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Sample records for phototransduction gene knockout

  1. Selection of Phototransduction Genes in Homo sapiens

    PubMed Central

    Christopher, Mark; Scheetz, Todd E.; Mullins, Robert F.; Abràmoff, Michael D.

    2013-01-01

    Purpose. We investigated the evidence of recent positive selection in the human phototransduction system at single nucleotide polymorphism (SNP) and gene level. Methods. SNP genotyping data from the International HapMap Project for European, Eastern Asian, and African populations was used to discover differences in haplotype length and allele frequency between these populations. Numeric selection metrics were computed for each SNP and aggregated into gene-level metrics to measure evidence of recent positive selection. The level of recent positive selection in phototransduction genes was evaluated and compared to a set of genes shown previously to be under recent selection, and a set of highly conserved genes as positive and negative controls, respectively. Results. Six of 20 phototransduction genes evaluated had gene-level selection metrics above the 90th percentile: RGS9, GNB1, RHO, PDE6G, GNAT1, and SLC24A1. The selection signal across these genes was found to be of similar magnitude to the positive control genes and much greater than the negative control genes. Conclusions. There is evidence for selective pressure in the genes involved in retinal phototransduction, and traces of this selective pressure can be demonstrated using SNP-level and gene-level metrics of allelic variation. We hypothesize that the selective pressure on these genes was related to their role in low light vision and retinal adaptation to ambient light changes. Uncovering the underlying genetics of evolutionary adaptations in phototransduction not only allows greater understanding of vision and visual diseases, but also the development of patient-specific diagnostic and intervention strategies. PMID:23868983

  2. 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.

  3. 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.

  4. Nr2e3-directed transcriptional regulation of genes involved in photoreceptor development and cell-type specific phototransduction.

    PubMed

    Haider, Neena B; Mollema, Nissa; Gaule, Meghan; Yuan, Yang; Sachs, Andrew J; Nystuen, Arne M; Naggert, Jürgen K; Nishina, Patsy M

    2009-09-01

    The retinal transcription factor Nr2e3 plays a key role in photoreceptor development and function. In this study we examine gene expression in the retina of Nr2e3(rd7/rd7) mutants with respect to wild-type control mice, to identify genes that are misregulated and hence potentially function in the Nr2e3 transcriptional network. Quantitative candidate gene real time PCR and subtractive hybridization approaches were used to identify transcripts that were misregulated in Nr2e3(rd7/rd7) mice. Chromatin immunoprecipitation assays were then used to determine which of the misregulated transcripts were direct targets of NR2E3. We identified 24 potential targets of NR2E3. In the developing retina, NR2E3 targets transcription factors such as Ror1, Rorg, and the nuclear hormone receptors Nr1d1 and Nr2c1. In the mature retina NR2E3 targets several genes including the rod specific gene Gnb1 and cone specific genes blue opsin, and two of the cone transducin subunits, Gnat2 and Gnb3. In addition, we identified 5 novel transcripts that are targeted by NR2E3. While mislocalization of proteins between rods and cones was not observed, we did observe diminished concentration of GNB1 protein in adult Nr2e3(rd7/rd7) retinas. These studies identified novel transcriptional pathways that are potentially targeted by Nr2e3 in the retina and specifically demonstrate a novel role for NR2E3 in regulating genes involved in phototransduction. PMID:19379737

  5. 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

  6. 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. PMID:25994633

  7. Targeted gene knockout in chickens mediated by TALENs.

    PubMed

    Park, Tae Sub; Lee, Hong Jo; Kim, Ki Hyun; Kim, Jin-Soo; Han, Jae Yong

    2014-09-01

    Genetically modified animals are used for industrial applications as well as scientific research, and studies on these animals contribute to a better understanding of biological mechanisms. Gene targeting techniques have been developed to edit specific gene loci in the genome, but the conventional strategy of homologous recombination with a gene-targeted vector has low efficiency and many technical complications. Here, we generated specific gene knockout chickens through the use of transcription activator-like effector nuclease (TALEN)-mediated gene targeting. In this study, we accomplished targeted knockout of the ovalbumin (OV) gene in the chicken primordial germ cells, and OV gene mutant offspring were generated through test-cross analysis. TALENs successfully induced nucleotide deletion mutations of ORF shifts, resulting in loss of chicken OV gene function. Our results demonstrate that the TALEN technique used in the chicken primordial germ cell line is a powerful strategy to create specific genome-edited chickens safely for practical applications. PMID:25139993

  8. Targeted gene knockout in chickens mediated by TALENs

    PubMed Central

    Park, Tae Sub; Lee, Hong Jo; Kim, Ki Hyun; Kim, Jin-Soo; Han, Jae Yong

    2014-01-01

    Genetically modified animals are used for industrial applications as well as scientific research, and studies on these animals contribute to a better understanding of biological mechanisms. Gene targeting techniques have been developed to edit specific gene loci in the genome, but the conventional strategy of homologous recombination with a gene-targeted vector has low efficiency and many technical complications. Here, we generated specific gene knockout chickens through the use of transcription activator-like effector nuclease (TALEN)-mediated gene targeting. In this study, we accomplished targeted knockout of the ovalbumin (OV) gene in the chicken primordial germ cells, and OV gene mutant offspring were generated through test-cross analysis. TALENs successfully induced nucleotide deletion mutations of ORF shifts, resulting in loss of chicken OV gene function. Our results demonstrate that the TALEN technique used in the chicken primordial germ cell line is a powerful strategy to create specific genome-edited chickens safely for practical applications. PMID:25139993

  9. 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.

