenzo[def,p]chrysene (DBC), and coal tar extract (CTE, SRM 1597a). Following 20 weeks of promotion with TPA the Cyp 1b1 null mice, initiated with DBC, exhibited reductions in incidence, multiplicity, and progression. None of these effects were observed with BaP or CTE. The mechanism of Cyp 1b1-dependent alteration of DBC skin carcinogenesis was further investigated by determining expression of select genes in skin from DBC-treated mice 2, 4 and 8 h post-initiation. A significant reduction in levels of Cyp 1a1, Nqo1 at 8 h and Akr 1c14 mRNA was observed in Cyp 1b1 null (but not wt or het) mice, whereas no impact was observed in Gst a1, Nqo 1 at 2 and 4 h or Akr 1c19 at any time point. Cyp 1b1 mRNA was not elevated by DBC. The major covalent DNA adducts, dibenzo[def,p]chrysene-(±)-11,12-dihydrodiol-cis and trans-13,14-epoxide-deoxyadenosine (DBCDE-dA) were quantified by UHPLC-MS/MS 8 h post-initiation. Loss of Cyp1 b1 expression reduced DBCDE-dA adducts in the skin but not to a statistically significant degree. The ratio of cis- to trans-DBCDE-dA adducts was higher in the skin than other target tissues such as the spleen, lung and liver (oral dosing). These results document that Cyp 1b1 plays a significant role in bioactivation and carcinogenesis of DBC in a two-stage mouse skin tumor model and that loss of Cyp 1b1 has little impact on tumor response with BaP or CTE as initiators. - Highlights: • Cyp1b1 null mice exhibit lower skin cancer sensitivity to DBC but not BaP or CTE. • Cyp1b1 expression impacts expression of other PAH metabolizing enzymes. • cis/trans-DBCDE-dA ratio significantly higher in the skin than the spleen, lung or liver • Potency of DBC and CTE in mouse skin is higher than predicted by RPFs.

  15. Identification of Novel Knockout Targets for Improving Terpenoids Biosynthesis in Saccharomyces cerevisiae

    PubMed Central

    Li, Jing; Wang, Jianfeng; Li, Qian; Wang, Yong; Zhang, Yansheng

    2014-01-01

    Many terpenoids have important pharmacological activity and commercial value; however, application of these terpenoids is often limited by problems associated with the production of sufficient amounts of these molecules. The use of Saccharomyces cerevisiae (S. cerevisiae) for the production of heterologous terpenoids has achieved some success. The objective of this study was to identify S. cerevisiae knockout targets for improving the synthesis of heterologous terpeniods. On the basis of computational analysis of the S. cerevisiae metabolic network, we identified the knockout sites with the potential to promote terpenoid production and the corresponding single mutant was constructed by molecular manipulations. The growth rates of these strains were measured and the results indicated that the gene deletion had no adverse effects. Using the expression of amorphadiene biosynthesis as a testing model, the gene deletion was assessed for its effect on the production of exogenous terpenoids. The results showed that the dysfunction of most genes led to increased production of amorphadiene. The yield of amorphadiene produced by most single mutants was 8–10-fold greater compared to the wild type, indicating that the knockout sites can be engineered to promote the synthesis of exogenous terpenoids. PMID:25386654

  16. Identification of novel knockout targets for improving terpenoids biosynthesis in Saccharomyces cerevisiae.

    PubMed

    Sun, Zhiqiang; Meng, Hailin; Li, Jing; Wang, Jianfeng; Li, Qian; Wang, Yong; Zhang, Yansheng

    2014-01-01

    Many terpenoids have important pharmacological activity and commercial value; however, application of these terpenoids is often limited by problems associated with the production of sufficient amounts of these molecules. The use of Saccharomyces cerevisiae (S. cerevisiae) for the production of heterologous terpenoids has achieved some success. The objective of this study was to identify S. cerevisiae knockout targets for improving the synthesis of heterologous terpeniods. On the basis of computational analysis of the S. cerevisiae metabolic network, we identified the knockout sites with the potential to promote terpenoid production and the corresponding single mutant was constructed by molecular manipulations. The growth rates of these strains were measured and the results indicated that the gene deletion had no adverse effects. Using the expression of amorphadiene biosynthesis as a testing model, the gene deletion was assessed for its effect on the production of exogenous terpenoids. The results showed that the dysfunction of most genes led to increased production of amorphadiene. The yield of amorphadiene produced by most single mutants was 8-10-fold greater compared to the wild type, indicating that the knockout sites can be engineered to promote the synthesis of exogenous terpenoids.

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

    PubMed Central

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

    2016-01-01

    Whole-genome knockout collections are invaluable for connecting gene sequence to function, yet traditionally, their construction has required an extraordinary technical effort. Here we report a method for the construction and purification of a curated whole-genome collection of single-gene transposon disruption mutants termed Knockout Sudoku. Using simple combinatorial pooling, a highly oversampled collection of mutants is condensed into a next-generation sequencing library in a single day, a 30- to 100-fold improvement over prior methods. The identities of the mutants in the collection are then solved by a probabilistic algorithm that uses internal self-consistency within the sequencing data set, followed by rapid algorithmically guided condensation to a minimal representative set of mutants, validation, and curation. Starting from a progenitor collection of 39,918 mutants, we compile a quality-controlled knockout collection of the electroactive microbe Shewanella oneidensis MR-1 containing representatives for 3,667 genes that is functionally validated by high-throughput kinetic measurements of quinone reduction. PMID:27830751

  18. Immunoglobulin knockout chickens via efficient homologous recombination in primordial germ cells

    PubMed Central

    Schusser, Benjamin; Collarini, Ellen J.; Yi, Henry; Izquierdo, Shelley Mettler; Fesler, Jeffrey; Pedersen, Darlene; Klasing, Kirk C.; Kaspers, Bernd; Harriman, William D.; van de Lavoir, Marie-Cecile; Etches, Robert J.; Leighton, Philip A.

    2013-01-01

    Gene targeting by homologous recombination or by sequence-specific nucleases allows the precise modification of genomes and genes to elucidate their functions. Although gene targeting has been used extensively to modify the genomes of mammals, fish, and amphibians, a targeting technology has not been available for the avian genome. Many of the principles of humoral immunity were discovered in chickens, yet the lack of gene targeting technologies in birds has limited biomedical research using this species. Here we describe targeting the joining (J) gene segment of the chicken Ig heavy chain gene by homologous recombination in primordial germ cells to establish fully transgenic chickens carrying the knockout. In homozygous knockouts, Ig heavy chain production is eliminated, and no antibody response is elicited on immunization. Migration of B-lineage precursors into the bursa of Fabricius is unaffected, whereas development into mature B cells and migration from the bursa are blocked in the mutants. Other cell types in the immune system appear normal. Chickens lacking the peripheral B-cell population will provide a unique experimental model to study avian immune responses to infectious disease. More generally, gene targeting in avian primordial germ cells will foster advances in diverse fields of biomedical research such as virology, stem cells, and developmental biology, and provide unique approaches in biotechnology, particularly in the field of antibody discovery. PMID:24282302

  19. Proteomic Analysis of Tissue from α1,3-galactosyltransferase Knockout Mice Reveals That a Wide Variety of Proteins and Protein Fragments Change Expression Level

    PubMed Central

    Thorlacius-Ussing, Louise; Ludvigsen, Maja; Kirkeby, Svend

    2013-01-01

    A barrier in a pig-to-man xenotransplantation is that the Galα1-3Galβ1-4GlcNAc-R carbohydrate (α-Gal epitope) expressed on pig endothelial cells reacts with naturally occurring antibodies in the recipient’s blood leading to rejection. Deletion of the α1,3-galactosyltransferase gene prevents the synthesis of the α-Gal epitope. Therefore, knockout models of the α1,3-galactosyltransferase gene are widely used to study xenotransplantation. We have performed proteomic studies on liver and pancreas tissues from wild type and α1,3-galactosyltransferase gene knockout mice. The tissues were analyzed by two-dimensional polyacrylamide gel electrophoresis and liquid chromatography - tandem mass spectrometry. The analyses revealed that a wide variety of proteins and protein fragments are differentially expressed suggesting that knockout of the α1,3-galactosyltransferase gene affects the expression of several other genes. PMID:24244699

  20. Oxytocin and behavior: Lessons from knockout mice.

    PubMed

    Caldwell, Heather K; Aulino, Elizabeth A; Freeman, Angela R; Miller, Travis V; Witchey, Shannah K

    2017-02-01

    It is well established that the nonapeptide oxytocin (Oxt) is important for the neural modulation of behaviors in many mammalian species. Since its discovery in 1906 and synthesis in the early 1950s, elegant pharmacological work has helped identify specific neural substrates on which Oxt exerts its effects. More recently, mice with targeted genetic disruptions of the Oxt system-i.e., both the peptide and its receptor (the Oxtr)-have further defined Oxt's actions and laid some important scientific groundwork for studies in other species. In this article, we highlight the scientific contributions that various mouse knockouts of the Oxt system have made to our understanding of Oxt's modulation of behavior. We specifically focus on how the use of these mice has shed light on our understanding of social recognition memory, maternal behavior, aggression, and several nonsocial behaviors. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 190-201, 2017.

  1. Brain penetration of WEB 2086 (Apafant) and dantrolene in Mdr1a (P-glycoprotein) and Bcrp knockout rats.

    PubMed

    Fuchs, Holger; Kishimoto, Wataru; Gansser, Dietmar; Tanswell, Paul; Ishiguro, Naoki

    2014-10-01

    Transporter gene knockout rat models are attracting increasing interest for mechanistic studies of new drugs as transporter substrates or inhibitors in vivo. However, limited data are available on the functional validity of such models at the blood-brain barrier. Therefore, the present study evaluated Mdr1a [P-glycoprotein (P-gp)], Bcrp, and combined Mdr1a/Bcrp knockout rat strains for the influence of P-gp and breast cancer resistance protein (BCRP) transport proteins on brain penetration of the selective test substrates [(14)C]WEB 2086 (3-[4-(2-chlorophenyl)-9-methyl-6H-thieno[3,2-f][1,2,4]triazolo-[4,3-a][1,4]-diazepin-2-yl]-1-(4-morpholinyl)-1-propanon) for P-gp and dantrolene for BCRP. Brain-to-plasma concentration ratios (BPR) were measured after intravenous coinfusions of 5.5 µmol/kg per hour [(14)C]WEB 2086 and 2 µmol/kg per hour dantrolene for 2 hours in groups of knockout or wild-type rats. Compared with wild-type controls, mean BPR of [(14)C]WEB 2086 increased 8-fold in Mdr1a knockouts, 9.5-fold in double Mdr1a/Bcrp knockouts, and 7.3-fold in zosuquidar-treated wild-type rats, but was unchanged in Bcrp knockout rats. Mean BPR of dantrolene increased 3.3-fold in Bcrp knockouts and 3.9-fold in double Mdr1a/Bcrp knockouts compared with wild type, but was unchanged in the Mdr1a knockouts. The human intestinal CaCo-2 cell bidirectional transport system in vitro confirmed the in vivo finding that [(14)C]WEB 2086 is a substrate of P-gp but not of BCRP. Therefore, Mdr1a, Bcrp, and combined Mdr1a/Bcrp knockout rats provide functional absence of these efflux transporters at the blood-brain barrier and are a suitable model for mechanistic studies on the brain penetration of drug candidates.

  2. Metabolic engineering of Escherichia coli: construction and characterization of a gltA (citrate synthase) knockout mutant.

    PubMed

    Vandedrinck, S; Deschamps, G; Sablon, E; Vandamme, E J

    2001-01-01

    E. coli is one of the most important host organisms for recombinant protein production. However, growth and recombinant protein production can be limited by acetate accumulation during high-cell-density fermentations. Some of the strategies used to overcome this problem are based on the alteration of the genotype of the host. This paper discusses the construction and characterization of an E. coli gltA- knockout mutant. The knockout of the gene was confirmed by the loss of citrate synthase activity in an enzyme assay. Also the growth rate of the mutant on Luria Broth and Luria Broth + acetate was reduced.

  3. Long-term potentiation in the hippocampus of fragile X knockout mice

    SciTech Connect

    Godfraind, J.M.; Reyniers, E.; De Boulle, K.

    1996-08-09

    To gain more insight in the physiological function of the fragile X gene (FMR1) and the mechanisms leading to fragile X syndrome, the Fmr1 gene has been inactivated in mice by gene targeting techniques. In the Morris water maze test, the Fmr1 knockout mice learn to find the hidden platform nearly as well as the control animals, but show impaired performance after the position of the platform has been modified. As malperformance in the Morris water maze test has been associated with impaired long-term potentiation (LTP), electrophysiological studies were performed in hippocampal slices of Fmr1 knockout mice to check for the presence of LTP. Judged by field extracellular excitatory postsynaptic potential recordings in the CA1 hippocampal area, Fmr1 knockout mice express LTP to a similar extent as their wild type littermates during the first 1-2 hr after high frequency stimulation. Also, short-term potentiation (STP) was similar in both types of mice. To investigate whether Fmr1 is involved in the latter stages of LTP as an immediate early gene, we compared Fmr1 mRNA quantities on northern blots after chemical induction of seizures. A transient increase in the transcription of immediate early genes is thought to be essential for the maintenance of LTP. As no increase in Fmr1 mRNA could be detected, neither in cortex nor in total brain, during the first 2{1/2} hr after pentylenetetrazol-induced seizures, it is unlikely that Fmr1 is an immediate early gene in mice. In conclusion, we found no evidence for a function of FMR1 in STP or LTP. 37 refs., 4 figs.

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

    PubMed

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

    2014-01-01

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

  5. 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.

  6. 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.

  7. Less is More: unveiling the functional core of hematopoietic stem cells through knockout mice

    PubMed Central

    Rossi, Lara; Lin, Kuanyin K.; Boles, Nathan C.; Yang, Liubin; King, Katherine Y.; Jeong, Mira; Mayle, Allison; Goodell, Margaret A.

    2012-01-01

    Summary Hematopoietic stem cells (HSCs) represent one of the first recognized somatic stem cells. As such, nearly 200 genes have been examined for roles in HSC function in knockout mice. In this review, we compile the majority of these reports to provide a broad overview of the functional modules revealed by these genetic analyses and highlight some key regulatory pathways involved, including cell cycle control, TGF-β signaling, Pten/AKT signaling, Wnt signaling, and cytokine signaling. Finally, we propose recommendations for characterization of HSC function in knockout mice to facilitate cross-study comparisons that would generate a more cohesive picture of HSC biology. In the field of design, the minimalist movement stripped down buildings and objects to their most basic features, a sentiment that architect Ludwig Mies van der Rohe summarized in his motto “less is more”. By depleting HSCs of specific genes, knockout studies transpose the minimalist approach into research biology, providing insights into the essential core of genetic features that is indispensable for a well-functioning hematopoietic system. PMID:22958929

  8. Metabolic flux analysis of Escherichia coli knockouts: lessons from the Keio collection and future outlook.

    PubMed

    Long, Christopher P; Antoniewicz, Maciek R

    2014-08-01

    Cellular metabolic and regulatory systems are of fundamental interest to biologists and engineers. Incomplete understanding of these complex systems remains an obstacle to progress in biotechnology and metabolic engineering. An established method for obtaining new information on network structure, regulation and dynamics is to study the cellular system following a perturbation such as a genetic knockout. The Keio collection of all viable Escherichia coli single-gene knockouts is facilitating a systematic investigation of the regulation and metabolism of E. coli. Of all omics measurements available, the metabolic flux profile (the fluxome) provides the most direct and relevant representation of the cellular phenotype. Recent advances in (13)C-metabolic flux analysis are now permitting highly precise and accurate flux measurements for investigating cellular systems and guiding metabolic engineering efforts.

  9. A norm knockout method on indirect reciprocity to reveal indispensable norms

    PubMed Central

    Yamamoto, Hitoshi; Okada, Isamu; Uchida, Satoshi; Sasaki, Tatsuya

    2017-01-01

    Although various norms for reciprocity-based cooperation have been suggested that are evolutionarily stable against invasion from free riders, the process of alternation of norms and the role of diversified norms remain unclear in the evolution of cooperation. We clarify the co-evolutionary dynamics of norms and cooperation in indirect reciprocity and also identify the indispensable norms for the evolution of cooperation. Inspired by the gene knockout method, a genetic engineering technique, we developed the norm knockout method and clarified the norms necessary for the establishment of cooperation. The results of numerical investigations revealed that the majority of norms gradually transitioned to tolerant norms after defectors are eliminated by strict norms. Furthermore, no cooperation emerges when specific norms that are intolerant to defectors are knocked out. PMID:28276485

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

    PubMed Central

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

    2014-01-01

    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. PMID:24675426

  11. A norm knockout method on indirect reciprocity to reveal indispensable norms

    NASA Astrophysics Data System (ADS)

    Yamamoto, Hitoshi; Okada, Isamu; Uchida, Satoshi; Sasaki, Tatsuya

    2017-03-01

    Although various norms for reciprocity-based cooperation have been suggested that are evolutionarily stable against invasion from free riders, the process of alternation of norms and the role of diversified norms remain unclear in the evolution of cooperation. We clarify the co-evolutionary dynamics of norms and cooperation in indirect reciprocity and also identify the indispensable norms for the evolution of cooperation. Inspired by the gene knockout method, a genetic engineering technique, we developed the norm knockout method and clarified the norms necessary for the establishment of cooperation. The results of numerical investigations revealed that the majority of norms gradually transitioned to tolerant norms after defectors are eliminated by strict norms. Furthermore, no cooperation emerges when specific norms that are intolerant to defectors are knocked out.

  12. 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.

  13. Gait Analysis in a Mecp2 Knockout Mouse Model of Rett Syndrome Reveals Early-Onset and Progressive Motor Deficits

    PubMed Central

    Riddell, John S.; Bailey, Mark E. S.; Cobb, Stuart R.

    2014-01-01

    Rett syndrome (RTT) is a genetic disorder characterized by a range of features including cognitive impairment, gait abnormalities and a reduction in purposeful hand skills. Mice harbouring knockout mutations in the Mecp2 gene display many RTT-like characteristics and are central to efforts to find novel therapies for the disorder. As hand stereotypies and gait abnormalities constitute major diagnostic criteria in RTT, it is clear that motor and gait-related phenotypes will be of importance in assessing preclinical therapeutic outcomes. We therefore aimed to assess gait properties over the prodromal phase in a functional knockout mouse model of RTT. In male Mecp2 knockout mice, we observed alterations in stride, coordination and balance parameters at 4 weeks of age, before the onset of other overt phenotypic changes as revealed by observational scoring. These data suggest that gait measures may be used as a robust and early marker of MeCP2-dysfunction in future preclinical therapeutic studies. PMID:25392929

  14. Nontransferrin-bound iron uptake by hepatocytes is increased in the Hfe knockout mouse model of hereditary hemochromatosis.

    PubMed

    Chua, Anita C G; Olynyk, John K; Leedman, Peter J; Trinder, Debbie

    2004-09-01

    Hereditary hemochromatosis (HH) is an iron-overload disorder caused by a C282Y mutation in the HFE gene. In HH, plasma nontransferrin-bound iron (NTBI) levels are increased and NTBI is bound mainly by citrate. The aim of this study was to examine the importance of NTBI in the pathogenesis of hepatic iron loading in Hfe knockout mice. Plasma NTBI levels were increased 2.5-fold in Hfe knockout mice compared with control mice. Total ferric citrate uptake by hepatocytes isolated from Hfe knockout mice (34.1 +/- 2.8 pmol Fe/mg protein/min) increased by 2-fold compared with control mice (17.8 +/- 2.7 pmol Fe/mg protein/min; P <.001; mean +/- SEM; n = 7). Ferrous ion chelators, bathophenanthroline disulfonate, and 2',2-bipyridine inhibited ferric citrate uptake by hepatocytes from both mouse types. Divalent metal ions inhibited ferric citrate uptake by hepatocytes, as did diferric transferrin. Divalent metal transporter 1 (DMT1) mRNA and protein expression was increased approximately 2-fold by hepatocytes from Hfe knockout mice. We conclude that NTBI uptake by hepatocytes from Hfe knockout mice contributed to hepatic iron loading. Ferric ion was reduced to ferrous ion and taken up by hepatocytes by a pathway shared with diferric transferrin. Inhibition of uptake by divalent metals and up-regulation of DMT1 expression suggested that NTBI uptake was mediated by DMT1.

  15. Production of CMAH Knockout Preimplantation Embryos Derived From Immortalized Porcine Cells Via TALE Nucleases.

    PubMed

    Moon, JoonHo; Lee, Choongil; Kim, Su Jin; Choi, Ji-Yei; Lee, Byeong Chun; Kim, Jin-Soo; Jang, Goo

    2014-05-27

    Although noncancerous immortalized cell lines have been developed by introducing genes into human and murine somatic cells, such cell lines have not been available in large domesticated animals like pigs. For immortalizing porcine cells, primary porcine fetal fibroblasts were isolated and cultured using the human telomerase reverse transcriptase (hTERT) gene. After selecting cells with neomycin for 2 weeks, outgrowing colonized cells were picked up and subcultured for expansion. Immortalized cells were cultured for more than 9 months without changing their doubling time (~24 hours) or their diameter (< 20 µm) while control cells became replicatively senescent during the same period. Even a single cell expanded to confluence in 100 mm dishes. Furthermore, to knockout the CMAH gene, designed plasmids encoding a transcription activator-like effector nuclease (TALENs) pairs were transfected into the immortalized cells. Each single colony was analyzed by the mutation-sensitive T7 endonuclease I assay, fluorescent PCR, and dideoxy sequencing to obtain three independent clonal populations of cells that contained biallelic modifications. One CMAH knockout clone was chosen and used for somatic cell nuclear transfer. Cloned embryos developed to the blastocyst stage. In conclusion, we demonstrated that immortalized porcine fibroblasts were successfully established using the human hTERT gene, and the TALENs enabled biallelic gene disruptions in these immortalized cells.

  16. Boolean Network Model Predicts Knockout Mutant Phenotypes of Fission Yeast

    PubMed Central

    Davidich, Maria I.; Bornholdt, Stefan

    2013-01-01

    Boolean networks (or: networks of switches) are extremely simple mathematical models of biochemical signaling networks. Under certain circumstances, Boolean networks, despite their simplicity, are capable of predicting dynamical activation patterns of gene regulatory networks in living cells. For example, the temporal sequence of cell cycle activation patterns in yeasts S. pombe and S. cerevisiae are faithfully reproduced by Boolean network models. An interesting question is whether this simple model class could also predict a more complex cellular phenomenology as, for example, the cell cycle dynamics under various knockout mutants instead of the wild type dynamics, only. Here we show that a Boolean network model for the cell cycle control network of yeast S. pombe correctly predicts viability of a large number of known mutants. So far this had been left to the more detailed differential equation models of the biochemical kinetics of the yeast cell cycle network and was commonly thought to be out of reach for models as simplistic as Boolean networks. The new results support our vision that Boolean networks may complement other mathematical models in systems biology to a larger extent than expected so far, and may fill a gap where simplicity of the model and a preference for an overall dynamical blueprint of cellular regulation, instead of biochemical details, are in the focus. PMID:24069138

  17. Bone growth and turnover in progesterone receptor knockout mice.

    SciTech Connect

    Rickard, David J.; Iwaniec, Urszula T.; Evans, Glenda; Hefferan, Theresa E.; Hunter, Jaime C.; Waters, Katrina M.; Lydon, John P.; O'Malley, Bert W.; Khosla, Sundeep; Spelsberg, Thomas C.; Turner, Russell T.

    2008-05-01

    The role of progesterone receptor (PR) signaling in skeletal metabolism is controversial. To address whether signaling through the PR is necessary for normal bone growth and turnover, we performed histomorphometric and mCT analyses of bone from homozygous female PR knockout (PRKO) mice at 6, 12, and 26 weeks of age. These mice possess a null mutation of the PR locus, which blocks the gene expression of A and B isoforms of PR. Body weight gain, uterine weight gain and tibia longitudinal bone growth was normal in PRKO mice. In contrast, total and cortical bone mass were increased in long bones of post-pubertal (12 and 26-week-old) PRKO mice, whereas cancellous bone mass was normal in the tibia but increased in the humerus. The striking 57% decrease in cancellous bone from the proximal tibia metaphysis which occurred between 6 and 26 weeks in WT mice was abolished in PRKO mice. The improved bone balance in aging PRKO mice was associated with elevated bone formation and a tendency toward reduced osteoclast perimeter. Taken together, these findings suggest that PR signaling in mice attenuates the accumulation of cortical bone mass during adolescence and is required for early age-related loss of cancellous bone.

  18. Lipid transport in cholecystokinin knockout mice.

    PubMed

    King, Alexandra; Yang, Qing; Huesman, Sarah; Rider, Therese; Lo, Chunmin C

    2015-11-01

    Cholecystokinin (CCK) is released in response to lipid feeding and regulates pancreatic digestive enzymes vital to the absorption of nutrients. Our previous reports demonstrated that cholecystokinin knockout (CCK-KO) mice fed for 10 weeks of HFD had reduced body fat mass, but comparable glucose uptake by white adipose tissues and skeletal muscles. We hypothesized that CCK is involved in energy homeostasis and lipid transport from the small intestine to tissues in response to acute treatment with dietary lipids. CCK-KO mice with comparable fat absorption had increased energy expenditure and were resistant to HFD-induced obesity. Using intraduodenal infusion of butter fat and intravenous infusion using Liposyn III, we determined the mechanism of lipid transport from the small intestine to deposition in lymph and adipocytes in CCK-KO mice. CCK-KO mice had delayed secretion of Apo B48-chylomicrons, lipid transport to the lymphatic system, and triglyceride (TG)-derived fatty acid uptake by epididymal fat in response to acute treatment of intraduodenal lipids. In contrast, CCK-KO mice had comparable TG clearance and lipid uptake by white adipocytes in response to TGs in chylomicron-like emulsion. Thus, we concluded that CCK is important for lipid transport and energy expenditure to control body weight in response to dietary lipid feeding.

  19. Complementation Test of Rpe65 Knockout and Tvrm148

    PubMed Central

    Wright, Charles B.; Chrenek, Micah A.; Foster, Stephanie L.; Duncan, Todd; Redmond, T. Michael; Pardue, Machelle T.; Boatright, Jeffrey H.; Nickerson, John M.

    2013-01-01

    Purpose. A mouse mutation, tvrm148, was previously reported as resulting in retinal degeneration. Tvrm148 and Rpe65 map between markers D3Mit147 and D3Mit19 on a genetic map, but the physical map places RPE65 outside the markers. We asked if Rpe65 or perhaps another nearby gene is mutated and if the mutant reduced 11-cis-retinal levels. We studied the impact of the tvrm148 mutation on visual function, morphology, and retinoid levels. Methods. Normal phase HPLC was used to measure retinoid levels. Rpe65+/+, tvrm148/+ (T+/−), tvrm148/tvrm148 (T−/−), RPE65KO/KO (Rpe65−/−), and Rpe65T/− mice visual function was measured by optokinetic tracking (OKT) and electroretinography (ERG). Morphology was assessed by light microscopy and transmission electron microscopy (TEM). qRT-PCR was used to measure Rpe65 mRNA levels. Immunoblotting measured the size and amount of RPE65 protein. Results. The knockout and tvrm148 alleles did not complement. No 11-cis-retinal was detected in T−/− or Rpe65−/− mice. Visual acuity in Rpe65+/+ and T+/− mouse was ∼0.382 c/d, but 0.037 c/d in T−/− mice at postnatal day 210 (P210). ERG response in T−/− mice was undetectable except at bright flash intensities. Outer nuclear layer (ONL) thickness in T−/− mice was ∼70% of Rpe65+/+ by P210. Rpe65 mRNA levels in T−/− mice were unchanged, yet 14.5% of Rpe65+/+ protein levels was detected. Protein size was unchanged. Conclusions. A complementation test revealed the RPE65 knockout and tvrm148 alleles do not complement, proving that the tvrm148 mutation is in Rpe65. Behavioral, physiological, molecular, biochemical, and histological approaches indicate that tvrm148 is a null allele of Rpe65. PMID:23778877

  20. Cerebellar defects in Pdss2 conditional knockout mice during embryonic development and in adulthood.

    PubMed

    Lu, Song; Lu, Lin-Yu; Liu, Meng-Fei; Yuan, Qiu-Ju; Sham, Mai-Har; Guan, Xin-Yuan; Huang, Jian-Dong

    2012-01-01

    PDSS2 is a gene that encodes one of the two subunits of trans-prenyl diphosphate synthase that is essential for ubiquinone biosynthesis. It is known that mutations in PDSS2 can cause primary ubiquinone deficiency in humans and a similar disease in mice. Cerebellum is the most often affected organ in ubiquinone deficiency, and cerebellar atrophy has been diagnosed in many infants with this disease. In this study, two Pdss2 conditional knockout mouse lines directed by Pax2-cre and Pcp2-cre were generated to investigate the effect of ubiquinone deficiency on cerebellum during embryonic development and in adulthood, respectively. The Pdss2(f/-); Pax2-cre mouse recapitulates some symptoms of ubiquinone deficiency in infants, including severe cerebellum hypoplasia and lipid accumulation in skeletal muscles at birth. During early cerebellum development (E12.5-14.5), Pdss2 knockout initially causes the delay of radial glial cell growth and neuron progenitor migration, so the growth of mutant cerebellum is retarded. During later development (E15.5-P0), increased ectopic apoptosis of neuroblasts and impaired cell proliferation result in the progression of cerebellum hypoplasia in the mutant. Thus, the mutant cerebellum contains fewer neurons at birth, and the cells are disorganized. The developmental defect of mutant cerebellum does not result from reduced Fgf8 expression before E12.5. Electron microscopy reveals mitochondrial defects and increased autophagic-like vacuolization that may arise in response to abnormal mitochondria in the mutant cerebellum. Nevertheless, the mutant mice die soon after birth probably due to cleft palate and micrognathia, which may result from Pdss2 knockout caused by ectopic Pax2-cre expression in the first branchial arch. On the other hand, the Pdss2(f/-); Pcp2-cre mouse is healthy at birth but gradually loses cerebellar Purkinje cells and develops ataxia-like symptoms at 9.5 months; thus this conditional knockout mouse may serve as a model for

  1. Axonal regeneration of optic nerve after crush in Nogo66 receptor knockout mice.

    PubMed

    Su, Ying; Wang, Feng; Teng, Yan; Zhao, Shi-Guang; Cui, Hao; Pan, Shang-Ha

    2009-09-04

    Mature retinal ganglion cells (RGCs) cannot regenerate injured axons because some neurite growth inhibitors, including the C-terminal of Nogo-A (Nogo66), myelin-associated glycoprotein (MAG) and Omgp, exert their effects on neuron regeneration through the Nogo receptor (NgR). In this study, the axonal regeneration of retinal ganglion cells (RGCs) after optic nerve (ON) crush was investigated both in vivo and in vitro in NgR knockout mice. We used NgR knockout mice as the experimental group, and C57BL/6 mice as the control group. Partial ON injury was induced by using a specially designed ON clip to pinch the ON 1mm behind the mouse eyeball with 40g pressure for 9s. NgR mRNA was studied by in situ hybridization (ISH). NgR protein was studied by Western blot. Growth Associated Protein 43 (GAP-43), a plasticity protein expressed highly during axon regeneration, was studied by immunofluorescence staining on the frozen sections. RGCs were cultured and purified. The axonal growth of RGCs was calculated by a computerized image analyzer. We found that compared with the control group, the GAP-43 expression was significantly higher and the axonal growth was significantly more active at every observation time point in the experimental group. These results indicate that NgR genes play an important role in the axonal regeneration after ON injury, while knockout of NgR is effective for eliminating this inhibition and enhancing axonal regeneration.

  2. Gomafu lncRNA knockout mice exhibit mild hyperactivity with enhanced responsiveness to the psychostimulant methamphetamine

    PubMed Central

    Ip, Joanna Y.; Sone, Masamitsu; Nashiki, Chieko; Pan, Qun; Kitaichi, Kiyoyuki; Yanaka, Kaori; Abe, Takaya; Takao, Keizo; Miyakawa, Tsuyoshi; Blencowe, Benjamin J.; Nakagawa, Shinichi

    2016-01-01

    The long noncoding RNA Gomafu/MIAT/Rncr2 is thought to function in retinal cell specification, stem cell differentiation and the control of alternative splicing. To further investigate physiological functions of Gomafu, we created mouse knockout (KO) model that completely lacks the Gomafu gene. The KO mice did not exhibit any developmental deficits. However, behavioral tests revealed that the KO mice are hyperactive. This hyperactive behavior was enhanced when the KO mice were treated with the psychostimulant methamphetamine, which was associated with an increase in dopamine release in the nucleus accumbens. RNA sequencing analyses identified a small number of genes affected by the deficiency of Gomafu, a subset of which are known to have important neurobiological functions. These observations suggest that Gomafu modifies mouse behavior thorough a mild modulation of gene expression and/or alternative splicing of target genes. PMID:27251103

  3. Sleep in Kcna2 knockout mice

    PubMed Central

    Douglas, Christopher L; Vyazovskiy, Vladyslav; Southard, Teresa; Chiu, Shing-Yan; Messing, Albee; Tononi, Giulio; Cirelli, Chiara

    2007-01-01

    Background Shaker codes for a Drosophila voltage-dependent potassium channel. Flies carrying Shaker null or hypomorphic mutations sleep 3–4 h/day instead of 8–14 h/day as their wild-type siblings do. Shaker-like channels are conserved across species but it is unknown whether they affect sleep in mammals. To address this issue, we studied sleep in Kcna2 knockout (KO) mice. Kcna2 codes for Kv1.2, the alpha subunit of a Shaker-like voltage-dependent potassium channel with high expression in the mammalian thalamocortical system. Results Continuous (24 h) electroencephalograph (EEG), electromyogram (EMG), and video recordings were used to measure sleep and waking in Kcna2 KO, heterozygous (HZ) and wild-type (WT) pups (P17) and HZ and WT adult mice (P67). Sleep stages were scored visually based on 4-s epochs. EEG power spectra (0–20 Hz) were calculated on consecutive 4-s epochs. KO pups die by P28 due to generalized seizures. At P17 seizures are either absent or very rare in KO pups (< 1% of the 24-h recording time), and abnormal EEG activity is only present during the seizure. KO pups have significantly less non-rapid eye movement (NREM) sleep (-23%) and significantly more waking (+21%) than HZ and WT siblings with no change in rapid eye movement (REM) sleep time. The decrease in NREM sleep is due to an increase in the number of waking episodes, with no change in number or duration of sleep episodes. Sleep patterns, daily amounts of sleep and waking, and the response to 6 h sleep deprivation are similar in HZ and WT adult mice. Conclusion Kv1.2, a mammalian homologue of Shaker, regulates neuronal excitability and affects NREM sleep. PMID:17925011

  4. Methylphenidate restores novel object recognition in DARPP-32 knockout mice.

    PubMed

    Heyser, Charles J; McNaughton, Caitlyn H; Vishnevetsky, Donna; Fienberg, Allen A

    2013-09-15

    Previously, we have shown that Dopamine- and cAMP-regulated phosphoprotein of 32kDa (DARPP-32) knockout mice required significantly more trials to reach criterion than wild-type mice in an operant reversal-learning task. The present study was conducted to examine adult male and female DARPP-32 knockout mice and wild-type controls in a novel object recognition test. Wild-type and knockout mice exhibited comparable behavior during the initial exploration trials. As expected, wild-type mice exhibited preferential exploration of the novel object during the substitution test, demonstrating recognition memory. In contrast, knockout mice did not show preferential exploration of the novel object, instead exhibiting an increase in exploration of all objects during the test trial. Given that the removal of DARPP-32 is an intracellular manipulation, it seemed possible to pharmacologically restore some cellular activity and behavior by stimulating dopamine receptors. Therefore, a second experiment was conducted examining the effect of methylphenidate. The results show that methylphenidate increased horizontal activity in both wild-type and knockout mice, though this increase was blunted in knockout mice. Pretreatment with methylphenidate significantly impaired novel object recognition in wild-type mice. In contrast, pretreatment with methylphenidate restored the behavior of DARPP-32 knockout mice to that observed in wild-type mice given saline. These results provide additional evidence for a functional role of DARPP-32 in the mediation of processes underlying learning and memory. These results also indicate that the behavioral deficits in DARPP-32 knockout mice may be restored by the administration of methylphenidate.

  5. Regulation of dopamine presynaptic markers and receptors in the striatum of DJ-1 and Pink1 knockout rats

    PubMed Central

    Sun, Jianjun; Kouranova, Evguenia; Cui, Xiaoxia; Mach, Robert H.; Xu, Jinbin

    2014-01-01

    Pathogenic autosomal recessive mutations in the DJ-1 (Park7) or the PTEN-induced putative kinase 1 (Pink1 or PARK6) genes are associated with familial Parkinson’s disease (PD). It is not well known regarding the pathological mechanisms involving the DJ-1 and Pink1 mutations. Here we characterized DJ-1 and Pink1 knockout rats both through expression profiling and using quantitative autoradiography to measure the densities of the dopamine D1, D2, D3 receptors, vesicular monoamine transporter type-2 (VMAT2) and dopamine transporter (DAT) in the striatum of transgenic rats and wild type controls. Expression profiling with a commercially available array of 84 genes known to be involved in PD indicated that only the target gene was significantly downregulated in each transgenic rat model. D1 receptor, VMAT2, and DAT were measured using [3H]SCH23390, [3H]dihydrotetrabenazine, and [3H]WIN35428, respectively. No significant changes were observed in the density of DAT in either model. Although the densities of VMAT2 and D1 receptor were unchanged in Pink1 knockout, but both were increased in DJ-1 knockout rats. The densities of D2 and D3 receptors, determined by mathematical analysis of binding of radioligands [3H]WC-10 and [3H]raclopride, were significantly increased in both knockout models. These distinctive changes in the expression of dopamine presynaptic markers and receptors in the striatum may reflect different compensatory regulation of dopamine system in DJ-1 versus Pink1 knockout rat models of familial PD. PMID:24157858

  6. [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.

  7. Establishment of MAGEC2-knockout cells and functional investigation of MAGEC2 in tumor cells.

    PubMed

    Wang, Jingjing; Song, Xiao; Guo, Chengli; Wang, Ying; Yin, Yanhui

    2016-12-01

    Cancer/testis antigen MAGEC2, a member of the type I melanoma-associated antigen family, is expressed in a wide variety of cancer types but not in normal somatic cells. MAGEC2 has long been recognized as a tumor-specific target, however, its functions remain largely unknown. In this study, we established MAGEC2-knockout A375 melanoma cell lines using the CRISPR/Cas9 system. Seven clonal cell lines were generated by using four single guide RNAs targeting the coding region of the MAGEC2 gene, which produced indels that abolished MAGEC2 protein expression. To identify the differentially expressed protein profiles associated with MAGEC2 loss, isobaric tag for relative quantitation-based comparative proteomics experiments were carried out on the MAGEC2-knockcout and control A375 cells. Mining of the proteomics data identified a total 224 (61.6% upregulated and 38.4% downregulated) proteins to be significantly altered in expression level in MAGEC2-knockcout cells. Ingenuity Pathway Analysis indicated that the significantly altered proteins were involved in critical neoplasia-related biological functions such as cell death, proliferation, and movement. Gene ontology analysis identified "apoptosis signaling" as the top-most upregulated pathway associated with MAGEC2 loss. We showed that knockout or knockdown of the MAGEC2 gene sensitized melanoma cells to tumor necrosis factor-α-induced apoptosis. Interestingly, actin-based motility by Rho and RhoA signaling, known to promote cell migration, were also identified as the top downregulated pathways in MAGEC2-knockout A375 cells. In short, our study provides a suitable cell model for exploring the biological functions of MAGEC2 in malignant cells, and sheds light on the molecular pathway by which MAGEC2 promotes tumor development.

  8. GONADAL HORMONE INDEPENDENT SEX DIFFERENCES IN STEROIDOGENIC FACTOR 1 KNOCKOUT MICE BRAIN

    PubMed Central

    Büdefeld, Tomaž; Tobet, Stuart A.; Majdič, Gregor

    2011-01-01

    Summary Sex differences in brain morphology have been described in a number of species including humans. Gonadal hormones were shown to provide a major influence on brain sexual differentiation more than 50 years ago. A growing number of studies is providing evidence for roles of genetic factors, in particular sex chromosome complement, on brain sexual differentiation in mammals. In this review, hormone-independent brain sexual differentiation, with the emphasis on mice with a disruption of the SF-1 gene (SF-1 knockout, SF-1 KO) are discussed. PMID:21887123

  9. Analysis of microsatellite polymorphism in inbred knockout mice.

    PubMed

    Zuo, Baofen; Du, Xiaoyan; Zhao, Jing; Yang, Huixin; Wang, Chao; Wu, Yanhua; Lu, Jing; Wang, Ying; Chen, Zhenwen

    2012-01-01

    Previously, we found that the genotype of 42 out of 198 mouse microsatellite loci, which are distributed among all chromosomes except the Y chromosome, changed from monomorphism to polymorphism (CMP) in a genetically modified inbred mouse strain. In this study, we further examined whether CMP also relates to the homologous recombination in gene knockout (KO) mouse strains. The same 42 microsatellite loci were analyzed by polymerase chain reaction (PCR) in 29 KO inbred mouse strains via short tandem sequence repeat (STR) scanning and direct sequence cloning to justify microsatellite polymorphisms. The C57BL/6J and 129 mouse strains, from which these 29 KO mice were derived, were chosen as the background controls. The results indicated that 10 out of 42 (23.8%) loci showed CMP in some of these mouse strains. Except for the trinucleotide repeat locus of D3Mit22, which had microsatellite CMP in strain number 9, the core sequences of the remaining 41 loci were dinucleotide repeats, and 9 out of 41 (21.95%) showed CMPs among detected mouse strains. However, 11 out of 29 (37.9%) KO mice strains were recognized as having CMPs. The popular dinucleotide motifs in CMP were (TG)(n) (50%, 2/4), followed by (GT)(n) (27.27%, 3/11) and (CA)(n) (23.08%, 3/13). The microsatellite CMP in (CT)(n) and (AG)(n) repeats were 20% (1/5). According to cloning sequencing results, 6 KO mouse strains showed insertions of nucleotides whereas 1 showed a deletion. Furthermore, 2 loci (D13Mit3 and D14Mit102) revealed CMP in 2 strains, and mouse strain number 9 showed CMPs in two loci (D3Mit22 and D13Mit3) simultaneously. Collectively, these results indicated that microsatellite polymorphisms were present in the examined inbred KO mice.

  10. Targeting cancer using KAT inhibitors to mimic lethal knockouts

    PubMed Central

    Brown, James A.L.; Bourke, Emer; Eriksson, Leif A.; Kerin, Michael J.

    2016-01-01

    Two opposing enzyme classes regulate fundamental elements of genome maintenance, gene regulation and metabolism, either through addition of an acetyl moiety by histone acetyltransferases (HATs) or its removal by histone de-acetyltransferases (HDAC), and are exciting targets for drug development. Importantly, dysfunctional acetylation has been implicated in numerous diseases, including cancer. Within the HAT superfamily the MYST family holds particular interest, as its members are directly involved in the DNA damage response and repair pathways and crucially, several members have been shown to be down-regulated in common cancers (such as breast and prostate). In the present study we focus on the development of lysine (K) acetyltransferase inhibitors (KATi) targeting the MYST family member Tip60 (Kat5), an essential protein, designed or discovered through screening libraries. Importantly, Tip60 has been demonstrated to be significantly down-regulated in many cancers which urgently require new treatment options. We highlight current and future efforts employing these KATi as cancer treatments and their ability to synergize and enhance current cancer treatments. We investigate the different methods of KATi production or discovery, their mechanisms and their validation models. Importantly, the utility of KATi is based on a key concept: using KATi to abrogate the activity of an already down-regulated essential protein (effectively creating a lethal knockout) provides another innovative mechanism for targeting cancer cells, while significantly minimizing any off-target effects to normal cells. This approach, combined with the rapidly developing interest in KATi, suggests that KATi have a bright future for providing truly personalized therapies. PMID:27528742

  11. Analysis of Microsatellite Polymorphism in Inbred Knockout Mice

    PubMed Central

    Zhao, Jing; Yang, Huixin; Wang, Chao; Wu, Yanhua; Lu, Jing; Wang, Ying; Chen, Zhenwen

    2012-01-01

    Previously, we found that the genotype of 42 out of 198 mouse microsatellite loci, which are distributed among all chromosomes except the Y chromosome, changed from monomorphism to polymorphism (CMP) in a genetically modified inbred mouse strain. In this study, we further examined whether CMP also relates to the homologous recombination in gene knockout (KO) mouse strains. The same 42 microsatellite loci were analyzed by polymerase chain reaction (PCR) in 29 KO inbred mouse strains via short tandem sequence repeat (STR) scanning and direct sequence cloning to justify microsatellite polymorphisms. The C57BL/6J and 129 mouse strains, from which these 29 KO mice were derived, were chosen as the background controls. The results indicated that 10 out of 42 (23.8%) loci showed CMP in some of these mouse strains. Except for the trinucleotide repeat locus of D3Mit22, which had microsatellite CMP in strain number 9, the core sequences of the remaining 41 loci were dinucleotide repeats, and 9 out of 41 (21.95%) showed CMPs among detected mouse strains. However, 11 out of 29 (37.9%) KO mice strains were recognized as having CMPs. The popular dinucleotide motifs in CMP were (TG)n (50%, 2/4), followed by (GT)n (27.27%, 3/11) and (CA)n (23.08%, 3/13). The microsatellite CMP in (CT)n and (AG)n repeats were 20% (1/5). According to cloning sequencing results, 6 KO mouse strains showed insertions of nucleotides whereas 1 showed a deletion. Furthermore, 2 loci (D13Mit3 and D14Mit102) revealed CMP in 2 strains, and mouse strain number 9 showed CMPs in two loci (D3Mit22 and D13Mit3) simultaneously. Collectively, these results indicated that microsatellite polymorphisms were present in the examined inbred KO mice. PMID:22509320

  12. Antibiotic-free segregational plasmid stabilization in Escherichia coli owing to the knockout of triosephosphate isomerase (tpiA)

    PubMed Central

    2014-01-01

    Background Segregational stability of plasmids is of major concern for recombinant bacterial production strains. One of the best strategies to counteract plasmid loss is the use of auxotrophic mutants which are complemented with the lacking gene along with the product-relevant ones. However, these knockout mutants often show unwanted growth in complex standard media or no growth at all under uncomplemented conditions. This led to the choice of a gene for knockout that only connects two essential arms of an essential metabolic pathway – the glycolysis. Results Triosephosphate isomerase was chosen because its knockout will have a tremendous effect on growth on glucose as well as on glycerol. On glycerol the effect is almost absolute whereas on glucose growth is still possible, but with considerably lower rate than usual. This feature is essential because it may render cloning easier. This enzymatic activity was successfully tested as an alternative to antibiotic-based plasmid selection. Expression of a model recombinant β-glucanase in continuous cultivation was possible with stable maintenance of the plasmid. In addition, the complementation of tpiA knockout strains by the corresponding plasmids and their growth characteristics were tested on a series of complex and synthetic media. The accumulation of methylglyoxal during the growth of tpiA-deficient strains was shown to be a possible cause for the growth disadvantage of these strains in comparison to the parent strain for the Keio Collection strain or the complemented knock-out strain. Conclusion Through the use of this new auxotrophic complementation system, antibiotic-free cloning and selection of recombinant plasmid were possible. Continuous cultivation and recombinant protein expression with high segregational stability over an extended time period was also demonstrated. PMID:24745552

  13. Phenotype of the Cyp1a1/1a2/1b1(−/−) Triple-Knockout Mouse*

    PubMed Central

    Dragin, Nadine; Shi, Zhanquan; Madan, Rajat; Karp, Christopher L.; Sartor, Maureen A.; Chen, Chi; Gonzalez, Frank J.; Nebert, Daniel W.

    2009-01-01

    Crossing the Cyp1a1/1a2(−/−) double-knockout mouse with the Cyp1b1(−/−) single-knockout mouse, we generated the Cyp1a1/1a2/1b1(−/−) triple-knockout mouse. In this triple-knockout mouse, statistically significant phenotypes (with incomplete penetrance) included slower weight gain and greater risk of embryolethality before gestational day 11, hydrocephalus, hermaphroditism, and cystic ovaries. Oral benzo[a]pyrene (BaP) daily for 18 days in the Cyp1a1/1a2(−/−) produced the same degree of marked immunosuppression as seen in the Cyp1a1(−/−) mouse; we believe this reflects the absence of intestinal CYP1A1. Oral BaP-treated Cyp1a1/1a2/1b1(−/−) mice showed the same “rescued” response as that seen in the Cyp1a1/1b1(−/−) mouse; we believe this reflects the absence of CYP1B1 in immune tissues. Urinary metabolite profiles were dramatically different between untreated triple-knockout and wild-type; principal components analysis showed that the shifts in urinary metabolite patterns in oral BaP-treated triple-knockout and wild-type mice were also strikingly different. Liver microarray cDNA differential expression (comparing triple-knockout with wild-type) revealed at least 89 genes up- and 62 genes down-regulated (P-value ≤0.00086). Gene Ontology “classes of genes” most perturbed in the untreated triple-knockout (compared with wild-type) include lipid, steroid, and cholesterol biosynthesis and metabolism; nucleosome and chromatin assembly; carboxylic and organic acid metabolism; metal-ion binding; and ion homeostasis. In the triple-knockout compared with the wild-type mice, response to zymosan-induced peritonitis was strikingly exaggerated, which may well reflect down-regulation of Socs2 expression. If a single common molecular pathway is responsible for all of these phenotypes, we suggest that functional effects of the loss of all three Cyp1 genes could be explained by perturbations in CYP1-mediated eicosanoid production, catabolism and

  14. The growth and reproduction performance of TALEN-mediated β-lactoglobulin-knockout bucks.

    PubMed

    Ge, Hengtao; Cui, Chenchen; Liu, Jun; Luo, Yan; Quan, Fusheng; Jin, Yaping; Zhang, Yong

    2016-10-01

    With the technological development of several engineered endonucleases (EENs), such as zinc-finger nucleases, transcription activator-like effector nucleases (TALENs) and CRISPR/Cas9, gene targeting by homologous recombination has been efficiently improved to generate site-specifically genetically modified livestock. However, few studies have been done to investigate the health and fertility of these animals. The purpose of the present study is to investigate if gene targeting events and a recloning procedure would affect the production traits of EEN-mediated gene targeted bucks. TALEN-mediated β-lactoglobulin (BLG) gene mono-allelic knockout (BLG (+/-)) goats and bi-allelic knockout (BLG (-/-)) buck produced by using sequential gene targeting combined with recloning in fibroblasts from BLG (+/-) buck were used to evaluate their health and fertility. Birth weight and postnatal growth of BLG (+/-) bucks were similar to the wild-type goats. None of the parameters for both fresh and frozen-thawed semen quality were significantly different in BLG (+/-) or BLG (-/-) bucks compared to their corresponding comparators. In vitro fertilization (IVF) test revealed that the proportion of IVF oocytes developing to the blastocyst stage was identical among BLG (+/-), BLG (-/-) and wild-type bucks. Conception rates of artificial insemination were respectively 42.3, 38.0 and 42.6 % for frozen-thawed semen from the BLG (+/-), BLG (-/-) and wild-type bucks. In addition, germline transmission of the targeted BLG modification was in accordance with Mendelian rules. These results demonstrated that the analyzed growth and reproductive traits were not impacted by targeting BLG gene and recloning, implicating the potential for dairy goat breeding of BLG (+/-) and BLG (-/-) bucks.