  10. 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. PMID:24553382

  11. 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

  12. IAP gene deletion and conditional knockout models.

    PubMed

    Silke, John; Vaux, David L

    2015-03-01

    Gene deletion studies have helped reveal the unique and overlapping roles played by IAP proteins. Crossing IAP mutant mice has helped unravel the complex feed-back regulatory circuits in which cIAP1, cIAP2 and XIAP allow innate defensive responses to microbial pathogens, without the development of auto-inflammatory syndromes. Deletion of genes for Survivin and its homologs in yeasts, invertebrates and mammals has shown that it functions differently, as it is not a regulator of innate immunity or apoptosis, but acts together with INCENP, aurora kinase B and Borealin to allow chromosome segregation during mitosis. PMID:25545814

  13. 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

  14. 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

  15. 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

  16. 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

  17. Evolution of Vertebrate Phototransduction: Cascade Activation.

    PubMed

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

    2016-08-01

    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

  18. 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

  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. PMID:26940866

  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. 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

  2. RGS Protein Regulation of Phototransduction

    PubMed Central

    Chen, Ching-Kang Jason

    2015-01-01

    First identified in yeast and worm and later in other species, the physiological importance of regulators of G-protein signaling (RGS) in mammals was first demonstrated at the turn of the century in mouse retinal photoreceptors, in which RGS9 is needed for timely recovery of rod phototransduction. The role of RGS in vision has been established a synapse away in retinal depolarizing bipolar cells (DBCs), where RGS7 and RGS11 work redundantly and in a complex with Gβ5-S as GAPs for Goα in the metabotropic glutamate receptor 6 pathway at DBC dendritic tips. Much less is known on how RGS protein subserves vision in the rest of the visual system. The research into the roles of RGS proteins in vision holds great potential for many exciting new discoveries. PMID:26123301

  3. 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

  4. Targeted ablation of the Pde6h gene in mice reveals cross-species differences in cone and rod phototransduction protein isoform inventory.

    PubMed

    Brennenstuhl, Christina; Tanimoto, Naoyuki; Burkard, Markus; Wagner, Rebecca; Bolz, Sylvia; Trifunovic, Dragana; Kabagema-Bilan, Clement; Paquet-Durand, Francois; Beck, Susanne C; Huber, Gesine; Seeliger, Mathias W; Ruth, Peter; Wissinger, Bernd; Lukowski, Robert

    2015-04-17

    Phosphodiesterase-6 (PDE6) is a multisubunit enzyme that plays a key role in the visual transduction cascade in rod and cone photoreceptors. Each type of photoreceptor utilizes discrete catalytic and inhibitory PDE6 subunits to fulfill its physiological tasks, i.e. the degradation of cyclic guanosine-3',5'-monophosphate at specifically tuned rates and kinetics. Recently, the human PDE6H gene was identified as a novel locus for autosomal recessive (incomplete) color blindness. However, the three different classes of cones were not affected to the same extent. Short wave cone function was more preserved than middle and long wave cone function indicating that some basic regulation of the PDE6 multisubunit enzyme was maintained albeit by a unknown mechanism. To study normal and disease-related functions of cone Pde6h in vivo, we generated Pde6h knock-out (Pde6h(-/-)) mice. Expression of PDE6H in murine eyes was restricted to both outer segments and synaptic terminals of short and long/middle cone photoreceptors, whereas Pde6h(-/-) retinae remained PDE6H-negative. Combined in vivo assessment of retinal morphology with histomorphological analyses revealed a normal overall integrity of the retinal organization and an unaltered distribution of the different cone photoreceptor subtypes upon Pde6h ablation. In contrast to human patients, our electroretinographic examinations of Pde6h(-/-) mice suggest no defects in cone/rod-driven retinal signaling and therefore preserved visual functions. To this end, we were able to demonstrate the presence of rod PDE6G in cones indicating functional substitution of PDE6. The disparities between human and murine phenotypes caused by mutant Pde6h/PDE6H suggest species-to-species differences in the vulnerability of biochemical and neurosensory pathways of the visual signal transduction system. PMID:25739440

  5. Targeted Ablation of the Pde6h Gene in Mice Reveals Cross-species Differences in Cone and Rod Phototransduction Protein Isoform Inventory*

    PubMed Central

    Brennenstuhl, Christina; Tanimoto, Naoyuki; Burkard, Markus; Wagner, Rebecca; Bolz, Sylvia; Trifunovic, Dragana; Kabagema-Bilan, Clement; Paquet-Durand, Francois; Beck, Susanne C.; Huber, Gesine; Seeliger, Mathias W.; Ruth, Peter; Wissinger, Bernd; Lukowski, Robert

    2015-01-01

    Phosphodiesterase-6 (PDE6) is a multisubunit enzyme that plays a key role in the visual transduction cascade in rod and cone photoreceptors. Each type of photoreceptor utilizes discrete catalytic and inhibitory PDE6 subunits to fulfill its physiological tasks, i.e. the degradation of cyclic guanosine-3′,5′-monophosphate at specifically tuned rates and kinetics. Recently, the human PDE6H gene was identified as a novel locus for autosomal recessive (incomplete) color blindness. However, the three different classes of cones were not affected to the same extent. Short wave cone function was more preserved than middle and long wave cone function indicating that some basic regulation of the PDE6 multisubunit enzyme was maintained albeit by a unknown mechanism. To study normal and disease-related functions of cone Pde6h in vivo, we generated Pde6h knock-out (Pde6h−/−) mice. Expression of PDE6H in murine eyes was restricted to both outer segments and synaptic terminals of short and long/middle cone photoreceptors, whereas Pde6h−/− retinae remained PDE6H-negative. Combined in vivo assessment of retinal morphology with histomorphological analyses revealed a normal overall integrity of the retinal organization and an unaltered distribution of the different cone photoreceptor subtypes upon Pde6h ablation. In contrast to human patients, our electroretinographic examinations of Pde6h−/− mice suggest no defects in cone/rod-driven retinal signaling and therefore preserved visual functions. To this end, we were able to demonstrate the presence of rod PDE6G in cones indicating functional substitution of PDE6. The disparities between human and murine phenotypes caused by mutant Pde6h/PDE6H suggest species-to-species differences in the vulnerability of biochemical and neurosensory pathways of the visual signal transduction system. PMID:25739440

  6. 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. PMID:26769100

  7. 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. PMID:26458835

  8. Characterization of physiological responses to 22 gene knockouts in Escherichia coli central carbon metabolism.

    PubMed

    Long, Christopher P; Gonzalez, Jacqueline E; Sandoval, Nicholas R; Antoniewicz, Maciek R

    2016-09-01

    Understanding the impact of gene knockouts on cellular physiology, and metabolism in particular, is centrally important to quantitative systems biology and metabolic engineering. Here, we present a comprehensive physiological characterization of wild-type Escherichia coli and 22 knockouts of enzymes in the upper part of central carbon metabolism, including the PTS system, glycolysis, pentose phosphate pathway and Entner-Doudoroff pathway. Our results reveal significant metabolic changes that are affected by specific gene knockouts. Analysis of collective trends and correlations in the data using principal component analysis (PCA) provide new, and sometimes surprising, insights into E. coli physiology. Additionally, by comparing the data-to-model predictions from constraint-based approaches such as FBA, MOMA and RELATCH we demonstrate the important role of less well-understood kinetic and regulatory effects in central carbon metabolism. PMID:27212692

  9. Development of a high-efficiency gene knockout system for Pochonia chlamydosporia.

    PubMed

    Shen, Baoming; Xiao, Jiling; Dai, Liangying; Huang, Yonghong; Mao, Zhenchuan; Lin, Runmao; Yao, Yurong; Xie, Bingyan

    2015-01-01

    The nematophagous fungus Pochonia chlamydosporia, which belongs to the family Clavicipitaceae (Ascomycota: Pezizomycotina: Sordariomycetes: Hypocreales), is a promising biological control agent for root-knot and cyst nematodes. Its biocontrol effect has been confirmed by pot and field trials. The genome sequence of the fungus was completed recently; therefore, genome-wide functional analyses will identify its infection-associated genes. Gene knockout techniques are useful molecular tools to study gene functions. However, cultures of P. chlamydosporia are resistant to high levels of a range of fungal inhibitors, which makes the gene knockout technique difficult in this fungus. Fortunately, we found that the wild P. chlamydosporia strain PC-170 could not grow on medium containing 150μgml(-1) G418 sulfate, representing a new selectable marker for P. chlamydosporia. The neomycin-resistance gene (neo), which was amplified from the plasmid pKOV21, conferred G418-resistance on the fungus; therefore, it was chosen as the marker gene. We subsequently developed a gene knockout system for P. chlamydosporia using split-marker homologous recombination cassettes with resistance selection and protoplast transformation. The split-marker cassettes were developed using fusion PCR, and involved only two rounds of PCR. The final products comprised two linear constructs. Each construct contained a flanking region of the target gene and two thirds of the neo gene. Alkaline serine protease and chitinase were confirmed to be produced by P. chlamydosporia during infection of nematode eggs and could participate in lysis of the eggshell of nematode eggs. Here, we knocked out one chitinase gene, VFPPC_01099, and two protease genes (VFPPC_10088, VFPPC_06535). We obtained approximately 100 suspected mutants after each transformation. After screening by PCR, the average rate of gene knockout was 13%: 11% (VFPPC_01099), 13% (VFPPC_10088) and 15% (VFPPC_06535). This efficient and convenient

  10. Hitting two birds with one stone: The unforeseen consequences of nested gene knockouts in Caenorhabditis elegans.

    PubMed

    Jovelin, Richard; Cutter, Asher D

    2016-01-01

    Nested genes represent an intriguing form of non-random genomic organization in which the boundaries of one gene are fully contained within another, longer host gene. The C. elegans genome contains over 10,000 nested genes, 92% of which are ncRNAs, which occur inside 16% of the protein coding gene complement. Host genes are longer than non-host coding genes, owing to their longer and more numerous introns. Indel alleles are available for nearly all of these host genes that simultaneously alter the nested gene, raising the possibility of nested gene disruption contributing to phenotypes that might be attributed to the host gene. Such dual-knockouts could represent a source of misinterpretation about host gene function. Dual-knockouts might also provide a novel source of synthetic phenotypes that reveal the functional effects of ncRNA genes, whereby the host gene disruption acts as a perturbed genetic background to help unmask ncRNA phenotypes. PMID:27386165

  11. Phototransduction in mouse rods and cones

    PubMed Central

    Fu, Yingbin; Yau, King-Wai

    2010-01-01

    Phototransduction is the process by which light triggers an electrical signal in a photoreceptor cell. Image-forming vision in vertebrates is mediated by two types of photoreceptors: the rods and the cones. In this review, we provide a summary of the success in which the mouse has served as a vertebrate model for studying rod phototransduction, with respect to both the activation and termination steps. Cones are still not as well-understood as rods partly because it is difficult to work with mouse cones due to their scarcity and fragility. The situation may change, however. PMID:17226052

  12. Efficient gene knockout in goats using CRISPR/Cas9 system.

    PubMed

    Ni, Wei; Qiao, Jun; Hu, Shengwei; Zhao, Xinxia; Regouski, Misha; Yang, Min; Polejaeva, Irina A; Chen, Chuangfu

    2014-01-01

    The CRISPR/Cas9 system has been adapted as an efficient genome editing tool in laboratory animals such as mice, rats, zebrafish and pigs. Here, we report that CRISPR/Cas9 mediated approach can efficiently induce monoallelic and biallelic gene knockout in goat primary fibroblasts. Four genes were disrupted simultaneously in goat fibroblasts by CRISPR/Cas9-mediated genome editing. The single-gene knockout fibroblasts were successfully used for somatic cell nuclear transfer (SCNT) and resulted in live-born goats harboring biallelic mutations. The CRISPR/Cas9 system represents a highly effective and facile platform for targeted editing of large animal genomes, which can be broadly applied to both biomedical and agricultural applications. PMID:25188313

  13. Have gene knockouts caused evolutionary reversals in the mammalian first arch?

    PubMed

    Smith, K K; Schneider, R A

    1998-03-01

    Many recent gene knockout experiments cause anatomical changes to the jaw region of mice that several investigators claim are evolutionary reversals. Here we evaluate these mutant phenotypes and the assertions of atavism. We argue that following the knockout of Hoxa-2, Dlx-2, MHox, Otx2, and RAR genes, ectopic cartilages arise as secondary consequences of disruptions in normal processes of cell specification, migration, or differentiation. These disruptions cause an excess of mesenchyme to accumulate in a region through which skeletal progenitor cells usually migrate, and at a site of condensation that is normally present in mammals but that is too small to chondrify. We find little evidence that these genes, when disrupted, cause a reversion to any primitive condition and although changes in their expression may have played a role in the evolution of the mammalian jaw, their function during morphogenesis is not sufficiently understood to confirm such hypotheses. PMID:9631652

  14. Targeted gene knockout by direct delivery of ZFN proteins

    PubMed Central

    Gaj, Thomas; Guo, Jing; Kato, Yoshio; Sirk, Shannon J.; Barbas, Carlos F.