  15. Food intake, tumor growth, and weight loss in EP2 receptor subtype knockout mice bearing PGE2-producing tumors

    PubMed Central

    Iresjö, Britt-Marie; Wang, Wenhua; Nilsberth, Camilla; Andersson, Marianne; Lönnroth, Christina; Smedh, Ulrika

    2015-01-01

    Previous studies in our laboratory have demonstrated that prostaglandin (PG) E2 is involved in anorexia/cachexia development in MCG 101 tumor-bearing mice. In the present study, we investigate the role of PGE receptor subtype EP2 in the development of anorexia after MCG 101 implantation in wild-type (EP2+/+) or EP2-receptor knockout (EP2−/−) mice. Our results showed that host absence of EP2 receptors attenuated tumor growth and development of anorexia in tumor-bearing EP2 knockout mice compared to tumor-bearing wild-type animals. Microarray profiling of the hypothalamus revealed a relative twofold change in expression of around 35 genes including mRNA transcripts coding for Phospholipase A2 and Prostaglandin D2 synthase (Ptgds) in EP2 receptor knockout mice compared to wild-type mice. Prostaglandin D2 synthase levels were increased significantly in EP2 receptor knockouts, suggesting that improved food intake may depend on altered balance of prostaglandin production in hypothalamus since PGE2 and PGD2 display opposing effects in feeding control. PMID:26197930

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

    SciTech Connect

    Antonson, P.; Omoto, Y.; Humire, P.; Gustafsson, J.-A.

    2012-08-10

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

  17. Large-Scale Screening for Targeted Knockouts in the Caenorhabditis elegans Genome

    PubMed Central

    2012-01-01

    The nematode Caenorhabditis elegans is a powerful model system to study contemporary biological problems. This system would be even more useful if we had mutations in all the genes of this multicellular metazoan. The combined efforts of the C. elegans Deletion Mutant Consortium and individuals within the worm community are moving us ever closer to this goal. At present, of the 20,377 protein-coding genes in this organism, 6764 genes with associated molecular lesions are either deletions or null mutations (WormBase WS220). Our three laboratories have contributed the majority of mutated genes, 6841 mutations in 6013 genes. The principal method we used to detect deletion mutations in the nematode utilizes polymerase chain reaction (PCR). More recently, we have used array comparative genome hybridization (aCGH) to detect deletions across the entire coding part of the genome and massively parallel short-read sequencing to identify nonsense, splicing, and missense defects in open reading frames. As deletion strains can be frozen and then thawed when needed, these strains will be an enduring community resource. Our combined molecular screening strategies have improved the overall throughput of our gene-knockout facilities and have broadened the types of mutations that we and others can identify. These multiple strategies should enable us to eventually identify a mutation in every gene in this multicellular organism. This knowledge will usher in a new age of metazoan genetics in which the contribution to any biological process can be assessed for all genes. PMID:23173093

  18. Biological characterization of gene response in Rpe65-/- mouse model of Leber's congenital amaurosis during progression of the disease.

    PubMed

    Cottet, Sandra; Michaut, Lydia; Boisset, Gaëlle; Schlecht, Ulrich; Gehring, Walter; Schorderet, Daniel F

    2006-10-01

    RPE65 is the retinal isomerase essential for conversion of all-trans-retinyl ester to 11-cis-retinol in the visual cycle. Leber's congenital amaurosis (LCA), an autosomal recessive form of RP resulting in blindness, is commonly caused by mutations in the Rpe65 gene. Whereas the molecular mechanisms by which these mutations contribute to retinal disease remain largely unresolved, affected patients show marked RPE damage and photoreceptor degeneration. We evaluated gene expression in Rpe65-/- mouse model of LCA before and at the onset of photoreceptor cell death in 2, 4, and 6 month old animals. Microarray analysis demonstrates altered expression of genes involved in phototransduction, apoptosis regulation, cytoskeleton organization, and extracellular matrix (ECM) constituents. Cone-specific phototransduction genes are strongly decreased, reflecting early loss of cones. In addition, remaining rods show modified expression of genes encoding components of the cytoskeleton and ECM. This may affect rod physiology and interaction with the adjacent RPE and lead to loss of survival signals, as reflected by the alteration of apoptosis-related genes Together, these results suggest that RPE65 defect triggers an overall remodeling of the neurosensitive retina that may, in turn, disrupt photoreceptor homeostasis and induce apoptosis signaling cascade toward retinal cell death.

  19. Generation of GGTA1 biallelic knockout pigs via zinc-finger nucleases and somatic cell nuclear transfer.

    PubMed

    Bao, Lei; Chen, HaiDe; Jong, UiMyong; Rim, CholHo; Li, WenLing; Lin, XiJuan; Zhang, Dan; Luo, Qiong; Cui, Chun; Huang, HeFeng; Zhang, Yan; Xiao, Lei; Fu, ZhiXin

    2014-02-01

    Genetically modified pigs are valuable models of human disease and donors of xenotransplanted organs. Conventional gene targeting in pig somatic cells is extremely inefficient. Zinc-finger nuclease (ZFN) technology has been shown to be a powerful tool for efficiently inducing mutations in the genome. However, ZFN-mediated targeting in pigs has rarely been achieved. Here, we used ZFNs to knock out the porcine α-1, 3-galactosyl-transferase (GGTA1) gene, which generates Gal epitopes that trigger hyperacute immune rejection in pig-to-human transplantation. Primary pig fibroblasts were transfected with ZFNs targeting the coding region of GGTA1. Eighteen mono-allelic and four biallelic knockout cell clones were obtained after drug selection with efficiencies of 23.4% and 5.2%, respectively. The biallelic cells were used to produce cloned pigs via somatic cell nuclear transfer (SCNT). Three GGTA1 null piglets were born, and one knockout primary fibroblast cell line was established from a cloned fetus. Gal epitopes on GGTA1 null pig cells were completely eliminated from the cell membrane. Functionally, GGTA1 knockout cells were protected from complement-mediated immune attacks when incubated with human serum. This study demonstrated that ZFN is an efficient tool in creating gene-modified pigs. GGTA1 null pigs and GGTA1 null fetal fibroblasts would benefit research and pig-to-human transplantation.

  20. Knockout of BRD7 results in impaired spermatogenesis and male infertility

    PubMed Central

    Wang, Heran; Zhao, Ran; Guo, Chi; Jiang, Shihe; Yang, Jing; Xu, Yang; Liu, Yukun; Fan, Liqing; Xiong, Wei; Ma, Jian; Peng, Shuping; Zeng, Zhaoyang; Zhou, Yanhong; Li, Xiayu; Li, Zheng; Li, Xiaoling; Schmitt, David C.; Tan, Ming; Li, Guiyuan; Zhou, Ming

    2016-01-01

    BRD7 was originally identified as a novel bromodomain gene and a potential transcriptional factor. BRD7 was found to be extensively expressed in multiple mouse tissues but was highly expressed in the testis. Furthermore, BRD7 was located in germ cells during multiple stages of spermatogenesis, ranging from the pachytene to the round spermatid stage. Homozygous knockout of BRD7 (BRD7−/−) resulted in complete male infertility and spermatogenesis defects, including deformed acrosomal formation, degenerative elongating spermatids and irregular head morphology in postmeiotic germ cells in the seminiferous epithelium, which led to the complete arrest of spermatogenesis at step 13. Moreover, a high ratio of apoptosis was determined by TUNEL analysis, which was supported by high levels of the apoptosis markers annexin V and p53 in knockout testes. Increased expression of the DNA damage maker λH2AX was also found in BRD7−/− mice, whereas DNA damage repair genes were down−regulated. Furthermore, no or lower expression of BRD7 was detected in the testes of azoospermia patients exhibiting spermatogenesis arrest than that in control group. These data demonstrate that BRD7 is involved in male infertility and spermatogenesis in mice, and BRD7 defect might be associated with the occurrence and development of human azoospermia. PMID:26878912

  1. A Knockout Experiment: Disciplinary Divides and Experimental Skill in Animal Behaviour Genetics

    PubMed Central

    Nelson, Nicole C.

    2015-01-01

    In the early 1990s, a set of new techniques for manipulating mouse DNA allowed researchers to ‘knock out’ specific genes and observe the effects of removing them on a live mouse. In animal behaviour genetics, questions about how to deploy these techniques to study the molecular basis of behaviour became quite controversial, with a number of key methodological issues dissecting the interdisciplinary research field along disciplinary lines. This paper examines debates that took place during the 1990s between a predominately North American group of molecular biologists and animal behaviourists around how to design, conduct, and interpret behavioural knockout experiments. Drawing from and extending Harry Collins’s work on how research communities negotiate what counts as a ‘well-done experiment,’ I argue that the positions practitioners took on questions of experimental skill reflected not only the experimental traditions they were trained in but also their differing ontological and epistemological commitments. Different assumptions about the nature of gene action, eg., were tied to different positions in the knockout mouse debates on how to implement experimental controls. I conclude by showing that examining representations of skill in the context of a community’s knowledge commitments sheds light on some of the contradictory ways in which contemporary animal behaviour geneticists talk about their own laboratory work as a highly skilled endeavour that also could be mechanised, as easy to perform and yet difficult to perform well. PMID:26090739

  2. CRISPR-Mediated VHL Knockout Generates an Improved Model for Metastatic Renal Cell Carcinoma.

    PubMed

    Schokrpur, Shiruyeh; Hu, Junhui; Moughon, Diana L; Liu, Peijun; Lin, Lucia C; Hermann, Kip; Mangul, Serghei; Guan, Wei; Pellegrini, Matteo; Xu, Hua; Wu, Lily

    2016-06-30

    Metastatic renal cell carcinoma (mRCC) is nearly incurable and accounts for most of the mortality associated with RCC. Von Hippel Lindau (VHL) is a tumour suppressor that is lost in the majority of clear cell RCC (ccRCC) cases. Its role in regulating hypoxia-inducible factors-1α (HIF-1α) and -2α (HIF-2α) is well-studied. Recent work has demonstrated that VHL knock down induces an epithelial-mesenchymal transition (EMT) phenotype. In this study we showed that a CRISPR/Cas9-mediated knock out of VHL in the RENCA model leads to morphologic and molecular changes indicative of EMT, which in turn drives increased metastasis to the lungs. RENCA cells deficient in HIF-1α failed to undergo EMT changes upon VHL knockout. RNA-seq revealed several HIF-1α-regulated genes that are upregulated in our VHL knockout cells and whose overexpression signifies an aggressive form of ccRCC in the cancer genome atlas (TCGA) database. Independent validation in a new clinical dataset confirms the upregulation of these genes in ccRCC samples compared to adjacent normal tissue. Our findings indicate that loss of VHL could be driving tumour cell dissemination through stabilization of HIF-1α in RCC. A better understanding of the mechanisms involved in this phenomenon can guide the search for more effective treatments to combat mRCC.

  3. CRISPR-Mediated VHL Knockout Generates an Improved Model for Metastatic Renal Cell Carcinoma

    PubMed Central

    Schokrpur, Shiruyeh; Hu, Junhui; Moughon, Diana L.; Liu, Peijun; Lin, Lucia C.; Hermann, Kip; Mangul, Serghei; Guan, Wei; Pellegrini, Matteo; Xu, Hua; Wu, Lily

    2016-01-01

    Metastatic renal cell carcinoma (mRCC) is nearly incurable and accounts for most of the mortality associated with RCC. Von Hippel Lindau (VHL) is a tumour suppressor that is lost in the majority of clear cell RCC (ccRCC) cases. Its role in regulating hypoxia-inducible factors-1α (HIF-1α) and -2α (HIF-2α) is well-studied. Recent work has demonstrated that VHL knock down induces an epithelial-mesenchymal transition (EMT) phenotype. In this study we showed that a CRISPR/Cas9-mediated knock out of VHL in the RENCA model leads to morphologic and molecular changes indicative of EMT, which in turn drives increased metastasis to the lungs. RENCA cells deficient in HIF-1α failed to undergo EMT changes upon VHL knockout. RNA-seq revealed several HIF-1α-regulated genes that are upregulated in our VHL knockout cells and whose overexpression signifies an aggressive form of ccRCC in the cancer genome atlas (TCGA) database. Independent validation in a new clinical dataset confirms the upregulation of these genes in ccRCC samples compared to adjacent normal tissue. Our findings indicate that loss of VHL could be driving tumour cell dissemination through stabilization of HIF-1α in RCC. A better understanding of the mechanisms involved in this phenomenon can guide the search for more effective treatments to combat mRCC. PMID:27358011

  4. Knockout of BRD7 results in impaired spermatogenesis and male infertility.

    PubMed

    Wang, Heran; Zhao, Ran; Guo, Chi; Jiang, Shihe; Yang, Jing; Xu, Yang; Liu, Yukun; Fan, Liqing; Xiong, Wei; Ma, Jian; Peng, Shuping; Zeng, Zhaoyang; Zhou, Yanhong; Li, Xiayu; Li, Zheng; Li, Xiaoling; Schmitt, David C; Tan, Ming; Li, Guiyuan; Zhou, Ming

    2016-02-16

    BRD7 was originally identified as a novel bromodomain gene and a potential transcriptional factor. BRD7 was found to be extensively expressed in multiple mouse tissues but was highly expressed in the testis. Furthermore, BRD7 was located in germ cells during multiple stages of spermatogenesis, ranging from the pachytene to the round spermatid stage. Homozygous knockout of BRD7 (BRD7(-/-)) resulted in complete male infertility and spermatogenesis defects, including deformed acrosomal formation, degenerative elongating spermatids and irregular head morphology in postmeiotic germ cells in the seminiferous epithelium, which led to the complete arrest of spermatogenesis at step 13. Moreover, a high ratio of apoptosis was determined by TUNEL analysis, which was supported by high levels of the apoptosis markers annexin V and p53 in knockout testes. Increased expression of the DNA damage maker λH2AX was also found in BRD7(-/-) mice, whereas DNA damage repair genes were down-regulated. Furthermore, no or lower expression of BRD7 was detected in the testes of azoospermia patients exhibiting spermatogenesis arrest than that in control group. These data demonstrate that BRD7 is involved in male infertility and spermatogenesis in mice, and BRD7 defect might be associated with the occurrence and development of human azoospermia.

  5. Sirh7/Ldoc1 knockout mice exhibit placental P4 overproduction and delayed parturition

    PubMed Central

    Naruse, Mie; Ono, Ryuichi; Irie, Masahito; Nakamura, Kenji; Furuse, Tamio; Hino, Toshiaki; Oda, Kanako; Kashimura, Misho; Yamada, Ikuko; Wakana, Shigeharu; Yokoyama, Minesuke; Ishino, Fumitoshi; Kaneko-Ishino, Tomoko

    2014-01-01

    Sirh7/Ldoc1 [sushi-ichi retrotransposon homolog 7/leucine zipper, downregulated in cancer 1, also called mammalian retrotransposon-derived 7 (Mart7)] is one of the newly acquired genes from LTR retrotransposons in eutherian mammals. Interestingly, Sirh7/Ldoc1 knockout (KO) mice exhibited abnormal placental cell differentiation/maturation, leading to an overproduction of placental progesterone (P4) and placental lactogen 1 (PL1) from trophoblast giant cells (TGCs). The placenta is an organ that is essential for mammalian viviparity and plays a major endocrinological role during pregnancy in addition to providing nutrients and oxygen to the fetus. P4 is an essential hormone in the preparation and maintenance of pregnancy and the determination of the timing of parturition in mammals; however, the biological significance of placental P4 in rodents is not properly recognized. Here, we demonstrate that mouse placentas do produce P4 in mid-gestation, coincident with a temporal reduction in ovarian P4, suggesting that it plays a role in the protection of the conceptuses specifically in this period. Pregnant Sirh7/Ldoc1 knockout females also displayed delayed parturition associated with a low pup weaning rate. All these results suggest that Sirh7/Ldoc1 has undergone positive selection during eutherian evolution as a eutherian-specific acquired gene because it impacts reproductive fitness via the regulation of placental endocrine function. PMID:25468940

  6. Sirh7/Ldoc1 knockout mice exhibit placental P4 overproduction and delayed parturition.

    PubMed

    Naruse, Mie; Ono, Ryuichi; Irie, Masahito; Nakamura, Kenji; Furuse, Tamio; Hino, Toshiaki; Oda, Kanako; Kashimura, Misho; Yamada, Ikuko; Wakana, Shigeharu; Yokoyama, Minesuke; Ishino, Fumitoshi; Kaneko-Ishino, Tomoko

    2014-12-01

    Sirh7/Ldoc1 [sushi-ichi retrotransposon homolog 7/leucine zipper, downregulated in cancer 1, also called mammalian retrotransposon-derived 7 (Mart7)] is one of the newly acquired genes from LTR retrotransposons in eutherian mammals. Interestingly, Sirh7/Ldoc1 knockout (KO) mice exhibited abnormal placental cell differentiation/maturation, leading to an overproduction of placental progesterone (P4) and placental lactogen 1 (PL1) from trophoblast giant cells (TGCs). The placenta is an organ that is essential for mammalian viviparity and plays a major endocrinological role during pregnancy in addition to providing nutrients and oxygen to the fetus. P4 is an essential hormone in the preparation and maintenance of pregnancy and the determination of the timing of parturition in mammals; however, the biological significance of placental P4 in rodents is not properly recognized. Here, we demonstrate that mouse placentas do produce P4 in mid-gestation, coincident with a temporal reduction in ovarian P4, suggesting that it plays a role in the protection of the conceptuses specifically in this period. Pregnant Sirh7/Ldoc1 knockout females also displayed delayed parturition associated with a low pup weaning rate. All these results suggest that Sirh7/Ldoc1 has undergone positive selection during eutherian evolution as a eutherian-specific acquired gene because it impacts reproductive fitness via the regulation of placental endocrine function.

  7. A Knockout Experiment: Disciplinary Divides and Experimental Skill in Animal Behaviour Genetics.

    PubMed

    Nelson, Nicole C

    2015-07-01

    In the early 1990s, a set of new techniques for manipulating mouse DNA allowed researchers to 'knock out' specific genes and observe the effects of removing them on a live mouse. In animal behaviour genetics, questions about how to deploy these techniques to study the molecular basis of behaviour became quite controversial, with a number of key methodological issues dissecting the interdisciplinary research field along disciplinary lines. This paper examines debates that took place during the 1990s between a predominately North American group of molecular biologists and animal behaviourists around how to design, conduct, and interpret behavioural knockout experiments. Drawing from and extending Harry Collins's work on how research communities negotiate what counts as a 'well-done experiment,' I argue that the positions practitioners took on questions of experimental skill reflected not only the experimental traditions they were trained in but also their differing ontological and epistemological commitments. Different assumptions about the nature of gene action, eg., were tied to different positions in the knockout mouse debates on how to implement experimental controls. I conclude by showing that examining representations of skill in the context of a community's knowledge commitments sheds light on some of the contradictory ways in which contemporary animal behaviour geneticists talk about their own laboratory work as a highly skilled endeavour that also could be mechanised, as easy to perform and yet difficult to perform well.

  8. Using engineered endonucleases to create knockout and knockin zebrafish models.

    PubMed

    Bedell, Victoria M; Ekker, Stephen C

    2015-01-01

    Over the last few years, the technology to create targeted knockout and knockin zebrafish animals has exploded. We have gained the ability to create targeted knockouts through the use of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR associated system (CRISPR/Cas). Furthermore, using the high-efficiency TALEN system, we were able to create knockin zebrafish using a single-stranded DNA (ssDNA) protocol described here. Through the use of these technologies, the zebrafish has become a valuable vertebrate model and an excellent bridge between the invertebrate and mammalian model systems for the study of human disease.

  9. Glutamate transporter type 3 knockout mice have a decreased isoflurane requirement to induce loss of righting reflex.

    PubMed

    Lee, S N; Li, L; Zuo, Z

    2010-12-15

    Excitatory amino acid transporters (EAAT) uptake extracellular glutamate, the major excitatory neurotransmitter in the brain. EAAT type 3 (EAAT3), the main neuronal EAAT, is expressed widely in the CNS. We have shown that the volatile anesthetic isoflurane increases EAAT3 activity and trafficking to the plasma membrane. Thus, we hypothesize that EAAT3 mediates isoflurane-induced anesthesia. To test this hypothesis, the potency of isoflurane to induce immobility and hypnosis, two major components of general anesthesia, was compared in the CD-1 wild-type mice and EAAT knockout mice that had a CD-1 strain gene background. Hypnosis was assessed by loss of righting reflex in this study. The expression of EAAT1 and EAAT2, two widely expressed EAATs in the CNS, in the cerebral cortex and spinal cord was not different between the EAAT3 knockout mice and wild-type mice. The concentration required for isoflurane to cause immobility to painful stimuli, a response involving primarily reflex loops in the spinal cord, was not changed by EAAT3 knockout. However, the EAAT3 knockout mice were more sensitive to isoflurane-induced hypnotic effects, which may be mediated by hypothalamic sleep neural circuits. Interestingly, the EAAT3 knockout mice did not have an altered sensitivity to the hypnotic effects caused by ketamine, an i.v. anesthetic that is a glutamate receptor antagonist and does not affect EAAT3 activity. These results suggest that EAAT3 modulates the sensitivity of neural circuits to isoflurane. These results, along with our previous findings which suggests that isoflurane increases EAAT3 activity, indicate that EAAT3 may regulate isoflurane-induced behavioral changes, including anesthesia.

  10. INTERPRETATION OF THE CANCER RESPONSE TO POTENTIAL RENTAL CARCINOGENS IN THE TSC2 KNOCKOUT (EKER) RAT IS DEPENDENT ON LENGTH OF TREATMENT.

    EPA Science Inventory

    INTERPRETATION OF THE CANCER RESPONSE TO POTENTIAL RENAL CARCINOGENS IN THE TSC2 KNOCKOUT (EKER) RAT IS DEPENDENT ON LENGTH OF TREATMENT.

    Genetically increasing the function of oncogenes or knocking out the function of a tumor supressor gene has dramatically increased the...

  11. A Knockout Mutation of a Constitutive GPCR in Tetrahymena Decreases Both G-Protein Activity and Chemoattraction

    PubMed Central

    Lampert, Thomas J.; Coleman, Kevin D.; Hennessey, Todd M.

    2011-01-01

    Although G-protein coupled receptors (GPCRs) are a common element in many chemosensory transduction pathways in eukaryotic cells, no GPCR or regulated G-protein activity has yet been shown in any ciliate. To study the possible role for a GPCR in the chemoresponses of the ciliate Tetrahymena, we have generated a number of macronuclear gene knockouts of putative GPCRs found in the Tetrahymena Genome database. One of these knockout mutants, called G6, is a complete knockout of a gene that we call GPCR6 (TTHERM_00925490). Based on sequence comparisons, the Gpcr6p protein belongs to the Rhodopsin Family of GPCRs. Notably, Gpcr6p shares highest amino acid sequence homologies to GPCRs from Paramecium and several plants. One of the phenotypes of the G6 mutant is a decreased responsiveness to the depolarizing ions Ba2+ and K+, suggesting a decrease in basal excitability (decrease in Ca2+ channel activity). The other major phenotype of G6 is a loss of chemoattraction to lysophosphatidic acid (LPA) and proteose peptone (PP), two known chemoattractants in Tetrahymena. Using microsomal [35S]GTPγS binding assays, we found that wild-type (CU427) have a prominent basal G-protein activity. This activity is decreased to the same level by pertussis toxin (a G-protein inhibitor), addition of chemoattractants, or the G6 mutant. Since the basal G-protein activity is decreased by the GPCR6 knockout, it is likely that this gene codes for a constitutively active GPCR in Tetrahymena. We propose that chemoattractants like LPA and PP cause attraction in Tetrahymena by decreasing the basal G-protein stimulating activity of Gpcr6p. This leads to decreased excitability in wild-type and longer runs of smooth forward swimming (less interrupted by direction changes) towards the attractant. Therefore, these attractants may work as inverse agonists through the constitutively active Gpcr6p coupled to a pertussis-sensitive G-protein. PMID:22140501

  12. Conventional murine gene targeting.

    PubMed

    Zimmermann, Albert G; Sun, Yue

    2013-01-01

    Murine gene knockout models engineered over the last two decades have continued to demonstrate their potential as invaluable tools in understanding the role of gene function in the context of normal human development and disease. The more recent elucidation of the human and mouse genomes through sequencing has opened up the capability to elucidate the function of every human gene. State-of-the-art mouse model generation allows, through a multitude of experimental steps requiring careful standardization, gene function to be reliably and predictably ablated in a live model system. The application of these standardized methodologies to directly target gene function through murine gene knockout has to date provided comprehensive and verifiable genetic models that have contributed tremendously to our understanding of the cellular and molecular pathways underlying normal and disease states in humans. The ensuing chapter provides an overview of the latest steps and procedures required to ablate gene function in a murine model.

  13. CRISPR/Cas9-Mediated Generation of Niemann-Pick C1 Knockout Cell Line.

    PubMed

    Du, Ximing; Lukmantara, Ivan; Yang, Hongyuan

    2017-01-01

    Generating a cholesterol storage phenotype of Niemann-Pick Type C (NPC) disease is important for investigating the mechanisms of intracellular cholesterol trafficking, as well as screening drugs for potential treatment of NPC disease. The use of the CRISPR/Cas9 technology to knockout specific genes within the genome of mammals has become routine in the past few years. Here, we describe a protocol for producing a cellular NPC cholesterol storage phenotype in HeLa cells using the CRISPR-Cas9 system to disrupt the NPC1 gene. The protocol details the steps for single guide RNA oligo cloning, cell colony selection, and cell line verification by filipin staining and immunoblotting.