    2012-01-01

    Zinc-finger nucleases (ZFNs) are versatile reagents that have redefined genome engineering. Realizing the full potential of this technology requires the development of safe and effective methods for delivering ZFNs into cells. We demonstrate the intrinsic cell-penetrating capabilities of the standard ZFN architecture and show that direct delivery of ZFNs as proteins leads to efficient endogenous gene disruption in a variety of mammalian cell types with minimal off-target effects. PMID:22751204

  15. CD47 gene knockout protects against transient focal cerebral ischemia in mice.

    PubMed

    Jin, Guang; Tsuji, Kiyoshi; Xing, Changhong; Yang, Yong-Guang; Wang, Xiaoying; Lo, Eng H

    2009-05-01

    CD47 is a cell surface glycoprotein that helps mediate neutrophil transmigration across blood vessels. The present study was performed to determine whether absence of the CD47 gene decreases focal ischemic brain damage. Mice were subjected to 90 min middle cerebral artery occlusion. CD47 knockout mice were compared against matching wildtype mice. CD47 expression was checked by Western blotting. Infarct volume and ischemic brain swelling were quantified with cresyl violet-stained brain sections at 24 and 72 h after ischemia. The tight junction protein claudin-5 was detected by imunohistochemistry. Two surrogate markers of neuroinflammation, brain levels of matrix metalloproteinase-9 (MMP-9) and infiltration of neutrophils, were assessed by immunohistochemistry. Western blots confirmed that CD47 was absent in knockout brains. Ischemia did not appear to upregulate total brain levels of CD47 in WT mice. In CD47 knockout mice, infarct volumes were reduced at 24 and 72 h after ischemia, and hemispheric swelling was decreased at 72 h. Loss of claudin-5 was observed in ischemic WT brain. This effect was ameliorated in CD47 knockout brains. Extravasation of neutrophils into the brain parenchyma was significantly reduced in CD47 knockout mice compared to wildtype mice. MMP-9 appeared to be upregulated in microvessels within ischemic brain. MMP-9 levels were markedly lower in CD47 knockout brains compared to wildtype brains. We conclude that CD47 is broadly involved in neuroinflammation, and this integrin-associated-protein plays a role in promoting MMP-9 upregulaton, neutrophil extravasation, brain swelling and progression of acute ischemic brain injury. PMID:19233173

  16. Germ line knockout of IGFBP-3 reveals influences of the gene on mammary gland neoplasia.

    PubMed

    Blouin, Marie-José; Bazile, Miguel; Birman, Elena; Zakikhani, Mahvash; Florianova, Livia; Aleynikova, Olga; Powell, David R; Pollak, Michael

    2015-02-01

    Insulin-like growth factor binding protein-3 (IGFBP-3) is an important carrier protein for insulin-like growth factors (IGFs) in the circulation. IGFBP-3 antagonizes the growth-promoting and anti-apoptotic activities of IGFs in experimental systems, but in certain contexts can increase IGF bioactivity, probably by increasing its half-life. The goal of this study was to investigate the role of IGFBP-3 in breast carcinogenesis and breast cancer metastasis. In the first part of the study, we exposed IGFBP-3 knockout and wild-type female mice to dimethylbenz[a]anthracene (DMBA) and followed them for appearance of primary tumors for up to 13 months. In the second part, mice of each genotype received an IV injection of 4T1 mammary carcinoma cells and then lung nodules were counted. Our results show that IGFBP-3 knockout mice developed breast tumors significantly earlier than the wild-type (13.9 ± 1.1 versus 22.5 ± 3.3 weeks, respectively, P = 0.0144), suggesting tumor suppression activity of IGFBP-3. In tumors of IGFBP-3 knockout mice, levels of phospho-AKT(Ser473) were increased compared to wild-type mice. The lung metastasis assay showed significantly more and larger lung nodules in IGFBP-3 knockout mice than in wild-type mice. While we observed increased levels of IGFBP-5 protein in the IGFBP-3 knockout mice, our findings suggest that this was not sufficient to completely compensate for the absence of IGFBP-3. Even though knockout of IGFBP-3 is associated with only a subtle phenotype under control conditions, our results reveal that loss of this gene has measurable effects on breast carcinogenesis and breast cancer metastasis. PMID:25614235

  17. Efficient TALEN-mediated gene knockout in livestock

    PubMed Central

    Carlson, Daniel F.; Tan, Wenfang; Lillico, Simon G.; Stverakova, Dana; Proudfoot, Chris; Christian, Michelle; Voytas, Daniel F.; Long, Charles R.; Whitelaw, C. Bruce A.; Fahrenkrug, Scott C.