  14. Unchanged survival rates of Shadoo knockout mice after infection with mouse-adapted scrapie

    PubMed Central

    Li, Sha; Ju, Chuanjing; Han, Chao; Li, Zhongyi; Liu, Wensen; Ye, Xuemin; Xu, Jing; Xulong, Liang; Wang, Xiong; Chen, Zhibao; Meng, Keyin; Wan, Jiayu

    2014-01-01

    Previous studies have demonstrated that Shadoo (Sho), a GPI-linked glycoprotein encoded by the Sprn gene with a membrane localization similar to PrPC, is reduced in the brains of rodents with terminal prion disease. To determine the functional significance of Sho in prion disease pathogenesis, Sho-deficient mice were generated by gene targeting. Sho knockout and control wild-type (WT) mice were infected with themouse-adapted scrapie strains 22L or RML. No significant differences in survival, the incubation period of prion disease or other disease features were observed between Sho mutant and WT mice. In this model of prion disease, Sho removal had no effect on disease pathogenesis. PMID:25495671

  15. Generation of myometrium-specific Bmal1 knockout mice for parturition analysis.

    PubMed

    Ratajczak, Christine K; Asada, Minoru; Allen, Gregg C; McMahon, Douglas G; Muglia, Lisa M; Smith, Donté; Bhattacharyya, Sandip; Muglia, Louis J

    2012-01-01

    Human and rodent studies indicate a role for circadian rhythmicity and associated clock gene expression in supporting normal parturition. The importance of clock gene expression in tissues besides the suprachiasmatic nucleus is emerging. Here, a Bmal1 conditional knockout mouse line and a novel Cre transgenic mouse line were used to examine the role of myometrial Bmal1 in parturition. Ninety-two percent (22/24) of control females but only 64% (14/22) of females with disrupted myometrial Bmal1 completed parturition during the expected time window of 5p.m. on Day 19 through to 9a.m. on Day 19.5 of gestation. However, neither serum progesterone levels nor uterine transcript expression of the contractile-associated proteins Connexin43 and Oxytocin receptor differed between females with disrupted myometrial Bmal1 and controls during late gestation. The data indicate a role for myometrial Bmal1 in maintaining normal time of day of parturition.

  16. One-neutron knockout from 51-55 Sc

    NASA Astrophysics Data System (ADS)

    Schwertel, S.; Maierbeck, P.; Krücken, R.; Gernhäuser, R.; Kröll, T.; Alvarez-Pol, H.; Aksouh, F.; Aumann, T.; Behr, K.; Benjamim, E. A.; Benlliure, J.; Bildstein, V.; Böhmer, M.; Boretzky, K.; Borge, M. J. G.; Brünle, A.; Bürger, A.; Caamaño, M.; Casarejos, E.; Chatillon, A.; Chulkov, L. V.; Cortina-Gil, D.; Enders, J.; Eppinger, K.; Faestermann, T.; Friese, J.; Fabbietti, L.; Gascón, M.; Geissel, H.; Gerl, J.; Gorska, M.; Hansen, P. G.; Jonson, B.; Kanungo, R.; Kiselev, O.; Kojouharov, I.; Klimkiewicz, A.; Kurtukian, T.; Kurz, N.; Larsson, K.; Le Bleis, T.; Mahata, K.; Maier, L.; Nilsson, T.; Nociforo, C.; Nyman, G.; Pascual-Izarra, C.; Perea, A.; Perez, D.; Prochazka, A.; Rodriguez-Tajes, C.; Rossi, D.; Schaffner, H.; Schrieder, G.; Simon, H.; Sitar, B.; Stanoiu, M.; Sümmerer, K.; Tengblad, O.; Weick, H.; Winkler, S.; Brown, B. A.; Otsuka, T.; Tostevin, J. A.; Rae, W. D. M.

    2012-12-01

    Results are presented from a one-neutron knockout experiment at relativistic energies of ≈ 420 A MeV on 51-55Sc using the GSI Fragment Separator as a two-stage magnetic spectrometer and the MINIBALL array for gamma-ray detection. Inclusive longitudinal momentum distributions and cross-sections were measured enabling the determination of the contributions corresponding to knockout from the ν p_{1/2} , ν p_{3/2} , ( L = 1 and ν f_{7/2} , ν f_{5/2} ( L = 3 neutron orbitals. The observed L = 1 and L = 3 contributions are compared with theoretical cross-sections using eikonal knockout theory and spectroscopic factors from shell model calculations using the GXPF1A interaction. The measured inclusive knockout cross-sections generally follow the trends expected theoretically and given by the spectroscopic strength predicted from the shell model calculations. However, the deduced L = 1 cross-sections are generally 30-40% higher while the L = 3 contributions are about a factor of two smaller than predicted. This points to a promotion of neutrons from the ν f_{7/2} to the ν p_{3/2} orbital indicating a weakening of the N = 28 shell gap in these nuclei. While this is not predicted for the phenomenological GXPF1A interaction such a weakening is predicted by recent calculations using realistic low-momentum interactions V_{low k} obtained by evolving a chiral N3LO nucleon-nucleon potential.

  17. Knockout mice in understanding the mechanism of action of lithium.

    PubMed

    Agam, Galila; Bersudsky, Yuly; Berry, Gerard T; Moechars, Diederik; Lavi-Avnon, Yael; Belmaker, R H

    2009-10-01

    Lithium inhibits IMPase (inositol monophosphatase) activity, as well as inositol transporter function. To determine whether one or more of these mechanisms might underlie lithium's behavioural effects, we studied Impa1 (encoding IMPase) and Smit1 (sodium-myo-inositol transporter 1)-knockout mice. In brains of adult homozygous Impa1-knockout mice, IMPase activity was found to be decreased; however, inositol levels were not found to be altered. Behavioural analysis indicated decreased immobility in the forced-swim test as well as a strongly increased sensitivity to pilocarpine-induced seizures. These are behaviours robustly induced by lithium. In homozygous Smit1-knockout mice, free inositol levels were decreased in the frontal cortex and hippocampus. These animals behave like lithium-treated animals in the model of pilocarpine seizures and in the Porsolt forced-swim test model of depression. In contrast with O'Brien et al. [O'Brien, Harper, Jove, Woodgett, Maretto, Piccolo and Klein (2004) J. Neurosci. 24, 6791-6798], we could not confirm that heterozygous Gsk3b (glycogen synthase kinase 3beta)-knockout mice exhibit decreased immobility in the Porsolt forced-swim test or decreased amphetamine-induced hyperactivity in a manner mimicking lithium's behavioural effects. These data support the role of inositol-related processes rather than GSK3beta in the mechanism of the therapeutic action of lithium.

  18. Cigarette smoke exposure aggravates air space enlargement and alveolar cell apoptosis in Smad3 knockout mice.

    PubMed

    Farkas, Laszlo; Farkas, Daniela; Warburton, David; Gauldie, Jack; Shi, Wei; Stampfli, Martin R; Voelkel, Norbert F; Kolb, Martin

    2011-10-01

    The concept of genetic susceptibility factors predisposing cigarette smokers to develop emphysema stems from the clinical observation that only a fraction of smokers develop clinically significant chronic obstructive pulmonary disease. We investigated whether Smad3 knockout mice, which develop spontaneous air space enlargement after birth because of a defect in transforming growth factor-β (TGF-β) signaling, develop enhanced alveolar cell apoptosis and air space enlargement following cigarette smoke exposure. We investigated Smad3(-/-) and Smad3(+/+) mice at different adult ages and determined air space enlargement, alveolar cell proliferation, and apoptosis. Furthermore, laser-capture microdissection and real-time PCR were used to measure compartment-specific gene expression. We then compared the effects of cigarette smoke exposure on Smad3(-/-) and littermate controls. Smad3 knockout resulted in the development of air space enlargement in the adult mouse and was associated with decreased alveolar VEGF levels and activity and increased alveolar cell apoptosis. Cigarette smoke exposure aggravated air space enlargement and alveolar cell apoptosis. We also found increased Smad2 protein expression and phosphorylation, which was enhanced following cigarette smoke exposure, in Smad3-knockout animals. Double immunofluorescence analysis revealed that endothelial apoptosis started before epithelial apoptosis. Our data indicate that balanced TGF-β signaling is not only important for regulation of extracellular matrix turnover, but also for alveolar cell homeostasis. Impaired signaling via the Smad3 pathway results in alveolar cell apoptosis and alveolar destruction, likely via increased Smad2 and reduced VEGF expression and might represent a predisposition for accelerated development of emphysema due to cigarette smoke exposure.

  19. Muscle developmental defects in heterogeneous nuclear Ribonucleoprotein A1 knockout mice

    PubMed Central

    Liu, Ting-Yuan; Chen, Yu-Chia; Jong, Yuh-Jyh; Tsai, Huai-Jen; Lee, Chien-Chin; Chang, Ya-Sian; Chang, Jan-Gowth

    2017-01-01

    Heterogeneous ribonucleoprotein A1 (hnRNP A1) is crucial for regulating alternative splicing. Its integrated function within an organism has not, however, been identified. We generated hnRNP A1 knockout mice to study the role of hnRNP A1 in vivo. The knockout mice, hnRNP A1−/−, showed embryonic lethality because of muscle developmental defects. The blood pressure and heart rate of the heterozygous mice were higher than those of the wild-type mice, indicating heart function defects. We performed mouse exon arrays to study the muscle development mechanism. The processes regulated by hnRNP A1 included cell adhesion and muscle contraction. The expression levels of muscle development-related genes in hnRNP A1+/− mice were significantly different from those in wild-type mice, as detected using qRT-PCR. We further confirmed the alternative splicing patterns of muscle development-related genes including mef2c, lrrfip1, usp28 and abcc9. Alternative mRNA isoforms of these genes were increased in hnRNP A1+/− mice compared with wild-type mice. Furthermore, we revealed that the functionally similar hnRNP A2/B1 did not compensate for the expression of hnRNP A1 in organisms. In summary, our study demonstrated that hnRNP A1 plays a critical and irreplaceable role in embryonic muscle development by regulating the expression and alternative splicing of muscle-related genes. PMID:28077597

  20. Knockout mutants as a tool to identify the subunit composition of Arabidopsis glutamine synthetase isoforms.

    PubMed

    Dragićević, Milan; Todorović, Slađana; Bogdanović, Milica; Filipović, Biljana; Mišić, Danijela; Simonović, Ana

    2014-06-01

    Glutamine synthetase (GS) is a key enzyme in nitrogen assimilation, which catalyzes the formation of glutamine from ammonia and glutamate. Plant GS isoforms are multimeric enzymes, recently shown to be decamers. The Arabidopsis genome encodes five cytosolic (GS1) proteins labeled as GLN1;1 through GLN1;5 and one chloroplastic (GS2) isoform, GLN2;0. However, as many as 11 GS activity bands were resolved from different Arabidopsis tissues by Native PAGE and activity staining. Western analysis showed that all 11 isoforms are composed exclusively of 40 kDa GS1 subunits. Of five GS1 genes, only GLN1;1, GLN1;2 and GLN1;3 transcripts accumulated to significant levels in vegetative tissues, indicating that only subunits encoded by these three genes produce the 11-band zymogram. Even though the GS2 gene also had significant expression, the corresponding activity was not detected, probably due to inactivation. To resolve the subunit composition of 11 active GS1 isoforms, homozygous knockout mutants deficient in the expression of different GS1 genes were selected from the progeny of T-DNA insertional SALK and SAIL lines. Comparison of GS isoenzyme patterns of the selected GS1 knockout mutants indicated that all of the detected isoforms consist of varying proportions of GLN1;1, GLN1;2 and GLN1;3 subunits, and that GLN1;1 and GLN1;3, as well as GLN1;2 and GLN1;3 and possibly GLN1;1 and GLN1;2 proteins combine in all proportions to form active homo- and heterodecamers.

  1. Altered Neurocircuitry in the Dopamine Transporter Knockout Mouse Brain

    PubMed Central

    Zhang, Xiaowei; Bearer, Elaine L.; Boulat, Benoit; Hall, F. Scott; Uhl, George R.; Jacobs, Russell E.

    2010-01-01

    The plasma membrane transporters for the monoamine neurotransmitters dopamine, serotonin, and norepinephrine modulate the dynamics of these monoamine neurotransmitters. Thus, activity of these transporters has significant consequences for monoamine activity throughout the brain and for a number of neurological and psychiatric disorders. Gene knockout (KO) mice that reduce or eliminate expression of each of these monoamine transporters have provided a wealth of new information about the function of these proteins at molecular, physiological and behavioral levels. In the present work we use the unique properties of magnetic resonance imaging (MRI) to probe the effects of altered dopaminergic dynamics on meso-scale neuronal circuitry and overall brain morphology, since changes at these levels of organization might help to account for some of the extensive pharmacological and behavioral differences observed in dopamine transporter (DAT) KO mice. Despite the smaller size of these animals, voxel-wise statistical comparison of high resolution structural MR images indicated little morphological change as a consequence of DAT KO. Likewise, proton magnetic resonance spectra recorded in the striatum indicated no significant changes in detectable metabolite concentrations between DAT KO and wild-type (WT) mice. In contrast, alterations in the circuitry from the prefrontal cortex to the mesocortical limbic system, an important brain component intimately tied to function of mesolimbic/mesocortical dopamine reward pathways, were revealed by manganese-enhanced MRI (MEMRI). Analysis of co-registered MEMRI images taken over the 26 hours after introduction of Mn2+ into the prefrontal cortex indicated that DAT KO mice have a truncated Mn2+ distribution within this circuitry with little accumulation beyond the thalamus or contralateral to the injection site. By contrast, WT littermates exhibit Mn2+ transport into more posterior midbrain nuclei and contralateral mesolimbic structures at

  2. Genome-scale CRISPR-Cas9 knockout screening in human cells.

    PubMed

    Shalem, Ophir; Sanjana, Neville E; Hartenian, Ella; Shi, Xi; Scott, David A; Mikkelsen, Tarjei S; Heckl, Dirk; Ebert, Benjamin L; Root, David E; Doench, John G; Zhang, Feng

    2014-01-03

    The simplicity of programming the CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease Cas9 to modify specific genomic loci suggests a new way to interrogate gene function on a genome-wide scale. We show that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells. First, we used the GeCKO library to identify genes essential for cell viability in cancer and pluripotent stem cells. Next, in a melanoma model, we screened for genes whose loss is involved in resistance to vemurafenib, a therapeutic RAF inhibitor. Our highest-ranking candidates include previously validated genes NF1 and MED12, as well as novel hits NF2, CUL3, TADA2B, and TADA1. We observe a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation, demonstrating the promise of genome-scale screening with Cas9.

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

    PubMed

    Wettstein, Rahel; Bodak, Maxime; Ciaudo, Constance

    2016-01-01

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

  4. Behavioral characterization of P311 knockout mice

    PubMed Central

    Taylor, Gregory A.; Rodriguiz, Ramona M.; Greene, Robert I.; Daniell, Xiaoju; Henry, Stanley C.; Crooks, Kristy R.; Kotloski, Robert; Tessarollo, Lino; Phillips, Lindsey E.; Wetsel, William C.

    2013-01-01

    P311 is an 8-kDa protein that is expressed in many brain regions, particularly the hippocampus, cerebellum and olfactory lobes, and is under stringent regulation by developmental, mitogenic and other physiological stimuli. P311 is thought to be involved in the transformation and motility of neural cells; however, its role in normal brain physiology is undefined. To address this point, P311-deficient mice were developed through gene targeting and their behaviors were characterized. Mutants displayed no overt abnormalities, bred normally and had normal survival rates. Additionally, no deficiencies were noted in motor co-ordination, balance, hearing or olfactory discrimination. Nevertheless, P311-deficient mice showed altered behavioral responses in learning and memory. These included impaired responses in social transmission of food preference, Morris water maze and contextual fear conditioning. Additionally, mutants displayed altered emotional responses as indicated by decreased freezing in contextual and cued fear conditioning and reduced fear-potentiated startle. Together, these data establish P311 as playing an important role in learning and memory processes and emotional responses. PMID:18616608

  5. RhoA knockout fibroblasts lose tumor-inhibitory capacity in vitro and promote tumor growth in vivo

    PubMed Central

    Alkasalias, Twana; Alexeyenko, Andrey; Hennig, Katharina; Danielsson, Frida; Lebbink, Robert Jan; Fielden, Matthew; Turunen, S. Pauliina; Lehti, Kaisa; Kashuba, Vladimir; Madapura, Harsha S.; Bozoky, Benedek; Lundberg, Emma; Balland, Martial; Guvén, Hayrettin; Klein, George; Gad, Annica K. B.; Pavlova, Tatiana

    2017-01-01

    Fibroblasts are a main player in the tumor-inhibitory microenvironment. Upon tumor initiation and progression, fibroblasts can lose their tumor-inhibitory capacity and promote tumor growth. The molecular mechanisms that underlie this switch have not been defined completely. Previously, we identified four proteins overexpressed in cancer-associated fibroblasts and linked to Rho GTPase signaling. Here, we show that knocking out the Ras homolog family member A (RhoA) gene in normal fibroblasts decreased their tumor-inhibitory capacity, as judged by neighbor suppression in vitro and accompanied by promotion of tumor growth in vivo. This also induced PC3 cancer cell motility and increased colony size in 2D cultures. RhoA knockout in fibroblasts induced vimentin intermediate filament reorganization, accompanied by reduced contractile force and increased stiffness of cells. There was also loss of wide F-actin stress fibers and large focal adhesions. In addition, we observed a significant loss of α-smooth muscle actin, which indicates a difference between RhoA knockout fibroblasts and classic cancer-associated fibroblasts. In 3D collagen matrix, RhoA knockout reduced fibroblast branching and meshwork formation and resulted in more compactly clustered tumor-cell colonies in coculture with PC3 cells, which might boost tumor stem-like properties. Coculturing RhoA knockout fibroblasts and PC3 cells induced expression of proinflammatory genes in both. Inflammatory mediators may induce tumor cell stemness. Network enrichment analysis of transcriptomic changes, however, revealed that the Rho signaling pathway per se was significantly triggered only after coculturing with tumor cells. Taken together, our findings in vivo and in vitro indicate that Rho signaling governs the inhibitory effects by fibroblasts on tumor-cell growth. PMID:28174275

  6. RhoA knockout fibroblasts lose tumor-inhibitory capacity in vitro and promote tumor growth in vivo.

    PubMed

    Alkasalias, Twana; Alexeyenko, Andrey; Hennig, Katharina; Danielsson, Frida; Lebbink, Robert Jan; Fielden, Matthew; Turunen, S Pauliina; Lehti, Kaisa; Kashuba, Vladimir; Madapura, Harsha S; Bozoky, Benedek; Lundberg, Emma; Balland, Martial; Guvén, Hayrettin; Klein, George; Gad, Annica K B; Pavlova, Tatiana

    2017-02-21

    Fibroblasts are a main player in the tumor-inhibitory microenvironment. Upon tumor initiation and progression, fibroblasts can lose their tumor-inhibitory capacity and promote tumor growth. The molecular mechanisms that underlie this switch have not been defined completely. Previously, we identified four proteins overexpressed in cancer-associated fibroblasts and linked to Rho GTPase signaling. Here, we show that knocking out the Ras homolog family member A (RhoA) gene in normal fibroblasts decreased their tumor-inhibitory capacity, as judged by neighbor suppression in vitro and accompanied by promotion of tumor growth in vivo. This also induced PC3 cancer cell motility and increased colony size in 2D cultures. RhoA knockout in fibroblasts induced vimentin intermediate filament reorganization, accompanied by reduced contractile force and increased stiffness of cells. There was also loss of wide F-actin stress fibers and large focal adhesions. In addition, we observed a significant loss of α-smooth muscle actin, which indicates a difference between RhoA knockout fibroblasts and classic cancer-associated fibroblasts. In 3D collagen matrix, RhoA knockout reduced fibroblast branching and meshwork formation and resulted in more compactly clustered tumor-cell colonies in coculture with PC3 cells, which might boost tumor stem-like properties. Coculturing RhoA knockout fibroblasts and PC3 cells induced expression of proinflammatory genes in both. Inflammatory mediators may induce tumor cell stemness. Network enrichment analysis of transcriptomic changes, however, revealed that the Rho signaling pathway per se was significantly triggered only after coculturing with tumor cells. Taken together, our findings in vivo and in vitro indicate that Rho signaling governs the inhibitory effects by fibroblasts on tumor-cell growth.

  7. Bile acid transport in sister of P-glycoprotein (ABCB11) knockout mice.

    PubMed

    Lam, Ping; Wang, Renxue; Ling, Victor

    2005-09-20

    In vertebrates, bile flow is essential for movement of water and solutes across liver canalicular membranes. In recent years, the molecular motor of canalicular bile acid secretion has been identified as a member of the ATP binding cassette transporter (ABC) superfamily, known as sister of P-glycoprotein (Spgp) or bile salt export pump (Bsep, ABCB11). In humans, mutations in the BSEP gene are associated with a very low level of bile acid secretion and severe cholestasis. However, as reported previously, because the spgp(-)(/)(-) knockout mice do not express severe cholestasis and have substantial bile acid secretion, we investigated the "alternative transport system" that allows these mice to be physiologically relatively normal. We examined the expression levels of several ABC transporters in spgp(-)(/)(-) mice and found that the level of multidrug resistance Mdr1 (P-glycoprotein) was strikingly increased while those of Mdr2, Mrp2, and Mrp3 were increased to only a moderate extent. We hypothesize that an elevated level of Mdr1 in the spgp(-)(/)(-) knockout mice functions as an alternative pathway to transport bile acids and protects hepatocytes from bile acid-induced cholestasis. In support of this hypothesis, we showed that plasma membrane vesicles isolated from a drug resistant cell line expressing high levels of P-glycoprotein were capable of transporting bile acids, albeit with a 5-fold lower affinity compared to Spgp. This finding is the first direct evidence that P-glycoprotein (Mdr1) is capable of transporting bile acids.

  8. Global Changes in Lipid Profiles of Mouse Cortex, Hippocampus, and Hypothalamus Upon p53 Knockout

    PubMed Central

    Lee, Sang Tak; Lee, Jong Cheol; Kim, Jong Whi; Cho, Soo Young; Seong, Je Kyung; Moon, Myeong Hee

    2016-01-01

    Comprehensive lipidomic profiling in three different brain tissues (cortex, hippocampus, and hypothalamus) of mouse with p53 deficiency was performed by nanoflow liquid chromatography-tandem mass spectrometry (nLC-MS/MS) and the profile was compared with that of the wild type. p53 gene is a well-known tumour suppressor that prevents genome mutations that can cause cancers. More than 300 lipids (among 455 identified species), including phospholipids (PLs), sphingolipids, ceramides (Cers), and triacylglycerols (TAGs) were quantitatively analysed by selective reaction monitoring (SRM) of nanoflow ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry (nUPLC-ESI-MS/MS). Among the three different neural tissues, hypothalamus demonstrated the most evident lipid profile changes upon p53 knockout. Alterations of PLs containing acyl chains of docosahexaenoic acid and arachidonic acid (highly enriched polyunsaturated fatty acids in the nervous system) were examined in relation to cell apoptosis upon p53 knockout. Comparison between sphingomyelins (SMs) and Cers showed that the conversion of SM to Cer did not effectively progress in the hypothalamus, resulting in the accumulation of SMs, possibly due to the inhibition of apoptosis caused by the lack of p53. Furthermore, TAGs were considerably decreased only in the hypothalamus, indicative of lipolysis that led to substantial weight loss of adipose tissue and muscles. PMID:27819311

  9. Retinal Ganglion Cell Loss is Delayed Following Optic Nerve Crush in NLRP3 Knockout Mice

    PubMed Central

    Puyang, Zhen; Feng, Liang; Chen, Hui; Liang, Peiji; Troy, John B.; Liu, Xiaorong

    2016-01-01

    The NLRP3 inflammasome, a sensor for a variety of pathogen- and host-derived threats, consists of the adaptor ASC (Apoptosis-associated Speck-like protein containing a Caspase Activation and Recruitment Domain (CARD)), pro-caspase-1, and NLRP3 (NOD-Like Receptor family Pyrin domain containing 3). NLRP3-induced neuroinflammation is implicated in the pathogenesis and progression of eye diseases, but it remains unclear whether activation of NLRP3 inflammasome contributes to retinal ganglion cell (RGC) death. Here we examined NLRP3-induced neuroinflammation and RGC survival following partial optic nerve crush (pONC) injury. We showed that NLRP3 was up-regulated in retinal microglial cells following pONC, propagating from the injury site to the optic nerve head and finally the entire retina within one day. Activation of NLRP3-ASC inflammasome led to the up-regulation of caspase-1 and a proinflammatory cytokine, interleukin-1β (IL-1β). In NLRP3 knockout mice, up-regulation of ASC, caspase-1, and IL-1β were all reduced, and, importantly, RGC and axon loss was substantially delayed following pONC injury. The average survival time of RGCs in NLRP3 knockout mice was about one week longer than for control animals. Taken together, our study demonstrated that ablating the NLRP3 gene significantly reduced neuroinflammation and delayed RGC loss after optic nerve crush injury. PMID:26893104

  10. Collecting duct-specific knockout of the endothelin B receptor causes hypertension and sodium retention.

    PubMed

    Ge, Yuqiang; Bagnall, Alan; Stricklett, Peter K; Strait, Kevin; Webb, David J; Kotelevtsev, Yuri; Kohan, Donald E

    2006-12-01

    Collecting duct (CD)-derived endothelin-1 (ET-1) inhibits renal Na reabsorption and its deficiency increases blood pressure (BP). The role of CD endothelin B (ETB) receptors in mediating these effects is unknown. CD-specific knockout of the ETB receptor was achieved using an aquaporin-2 promoter-Cre recombinase transgene and the loxP-flanked ETB receptor gene (CD ETB KO). Systolic BP in mice with CD-specific knockout of the ETB receptor, ETA receptor (CD ETA KO) and ET-1 (CD ET-1 KO), and their respective controls were compared during normal- and high-salt diet. On a normal-sodium diet, CD ETB KO mice had elevated BP, which increased further during high salt feeding. However, the degree of hypertension in CD ETB KO mice and the further increase in BP during salt feeding were lower than that of CD ET-1 KO mice, whereas CD ETA KO mice were normotensive. CD ETB KO mice had impaired sodium excretion following acute sodium loading. Aldosterone and plasma renin activity were decreased in CD ETB KO mice on normal- and high-sodium diets, while plasma and urinary ET-1 levels did not differ from controls. In conclusion, the CD ETB receptor partially mediates the antihypertensive and natriuretic effects of ET-1. CD ETA and ETB receptors do not fully account for the antihypertensive and natriuretic effects of CD-derived ET-1, suggesting paracrine effects of this peptide.