    2012-01-01

    Transcription activator-like effector nucleases (TALENs) are programmable nucleases that join FokI endonuclease with the modular DNA-binding domain of TALEs. Although zinc-finger nucleases enable a variety of genome modifications, their application to genetic engineering of livestock has been slowed by technical limitations of embryo-injection, culture of primary cells, and difficulty in producing reliable reagents with a limited budget. In contrast, we found that TALENs could easily be manufactured and that over half (23/36, 64%) demonstrate high activity in primary cells. Cytoplasmic injections of TALEN mRNAs into livestock zygotes were capable of inducing gene KO in up to 75% of embryos analyzed, a portion of which harbored biallelic modification. We also developed a simple transposon coselection strategy for TALEN-mediated gene modification in primary fibroblasts that enabled both enrichment for modified cells and efficient isolation of modified colonies. Coselection after treatment with a single TALEN-pair enabled isolation of colonies with mono- and biallelic modification in up to 54% and 17% of colonies, respectively. Coselection after treatment with two TALEN-pairs directed against the same chromosome enabled the isolation of colonies harboring large chromosomal deletions and inversions (10% and 4% of colonies, respectively). TALEN-modified Ossabaw swine fetal fibroblasts were effective nuclear donors for cloning, resulting in the creation of miniature swine containing mono- and biallelic mutations of the LDL receptor gene as models of familial hypercholesterolemia. TALENs thus appear to represent a highly facile platform for the modification of livestock genomes for both biomedical and agricultural applications. PMID:23027955

  18. Determination of antibiotic hypersensitivity among 4,000 single-gene-knockout mutants of Escherichia coli.

    PubMed

    Tamae, Cindy; Liu, Anne; Kim, Katherine; Sitz, Daniel; Hong, Jeeyoon; Becket, Elinne; Bui, Ann; Solaimani, Parrisa; Tran, Katherine P; Yang, Hanjing; Miller, Jeffrey H

    2008-09-01

    We have tested the entire Keio collection of close to 4,000 single-gene knockouts in Escherichia coli for increased susceptibility to one of seven different antibiotics (ciprofloxacin, rifampin, vancomycin, ampicillin, sulfamethoxazole, gentamicin, or metronidazole). We used high-throughput screening of several subinhibitory concentrations of each antibiotic and reduced more than 65,000 data points to a set of 140 strains that display significantly increased sensitivities to at least one of the antibiotics, determining the MIC in each case. These data provide targets for the design of "codrugs" that can potentiate existing antibiotics. We have made a number of double mutants with greatly increased sensitivity to ciprofloxacin, and these overcome the resistance generated by certain gyrA mutations. Many of the gene knockouts in E. coli are hypersensitive to more than one antibiotic. Together, all of these data allow us to outline the cell's "intrinsic resistome," which provides innate resistance to antibiotics. PMID:18621901

  19. 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

  20. CRISPR/Cas9-mediated gene knockout in the mouse brain using in utero electroporation

    PubMed Central

    Shinmyo, Yohei; Tanaka, Satoshi; Tsunoda, Shinichi; Hosomichi, Kazuyoshi; Tajima, Atsushi; Kawasaki, Hiroshi

    2016-01-01

    The CRISPR/Cas9 system has recently been adapted for generating knockout mice to investigate physiological functions and pathological mechanisms. Here, we report a highly efficient procedure for brain-specific disruption of genes of interest in vivo. We constructed pX330 plasmids expressing humanized Cas9 and single-guide RNAs (sgRNAs) against the Satb2 gene, which encodes an AT-rich DNA-binding transcription factor and is responsible for callosal axon projections in the developing mouse brain. We first confirmed that these constructs efficiently induced double-strand breaks (DSBs) in target sites of exogenous plasmids both in vitro and in vivo. We then found that the introduction of pX330-Satb2 into the developing mouse brain using in utero electroporation led to a dramatic reduction of Satb2 expression in the transfected cerebral cortex, suggesting DSBs had occurred in the Satb2 gene with high efficiency. Furthermore, we found that Cas9-mediated targeting of the Satb2 gene induced abnormalities in axonal projection patterns, which is consistent with the phenotypes previously observed in Satb2 mutant mice. Introduction of pX330-NeuN using our procedure also resulted in the efficient disruption of the NeuN gene. Thus, our procedure combining the CRISPR/Cas9 system and in utero electroporation is an effective and rapid approach to achieve brain-specific gene knockout in vivo. PMID:26857612

  1. Up-regulation of the interferon-related genes in BRCA2 knockout epithelial cells

    PubMed Central

    Xu, Hong; Xian, Jian; Vire, Emmanuelle; McKinney, Steven; Wong, Jason; Wei, Vivien; Tong, Rebecca; Kouzarides, Tony; Caldas, Carlos; Aparicio, Samuel

    2016-01-01

    BRCA2 mutations are significantly associated with early onset breast cancer, and the tumour suppressing function of BRCA2 has been attributed to its involvement in homologous recombination [1]-mediated DNA repair. In order to identify additional functions of BRCA2, we generated BRCA2-knockout HCT116 human colorectal carcinoma cells. Using genome-wide microarray analyses, we have discovered a link between the loss of BRCA2 and the up-regulation of a subset of interferon (IFN)-related genes, including APOBEC3F and APOBEC3G. The over-expression of IFN-related genes was confirmed in different human BRCA2−/− and mouse Brca2−/− tumour cell lines, and was independent of either senescence or apoptosis. In isogenic wild type BRCA2 cells, we observed over-expression of IFN-related genes after treatment with DNA-damaging agents, and following ionizing radiation. Cells with endogenous DNA damage because of defective BRCA1 or RAD51 also exhibited over-expression of IFN-related genes. Transcriptional activity of the IFN-stimulated response element (ISRE) was increased in BRCA2 knockout cells, and the expression of BRCA2 greatly decreased IFN-α stimulated ISRE reporter activity, suggesting that BRCA2 directly represses the expression of IFN-related genes through the ISRE. Finally, the colony forming capacity of BRCA2 knockout cells was significantly reduced in the presence of either IFN-β or IFN-γ, suggesting that IFNs may have potential as therapeutic agents in cancer cells with BRCA2 mutations. PMID:25043256