  11. Generation of myostatin B knockout yellow catfish (Tachysurus fulvidraco) using transcription activator-like effector nucleases.

    PubMed

    Dong, Zhangji; Ge, Jiachun; Xu, Zhiqiang; Dong, Xiaohua; Cao, Shasha; Pan, Jianlin; Zhao, Qingshun

    2014-06-01

    Myostatin (Mstn), a member of the transforming growth factor β superfamily, plays an inhibiting role in mammalian muscle growth. Mammals like human, cattle, mouse, sheep, and dog carrying null alleles of Mstn display a double-muscle phenotype. Mstn is conserved in fish; however, little is known whether the fish with mutated mstn display a similar phenotype to mammals because of the lack of mutant fish with mstn null alleles. Previously, we knocked out one of the duplicated copies of myostatin gene (mstna) in yellow catfish using zinc-finger nucleases. In this study, we report the identification of the second myostatin gene (mstnb) and knockout of mstnb in yellow catfish. The gene comprises three exons. It is predicted to encode 373 amino acid residues. The predicted protein exhibits 59.3% identity with yellow catfish Mstna and 57.3% identity with human MSTN. Employing TALEN (transcription activator-like effector nucleases) technology, we obtained two founders (from four randomly selected founders) of yellow catfish carrying the mutated mstnb gene in their germ cells. Totally, six mutated alleles of mstnb were obtained from the founders. Among the six alleles, four are nonframeshift and two are frameshift mutation. The frameshift mutated alleles include mstnb(nju22), an 8 bp deletion, and mstnb(nju24), a complex type of mutation comprising a 7 bp deletion and a 12 bp insertion. They are predicted to encode function null Mstnb. Our results will help to understand the roles of mstn genes in fish growth.

  12. Knockout driven reactions in complex molecules and their clusters

    NASA Astrophysics Data System (ADS)

    Gatchell, Michael; Zettergren, Henning

    2016-08-01

    Energetic ions lose some of their kinetic energy when interacting with electrons or nuclei in matter. Here, we discuss combined experimental and theoretical studies on such impulse driven reactions in polycyclic aromatic hydrocarbons (PAHs), fullerenes, and pure or mixed clusters of these molecules. These studies show that the nature of excitation is important for how complex molecular systems respond to ion/atom impact. Rutherford-like nuclear scattering processes may lead to prompt atom knockout and formation of highly reactive fragments, while heating of the molecular electron clouds in general lead to formation of more stable and less reactive fragments. In this topical review, we focus on recent studies of knockout driven reactions, and present new calculations of the angular dependent threshold (displacement) energies for such processes in PAHs. The so-formed fragments may efficiently form covalent bonds with neighboring molecules in clusters. These unique molecular growth processes may be important in astrophysical environments such as low velocity shock waves.

  13. A STAT-1 knockout mouse model for Machupo virus pathogenesis

    PubMed Central

    2011-01-01

    Background Machupo virus (MACV), a member of the Arenaviridae, causes Bolivian hemorrhagic fever, with ~20% lethality in humans. The pathogenesis of MACV infection is poorly understood, and there are no clinically proven treatments for disease. This is due, in part, to a paucity of small animal models for MACV infection in which to discover and explore candidate therapeutics. Methods Mice lacking signal transducer and activator of transcription 1 (STAT-1) were infected with MACV. Lethality, viral replication, metabolic changes, hematology, histopathology, and systemic cytokine expression were analyzed throughout the course of infection. Results We report here that STAT-1 knockout mice succumbed to MACV infection within 7-8 days, and presented some relevant clinical and histopathological manifestations of disease. Furthermore, the model was used to validate the efficacy of ribavirin in protection against infection. Conclusions The STAT-1 knockout mouse model can be a useful small animal model for drug testing and preliminary immunological analysis of lethal MACV infection. PMID:21672221

  14. Using engineered endonucleases to create knockout and knockin zebrafish models

    PubMed Central

    Bedell, Victoria M.; Ekker, Stephen C.

    2015-01-01

    Summary Over the last few years, the technology to create targeted knockout and knockin zebrafish animals has exploded. We have gained the ability to create targeted knockouts through the use of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR associated system (CRISPR/Cas). Furthermore, using the high-efficiency TALEN system, we were able to create knockin zebrafish using a single-stranded DNA (ssDNA) protocol described here. Through the use of these technologies, the zebrafish has become a valuable vertebrate model and an excellent bridge between the invertebrate and mammalian model systems for the study of human disease. PMID:25408414

  15. In Silico Knockout Studies of Xenophagic Capturing of Salmonella

    PubMed Central

    Scheidel, Jennifer; Amstein, Leonie; Ackermann, Jörg; Dikic, Ivan; Koch, Ina

    2016-01-01

    The degradation of cytosol-invading pathogens by autophagy, a process known as xenophagy, is an important mechanism of the innate immune system. Inside the host, Salmonella Typhimurium invades epithelial cells and resides within a specialized intracellular compartment, the Salmonella-containing vacuole. A fraction of these bacteria does not persist inside the vacuole and enters the host cytosol. Salmonella Typhimurium that invades the host cytosol becomes a target of the autophagy machinery for degradation. The xenophagy pathway has recently been discovered, and the exact molecular processes are not entirely characterized. Complete kinetic data for each molecular process is not available, so far. We developed a mathematical model of the xenophagy pathway to investigate this key defense mechanism. In this paper, we present a Petri net model of Salmonella xenophagy in epithelial cells. The model is based on functional information derived from literature data. It comprises the molecular mechanism of galectin-8-dependent and ubiquitin-dependent autophagy, including regulatory processes, like nutrient-dependent regulation of autophagy and TBK1-dependent activation of the autophagy receptor, OPTN. To model the activation of TBK1, we proposed a new mechanism of TBK1 activation, suggesting a spatial and temporal regulation of this process. Using standard Petri net analysis techniques, we found basic functional modules, which describe different pathways of the autophagic capture of Salmonella and reflect the basic dynamics of the system. To verify the model, we performed in silico knockout experiments. We introduced a new concept of knockout analysis to systematically compute and visualize the results, using an in silico knockout matrix. The results of the in silico knockout analyses were consistent with published experimental results and provide a basis for future investigations of the Salmonella xenophagy pathway. PMID:27906974

  16. PPAR-gamma knockout in pancreatic epithelial cells abolishes the inhibitory effect of rosiglitazone on caerulein-induced acute pancreatitis.

    PubMed

    Ivashchenko, C Y; Duan, S Z; Usher, M G; Mortensen, R M

    2007-07-01

    Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonists, such as the thiazolidinediones (TZDs), decrease acute inflammation in both pancreatic cell lines and mouse models of acute pancreatitis. Since PPAR-gamma agonists have been shown to exert some of their actions independent of PPAR-gamma, the role of PPAR-gamma in pancreatic inflammation has not been directly tested. Furthermore, the differential role of PPAR-gamma in endodermal derivatives (acini, ductal cells, and islets) as opposed to the endothelial or inflammatory cells is unknown. To determine whether the effects of a TZD, rosiglitazone, on caerulein-induced acute pancreatitis are dependent on PPAR-gamma in the endodermal derivatives, we created a cell-type specific knock out of PPAR-gamma in pancreatic acini, ducts, and islets. PPAR-gamma knockout animals show a greater response in some inflammatory genes after caerulein challenge. The anti-inflammatory effect of rosiglitazone on edema, macrophage infiltration, and expression of the proinflammatory cytokines is significantly decreased in pancreata of the knockout animals compared with control animals. However, rosiglitazone retains its effect in the lungs of the pancreatic-specific PPAR-gamma knockout animals, likely due to direct anti-inflammatory effect on lung parenchyma. These data show that the PPAR-gamma in the pancreatic epithelia and islets is important in suppressing inflammation and is required for the anti-inflammatory effects of TZDs in acute pancreatitis.

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

    PubMed Central

    Park, Rackhyun; Li, Liqing; Jang, Minsu; Morris, Andrew J.; Huang, Cai

    2017-01-01

    Increased expression of NOX4 protein is associated with cancer progression and metastasis but the role of NOX4 in cell proliferation and invasion is not fully understood. We generated NOX4 knockout HeLa cell lines using the CRISPR-Cas9 gene editing system to explore the cellular functions of NOX4. After transfection of CRISPR-Cas9 construct, we performed T7 endonuclease 1 assays and DNA sequencing to generate and identify insertion and deletion of the NOX4 locus. We confirmed the knockout of NOX4 by Western blotting. NOX4 knockout cell lines showed reduced cell proliferation with an increase of sub-G1 cell population and the decrease of S/G2/M population. Moreover, NOX4 deficiency resulted in a dramatic decrease in invadopodium formation and the invasive activity. In addition, NOX4 deficiency also caused a decrease in focal adhesions and cell migration in HeLa cells. These results suggest that NOX4 is required for both efficient proliferation and invasion of HeLa cells. PMID:28099519

  18. Phenotypic and Molecular Alterations in the Mammary Tissue of R-Spondin1 Knock-Out Mice during Pregnancy

    PubMed Central

    Chadi, Sead; Polyte, Jacqueline; Lefevre, Lucas; Castille, Johan; Ehanno, Aude; Laubier, Johann; Jaffrézic, Florence; Le Provost, Fabienne

    2016-01-01

    R-spondin1 (Rspo1) is a member of a secreted protein family which has pleiotropic functions in development and stem cell growth. Rspo1 knock-out mice are sex-reversed, but some remain sub-fertile, so they fail to nurse their pups. A lack of Rspo1 expression in the mammary gland results in an absence of duct side-branching development and defective alveolar formation. The aim of this study was to characterize the phenotypic and molecular alterations of mammary gland due to Rspo1 knock-out. Using the transcriptional profiling of mammary tissues, we identified misregulated genes in the mammary gland of Rspo1 knock-out mice during pregnancy. A stronger expression of mesenchymal markers was observed, without modifications to the structure of mammary epithelial tissue. Mammary epithelial cell immunohistochemical analysis revealed a persistence of virgin markers, which signify a delay in cell differentiation. Moreover, serial transplantation experiments showed that Rspo1 is associated with a regenerative potential of mammary epithelial cell control. Our finding also highlights the negatively regulated expression of Rspo1’s partners, Lgr4 and RNF43, in the mammary gland during pregnancy. Moreover, we offer evidence that Tgf-β signalling is modified in the absence of Rspo1. Taken together, our results show an abrupt halt or delay to mammary development during pregnancy due to the loss of a further differentiated function. PMID:27611670

  19. A satellite cell-specific knockout of the androgen receptor reveals myostatin as a direct androgen target in skeletal muscle.

    PubMed

    Dubois, Vanessa; Laurent, Michaël R; Sinnesael, Mieke; Cielen, Nele; Helsen, Christine; Clinckemalie, Liesbeth; Spans, Lien; Gayan-Ramirez, Ghislaine; Deldicque, Louise; Hespel, Peter; Carmeliet, Geert; Vanderschueren, Dirk; Claessens, Frank

    2014-07-01

    Androgens have well-established anabolic actions on skeletal muscle, although the direct effects of the androgen receptor (AR) in muscle remain unclear. We generated satellite cell-specific AR-knockout (satARKO) mice in which the AR is selectively ablated in satellite cells, the muscle precursor cells. Total-limb maximal grip strength is decreased by 7% in satARKO mice, with soleus muscles containing ∼10% more type I fibers and 10% less type IIa fibers than the corresponding control littermates. The weight of the perineal levator ani muscle is markedly reduced (-52%). Thus, muscle AR is involved in fiber-type distribution and force production of the limb muscles, while it is a major determinant of the perineal muscle mass. Surprisingly, myostatin (Mstn), a strong inhibitor of skeletal muscle growth, is one of the most androgen-responsive genes (6-fold reduction in satARKO) through direct transcription activation by the AR. Consequently, muscle hypertrophy in response to androgens is augmented in Mstn-knockout mice. Our finding that androgens induce Mstn signaling to restrain their own anabolic actions has implications for the treatment of muscle wasting disorders.-Dubois, V., Laurent, M. R., Sinnesael, M., Cielen, N., Helsen, C., Clinckemalie, L., Spans, L., Gayan-Ramirez, G., Deldicque, L., Hespel, P., Carmeliet, G., Vanderschueren, D., and Claessens, F. A satellite cell-specific knockout of the androgen receptor reveals myostatin as a direct androgen target in skeletal muscle.

  20. Effects of clonidine and methylphenidate on motor activity in Fmr1 knockout mice.

    PubMed

    Wrenn, Craige C; Heitzer, Andrew M; Roth, Alexandra K; Nawrocki, Lauren; Valdovinos, Maria G

    2015-01-12

    Fragile X syndrome (FXS), a disorder caused by a mutation in the FMR1 gene, is often associated with Attention Deficit Hyperactivity Disorder (ADHD). Common treatments for the hyperactivity often seen in ADHD involve the use of stimulants and α2-adrenergic agonists. The Fmr1 knockout (KO) mouse has been found to be a valid model for FXS both biologically and behaviorally. Of particular interest to our research, the Fmr1 KO mouse has been demonstrated to show increased locomotion in comparison to wild type (WT) littermates. In the present study, we assessed the effects of clonidine (0.05 mg/kg) and methylphenidate (5 mg/kg) on motor activity in Fmr1 KO mice and their WT littermates in the open field test. Results showed that methylphenidate increased motor activity in both genotypes. Clonidine decreased motor activity in both genotypes, but the effect was delayed in the Fmr1 KO mice.

  1. Homozygous and Heterozygous p53 Knockout Rats Develop Metastasizing Sarcomas with High Frequency

    PubMed Central

    van Boxtel, Ruben; Kuiper, Raoul V.; Toonen, Pim W.; van Heesch, Sebastiaan; Hermsen, Roel; de Bruin, Alain; Cuppen, Edwin

    2011-01-01

    The TP53 tumor suppressor gene is mutated in the majority of human cancers. Inactivation of p53 in a variety of animal models results in early-onset tumorigenesis, reflecting the importance of p53 as a gatekeeper tumor suppressor. We generated a mutant Tp53 allele in the rat using a target-selected mutagenesis approach. Here, we report that homozygosity for this allele results in complete loss of p53 function. Homozygous mutant rats predominantly develop sarcomas with an onset of 4 months of age with a high occurrence of pulmonary metastases. Heterozygous rats develop sarcomas starting at 8 months of age. Molecular analysis revealed that these tumors exhibit a loss-of-heterozygosity of the wild-type Tp53 allele. These unique features make this rat highly complementary to other rodent p53 knockout models and a versatile tool for investigating tumorigenesis processes as well as genotoxic studies. PMID:21854749

  2. Liver-specific knockout of arginase-1 leads to a profound phenotype similar to inducible whole body arginase-1 deficiency.

    PubMed

    Ballantyne, Laurel L; Sin, Yuan Yan; Al-Dirbashi, Osama Y; Li, Xinzhi; Hurlbut, David J; Funk, Colin D

    2016-12-01

    Arginase-1 (Arg1) converts arginine to urea and ornithine in the distal step of the urea cycle in liver. We previously generated a tamoxifen-inducible Arg1 deficient mouse model (Arg1-Cre) that disrupts Arg1 expression throughout the whole body and leads to lethality ≈ 2 weeks after gene disruption. Here, we evaluate if liver-selective Arg1 loss is sufficient to recapitulate the phenotype observed in global Arg1 knockout mice, as well as to gauge the effectiveness of gene delivery or hepatocyte transplantation to rescue the phenotype. Liver-selective Arg1 deletion was induced by using an adeno-associated viral (AAV)-thyroxine binding globulin (TBG) promoter-Cre recombinase vector administered to Arg1 "floxed" mice; Arg1(fl/fl) ). An AAV vector expressing an Arg1-enhanced green fluorescent protein (Arg1-eGFP) transgene was used for gene delivery, while intrasplenic injection of wild-type (WT) C57BL/6 hepatocytes after partial hepatectomy was used for cell delivery to "rescue" tamoxifen-treated Arg1-Cre mice. The results indicate that liver-selective loss of Arg1 (> 90% deficient) leads to a phenotype resembling the whole body knockout of Arg1 with lethality ≈ 3 weeks after Cre-induced gene disruption. Delivery of Arg1-eGFP AAV rescues more than half of Arg1 global knockout male mice (survival > 4 months) but a significant proportion still succumb to the enzyme deficiency even though liver expression and enzyme activity of the fusion protein reach levels observed in WT animals. Significant Arg1 enzyme activity from engrafted WT hepatocytes into knockout livers can be achieved but not sufficient for rescuing the lethal phenotype. This raises a conundrum relating to liver-specific expression of Arg1. On the one hand, loss of expression in this organ appears to be both necessary and sufficient to explain the lethal phenotype of the genetic disorder in mice. On the other hand, gene and cell-directed therapies suggest that rescue of extra-hepatic Arg1

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

    PubMed

    Raymond, Karine; Kreft, Maaike; Janssen, Hans; Calafat, Jero; Sonnenberg, Arnoud

    2005-03-01

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

  4. Does murine spermatogenesis require WNT signalling? A lesson from Gpr177 conditional knockout mouse models.

    PubMed

    Chen, Su-Ren; Tang, J-X; Cheng, J-M; Hao, X-X; Wang, Y-Q; Wang, X-X; Liu, Y-X

    2016-06-30

    Wingless-related MMTV integration site (WNT) proteins and several other components of the WNT signalling pathway are expressed in the murine testes. However, mice mutant for WNT signalling effector β-catenin using different Cre drivers have phenotypes that are inconsistent with each other. The complexity and overlapping expression of WNT signalling cascades have prevented researchers from dissecting their function in spermatogenesis. Depletion of the Gpr177 gene (the mouse orthologue of Drosophila Wntless), which is required for the secretion of various WNTs, makes it possible to genetically dissect the overall effect of WNTs in testis development. In this study, the Gpr177 gene was conditionally depleted in germ cells (Gpr177(flox/flox), Mvh-Cre; Gpr177(flox/flox), Stra8-Cre) and Sertoli cells (Gpr177(flox/flox), Amh-Cre). No obvious defects in fertility and spermatogenesis were observed in these three Gpr177 conditional knockout (cKO) mice at 8 weeks. However, late-onset testicular atrophy and fertility decline in two germ cell-specific Gpr177 deletion mice were noted at 8 months. In contrast, we did not observe any abnormalities of spermatogenesis and fertility, even in 8-month-old Gpr177(flox/flox), Amh-Cre mice. Elevation of reactive oxygen species (ROS) was detected in Gpr177 cKO germ cells and Sertoli cells and exhibited an age-dependent manner. However, significant increase in the activity of Caspase 3 was only observed in germ cells from 8-month-old germ cell-specific Gpr177 knockout mice. In conclusion, GPR177 in Sertoli cells had no apparent influence on spermatogenesis, whereas loss of GPR177 in germ cells disrupted spermatogenesis in an age-dependent manner via elevating ROS levels and triggering germ cell apoptosis.

  5. Does murine spermatogenesis require WNT signalling? A lesson from Gpr177 conditional knockout mouse models

    PubMed Central

    Chen, Su-Ren; Tang, J-X; Cheng, J-M; Hao, X-X; Wang, Y-Q; Wang, X-X; Liu, Y-X

    2016-01-01

    Wingless-related MMTV integration site (WNT) proteins and several other components of the WNT signalling pathway are expressed in the murine testes. However, mice mutant for WNT signalling effector β-catenin using different Cre drivers have phenotypes that are inconsistent with each other. The complexity and overlapping expression of WNT signalling cascades have prevented researchers from dissecting their function in spermatogenesis. Depletion of the Gpr177 gene (the mouse orthologue of Drosophila Wntless), which is required for the secretion of various WNTs, makes it possible to genetically dissect the overall effect of WNTs in testis development. In this study, the Gpr177 gene was conditionally depleted in germ cells (Gpr177flox/flox, Mvh-Cre; Gpr177flox/flox, Stra8-Cre) and Sertoli cells (Gpr177flox/flox, Amh-Cre). No obvious defects in fertility and spermatogenesis were observed in these three Gpr177 conditional knockout (cKO) mice at 8 weeks. However, late-onset testicular atrophy and fertility decline in two germ cell-specific Gpr177 deletion mice were noted at 8 months. In contrast, we did not observe any abnormalities of spermatogenesis and fertility, even in 8-month-old Gpr177flox/flox, Amh-Cre mice. Elevation of reactive oxygen species (ROS) was detected in Gpr177 cKO germ cells and Sertoli cells and exhibited an age-dependent manner. However, significant increase in the activity of Caspase 3 was only observed in germ cells from 8-month-old germ cell-specific Gpr177 knockout mice. In conclusion, GPR177 in Sertoli cells had no apparent influence on spermatogenesis, whereas loss of GPR177 in germ cells disrupted spermatogenesis in an age-dependent manner via elevating ROS levels and triggering germ cell apoptosis. PMID:27362799

  6. Cadherin 13: Human cis-Regulation and Selectively Altered Addiction Phenotypes and Cerebral Cortical Dopamine in Knockout Mice

    PubMed Central

    Drgonova, Jana; Walther, Donna; Hartstein, G Luke; Bukhari, Mohammad O; Baumann, Michael H; Katz, Jonathan; Hall, F Scott; Arnold, Elizabeth R; Flax, Shaun; Riley, Anthony; Rivero, Olga; Lesch, Klaus-Peter; Troncoso, Juan; Ranscht, Barbara; Uhl, George R

    2016-01-01

    The cadherin 13 (CDH13) gene encodes a cell adhesion molecule likely to influence development and connections of brain circuits that modulate addiction, locomotion and cognition, including those that involve midbrain dopamine neurons. Human CDH13 mRNA expression differs by more than 80% in postmortem cerebral cortical samples from individuals with different CDH13 genotypes, supporting examination of mice with altered CDH13 expression as models for common human variation at this locus. Constitutive CDH13 knockout mice display evidence for changed cocaine reward: shifted dose response relationship in tests of cocaine-conditioned place preference using doses that do not alter cocaine-conditioned taste aversion. Reduced adult CDH13 expression in conditional knockouts also alters cocaine reward in ways that correlate with individual differences in cortical CDH13 mRNA levels. In control and comparison behavioral assessments, knockout mice display modestly quicker acquisition of rotarod and water maze tasks, with a trend toward faster acquisition of 5-choice serial reaction time tasks that otherwise displayed no genotype-related differences. They display significant differences in locomotion in some settings, with larger effects in males. In assessments of brain changes that might contribute to these behavioral differences, there are selective alterations of dopamine levels, dopamine/metabolite ratios, dopaminergic fiber densities and mRNA encoding the activity dependent transcription factor npas4 in cerebral cortex of knockout mice. These novel data and previously reported human associations of CDH13 variants with addiction, individual differences in responses to stimulant administration and attention deficit hyperactivity disorder (ADHD) phenotypes suggest that levels of CDH13 expression, through mechanisms likely to include effects on mesocortical dopamine, influence stimulant reward and may contribute modestly to cognitive and locomotor phenotypes relevant to ADHD

  7. 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

  8. Knockout of a transgene by transcription activator-like effector nucleases (TALENs) in the sawfly, Athalia rosae (Hymenoptera) and the ladybird beetle, Harmonia axyridis (Coleoptera).

    PubMed

    Hatakeyama, M; Yatomi, J; Sumitani, M; Takasu, Y; Sekiné, K; Niimi, T; Sezutsu, H

    2016-02-01

    Transcription activator-like effector nucleases (TALENs) are efficient tools for targeted genome editing and have been utilized in a number of insects. Here, we demonstrate the gene disruption (knockout) caused by TALENs targeting a transgene, 3xP3-driven enhanced green fluorescence protein (EGFP), that is integrated in the genome of two species, the sawfly Athalia rosae (Hymenoptera) and the ladybird beetle Harmonia axyridis (Coleoptera). Messenger RNAs of TALENs targeting the sequences adjacent to the chromophore region were microinjected into the eggs/embryos of each species. In At. rosae, when microinjection was performed at the posterior end of eggs, 15% of G(0) individuals showed a somatic mosaic phenotype for eye EGFP fluorescence. Three-quarters of the somatic mosaics produced EGFP-negative G(1) progeny. When eggs were injected at the anterior end, 63% of the G(0) individuals showed somatic mosaicism, and 17% of them produced EGFP-negative G(1) progeny. In H. axyridis, 25% of posterior-injected and 8% of anterior-injected G(0) individuals produced EGFP-negative G(1) progeny. In both species, the EGFP-negative progeny retained the EGFP gene, and various deletions were detected in the target sequences, indicating that gene disruption was successfully induced. Finally, for both species, 18-21% of G(0) founders produced gene knockout progeny sufficient for establishing knockout strains.

  9. Pre-Equilibrium Cluster Emission with Pickup and Knockout

    SciTech Connect

    Betak, E.

    2005-05-24

    We present a generalization of the Iwamoto-Harada-Bisplinghoff pre-equilibrium model of light cluster formation and emission, which is enhanced by allowing for possible admixtures of knockout for strongly coupled ejectiles, like {alpha}'s. The model is able to attain the Weisskopf-Ewing formula for compound-nucleus decay at long-time limit; it keeps the philosophy of pre-equilibrium decay during the equilibration stage and it describes the initial phase of a reaction as direct process(es) expressed using the language of the exciton model.

  10. Normal Taste Acceptance and Preference of PANX1 Knockout Mice

    PubMed Central

    Aleman, Tiffany R.; Ellis, Hillary T.; Ohmoto, Makoto; Matsumoto, Ichiro; Shestopalov, Val I.; Mitchell, Claire H.; Foskett, J. Kevin; Poole, Rachel L.

    2015-01-01

    Taste compounds detected by G protein-coupled receptors on the apical surface of Type 2 taste cells initiate an intracellular molecular cascade culminating in the release of ATP. It has been suggested that this ATP release is accomplished by pannexin 1 (PANX1). However, we report here that PANX1 knockout mice do not differ from wild-type controls in response to representative taste solutions, measured using 5-s brief-access tests or 48-h two-bottle choice tests. This implies that PANX1 is unnecessary for taste detection and consequently that ATP release from Type 2 taste cells does not require PANX1. PMID:25987548

  11. Acceleration of intestinal polyposis through prostaglandin receptor EP2 in Apc(Delta 716) knockout mice.

    PubMed

    Sonoshita, M; Takaku, K; Sasaki, N; Sugimoto, Y; Ushikubi, F; Narumiya, S; Oshima, M; Taketo, M M

    2001-09-01

    Arachidonic acid is metabolized to prostaglandin H(2) (PGH(2)) by cyclooxygenase (COX). COX-2, the inducible COX isozyme, has a key role in intestinal polyposis. Among the metabolites of PGH(2), PGE(2) is implicated in tumorigenesis because its level is markedly elevated in tissues of intestinal adenoma and colon cancer. Here we show that homozygous deletion of the gene encoding a cell-surface receptor of PGE(2), EP2, causes decreases in number and size of intestinal polyps in Apc(Delta 716) mice (a mouse model for human familial adenomatous polyposis). This effect is similar to that of COX-2 gene disruption. We also show that COX-2 expression is boosted by PGE(2) through the EP2 receptor via a positive feedback loop. Homozygous gene knockout for other PGE(2) receptors, EP1 or EP3, did not affect intestinal polyp formation in Apc(Delta 716) mice. We conclude that EP2 is the major receptor mediating the PGE2 signal generated by COX-2 upregulation in intestinal polyposis, and that increased cellular cAMP stimulates expression of more COX-2 and vascular endothelial growth factor in the polyp stroma.

  12. alpha7 Nicotinic acetylcholine receptor knockout selectively enhances ethanol-, but not beta-amyloid-induced neurotoxicity.

    PubMed

    de Fiebre, Nancyellen C; de Fiebre, Christopher M

    2005-01-03

    The alpha7 subtype of nicotinic acetylcholine receptor (nAChR) has been implicated as a potential site of action for two neurotoxins, ethanol and the Alzheimer's disease related peptide, beta-amyloid. Here, we utilized primary neuronal cultures of cerebral cortex from alpha7 nAChR null mutant mice to examine the role of this receptor in modulating the neurotoxic properties of subchronic, "binge" ethanol and beta-amyloid. Knockout of the alpha7 nAChR gene selectively enhanced ethanol-induced neurotoxicity in a gene dosage-related fashion. Susceptibility of cultures to beta-amyloid induced toxicity, however, was unaffected by alpha7 nAChR gene null mutation. Further, beta-amyloid did not inhibit the binding of the highly alpha7-selective radioligand, [(125)I]alpha-bungarotoxin. On the other hand, in studies in Xenopus oocytes ethanol efficaciously inhibited alpha7 nAChR function. These data suggest that alpha7 nAChRs modulate the neurotoxic effects of binge ethanol, but not the neurotoxicity produced by beta-amyloid. It is hypothesized that inhibition of alpha7 nAChRs by ethanol provides partial protection against the neurotoxic properties of subchronic ethanol.

  13. Knockout of Drosophila RNase ZL impairs mitochondrial transcript processing, respiration and cell cycle progression

    PubMed Central

    Xie, Xie; Dubrovsky, Edward B.

    2015-01-01

    RNase ZL is a highly conserved tRNA 3′-end processing endoribonuclease. Similar to its mammalian counterpart, Drosophila RNase ZL (dRNaseZ) has a mitochondria targeting signal (MTS) flanked by two methionines at the N-terminus. Alternative translation initiation yields two protein forms: the long one is mitochondrial, and the short one may localize in the nucleus or cytosol. Here, we have generated a mitochondria specific knockout of the dRNaseZ gene. In this in vivo model, cells deprived of dRNaseZ activity display impaired mitochondrial polycistronic transcript processing, increased reactive oxygen species (ROS) and a switch to aerobic glycolysis compensating for cellular ATP. Damaged mitochondria impose a cell cycle delay at the G2 phase disrupting cell proliferation without affecting cell viability. Antioxidants attenuate genotoxic stress and rescue cell proliferation, implying a critical role for ROS. We suggest that under a low-stress condition, ROS activate tumor suppressor p53, which modulates cell cycle progression and promotes cell survival. Transcriptional profiling of p53 targets confirms upregulation of antioxidant and cycB-Cdk1 inhibitor genes without induction of apoptotic genes. This study implicates Drosophila RNase ZL in a novel retrograde signaling pathway initiated by the damage in mitochondria and manifested in a cell cycle delay before the mitotic entry. PMID:26553808

  14. 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…

  15. The International Mouse Phenotyping Consortium Web Portal, a unified point of access for knockout mice and related phenotyping data

    PubMed Central

    Koscielny, Gautier; Yaikhom, Gagarine; Iyer, Vivek; Meehan, Terrence F.; Morgan, Hugh; Atienza-Herrero, Julian; Blake, Andrew; Chen, Chao-Kung; Easty, Richard; Di Fenza, Armida; Fiegel, Tanja; Grifiths, Mark; Horne, Alan; Karp, Natasha A.; Kurbatova, Natalja; Mason, Jeremy C.; Matthews, Peter; Oakley, Darren J.; Qazi, Asfand; Regnart, Jack; Retha, Ahmad; Santos, Luis A.; Sneddon, Duncan J.; Warren, Jonathan; Westerberg, Henrik; Wilson, Robert J.; Melvin, David G.; Smedley, Damian; Brown, Steve D. M.; Flicek, Paul; Skarnes, William C.; Mallon, Ann-Marie; Parkinson, Helen

    2014-01-01

    The International Mouse Phenotyping Consortium (IMPC) web portal (http://www.mousephenotype.org) provides the biomedical community with a unified point of access to mutant mice and rich collection of related emerging and existing mouse phenotype data. IMPC mouse clinics worldwide follow rigorous highly structured and standardized protocols for the experimentation, collection and dissemination of data. Dedicated ‘data wranglers’ work with each phenotyping center to collate data and perform quality control of data. An automated statistical analysis pipeline has been developed to identify knockout strains with a significant change in the phenotype parameters. Annotation with biomedical ontologies allows biologists and clinicians to easily find mouse strains with phenotypic traits relevant to their research. Data integration with other resources will provide insights into mammalian gene function and human disease. As phenotype data become available for every gene in the mouse, the IMPC web portal will become an invaluable tool for researchers studying the genetic contributions of genes to human diseases. PMID:24194600

  16. The International Mouse Phenotyping Consortium Web Portal, a unified point of access for knockout mice and related phenotyping data.

    PubMed

    Koscielny, Gautier; Yaikhom, Gagarine; Iyer, Vivek; Meehan, Terrence F; Morgan, Hugh; Atienza-Herrero, Julian; Blake, Andrew; Chen, Chao-Kung; Easty, Richard; Di Fenza, Armida; Fiegel, Tanja; Grifiths, Mark; Horne, Alan; Karp, Natasha A; Kurbatova, Natalja; Mason, Jeremy C; Matthews, Peter; Oakley, Darren J; Qazi, Asfand; Regnart, Jack; Retha, Ahmad; Santos, Luis A; Sneddon, Duncan J; Warren, Jonathan; Westerberg, Henrik; Wilson, Robert J; Melvin, David G; Smedley, Damian; Brown, Steve D M; Flicek, Paul; Skarnes, William C; Mallon, Ann-Marie; Parkinson, Helen

    2014-01-01

    The International Mouse Phenotyping Consortium (IMPC) web portal (http://www.mousephenotype.org) provides the biomedical community with a unified point of access to mutant mice and rich collection of related emerging and existing mouse phenotype data. IMPC mouse clinics worldwide follow rigorous highly structured and standardized protocols for the experimentation, collection and dissemination of data. Dedicated 'data wranglers' work with each phenotyping center to collate data and perform quality control of data. An automated statistical analysis pipeline has been developed to identify knockout strains with a significant change in the phenotype parameters. Annotation with biomedical ontologies allows biologists and clinicians to easily find mouse strains with phenotypic traits relevant to their research. Data integration with other resources will provide insights into mammalian gene function and human disease. As phenotype data become available for every gene in the mouse, the IMPC web portal will become an invaluable tool for researchers studying the genetic contributions of genes to human diseases.

  17. A tissue-specific knockout reveals that Gata1 is not essential for Sertoli cell function in the mouse

    PubMed Central

    Lindeboom, Fokke; Gillemans, Nynke; Karis, Alar; Jaegle, Martine; Meijer, Dies; Grosveld, Frank; Philipsen, Sjaak

    2003-01-01

    The transcription factor Gata1 is essential for the development of erythroid cells. Consequently, Gata1 null mutants die in utero due to severe anaemia. Outside the haematopoietic system, Gata1 is only expressed in the Sertoli cells of the testis. To elucidate the function of Gata1 in the testis, we made a Sertoli cell-specific knockout of the Gata1 gene in the mouse. We deleted a normally functioning ‘floxed’ Gata1 gene in pre-Sertoli cells in vivo through the expression of Cre from a transgene driven by the Desert Hedgehog promoter. Sur prisingly, Gata1 null testes developed to be morphologically normal, spermatogenesis was not obviously affected and expression levels of putative Gata1 target genes, and other Gata factors, were not altered. We conclude that expression of Gata1 in Sertoli cells is not essential for testis development or spermatogenesis in the mouse. PMID:12954777

  18. Zinc Finger Nuclease Mediated Knockout of ADP-Dependent Glucokinase in Cancer Cell Lines: Effects on Cell Survival and Mitochondrial Oxidative Metabolism

    PubMed Central

    Richter, Susan; Morrison, Shona; Connor, Tim; Su, Jiechuang; Print, Cristin G.; Ronimus, Ron S.; McGee, Sean L.; Wilson, William R.

    2013-01-01

    Zinc finger nucleases (ZFN) are powerful tools for editing genes in cells. Here we use ZFNs to interrogate the biological function of ADPGK, which encodes an ADP-dependent glucokinase (ADPGK), in human tumour cell lines. The hypothesis we tested is that ADPGK utilises ADP to phosphorylate glucose under conditions where ATP becomes limiting, such as hypoxia. We characterised two ZFN knockout clones in each of two lines (H460 and HCT116). All four clones had frameshift mutations in all alleles at the target site in exon 1 of ADPGK, and were ADPGK-null by immunoblotting. ADPGK knockout had little or no effect on cell proliferation, but compromised the ability of H460 cells to survive siRNA silencing of hexokinase-2 under oxic conditions, with clonogenic survival falling from 21±3% for the parental line to 6.4±0.8% (p = 0.002) and 4.3±0.8% (p = 0.001) for the two knockouts. A similar increased sensitivity to clonogenic cell killing was observed under anoxia. No such changes were found when ADPGK was knocked out in HCT116 cells, for which the parental line was less sensitive than H460 to anoxia and to hexokinase-2 silencing. While knockout of ADPGK in HCT116 cells caused few changes in global gene expression, knockout of ADPGK in H460 cells caused notable up-regulation of mRNAs encoding cell adhesion proteins. Surprisingly, we could discern no consistent effect on glycolysis as measured by glucose consumption or lactate formation under anoxia, or extracellular acidification rate (Seahorse XF analyser) under oxic conditions in a variety of media. However, oxygen consumption rates were generally lower in the ADPGK knockouts, in some cases markedly so. Collectively, the results demonstrate that ADPGK can contribute to tumour cell survival under conditions of high glycolytic dependence, but the phenotype resulting from knockout of ADPGK is cell line dependent and appears to be unrelated to priming of glycolysis in these lines. PMID:23799003

  19. Genes

    MedlinePlus

    ... Search Search MedlinePlus GO GO About MedlinePlus Site Map FAQs Customer Support Health Topics Drugs & Supplements Videos & Tools Español You Are Here: Home → Medical Encyclopedia → Genes URL of this page: //medlineplus.gov/ency/article/ ...

  20. Norepinephrine transporter heterozygous knockout mice exhibit altered transport and behavior.

    PubMed

    Fentress, H M; Klar, R; Krueger, J J; Sabb, T; Redmon, S N; Wallace, N M; Shirey-Rice, J K; Hahn, M K

    2013-11-01

    The norepinephrine (NE) transporter (NET) regulates synaptic NE availability for noradrenergic signaling in the brain and sympathetic nervous system. Although genetic variation leading to a loss of NET expression has been implicated in psychiatric and cardiovascular disorders, complete NET deficiency has not been found in people, limiting the utility of NET knockout mice as a model for genetically driven NET dysfunction. Here, we investigate NET expression in NET heterozygous knockout male mice (NET(+/-) ), demonstrating that they display an approximately 50% reduction in NET protein levels. Surprisingly, these mice display no significant deficit in NET activity assessed in hippocampal and cortical synaptosomes. We found that this compensation in NET activity was due to enhanced activity of surface-resident transporters, as opposed to surface recruitment of NET protein or compensation through other transport mechanisms, including serotonin, dopamine or organic cation transporters. We hypothesize that loss of NET protein in the NET(+/-) mouse establishes an activated state of existing surface NET proteins. The NET(+/-) mice exhibit increased anxiety in the open field and light-dark box and display deficits in reversal learning in the Morris water maze. These data suggest that recovery of near basal activity in NET(+/-) mice appears to be insufficient to limit anxiety responses or support cognitive performance that might involve noradrenergic neurotransmission. The NET(+/-) mice represent a unique model to study the loss and resultant compensatory changes in NET that may be relevant to behavior and physiology in human NET deficiency disorders.

  1. RAG1/2 knockout pigs with severe combined immunodeficiency.

    PubMed

    Huang, Jiao; Guo, Xiaogang; Fan, Nana; Song, Jun; Zhao, Bentian; Ouyang, Zhen; Liu, Zhaoming; Zhao, Yu; Yan, Quanmei; Yi, Xiaoling; Schambach, Axel; Frampton, Jon; Esteban, Miguel A; Yang, Dongshan; Yang, Huaqiang; Lai, Liangxue

    2014-08-01

    Pigs share many physiological, biochemical, and anatomical similarities with humans and have emerged as valuable large animal models for biomedical research. Considering the advantages in immune system resemblance, suitable size, and longevity for clinical practical and monitoring purpose, SCID pigs bearing dysfunctional RAG could serve as important experimental tools for regenerative medicine, allograft and xenograft transplantation, and reconstitution experiments related to the immune system. In this study, we report the generation and phenotypic characterization of RAG1 and RAG2 knockout pigs using transcription activator-like effector nucleases. Porcine fetal fibroblasts were genetically engineered using transcription activator-like effector nucleases and then used to provide donor nuclei for somatic cell nuclear transfer. We obtained 27 live cloned piglets; among these piglets, 9 were targeted with biallelic mutations in RAG1, 3 were targeted with biallelic mutations in RAG2, and 10 were targeted with a monoallelic mutation in RAG2. Piglets with biallelic mutations in either RAG1 or RAG2 exhibited hypoplasia of immune organs, failed to perform V(D)J rearrangement, and lost mature B and T cells. These immunodeficient RAG1/2 knockout pigs are promising tools for biomedical and translational research.

  2. Norepinephrine Transporter Heterozygous Knockout Mice Exhibit Altered Transport and Behavior

    PubMed Central

    Fentress, HM; Klar, R; Krueger, JK; Sabb, T; Redmon, SN; Wallace, NM; Shirey-Rice, JK; Hahn, MK

    2013-01-01

    The norepinephrine (NE) transporter (NET) regulates synaptic NE availability for noradrenergic signaling in the brain and sympathetic nervous system. Although genetic variation leading to a loss of NET expression has been implicated in psychiatric and cardiovascular disorders, complete NET deficiency has not been found in people, limiting the utility of NET knockout mice as a model for genetically-driven NET dysfunction. Here, we investigate NET expression in NET heterozygous knockout male mice (NET+/−), demonstrating that they display an ~50% reduction in NET protein levels. Surprisingly, these mice display no significant deficit in NET activity, assessed in hippocampal and cortical synaptosomes. We found that this compensation in NET activity was due to enhanced activity of surface-resident transporters, as opposed to surface recruitment of NET protein or compensation through other transport mechanisms, including serotonin, dopamine or organic cation transporters. We hypothesize that loss of NET protein in the NET+/− mouse establishes an activated state of existing, surface NET proteins. NET+/− mice exhibit increased anxiety in the open field and light-dark box and display deficits in reversal learning in the Morris Water Maze. These data suggest recovery of near basal activity in NET+/− mice appears to be insufficient to limit anxiety responses or support cognitive performance that might involve noradrenergic neurotransmission. The NET+/− mice represent a unique model to study the loss and resultant compensatory changes in NET that may be relevant to behavior and physiology in human NET deficiency disorders. PMID:24102798

  3. p21{sup WAF1/Cip1/Sdi1} knockout mice respond to doxorubicin with reduced cardiotoxicity

    SciTech Connect

    Terrand, Jerome; Xu, Beibei; Morrissy, Steve; Dinh, Thai Nho; Williams, Stuart; Chen, Qin M.

    2011-11-15

    Doxorubicin (Dox) is an antineoplastic agent that can cause cardiomyopathy in humans and experimental animals. As an inducer of reactive oxygen species and a DNA damaging agent, Dox causes elevated expression of p21{sup WAF1/Cip1/Sdi1} (p21) gene. Elevated levels of p21 mRNA and p21 protein have been detected in the myocardium of mice following Dox treatment. With chronic treatment of Dox, wild type (WT) animals develop cardiomyopathy evidenced by elongated nuclei, mitochondrial swelling, myofilamental disarray, reduced cardiac output, reduced ejection fraction, reduced left ventricular contractility, and elevated expression of ANF gene. In contrast, p21 knockout (p21KO) mice did not show significant changes in the same parameters in response to Dox treatment. In an effort to understand the mechanism of the resistance against Dox induced cardiomyopathy, we measured levels of antioxidant enzymes and found that p21KO mice did not contain elevated basal or inducible levels of glutathione peroxidase and catalase. Measurements of 6 circulating cytokines indicated elevation of IL-6, IL-12, IFN{gamma} and TNF{alpha} in Dox treated WT mice but not p21KO mice. Dox induced elevation of IL-6 mRNA was detected in the myocardium of WT mice but not p21KO mice. While the mechanism of the resistance against Dox induced cardiomyopathy remains unclear, lack of inflammatory response may contribute to the observed cardiac protection in p21KO mice. -- Highlights: Black-Right-Pointing-Pointer Doxorubicin induces p21 elevation in the myocardium. Black-Right-Pointing-Pointer Doxorubicin causes dilated cardiomyopathy in wild type mice. Black-Right-Pointing-Pointer p21 Knockout mice are resistant against doxorubicin induced cardiomyopathy. Black-Right-Pointing-Pointer Lack of inflammatory response correlates with the resistance in p21 knockout mice.

  4. Auxin/AID versus conventional knockouts: distinguishing the roles of CENP-T/W in mitotic kinetochore assembly and stability

    PubMed Central

    Wood, Laura; Booth, Daniel G.; Vargiu, Giulia; Ohta, Shinya; deLima Alves, Flavia; Samejima, Kumiko; Fukagawa, Tatsuo; Rappsilber, Juri; Earnshaw, William C.

    2016-01-01

    Most studies using knockout technologies to examine protein function have relied either on shutting off transcription (conventional conditional knockouts with tetracycline-regulated gene expression or gene disruption) or destroying the mature mRNA (RNAi technology). In both cases, the target protein is lost at a rate determined by its intrinsic half-life. Thus, protein levels typically fall over at least 1–3 days, and cells continue to cycle while exposed to a decreasing concentration of the protein. Here we characterise the kinetochore proteome of mitotic chromosomes isolated from a cell line in which the essential kinetochore protein CENP-T is present as an auxin-inducible degron (AID) fusion protein that is fully functional and able to support the viability of the cells. Stripping of the protein from chromosomes in early mitosis via targeted proteasomal degradation reveals the dependency of other proteins on CENP-T for their maintenance in kinetochores. We compare these results with the kinetochore proteome of conventional CENP-T/W knockouts. As the cell cycle is mostly formed from G1, S and G2 phases a gradual loss of CENP-T/W levels is more likely to reflect dependencies associated with kinetochore assembly pre-mitosis and upon entry into mitosis. Interestingly, a putative super-complex involving Rod-Zw10-zwilch (RZZ complex), Spindly, Mad1/Mad2 and CENP-E requires the function of CENP-T/W during kinetochore assembly for its stable association with the outer kinetochore, but once assembled remains associated with chromosomes after stripping of CENP-T during mitosis. This study highlights the different roles core kinetochore components may play in the assembly of kinetochores (upon entry into mitosis) versus the maintenance of specific components (during mitosis). PMID:26791246

  5. FKBP5 Moderates Alcohol Withdrawal Severity: Human Genetic Association and Functional Validation in Knockout Mice

    PubMed Central

    Huang, Ming-Chyi; Schwandt, Melanie L; Chester, Julia A; Kirchhoff, Aaron M; Kao, Chung-Feng; Liang, Tiebing; Tapocik, Jenica D; Ramchandani, Vijay A; George, David T; Hodgkinson, Colin A; Goldman, David; Heilig, Markus

    2014-01-01

    Alcohol withdrawal is associated with hypothalamic–pituitary–adrenal (HPA) axis dysfunction. The FKBP5 gene codes for a co-chaperone, FK506-binding protein 5, that exerts negative feedback on HPA axis function. This study aimed to examine the effects of single-nucleotide polymorphisms (SNPs) of the FKBP5 gene in humans and the effect of Fkbp5 gene deletion in mice on alcohol withdrawal severity. We genotyped six FKBP5 SNPs (rs3800373, rs9296158, rs3777747, rs9380524, rs1360780, and rs9470080) in 399 alcohol-dependent inpatients with alcohol consumption 48 h before admission and recorded scores from the Clinical Institute Withdrawal Assessment-Alcohol revised (CIWA-Ar). Fkbp5 gene knockout (KO) and wild-type (WT) mice were assessed for alcohol withdrawal using handling-induced convulsions (HICs) following both acute and chronic alcohol exposure. We found the minor alleles of rs3800373 (G), rs9296158 (A), rs1360780 (T), and rs9470080 (T) were significantly associated with lower CIWA-Ar scores whereas the minor alleles of rs3777747 (G) and rs9380524 (A) were associated with higher scores. The haplotype-based analyses also showed an association with alcohol withdrawal severity. Fkbp5 KO mice showed significantly greater HICs during withdrawal from chronic alcohol exposure compared with WT controls. This study is the first to show a genetic effect of FKBP5 on the severity of alcohol withdrawal syndrome. In mice, the absence of the Fkbp5 gene enhances sensitivity to alcohol withdrawal. We suggest that FKBP5 variants may trigger different adaptive changes in HPA axis regulation during alcohol withdrawal with concomitant effects on withdrawal severity. PMID:24603855

  6. The "Goldilocks Effect" in Cystic Fibrosis: identification of a lung phenotype in the cftr knockout and heterozygous mouse

    PubMed Central

    Craig Cohen, J; Lundblad, Lennart KA; Bates, Jason HT; Levitzky, Michael; Larson, Janet E

    2004-01-01

    Background Cystic Fibrosis is a pleiotropic disease in humans with primary morbidity and mortality associated with a lung disease phenotype. However, knockout in the mouse of cftr, the gene whose mutant alleles are responsible for cystic fibrosis, has previously failed to produce a readily, quantifiable lung phenotype. Results Using measurements of pulmonary mechanics, a definitive lung phenotype was demonstrated in the cftr-/- mouse. Lungs showed decreased compliance and increased airway resistance in young animals as compared to cftr+/+ littermates. These changes were noted in animals less than 60 days old, prior to any long term inflammatory effects that might occur, and are consistent with structural differences in the cftr-/- lungs. Surprisingly, the cftr+/- animals exhibited a lung phenotype distinct from either the homozygous normal or knockout genotypes. The heterozygous mice showed increased lung compliance and decreased airway resistance when compared to either homozygous phenotype, suggesting a heterozygous advantage that might explain the high frequency of this mutation in certain populations. Conclusions In the mouse the gene dosage of cftr results in distinct differences in pulmonary mechanics of the adult. Distinct phenotypes were demonstrated in each genotype, cftr-/-, cftr +/-, and cftr+/+. These results are consistent with a developmental role for CFTR in the lung. PMID:15279681

  7. Phototransduction and the Evolution of Photoreceptors

    PubMed Central

    Fain, Gordon L.; Hardie, Roger; Laughlin, Simon B.