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

    PubMed

    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

  3. Genes critical to vasculogenesis as defined by systematic analysis of vascular defects in knockout mice.

    PubMed

    Argraves, W Scott; Drake, Christopher J

    2005-10-01

    To identify genes important to the process of vasculogenesis, we evaluated embryonic vascular anomalies from 100 mouse knockout studies using a novel meta-analysis approach. By applying this method, termed approach for ranking of embryonic vascular anomalies (AREVA), rank scores were calculated for each knockout based on the occurrence of vascular defects during periods of vasculogenesis in specific embryonic regions. As a result, 12 genes (fibronectin, VEGFR-1/Flt-1, VEGFR-2/Flk-1, alpha 5 integrin, Tek/Tie2, VE-cadherin, VEGFA, connexin 45, ShcA, cytochrome P450 reductase, CD148/DEP-1, and EphrinB2) were determined to play critical roles in vasculogenesis. Functional categorization of these genes revealed the fundamental importance of VEGF signaling since 10 of the 12 genes (fibronectin, VEGFR-1/Flt-1, VEGFR-2/Flk-1, alpha 5 integrin, VE-cadherin, VEGFA, ShcA, cytochrome P450 reductase, CD148/DEP-1, and EphrinB2) relate to this pathway. Furthermore, the findings highlight a potential network for regulating VEGF signaling involving integration of fibronectin, EphrinB2, Tie2, and connexin 45 signaling pathways via the ShcA/Ras/Raf/Mek/Erk cascade. In addition to retrospective application of AREVA as done herein, AREVA can be used prospectively to determine the relevancy to vasculogenesis of newly inactivated genes. PMID:16114069

  4. 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

  5. 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

  6. 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. PMID:27458822

  7. 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

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

    SciTech Connect

    Watanabe, Masahito; Umeyama, Kazuhiro; Matsunari, Hitomi; Takayanagi, Shuko; Haruyama, Erika; Nakano, Kazuaki; Fujiwara, Tsukasa; Ikezawa, Yuka; Nakauchi, Hiromitsu; and others

    2010-11-05

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

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

    PubMed

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

    2016-08-01

    Conditional gene knockout in postmitotic cells is a valuable technique which allows the study of gene function with spatiotemporal control. Surprisingly, in contrast to its long-term and extensive use in mouse studies, this technology is lacking in Drosophila. Here, we use a novel method for generating complete loss of eyes absent (eya) or sine oculis (so) function in postmitotic cells posterior to the morphogenetic furrow (MF). Specifically, genomic rescue constructs with flippase recognition target (FRT) sequences flanking essential exons are used to generate conditional null alleles. By removing gene function in differentiating cells, we show that eya and so are dispensable for larval photoreceptor differentiation, but are required for differentiation during pupal development. Both eya and so are necessary for photoreceptor survival and the apoptosis caused by loss of eya or so function is likely a secondary consequence of inappropriate differentiation. We also confirm their requirement for cone cell development and reveal a novel role in interommatidial bristle (IOB) formation. In addition, so is required for normal eye disc morphology. This is the first report of a knockout method to study eya and so function in postmitotic cells. This technology will open the door to a large array of new functional studies in virtually any tissue and at any stage of development or in adults. PMID:27257739

  10. 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

  11. 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. PMID:25846201

  12. Foxn1 gene knockout suppresses sexual attractiveness and pheromonal components of male urine in inbred mice.

    PubMed

    Zhang, Jian-Xu; Sun, Lixing; Zhang, Yao-Hua

    2010-01-01

    The immunocompetence handicap hypothesis (ICHH) posits that females prefer signals emitted by immunocompetent males over immunocompromised males and that these signals are honest. However, mechanisms of mate choice under an ICHH model may be impacted by levels of genetic variation (inbred animals vs. outbred animals). Here, we conducted 2-choice female preference experiments and chemical analyses of male urine in inbred BALB/c and outbred CD-1 mice, both of which have immunocompromised nude (nu) strains resulting from a Foxn1 gene knockout. We found that inbred BALB/c females but not outbred CD-1 females preferred the urine of healthy males over that of immunocompromised males despite measured differences in the qualities of their urine. There was a clear increase in female-attracting pheromones (such as farnesenes) in the preputial glands and urine metabolites in healthy BALB/c males but no such difference between CD-1 and CD-1 nu males. Therefore, CD-1 male urine failed to provide an honest mate-choice cue for females. Our results suggest that deleterious traits associated with male odor in mice might be jointly affected by the level of inbreeding and immunodeficiency caused by a single-gene knockout. PMID:20019156

  13. 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.

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

    PubMed

    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

  15. 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

  16. A conditional knockout resource for the genome–wide study of mouse gene function

    PubMed Central

    Skarnes, William C.; Rosen, Barry; West, Anthony P.; Koutsourakis, Manousos; Bushell, Wendy; Iyer, Vivek; Mujica, Alejandro O.; Thomas, Mark; Harrow, Jennifer; Cox, Tony; Jackson, David; Severin, Jessica; Biggs, Patrick; Fu, Jun; Nefedov, Michael; de Jong, Pieter J.; Stewart, A. Francis; Bradley, Allan

    2013-01-01

    Gene targeting in embryonic stem cells has become the principal technology for manipulation of the mouse genome, offering unrivalled accuracy in allele design and access to conditional mutagenesis. To bring these advantages to the wider research community, large-scale mouse knockout programmes are producing a permanent resource of targeted mutations in all protein-coding genes. Here we report the establishment of a high-throughput gene-targeting pipeline for the generation of reporter-tagged, conditional alleles. Computational allele design, 96-well modular vector construction and high-efficiency gene-targeting strategies have been combined to mutate genes on an unprecedented scale. So far, more than 12,000 vectors and 9,000 conditional targeted alleles have been produced in highly germline-competent C57BL/6N embryonic stem cells. High-throughput genome engineering highlighted by this study is broadly applicable to rat and human stem cells and provides a foundation for future genome-wide efforts aimed at deciphering the function of all genes encoded by the mammalian genome. PMID:21677750

  17. 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 ...