    2010-01-01

    Photoreceptors in metazoans can be grouped into two classes, with their photoreceptive membrane derived either from cilia or microvilli. Both classes use some form of the visual pigment protein opsin, which together with 11-cis retinaldehyde absorbs light and activates a G-protein cascade, resulting in the opening or closing of ion channels. Considerable attention has recently been given to the molecular evolution of the opsins and other photoreceptor proteins; much is also known about transduction in the various photoreceptor types. Here we combine this knowledge in an attempt to understand why certain photoreceptors might have conferred particular selective advantages during evolution. We suggest that microvillar photoreceptors became predominant in most invertebrate species because of their single-photon sensitivity, high temporal resolution, and large dynamic range, and that rods and a duplex retina provided primitive chordates and vertebrates with similar sensitivity and dynamic range, but with a smaller expenditure of ATP. PMID:20144772

  8. Characterization and mapping of the human rhodopsin kinase gene and screening of the gene for mutations in patients with retinitis pigmentosa

    SciTech Connect

    Khani, S.C.; Lin, D.; Magovcevic, I.

    1994-09-01

    Rhodopsin kinase (RK) is a cytosolic enzyme in rod photoreceptors that initiates the deactivation of the phototransductions cascade by phosphorylating photoactivated rhodopsin. Although the cDNA sequence of bovine RK has been determined previously, no human cDNA or genomic sequence has thus far been available for genetic studies. In order to investigate the possible role of this candidate gene in retinitis pigmentosa (RP) and allied diseases, we have isolated and characterized human cDNA and genomic clones derived from the RK locus. The coding sequence of the human gene is 1692 nucleotides in length and is split into seven exons. The human and the bovine sequence show 84% identity at the nucleotide level and 92% identity at the amino acid level. Thus far, the intronic sequences flanking each exon except for one have been determined. We have also mapped the human RK gene to chromosome 13q34 using fluorescence in situ hybridization. To our knowledge, no RP gene has as yet been linked to this region. However, since the substrate for RK (rhodopsin) and other members of the phototransduction cascade have been implicated in the pathogenesis of RP, it is conceivable that defects in RK can also cause some forms of this disease. We are evaluating this possibility by screening DNA from 173 patients with autosomal recessive RP and 190 patients with autosomal dominant RP. So far, we have found 11 patients with variant bands. In one patient with autosomal dominant RP we discovered the missense change Ser536Leu. Cosegregation studies and further sequencing of the variant bands are currently underway.

  9. Dnd knockout ablates germ cells and demonstrates germ cell independent sex differentiation in Atlantic salmon.

    PubMed

    Wargelius, Anna; Leininger, Sven; Skaftnesmo, Kai Ove; Kleppe, Lene; Andersson, Eva; Taranger, Geir Lasse; Schulz, Rüdiger W; Edvardsen, Rolf B

    2016-02-18

    Introgression of farmed salmon escapees into wild stocks is a major threat to the genetic integrity of wild populations. Using germ cell-free fish in aquaculture may mitigate this problem. Our study investigated whether it is possible to produce germ cell-free salmon in F0 by using CRISPR-Cas9 to knock out dnd, a factor required for germ cell survival in vertebrates. To avoid studying mosaic animals, sgRNA targeting alb was simultaneously used as a visual tracer since the phenotype of alb KO is complete loss of pigmentation. Induced mutations for the tracer (alb) and the target (dnd) genes were highly correlated and produced germ cell-less fish lacking pigmentation, underlining the suitability of alb KO to serve as tracer for targeted double allelic mutations in F0 animals in species with prohibitively long generation times. This is also the first report describing dnd knockout in any fish species. Analyzing gene expression and histology of dnd KO fish revealed that sex differentiation of the somatic compartment does not depend on the presence of germ cells. However, the organization of the ovarian somatic compartment seems compromised in mutant fish.

  10. Dnd knockout ablates germ cells and demonstrates germ cell independent sex differentiation in Atlantic salmon

    PubMed Central

    Wargelius, Anna; Leininger, Sven; Skaftnesmo, Kai Ove; Kleppe, Lene; Andersson, Eva; Taranger, Geir Lasse; Schulz, Rüdiger W; Edvardsen, Rolf B

    2016-01-01

    Introgression of farmed salmon escapees into wild stocks is a major threat to the genetic integrity of wild populations. Using germ cell-free fish in aquaculture may mitigate this problem. Our study investigated whether it is possible to produce germ cell-free salmon in F0 by using CRISPR-Cas9 to knock out dnd, a factor required for germ cell survival in vertebrates. To avoid studying mosaic animals, sgRNA targeting alb was simultaneously used as a visual tracer since the phenotype of alb KO is complete loss of pigmentation. Induced mutations for the tracer (alb) and the target (dnd) genes were highly correlated and produced germ cell-less fish lacking pigmentation, underlining the suitability of alb KO to serve as tracer for targeted double allelic mutations in F0 animals in species with prohibitively long generation times. This is also the first report describing dnd knockout in any fish species. Analyzing gene expression and histology of dnd KO fish revealed that sex differentiation of the somatic compartment does not depend on the presence of germ cells. However, the organization of the ovarian somatic compartment seems compromised in mutant fish. PMID:26888627

  11. Altered thalamocortical development in the SAP102 knockout model of intellectual disability

    PubMed Central

    Crocker-Buque, Alex; Currie, Stephen P.; Luz, Liliana L.; Grant, Seth G.; Duffy, Kevin R.; Kind, Peter C.; Daw, Michael I.

    2016-01-01

    Genetic mutations known to cause intellectual disabilities (IDs) are concentrated in specific sets of genes including both those encoding synaptic proteins and those expressed during early development. We have characterized the effect of genetic deletion of Dlg3, an ID-related gene encoding the synaptic NMDA-receptor interacting protein synapse-associated protein 102 (SAP102), on development of the mouse somatosensory cortex. SAP102 is the main representative of the PSD-95 family of postsynaptic MAGUK proteins during early development and is proposed to play a role in stabilizing receptors at immature synapses. Genetic deletion of SAP102 caused a reduction in the total number of thalamocortical (TC) axons innervating the somatosensory cortex, but did not affect the segregation of barrels. On a synaptic level SAP102 knockout mice display a transient speeding of NMDA receptor kinetics during the critical period for TC plasticity, despite no reduction in GluN2B-mediated component of synaptic transmission. These data indicated an interesting dissociation between receptor kinetics and NMDA subunit expression. Following the critical period NMDA receptor function was unaffected by loss of SAP102 but there was a reduction in the divergence of TC connectivity. These data suggest that changes in synaptic function early in development caused by mutations in SAP102 result in changes in network connectivity later in life. PMID:27466188

  12. Rescue of the Friedreich's ataxia knockout mouse by human YAC transgenesis.

    PubMed

    Pook, M A; Al-Mahdawi, S; Carroll, C J; Cossée, M; Puccio, H; Lawrence, L; Clark, P; Lowrie, M B; Bradley, J L; Cooper, J M; Koenig, M; Chamberlain, S

    2001-10-01

    We have generated and characterised transgenic mice that contain the entire Friedreich's ataxia gene (FRDA) within a human YAC clone of 370 kb. In an effort to overcome the embryonic lethality of homozygous Frda knockout mice and to study the behaviour of human frataxin in a mouse cellular environment, we bred the FRDA YAC transgene onto the null mouse background. Phenotypically normal offspring that express only YAC-derived human frataxin were identified. The human frataxin was expressed in the appropriate tissues at levels comparable to the endogenous mouse frataxin, and it was correctly processed and localised to mitochondria. Biochemical analysis of heart tissue demonstrated preservation of mitochondrial respiratory chain function, together with some increase in citrate synthase and aconitase activities. Thus, we have demonstrated that human frataxin can effectively substitute for endogenous murine frataxin in the null mutant. Our studies are of immediate consequence for the generation of Friedreich's ataxia transgenic mouse models, and further contribute to the accumulating knowledge of human-mouse functional gene replacement systems.

  13. Autozygome Sequencing Expands the Horizon of Human Knockout Research and Provides Novel Insights into Human Phenotypic Variation

    PubMed Central

    Anazi, Shamsa; Alshamekh, Shomoukh; Alkuraya, Fowzan S.

    2013-01-01

    The use of autozygosity as a mapping tool in the search for autosomal recessive disease genes is well established. We hypothesized that autozygosity not only unmasks the recessiveness of disease causing variants, but can also reveal natural knockouts of genes with less obvious phenotypic consequences. To test this hypothesis, we exome sequenced 77 well phenotyped individuals born to first cousin parents in search of genes that are biallelically inactivated. Using a very conservative estimate, we show that each of these individuals carries biallelic inactivation of 22.8 genes on average. For many of the 169 genes that appear to be biallelically inactivated, available data support involvement in modulating metabolism, immunity, perception, external appearance and other phenotypic aspects, and appear therefore to contribute to human phenotypic variation. Other genes with biallelic inactivation may contribute in yet unknown mechanisms or may be on their way to conversion into pseudogenes due to true recent dispensability. We conclude that sequencing the autozygome is an efficient way to map the contribution of genes to human phenotypic variation that goes beyond the classical definition of disease. PMID:24367280

  14. Organ-dependent susceptibility of p53 knockout mice to 2-amino-3-methylimidazo[4,5-f]quinoline (IQ).

    PubMed

    Hirata, Akihiro; Tsukamoto, Tetsuya; Yamamoto, Masami; Takasu, Shinji; Sakai, Hiroki; Ban, Hisayo; Yanai, Tokuma; Masegi, Toshiaki; Donehower, Lawrence A; Tatematsu, Masae

    2007-08-01

    p53 knockout mice are now being frequently used to identify the carcinogenic potential of chemicals, thus it is important to precisely assess the susceptibility of the animals to various test chemicals. In the present study the susceptibility of p53 nullizygous((-/-)), heterozygous((+/-)), and wild-type((+/+)) mice to 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was investigated. Mice of all three genotypes were first fed a diet containing 100 or 300 p.p.m. IQ for 15 weeks in a short-term experiment. p53((+/-)) and ((+/+)) mice were then treated with IQ for 40 weeks and maintained without further treatment for an additional 12 weeks in the long-term experiment. In the forestomach, the incidence of squamous cell hyperplasia was significantly higher in p53((-/-)) than in ((+/-)) and ((+/+)) mice at 15 weeks and higher in ((+/-)) mice than ((+/+)) mice with long-term IQ treatment, indicating an elevated susceptibility of p53 knockout mice. In contrast, in the liver, various hepatocellular lesions developed mainly in female mice with long-term IQ exposure but no significant differences were evident between p53 knockout and wild-type mice, indicating a lack of elevated susceptibility in the knockout animals. Furthermore, polymerase chain reaction-single strand conformation polymorphism and sequencing analysis revealed relatively high (13/30) and low (1/15) incidences of p53 mutations (exons 5-8) in squamous cell hyperplasia and hepatocellular tumors, respectively. These results clearly indicate that the susceptibility of p53 knockout mice is organ-dependent, coinciding to some extent with the likelihood of p53 gene alteration in the induced tumors.

  15. Impaired Long-term Potentiation and Enhanced Neuronal Excitability in the Amygdala of CaV1.3 Knockout Mice

    PubMed Central

    McKinney, Brandon C.; Sze, Wilson; Lee, Benjamin; Murphy, Geoffrey G.

    2009-01-01

    Previously, we demonstrated that mice in which the gene for the L-type voltage-gated calcium channel CaV1.3 is deleted (CaV1.3 knockout mice) exhibit an impaired ability to consolidate contextually-conditioned fear. Given that this form of Pavlovian fear conditioning is critically dependent on the basolateral complex of the amygdala (BLA), we were interested in the mechanisms by which CaV1.3 contributes to BLA neurophysiology. In the present study, we used in vitro amygdala slices prepared from CaV1.3 knockout mice and wild-type littermates to explore the role of CaV1.3 in long-term potentiation (LTP) and intrinsic neuronal excitability in the BLA. We found that LTP in the lateral nucleus (LA) of the BLA, induced by high-frequency stimulation of the external capsule, was significantly reduced in CaV1.3 knockout mice. Additionally, we found that BLA principal neurons from CaV1.3 knockout mice were hyperexcitable, exhibiting significant increases in firing rates and decreased interspike intervals in response to prolonged somatic depolarization. This aberrant increase in neuronal excitability appears to be at least in part due to a concomitant reduction in the slow component of the post-burst afterhyperpolarization. Together, these results demonstrate altered neuronal function in the BLA of CaV1.3 knockout mice which may account for the impaired ability of these mice to consolidate contextually-conditioned fear. PMID:19595780

  16. Derivation of rat embryonic stem cells and generation of protease-activated receptor-2 knockout rats.

    PubMed

    Yamamoto, Satoshi; Nakata, Mitsugu; Sasada, Reiko; Ooshima, Yuki; Yano, Takashi; Shinozawa, Tadahiro; Tsukimi, Yasuhiro; Takeyama, Michiyasu; Matsumoto, Yoshio; Hashimoto, Tadatoshi

    2012-08-01

    One of the remarkable achievements in knockout (KO) rat production reported during the period 2008-2010 is the derivation of authentic embryonic stem (ES) cells from rat blastocysts using a novel culture medium containing glycogen synthase kinase 3 and mitogen-activated protein kinase kinase inhibitors (2i medium). Here, we report gene-targeting technology via homologous recombination in rat ES cells, demonstrating its use through production of a protease-activated receptor-2 gene (Par-2) KO rat. We began by generating germline-competent ES cells from Dark Agouti rats using 2i medium. These ES cells, which differentiate into cardiomyocytes in vitro, can produce chimeras with high ES cell contribution when injected into blastocysts. We then introduced a targeting vector with a neomycin-resistant gene driven by the CAG promoter to disrupt Par-2. After a 7-day drug selection, 489 neomycin-resistant colonies were obtained. Following screening by polymerase chain reaction (PCR) genotyping and quantitative PCR analysis, we confirmed three homologous recombinant clones, resulting in chimeras that transmitted the Par-2 targeted allele to offspring. Par-2 KO rats showed a loss of Par-2 messenger RNA expression in their stomach cells and a lack of PAR-2 mediated smooth muscle relaxation in the aorta as indicated by pharmacological testing. Compared with mice, rats offer many advantages in biomedical research, including a larger body size; consequently, they are widely used in scientific investigation. Thus, the establishment of a gene-targeting technology using rat ES cells will be a valuable tool in human disease model production and drug discovery.

  17. STAT2 Knockout Syrian Hamsters Support Enhanced Replication and Pathogenicity of Human Adenovirus, Revealing an Important Role of Type I Interferon Response in Viral Control.

    PubMed

    Toth, Karoly; Lee, Sang R; Ying, Baoling; Spencer, Jacqueline F; Tollefson, Ann E; Sagartz, John E; Kong, Il-Keun; Wang, Zhongde; Wold, William S M

    2015-08-01

    Human adenoviruses have been studied extensively in cell culture and have been a model for studies in molecular, cellular, and medical biology. However, much less is known about adenovirus replication and pathogenesis in vivo in a permissive host because of the lack of an adequate animal model. Presently, the most frequently used permissive immunocompetent animal model for human adenovirus infection is the Syrian hamster. Species C human adenoviruses replicate in these animals and cause pathology that is similar to that seen with humans. Here, we report findings with a new Syrian hamster strain in which the STAT2 gene was functionally knocked out by site-specific gene targeting. Adenovirus-infected STAT2 knockout hamsters demonstrated an accentuated pathology compared to the wild-type control animals, and the virus load in the organs of STAT2 knockout animals was 100- to 1000-fold higher than that in wild-type hamsters. Notably, the adaptive immune response to adenovirus is not adversely affected in STAT2 knockout hamsters, and surviving hamsters cleared the infection by 7 to 10 days post challenge. We show that the Type I interferon pathway is disrupted in these hamsters, revealing the critical role of interferon-stimulated genes in controlling adenovirus infection. This is the first study to report findings with a genetically modified Syrian hamster infected with a virus. Further, this is the first study to show that the Type I interferon pathway plays a role in inhibiting human adenovirus replication in a permissive animal model. Besides providing an insight into adenovirus infection in humans, our results are also interesting from the perspective of the animal model: STAT2 knockout Syrian hamster may also be an important animal model for studying other viral infections, including Ebola-, hanta-, and dengue viruses, where Type I interferon-mediated innate immunity prevents wild type hamsters from being effectively infected to be used as animal models.

  18. Delayed liver regeneration after partial hepatectomy in adiponectin knockout mice

    SciTech Connect

    Ezaki, Hisao; Yoshida, Yuichi; Saji, Yukiko; Takemura, Takayo; Fukushima, Juichi; Matsumoto, Hitoshi; Kamada, Yoshihiro; Wada, Akira; Igura, Takumi; Kihara, Shinji; Funahashi, Tohru; Shimomura, Iichiro; Tamura, Shinji; Kiso, Shinichi Hayashi, Norio

    2009-01-02

    We previously demonstrated that adiponectin has anti-fibrogenic and anti-inflammatory effects in the liver of mouse models of various liver diseases. However, its role in liver regeneration remains unclear. The aim of this study was to determine the role of adiponectin in liver regeneration. We assessed liver regeneration after partial hepatectomy in wild-type (WT) and adiponectin knockout (KO) mice. We analyzed DNA replication and various signaling pathways involved in cell proliferation and metabolism. Adiponectin KO mice exhibited delayed DNA replication and increased lipid accumulation in the regenerating liver. The expression levels of peroxisome proliferator-activated receptor (PPAR) {alpha} and carnitine palmitoyltransferase-1 (CPT-1), a key enzyme in mitochondrial fatty acid oxidation, were decreased in adiponectin KO mice, suggesting possible contribution of altered fat metabolism to these phenomena. Collectively, the present results highlight a new role for adiponectin in the process of liver regeneration.

  19. Progranulin Knockout Accelerates Intervertebral Disc Degeneration in Aging Mice

    PubMed Central

    Zhao, Yun-peng; Tian, Qing-yun; Liu, Ben; Cuellar, Jason; Richbourgh, Brendon; Jia, Tang-hong; Liu, Chuan-ju

    2015-01-01

    Intervertebral disc (IVD) degeneration is a common degenerative disease, yet much is unknown about the mechanisms during its pathogenesis. Herein we investigated whether progranulin (PGRN), a chondroprotective growth factor, is associated with IVD degeneration. PGRN was detectable in both human and murine IVD. The levels of PGRN were upregulated in murine IVD tissue during aging process. Loss of PGRN resulted in an early onset of degenerative changes in the IVD tissue and altered expressions of the degeneration-associated molecules in the mouse IVD tissue. Moreover, PGRN knockout mice exhibited accelerated IVD matrix degeneration, abnormal bone formation and exaggerated bone resorption in vertebra with aging. The acceleration of IVD degeneration observed in PGRN null mice was probably due to the enhanced activation of NF-κB signaling and β-catenin signaling. Taken together, PGRN may play a critical role in homeostasis of IVD, and may serve as a potential molecular target for prevention and treatment of disc degenerative diseases. PMID:25777988

  20. Preaxial Polydactyly in Sost/Sostdc1 Double Knockouts

    SciTech Connect

    Yee, C M; Collette, N M; Loots, G G

    2011-07-29

    In the United States, {approx}5% are born with congenital birth defects due to abnormal function of cellular processes and interactions. Sclerosteosis, a rare autosomal recessive disease, causes hyperostosis of the axial and appendicular skeleton, and patients present radial deviation, digit syndactyly, nail dysplasia, and overall high bone mineral density. Sclerosteosis is due to a loss of function of sclerostin (Sost). Sost is a Wnt (abbrev.) antagonist; when mutated, nonfunctional Sost results in hyperactive osteoblast activity which leads to abnormal high bone mass. Previous studies have shown that Sost overexpression in transgenic mice causes reduced bone mineral density and a variety of limb phenotypes ranging from lost, fused, and split phalanges. Consistent with clinical manifestations of Sclerosteosis, Sost knockout mice exhibit increased generalized bone mineral density and syndactyly of the digits. Sostdc1 is a paralog of Sost that has also been described as an antagonist of Wnt signaling, in developing tooth buds. Unlike Sost knockouts, Sostdc1 null mice do not display any limb abnormalities. To determine if Sost and Sostdc1 have redundant functions during limb patterning, we examined Sost; Sostdc1 mice determined that they exhibit a novel preaxial polydactyly phenotype with a low penetrance. LacZ staining, skeletal preparations, and in situ hybridization experiments were used to help characterize this novel phenotype and understand how this phenotype develops. We find Sost and Sostdc1 to have complementary expression patterns during limb development, and the loss of their expression alters the transcription of several key limb regulators, such as Fgf8, Shh and Grem.

  1. Intercellular Adhesion Molecule 1 Knockout Abrogates Radiation Induced Pulmonary Inflammation

    NASA Astrophysics Data System (ADS)

    Hallahan, Dennis E.; Virudachalam, Subbulakshmi

    1997-06-01

    Increased expression of intercellular adhesion molecule 1 (ICAM-1; CD54) is induced by exposure to ionizing radiation. The lung was used as a model to study the role of ICAM-1 in the pathogenesis of the radiation-induced inflammation-like response. ICAM-1 expression increased in the pulmonary microvascular endothelium and not in the endothelium of larger pulmonary vessels following treatment of mice with thoracic irradiation. To quantify radiation-induced ICAM-1 expression, we utilized fluorescence-activated cell sorting analysis of anti-ICAM-1 antibody labeling of pulmonary microvascular endothelial cells from human cadaver donors (HMVEC-L cells). Fluorochrome conjugates and UV microscopy were used to quantify the fluorescence intensity of ICAM in the irradiated lung. These studies showed a dose- and time-dependent increase in ICAM-1 expression in the pulmonary microvascular endothelium. Peak expression occurred at 24 h, while threshold dose was as low as 2 Gy. To determine whether ICAM-1 is required for inflammatory cell infiltration into the irradiated lung, the anti-ICAM-1 blocking antibody was administered by tail vein injection to mice following thoracic irradiation. Inflammatory cells were quantified by immunofluorescence for leukocyte common antigen (CD45). Mice treated with the anti-ICAM-1 blocking antibody showed attenuation of inflammatory cell infiltration into the lung in response to ionizing radiation exposure. To verify the requirement of ICAM-1 in the inflammation-like radiation response, we utilized the ICAM-1 knockout mouse. ICAM-1 was not expressed in the lungs of ICAM-1-deficient mice following treatment with thoracic irradiation. ICAM-1 knockout mice had no increase in the inflammatory cell infiltration into the lung in response to thoracic irradiation. These studies demonstrate a radiation dose-dependent increase in ICAM-1 expression in the pulmonary microvascular endothelium, and show that ICAM-1 is required for inflammatory cell infiltration

  2. 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…

  3. Electrophysiological and Ultrastructural Characterization of Neuromuscular Junctions in Diaphragm Muscle of Acetylcholinesterase Knockout Mice

    DTIC Science & Technology

    2008-04-01

    Electrophysiological and Ultrastructural Characterization of Neuromuscular Junctions in 5a. CONTRACT NUMBER Diaphragm Muscle of Acetylcholinesterase Knockout Mice...AChE +/+) and acetylcholinesterase knockout (AChE -/-) mice to determine the compensatory mechanism manifested by the neuromuscular junction to...had smaller nerve terminals and diminished pre- and postsynaptic surface contacts relative to neuromuscular junctions of AChE +/+ mice. The

  4. Adh1 and Adh1/4 knockout mice as possible rodent models for presymptomatic Parkinson's disease.

    PubMed

    Anvret, Anna; Ran, Caroline; Westerlund, Marie; Gellhaar, Sandra; Lindqvist, Eva; Pernold, Karin; Lundströmer, Karin; Duester, Gregg; Felder, Michael R; Galter, Dagmar; Belin, Andrea Carmine

    2012-02-01

    Alcohol dehydrogenases (ADH) catalyze the reversible metabolism of many types of alcohols and aldehydes to prevent the possible toxic accumulation of these compounds. ADHs are of interest in Parkinson's disease (PD) since these compounds can be harmful to dopamine (DA) neurons. Genetic variants in ADH1C and ADH4 have been found to associate with PD and lack of Adh4 gene activity in a mouse model has recently been reported to induce changes in the DA system. Adh1 knockout (Adh1-/-) and Adh1/4 double knockout (Adh1/4-/-) mice were investigated for possible changes in DA system related activity, biochemical parameters and olfactory function compared to wild-type (WT) mice. Locomotor activity was tested at ∼7 (adult) and >15 months of age to mimic the late onset of PD. Adh1-/- and Adh1/4-/- mice displayed a significantly higher spontaneous locomotor activity than WT littermates. Both apomorphine and d-amphetamine increased total distance activity in Adh1-/- mice at both age intervals and in Adh1/4-/- mice at 7 months of age compared to WT mice. No significant changes were found regarding olfactory function, however biochemical data showed decreased 3,4-dihydroxyphenylacetic acid (DOPAC)/DA ratios in the olfactory bulb and decreased homovanillic acid (HVA)/DA ratios in the olfactory bulb, frontal cortex and striatum of Adh1/4-/- mice compared to WT mice. Our results suggest that lack of Adh1 alone or Adh1 and Adh4 together lead to changes in DA system related behavior, and that these knockout mice might be possible rodent models to study presymptomatic PD.

  5. BACE knockout mice are healthy despite lacking the primary beta-secretase activity in brain: implications for Alzheimer's disease therapeutics.