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

    PubMed

    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

  19. 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

  20. Generation of a conditional knockout allele for the NFAT5 gene in mice.

    PubMed

    Küper, Christoph; Beck, Franz-Xaver; Neuhofer, Wolfgang

    2014-01-01

    The osmosensitive transcription factor nuclear factor of activated T-cells 5 (NFAT5), also known as tonicity enhancer element binding protein (TonEBP) plays a crucial role in protection of renal medullary cells against hyperosmotic stress, urinary concentration, the adaptive immune response, and other physiological systems. Since it is also important for development, conventional homozygous-null mutations result in perinatal death, which hinders the analysis of NFAT5 function in specific tissues in vivo. Here we describe the generation of mice with a conditional-null allele, in which loxP sites are inserted around exon 4. Mice harboring the floxed allele (NFAT5(flx) ) were mated to a strain expressing a tamoxifen-inducible derivative of the Cre-recombinase (Cre (+)) under the control of the ubiqitinC promoter. The resultant homozygous conditional knockout mice (Cre (+) NFAT5 (flx/flx) ) are viable, fertile, and show normal expression of NFAT5 and NFAT5 target genes, indicating that the conditional alleles retain their wild-type function. Induction of Cre-mediated recombination by administration of tamoxifen in 8-week-old mice resulted in a decrease in NFAT5 expression of about 70-90% in all tested tissues (renal cortex, renal outer medulla, renal inner medulla, heart, lung, spleen, skeletal muscle). Accordingly, the expression of the NFAT5 target genes aldose reductase and heat shock protein 70 in the renal medulla was also significantly decreased. Mice harboring this conditional knockout allele should be useful in future studies for gaining a better understanding of tissue and cell-type specific functions of NFAT5 in adult animals under physiological and pathophysiological conditions. PMID:25601839

  1. Effect of arylamine acetyltransferase Nat3 gene knockout on N-acetylation in the mouse.

    PubMed

    Sugamori, K S; Brenneman, D; Wong, S; Gaedigk, A; Yu, V; Abramovici, H; Rozmahel, R; Grant, D M

    2007-07-01

    Arylamine N-acetyltransferases (NAT) catalyze the biotransformation of many important arylamine drugs and procarcinogens. NAT can either detoxify or activate procarcinogens, complicating the manner in which these enzymes may participate in enhancing or preventing toxic responses to particular agents. Mice possess three NAT isoenzymes: Nat1, Nat2, and Nat3. Whereas Nat1 and Nat2 can efficiently acetylate many arylamines, few substrates appear to be appreciably metabolized by Nat3. We generated a Nat3 knockout mouse strain and used it along with our double Nat1/2(-/-) knockout strain to further investigate the functional role of Nat3. Nat3(-/-) mice showed normal viability and reproductive capacity. Nat3 expression was very low in wild-type animals and completely undetectable in Nat3(-/-) mice. In contrast, greatly elevated expression of Nat3 transcript was observed in Nat1/2(-/-) mice. We used a transcribed marker polymorphism approach to establish that the increased expression of Nat3 in Nat1/2(-/-) mice is a positional artifact of insertion of the phosphoglycerate kinase-neomycin resistance cassette in place of the Nat1/Nat2 gene region and upstream of the intact Nat3 gene, rather than a biological compensatory mechanism. Despite the increase in Nat3 transcript, the N-acetylation of p-aminosalicylate, sulfamethazine, 2-aminofluorene, and 4-aminobiphenyl was undetectable either in vivo or in vitro in Nat1/2(-/-) animals. In parallel, no difference was observed in the in vivo clearance or in vitro metabolism of any of these substrates between wild-type and Nat3(-/-) mice. Thus, Nat3 is unlikely to play a significant role in the N-acetylation of arylamines either in wild-type mice or in mice lacking Nat1 and Nat2 activities. PMID:17403913

  2. 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...

  3. CRISPR-Cas9-mediated gene knockout in primary human airway epithelial cells reveals a proinflammatory role for MUC18.

    PubMed

    Chu, H W; Rios, C; Huang, C; Wesolowska-Andersen, A; Burchard, E G; O'Connor, B P; Fingerlin, T E; Nichols, D; Reynolds, S D; Seibold, M A

    2015-10-01

    Targeted knockout of genes in primary human cells using CRISPR-Cas9-mediated genome-editing represents a powerful approach to study gene function and to discern molecular mechanisms underlying complex human diseases. We used lentiviral delivery of CRISPR-Cas9 machinery and conditional reprogramming culture methods to knockout the MUC18 gene in human primary nasal airway epithelial cells (AECs). Massively parallel sequencing technology was used to confirm that the genome of essentially all cells in the edited AEC populations contained coding region insertions and deletions (indels). Correspondingly, we found mRNA expression of MUC18 was greatly reduced and protein expression was absent. Characterization of MUC18 knockout cell populations stimulated with TLR2, 3 and 4 agonists revealed that IL-8 (a proinflammatory chemokine) responses of AECs were greatly reduced in the absence of functional MUC18 protein. Our results show the feasibility of CRISPR-Cas9-mediated gene knockouts in AEC culture (both submerged and polarized), and suggest a proinflammatory role for MUC18 in airway epithelial response to bacterial and viral stimuli. PMID:26043872

  4. 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. PMID:27565865

  5. Determination of Hypersensitivity to Genotoxic Agents Among Escherichia coli Single Gene Knockout Mutants

    PubMed Central

    Becket, Elinne; Chen, Frank; Tamae, Cindy; Miller, Jeffrey H.

    2010-01-01

    Summary We have tested the KEIO collection of 3985 different viable single gene knockouts in Escherichia coli to identify genes whose loss increases sensitivity to one or more of six different chemotherapeutic agents and mutagens: Bleomycin (BLM), Cisplatin (CPT), ICR-191 (ICR), 5-azacytidine (5AZ), Zebularine (ZEB), and 5-bromo-2’-deoxyuridine (5BdU). We discovered a set of 156 strains that display a significant increase in sensitivity to at least one of the agents tested. Each genotoxic agent generates a distinct “sensitivity profile” that is characteristic of the agent. Comparison with an independent study of sensitivity profiles for an extensive set of antibiotics pinpoints those effects that are relatively specific for each agent. In some cases engineered double mutants have greatly increased effects. These results provide insight into the mechanism of action of each agent, and define targets for the design of co-drugs that can potentiate these agents. An example is the finding that mutants lacking one of several genes in the folate biosynthetic pathway are hypersensitive to ZEB, leading to a demonstration of synergy between trimethoprim and ZEB. PMID:20674514