    PubMed

    Roberds, S L; Anderson, J; Basi, G; Bienkowski, M J; Branstetter, D G; Chen, K S; Freedman, S B; Frigon, N L; Games, D; Hu, K; Johnson-Wood, K; Kappenman, K E; Kawabe, T T; Kola, I; Kuehn, R; Lee, M; Liu, W; Motter, R; Nichols, N F; Power, M; Robertson, D W; Schenk, D; Schoor, M; Shopp, G M; Shuck, M E; Sinha, S; Svensson, K A; Tatsuno, G; Tintrup, H; Wijsman, J; Wright, S; McConlogue, L

    2001-06-01

    Alzheimer's disease (AD) is a neurodegenerative disorder characterized by accumulation of amyloid plaques and neurofibrillary tangles in the brain. The major components of plaque, beta-amyloid peptides (Abetas), are produced from amyloid precursor protein (APP) by the activity of beta- and gamma-secretases. beta-secretase activity cleaves APP to define the N-terminus of the Abeta1-x peptides and, therefore, has been a long- sought therapeutic target for treatment of AD. The gene encoding a beta-secretase for beta-site APP cleaving enzyme (BACE) was identified recently. However, it was not known whether BACE was the primary beta-secretase in mammalian brain nor whether inhibition of beta-secretase might have effects in mammals that would preclude its utility as a therapeutic target. In the work described herein, we generated two lines of BACE knockout mice and characterized them for pathology, beta-secretase activity and Abeta production. These mice appeared to develop normally and showed no consistent phenotypic differences from their wild-type littermates, including overall normal tissue morphology and brain histochemistry, normal blood and urine chemistries, normal blood-cell composition, and no overt behavioral and neuromuscular effects. Brain and primary cortical cultures from BACE knockout mice showed no detectable beta-secretase activity, and primary cortical cultures from BACE knockout mice produced much less Abeta from APP. The findings that BACE is the primary beta-secretase activity in brain and that loss of beta-secretase activity produces no profound phenotypic defects with a concomitant reduction in beta-amyloid peptide clearly indicate that BACE is an excellent therapeutic target for treatment of AD.

  6. Studies of an Androgen-Binding Protein Knockout Corroborate a Role for Salivary ABP in Mouse Communication.

    PubMed

    Chung, Amanda G; Belone, Phillip M; Vošlajerová Bímová, Barbora; Karn, Robert M; Laukaitis, Christina M

    2017-02-03

    The house mouse Androgen-binding protein (Abp) gene family is comprised of 64 paralogs, 30 Abpa and 34 Abpbg, encoding the alpha (ABPA) and betagamma (ABPBG) protein subunits that are disulfide-bridged to form dimers in secretions. Only 14 Abp genes are expressed in distinct patterns in the lacrimal (11) and submandibular glands (3). We created a knockout mouse line lacking two of the three genes expressed in submandibular glands, Abpa27 and Abpbg27, by replacing them with the neomycin resistance gene. The knockout genotype (-/-) showed no Abpa27 or Abpbg27 transcripts in submandibular gland cDNA libraries and there was a concomitant lack of protein expression of ABPA27 and ABPBG27 in the -/- genotype saliva, shown by elimination of these two proteins from the saliva proteome and the loss of cross-reactive material in the acinar cells of the submandibular glands. We also observed a decrease in BG26 protein in the -/- animals, suggesting monomer instability. Overall, we observed no major phenotypic changes in the -/- genotype, compared with their +/+ and +/- siblings raised in a laboratory setting, including normal growth curves, tissue histology, fecundity and longevity. The only difference is that male and female C57BL/6 mice preferred saliva of the opposite sex containing ABP statistically significantly more than saliva of the opposite sex without ABP in a Y-maze test. These results show for the first time that mice can sense the presence of ABP between saliva targets with and without ABPs, and that they spend more time investigating the target containing ABP.

  7. Generation of Knockout Rats with X-Linked Severe Combined Immunodeficiency (X-SCID) Using Zinc-Finger Nucleases

    PubMed Central

    Mashimo, Tomoji; Takizawa, Akiko; Voigt, Birger; Yoshimi, Kazuto; Hiai, Hiroshi; Kuramoto, Takashi; Serikawa, Tadao

    2010-01-01

    Background Although the rat is extensively used as a laboratory model, the inability to utilize germ line-competent rat embryonic stem (ES) cells has been a major drawback for studies that aim to elucidate gene functions. Recently, zinc-finger nucleases (ZFNs) were successfully used to create genome-specific double-stranded breaks and thereby induce targeted gene mutations in a wide variety of organisms including plants, drosophila, zebrafish, etc. Methodology/Principal Findings We report here on ZFN-induced gene targeting of the rat interleukin 2 receptor gamma (Il2rg) locus, where orthologous human and mouse mutations cause X-linked severe combined immune deficiency (X-SCID). Co-injection of mRNAs encoding custom-designed ZFNs into the pronucleus of fertilized oocytes yielded genetically modified offspring at rates greater than 20%, which possessed a wide variety of deletion/insertion mutations. ZFN-modified founders faithfully transmitted their genetic changes to the next generation along with the severe combined immune deficiency phenotype. Conclusions and Significance The efficient and rapid generation of gene knockout rats shows that using ZFN technology is a new strategy for creating gene-targeted rat models of human diseases. In addition, the X-SCID rats that were established in this study will be valuable in vivo tools for evaluating drug treatment or gene therapy as well as model systems for examining the treatment of xenotransplanted malignancies. PMID:20111598

  8. Highly efficient generation of GGTA1 biallelic knockout inbred mini-pigs with TALENs.

    PubMed

    Xin, Jige; Yang, Huaqiang; Fan, Nana; Zhao, Bentian; Ouyang, Zhen; Liu, Zhaoming; Zhao, Yu; Li, Xiaoping; Song, Jun; Yang, Yi; Zou, Qingjian; Yan, Quanmei; Zeng, Yangzhi; Lai, Liangxue

    2013-01-01

    Inbred mini-pigs are ideal organ donors for future human xenotransplantations because of their clear genetic background, high homozygosity, and high inbreeding endurance. In this study, we chose fibroblast cells from a highly inbred pig line called Banna mini-pig inbred line (BMI) as donor nuclei for nuclear transfer, combining with transcription activator-like effector nucleases (TALENs) and successfully generated α-1,3-galactosyltransferase (GGTA1) gene biallelic knockout (KO) pigs. To validate the efficiency of TALEN vectors, in vitro-transcribed TALEN mRNAs were microinjected into one-cell stage parthenogenetically activated porcine embryos. The efficiency of indel mutations at the GGTA1-targeting loci was as high as 73.1% (19/26) among the parthenogenetic blastocysts. TALENs were co-transfected into porcine fetal fibroblasts of BMI with a plasmid containing neomycin gene. The targeting efficiency reached 89.5% (187/209) among the survived cell clones after a 10 d selection. More remarkably 27.8% (58/209) of colonies were biallelic KO. Five fibroblast cell lines with biallelic KO were chosen as nuclear donors for somatic cell nuclear transfer (SCNT). Three miniature piglets with biallelic mutations of the GGTA1 gene were achieved. Gal epitopes on the surface of cells from all the three biallelic KO piglets were completely absent. The fibroblasts from the GGTA1 null piglets were more resistant to lysis by pooled complement-preserved normal human serum than those from wild-type pigs. These results indicate that a combination of TALENs technology with SCNT can generate biallelic KO pigs directly with high efficiency. The GGTA1 null piglets with inbred features created in this study can provide a new organ source for xenotransplantation research.

  9. Conditional (intestinal-specific) knockout of the riboflavin transporter-3 (RFVT-3) impairs riboflavin absorption.

    PubMed

    Subramanian, Veedamali S; Lambrecht, Nils; Lytle, Christian; Said, Hamid M

    2016-02-15

    Riboflavin (RF) is indispensable for normal cell metabolism, proliferation, and growth. The RFVT-3 protein (product of the Slc52a3 gene) is expressed in the gut with the expression being restricted to the apical membrane domain of the polarized intestinal epithelial cells. The relative contribution of RFVT-3 to total carrier-mediated RF uptake in the native intestine, however, is not clear. We addressed this issue in the current investigation using a conditional (intestinal-specific) RFVT-3 knockout (cKO) mouse model developed by the Cre/Lox approach. All RFVT-3 cKO mice were found to be RF deficient and showed a significant growth and development retardation; also, nearly two-thirds of them died prematurely between the age of 6 and 12 wk. In vivo (intestinal and colonic loops) and in vitro (native isolated intestinal epithelial cells) uptake studies showed a severe inhibition in carrier-mediated RF uptake in the cKO mice compared with control littermates. We also observed a significant increase in the level of expression of oxidative stress-responsive genes in the intestine of the cKO mice compared with control littermates. Supplementation of the RFVT-3 cKO mice with pharmacological doses of RF led to a complete correction of the growth retardation and to normalization in the level of expression of the oxidative stress-responsive genes in the gut. These results show, for the first time, that the RFVT-3 system is the main transporter involved in carrier-mediated RF uptake in the native mouse small and large intestine, and that its dysfunction impairs normal RF body homeostasis.

  10. Systemic and Cerebral Iron Homeostasis in Ferritin Knock-Out Mice

    PubMed Central

    Li, Wei; Garringer, Holly J.; Goodwin, Charles B.; Richine, Briana; Acton, Anthony; VanDuyn, Natalia; Muhoberac, Barry B.; Irimia-Dominguez, Jose; Chan, Rebecca J.; Peacock, Munro; Nass, Richard; Ghetti, Bernardino; Vidal, Ruben

    2015-01-01

    Ferritin, a 24-mer heteropolymer of heavy (H) and light (L) subunits, is the main cellular iron storage protein and plays a pivotal role in iron homeostasis by modulating free iron levels thus reducing radical-mediated damage. The H subunit has ferroxidase activity (converting Fe(II) to Fe(III)), while the L subunit promotes iron nucleation and increases ferritin stability. Previous studies on the H gene (Fth) in mice have shown that complete inactivation of Fth is lethal during embryonic development, without ability to compensate by the L subunit. In humans, homozygous loss of the L gene (FTL) is associated with generalized seizure and atypical restless leg syndrome, while mutations in FTL cause a form of neurodegeneration with brain iron accumulation. Here we generated mice with genetic ablation of the Fth and Ftl genes. As previously reported, homozygous loss of the Fth allele on a wild-type Ftl background was embryonic lethal, whereas knock-out of the Ftl allele (Ftl-/-) led to a significant decrease in the percentage of Ftl-/- newborn mice. Analysis of Ftl-/- mice revealed systemic and brain iron dyshomeostasis, without any noticeable signs of neurodegeneration. Our findings indicate that expression of the H subunit can rescue the loss of the L subunit and that H ferritin homopolymers have the capacity to sequester iron in vivo. We also observed that a single allele expressing the H subunit is not sufficient for survival when both alleles encoding the L subunit are absent, suggesting the need of some degree of complementation between the subunits as well as a dosage effect. PMID:25629408

  11. MOMDIS: a Glauber model computer code for knockout reactions

    NASA Astrophysics Data System (ADS)

    Bertulani, C. A.; Gade, A.

    2006-09-01

    A computer program is described to calculate momentum distributions in stripping and diffraction dissociation reactions. A Glauber model is used with the scattering wavefunctions calculated in the eikonal approximation. The program is appropriate for knockout reactions at intermediate energy collisions ( 30 MeV⩽E/nucleon⩽2000 MeV). It is particularly useful for reactions involving unstable nuclear beams, or exotic nuclei (e.g., neutron-rich nuclei), and studies of single-particle occupancy probabilities (spectroscopic factors) and other related physical observables. Such studies are an essential part of the scientific program of radioactive beam facilities, as in for instance the proposed RIA (Rare Isotope Accelerator) facility in the US. Program summaryTitle of program: MOMDIS (MOMentum DIStributions) Catalogue identifier:ADXZ_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/ADXZ_v1_0 Computers: The code has been created on an IBM-PC, but also runs on UNIX or LINUX machines Operating systems: WINDOWS or UNIX Program language used: Fortran-77 Memory required to execute with typical data: 16 Mbytes of RAM memory and 2 MB of hard disk space No. of lines in distributed program, including test data, etc.: 6255 No. of bytes in distributed program, including test data, etc.: 63 568 Distribution format: tar.gz Nature of physical problem: The program calculates bound wavefunctions, eikonal S-matrices, total cross-sections and momentum distributions of interest in nuclear knockout reactions at intermediate energies. Method of solution: Solves the radial Schrödinger equation for bound states. A Numerov integration is used outwardly and inwardly and a matching at the nuclear surface is done to obtain the energy and the bound state wavefunction with good accuracy. The S-matrices are obtained using eikonal wavefunctions and the "t- ρρ" method to obtain the eikonal phase-shifts. The momentum distributions are obtained by means of a Gaussian expansion of

  12. Analysis of knockout mutants reveals non-redundant functions of poly(ADP-ribose)polymerase isoforms in Arabidopsis.

    PubMed

    Pham, Phuong Anh; Wahl, Vanessa; Tohge, Takayuki; de Souza, Laise Rosado; Zhang, Youjun; Do, Phuc Thi; Olas, Justyna J; Stitt, Mark; Araújo, Wagner L; Fernie, Alisdair R

    2015-11-01

    The enzyme poly(ADP-ribose)polymerase (PARP) has a dual function being involved both in the poly(ADP-ribosyl)ation and being a constituent of the NAD(+) salvage pathway. To date most studies, both in plant and non-plant systems, have focused on the signaling role of PARP in poly(ADP-ribosyl)ation rather than any role that can be ascribed to its metabolic function. In order to address this question we here used a combination of expression, transcript and protein localization studies of all three PARP isoforms of Arabidopsis alongside physiological analysis of the corresponding mutants. Our analyses indicated that whilst all isoforms of PARP were localized to the nucleus they are also present in non-nuclear locations with parp1 and parp3 also localised in the cytosol, and parp2 also present in the mitochondria. We next isolated and characterized insertional knockout mutants of all three isoforms confirming a complete knockout in the full length transcript levels of the target genes as well as a reduced total leaf NAD hydrolase activity in the two isoforms (PARP1, PARP2) that are highly expressed in leaves. Physiological evaluation of the mutant lines revealed that they displayed distinctive metabolic and root growth characteristics albeit unaltered leaf morphology under optimal growth conditions. We therefore conclude that the PARP isoforms play non-redundant non-nuclear metabolic roles and that their function is highly important in rapidly growing tissues such as the shoot apical meristem, roots and seeds.

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

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

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

  14. Physcomitrella patens auxin conjugate synthetase (GH3) double knockout mutants are more resistant to Pythium infection than wild type.

    PubMed

    Mittag, Jennifer; Šola, Ivana; Rusak, Gordana; Ludwig-Müller, Jutta

    2015-07-01

    Auxin homeostasis is involved in many different plant developmental and stress responses. The auxin amino acid conjugate synthetases belonging to the GH3 family play major roles in the regulation of free indole-3-acetic acid (IAA) levels and the moss Physcomitrella patens has two GH3 genes in its genome. A role for IAA in several angiosperm--pathogen interactions was reported, however, in a moss--oomycete pathosystem it had not been published so far. Using GH3 double knockout lines we have investigated the role of auxin homeostasis during the infection of P. patens with the two oomycete species, Pythium debaryanum and Pythium irregulare. We show that infection with P. debaryanum caused stronger disease symptoms than with P. irregulare. Also, P. patens lines harboring fusion constructs of an auxin-inducible promoter from soybean (GmGH3) with a reporter (ß-glucuronidase) showed higher promoter induction after P. debaryanum infection than after P. irregulare, indicating a differential induction of the auxin response. Free IAA was induced upon P. debaryanum infection in wild type by 1.6-fold and in two GH3 double knockout (GH3-doKO) mutants by 4- to 5-fold. All GH3-doKO lines showed a reduced disease symptom progression compared to wild type. Since P. debaryanum can be inhibited in growth on medium containing IAA, these data might indicate that endogenous high auxin levels in P. patens GH3-doKO mutants lead to higher resistance against the oomycete.

  15. Arabidopsis serotonin N-acetyltransferase knockout mutant plants exhibit decreased melatonin and salicylic acid levels resulting in susceptibility to an avirulent pathogen.

    PubMed

    Lee, Hyoung Yool; Byeon, Yeong; Tan, Dun-Xian; Reiter, Russel J; Back, Kyoungwhan

    2015-04-01

    Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme in the melatonin biosynthesis pathway in plants. We examined the effects of SNAT gene inactivation in two Arabidopsis T-DNA insertion mutant lines. After inoculation with the avirulent pathogen Pseudomonas syringe pv. tomato DC3000 harboring the elicitor avrRpt2 (Pst-avrRpt2), melatonin levels in the snat knockout mutant lines were 50% less than in wild-type Arabidopsis Col-0 plants. The snat knockout mutant lines exhibited susceptibility to pathogen infection that coincided with decreased induction of defense genes including PR1, ICS1, and PDF1.2. Because melatonin acts upstream of salicylic acid (SA) synthesis, the reduced melatonin levels in the snat mutant lines led to decreased SA levels compared to wild-type, suggesting that the increased pathogen susceptibility of the snat mutant lines could be attributed to decreased SA levels and subsequent attenuation of defense gene induction. Exogenous melatonin treatment failed to induce defense gene expression in nahG Arabidopsis plants, but restored the induction of defense gene expression in the snat mutant lines. In addition, melatonin caused translocation of NPR1 (nonexpressor of PR1) protein from the cytoplasm into the nucleus indicating that melatonin-elicited pathogen resistance in response to avirulent pathogen attack is SA-dependent in Arabidopsis.

  16. Retinal Pre-Conditioning by CD59a Knockout Protects against Light-Induced Photoreceptor Degeneration

    PubMed Central

    Wilson, Brooks; Zhao, Liangliang; Bhuyan, Rupak; Bandyopadhyay, Mausumi; Lyubarsky, Arkady; Yu, Chen; Li, Yafeng; Kanu, Levi; Miwa, Takashi; Song, Wen-Chao; Finnemann, Silvia C.; Rohrer, Bärbel; Dunaief, Joshua L.

    2016-01-01

    Complement dysregulation plays a key role in the pathogenesis of age-related macular degeneration (AMD), but the specific mechanisms are incompletely understood. Complement also potentiates retinal degeneration in the murine light damage model. To test the retinal function of CD59a, a complement inhibitor, CD59a knockout (KO) mice were used for light damage (LD) experiments. Retinal degeneration and function were compared in WT versus KO mice following light damage. Gene expression changes, endoplasmic reticulum (ER) stress, and glial cell activation were also compared. At baseline, the ERG responses and rhodopsin levels were lower in CD59aKO compared to wild-type (WT) mice. Following LD, the ERG responses were better preserved in CD59aKO compared to WT mice. Correspondingly, the number of photoreceptors was higher in CD59aKO retinas than WT controls after LD. Under normal light conditions, CD59aKO mice had higher levels than WT for GFAP immunostaining in Müller cells, mRNA and protein levels of two ER-stress markers, and neurotrophic factors. The reduction in photon capture, together with the neurotrophic factor upregulation, may explain the structural and functional protection against LD in the CD59aKO. PMID:27893831

  17. Modeling Parkinson's disease genetics: altered function of the dopamine system in Adh4 knockout mice.

    PubMed

    Belin, Andrea Carmine; Westerlund, Marie; Anvret, Anna; Lindqvist, Eva; Pernold, Karin; Ogren, Sven Ove; Duester, Gregg; Galter, Dagmar

    2011-03-01

    Class IV alcohol dehydrogenase (ADH4) efficiently reduces aldehydes produced during lipid peroxidation, and may thus serve to protect from toxic effects of aldehydes e.g. on neurons. We hypothesized that ADH4 dysfunction may increase risk for Parkinson's disease (PD) and previously reported association of an ADH4 allele with PD. We found that a promoter polymorphism in this allele induced a 25-30% reduction of transcriptional activity. Based on these findings, we have now investigated whether Adh4 homo- (Adh4-/-) or heterozygous (Adh4+/-) knockout mice display any dopamine system-related changes in behavior, biochemical parameters or olfaction compared to wild-type mice. The spontaneous locomotor activity was found to be similar in the three groups, whereas administration of d-amphetamine or apomorphine induced a significant increase in horizontal activity in the Adh4-/- mice compared to wild-type mice. We measured levels of monoamines and their metabolites in striatum, frontal cortex and substantia nigra and found increased levels of dopamine and DOPAC in substantia nigra of Adh4-/- mice. Investigation of olfactory function revealed a reduced sense of smell in Adh4-/- mice accompanied by alterations in dopamine metabolite levels in the olfactory bulb. Taken together, our results suggest that lack of Adh4 gene activity induces changes in the function of the dopamine system, findings which are compatible with a role of loss-of-function mutations in ADH4 as possible risk factors for PD.

  18. Deficient Cholesterol Esterification in Plasma of apoc2 Knockout Zebrafish and Familial Chylomicronemia Patients

    PubMed Central

    Liu, Chao; Gaudet, Daniel; Miller, Yury I.

    2017-01-01

    Hypertriglyceridemia is an independent risk factor for cardiovascular disease. Apolipoprotein C-II (APOC2) is an obligatory cofactor for lipoprotein lipase (LPL), the major enzyme catalyzing plasma triglyceride hydrolysis. We have created an apoc2 knockout zebrafish model, which mimics the familial chylomicronemia syndrome (FCS) in human patients with a defect in the APOC2 or LPL gene. In this study, we measured plasma levels of free cholesterol (FC) and cholesterol esters (CE) and found that apoc2 mutant zebrafish have a significantly higher FC to CE ratio (FC/CE), when compared to the wild type. Feeding apoc2 mutant zebrafish a low-fat diet reduced triglyceride levels but not the FC/CE ratio. In situ hybridization and qPCR results demonstrated that the hepatic expression of lecithin-cholesterol acyltransferase (lcat), the enzyme responsible for esterifying plasma FC to CE, and of apolipoprotein A-I, a major protein component of HDL, were dramatically decreased in apoc2 mutants. Furthermore, the FC/CE ratio was significantly increased in the whole plasma and in a chylomicron-depleted fraction of human FCS patients. The FCS plasma LCAT activity was significantly lower than that of healthy controls. In summary, this study, using a zebrafish model and human patient samples, reports for the first time the defect in plasma cholesterol esterification associated with LPL deficiency. PMID:28107429

  19. Investigations of Salmonella enterica serovar newport infections of oysters by using immunohistochemistry and knockout mutagenesis.

    PubMed

    Morrison, Christopher M; Dial, Sharon M; Day, William A; Joens, Lynn A

    2012-04-01

    The consumption of raw oysters is an important risk factor in the acquisition of food-borne disease, with Salmonella being one of a number of pathogens that have been found in market oysters. Previous work by our lab found that Salmonella was capable of surviving in oysters for over 2 months under laboratory conditions, and this study sought to further investigate Salmonella's tissue affinity and mechanism of persistence within the oysters. Immunohistochemistry was used to show that Salmonella was capable of breaching the epithelial barriers, infecting the deeper connective tissues of the oysters, and evading destruction by the oysters' phagocytic hemocytes. To further investigate the mechanism of these infections, genes vital to the function of Salmonella's two main type III secretion systems were disrupted and the survivability of these knockout mutants within oysters was assayed. When the Salmonella pathogenicity island 1 and 2 mutant strains were exposed to oysters, there were no detectable deficiencies in their abilities to survive, suggesting that Salmonella's long-term infection of oysters does not rely upon these two important pathogenicity islands and must be due to some other, currently unknown, mechanism.

  20. A FANCD2/FANCI-Associated Nuclease 1-Knockout Model Develops Karyomegalic Interstitial Nephritis.

    PubMed

    Airik, Rannar; Schueler, Markus; Airik, Merlin; Cho, Jang; Porath, Jonathan D; Mukherjee, Elina; Sims-Lucas, Sunder; Hildebrandt, Friedhelm

    2016-12-01

    Karyomegalic interstitial nephritis (KIN) is a chronic interstitial nephropathy characterized by tubulointerstitial nephritis and formation of enlarged nuclei in the kidneys and other tissues. We recently reported that recessive mutations in the gene encoding FANCD2/FANCI-associated nuclease 1 (FAN1) cause KIN in humans. FAN1 is a major component of the Fanconi anemia-related pathway of DNA damage response (DDR) signaling. To study the pathogenesis of KIN, we generated a Fan1 knockout mouse model, with abrogation of Fan1 expression confirmed by quantitative RT-PCR. Challenging Fan1(-/-) and wild-type mice with 20 mg/kg cisplatin caused AKI in both genotypes. In contrast, chronic injection of cisplatin at 2 mg/kg induced KIN that led to renal failure within 5 weeks in Fan1(-/-) mice but not in wild-type mice. Cell culture studies showed decreased survival and reduced colony formation of Fan1(-/-) mouse embryonic fibroblasts and bone marrow mesenchymal stem cells compared with wild-type counterparts in response to treatment with genotoxic agents, suggesting that FAN1 mutations cause chemosensitivity and bone marrow failure. Our data show that Fan1 is involved in the physiologic response of kidney tubular cells to DNA damage, which contributes to the pathogenesis of CKD. Moreover, Fan1(-/-) mice provide a new model with which to study the pathomechanisms of CKD.

  1. Abnormal spermatogenesis and male infertility in testicular zinc finger protein Zfp318-knockout mice.

    PubMed

    Ishizuka, Masamichi; Ohtsuka, Eri; Inoue, Atsuto; Odaka, Mirei; Ohshima, Hirotaka; Tamura, Norihisa; Yoshida, Kaoru; Sako, Norihisa; Baba, Tadashi; Kashiwabara, Shin-Ichi; Okabe, Masaru; Noguchi, Junko; Hagiwara, Hiromi

    2016-09-01

    Zfp318, a mouse gene with a Cys2/His2 zinc finger motif, is mainly expressed in germ cells in the testis. It encodes two alternative transcripts, which regulate androgen receptor-mediated transcriptional activation or repression by overexpression of them. However, the role of Zfp318 is still obscure in vivo, especially in spermatogenesis. To elucidate the role of Zfp318 during gamete production, we established a knockout mouse line. Zfp318-null male mice exhibited infertility, whereas Zfp318-null female mice displayed normal fertility. ZFP318 was expressed during multiple stages of spermatogenesis, from spermatocytes to round spermatids. The nuclei of secondary spermatocytes showed high levels of expression. Histological analysis and quantitative analysis of DNA content showed decreased numbers of both spermatids in the seminiferous tubules and mature spermatozoa in the epididymides of Zfp318-null mice. These results suggest that Zfp318 is expressed as a functional protein in testicular germ cells and plays an important role in meiosis during spermatogenesis.

  2. Interactions of the opioid and cannabinoid systems in reward: Insights from knockout studies

    PubMed Central