  6. Determination of hypersensitivity to genotoxic agents among Escherichia coli single gene knockout mutants.

    PubMed

    Becket, Elinne; Chen, Frank; Tamae, Cindy; Miller, Jeffrey H

    2010-09-01

    We have tested the KEIO collection of 3985 different viable single gene knockouts in Escherichia coli to identify genes whose loss increases sensitivity to one or more of six different chemotherapeutic agents and mutagens: Bleomycin (BLM), Cisplatin (CPT), ICR-191 (ICR), 5-azacytidine (5AZ), Zebularine (ZEB), and 5-bromo-2'-deoxyuridine (5BdU). We discovered a set of 156 strains that display a significant increase in sensitivity to at least one of the agents tested. Each genotoxic agent generates a distinct "sensitivity profile" that is characteristic of the agent. Comparison with an independent study of sensitivity profiles for an extensive set of antibiotics pinpoints those effects that are relatively specific for each agent. In some cases engineered double mutants have greatly increased effects. These results provide insight into the mechanism of action of each agent, and define targets for the design of co-drugs that can potentiate these agents. An example is the finding that mutants lacking one of several genes in the folate biosynthetic pathway are hypersensitive to ZEB, leading to a demonstration of synergy between trimethoprim and ZEB. PMID:20674514

  7. Benefits of a "vulnerability gene"? A study in serotonin transporter knockout mice.

    PubMed

    Kästner, Niklas; Richter, S Helene; Lesch, Klaus-Peter; Schreiber, Rebecca S; Kaiser, Sylvia; Sachser, Norbert

    2015-04-15

    Over the past years, certain "vulnerability genes" have been identified that play a key role in the development of mood and anxiety disorders. In particular, a low-expressing variant of the human serotonin transporter (5-HTT) gene has been described that renders individuals more susceptible to adverse experience and hence to the development of psychiatric diseases. However, some authors have recently argued that lower 5-HTT expression not only increases vulnerability to adverse experiences, but also enhances susceptibility to beneficial experiences, thus promoting phenotypic plasticity. The aim of the present study was to assess the effects of 5-HTT expression on susceptibility to beneficial experience in a hypothesis-driven experimental approach. Using a well-established rodent model for the human polymorphism, male heterozygous 5-HTT knockout (HET) and 5-HTT wildtype (WT) mice were either provided with the beneficial experience of cohabitation with a female (mating experience) or kept as naïve controls in single-housing conditions. Following the experimental treatment, they were tested for their anxiety-like behaviour and exploratory locomotion in three widely used behavioural tests. Interestingly, while cohabitation reduced anxiety-like behaviour and increased exploratory locomotion in the open field test in HET mice, it did not affect WT mice, pointing to a genotype-dependent susceptibility to the beneficial experience. Thus, our results might support the view of the low expressing version of the 5-HTT gene as a "plasticity" rather than a "vulnerability" variant. PMID:25629942

  8. Melanopsin phototransduction: slowly emerging from the dark.

    PubMed

    Hughes, Steven; Hankins, Mark W; Foster, Russell G; Peirson, Stuart N

    2012-01-01

    Melanopsin expressing retinal ganglion cells represent a third class of ocular photoreceptors and are involved in irradiance detection and non-image-forming responses to light including pupil constriction, circadian entrainment, and regulation of sleep. Over recent years, there has been a rapid increase in our understanding of the anatomical variety of pRGC subtypes, the regions of the brain which they innervate, and the behavioral responses of melanopsin-based light detection. However, by contrast, our understanding of the intracellular signaling cascade initiated following activation of melanopsin has, until recently, remained poorly characterized. This chapter focus on the melanopsin signaling pathway, detailing the cellular mechanisms of phototransduction that occur within pRGCs, highlighting recent advances, but also the gaps in our understanding of this important light detecting system. PMID:22877657

  9. Caveolin-1 gene knockout impairs nitrergic function in mouse small intestine

    PubMed Central

    El-Yazbi, Ahmed F; Cho, Woo-Jung; Boddy, Geoffrey; Daniel, Edwin E

    2005-01-01

    Caveolin-1 is a plasma membrane-associated protein that is responsible for caveolae formation. It plays an important role in the regulation of the function of different signaling molecules, among which are the different isoforms of nitric oxide synthase (NOS). Nitric oxide (NO) is known to be an important inhibitory mediator in the mouse gut. Caveolin-1 knockout mice (Cav1−/−) were used to examine the effect of caveolin-1 absence on the NO function in the mouse small intestine (ileum and jejunum) compared to their genetic controls and BALB/c controls. Immunohistochemical staining showed loss of caveolin-1 and NOS in the jejunal smooth muscles and myenteric plexus interstitial cells of Cajal (ICC) of Cav1−/− mice; however, nNOS immunoreactive nerves were still present in myenteric ganglia. Under nonadrenergic noncholinergic (NANC) conditions, small intestinal tissues from Cav1−/− mice relaxed to electrical field stimulation (EFS), as did tissues from control mice. Relaxation of tissues from control mice was markedly reduced by N-omega-nitro-L-arginine (10−4 M), but relaxation of Cav1−/− animals was affected much less. Also, Cav1−/− mice tissues showed reduced relaxation responses to sodium nitroprusside (100 μM) compared to controls; yet there were no significant differences in the relaxation responses to 8-bromoguanosine-3′ : 5′-cyclic monophosphate (100 μM). Apamin (10−6 M) significantly reduced relaxations to EFS in NANC conditions in Cav1−/− mice, but not in controls. The data from this study suggest that caveolin-1 gene knockout causes alterations in the smooth muscles and the ICC, leading to an impaired NO function in the mouse small intestine that could possibly be compensated by apamin-sensitive inhibitory mediators. PMID:15937515

  10. 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

  11. 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. PMID:26977770

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

    PubMed

    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

    2016-02-26

    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. 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

  14. Site-Directed Genome Knockout in Chicken Cell Line and Embryos Can Use CRISPR/Cas Gene Editing Technology

    PubMed Central

    Zuo, Qisheng; Wang, Yinjie; Cheng, Shaoze; Lian, Chao; Tang, Beibei; Wang, Fei; Lu, Zhenyu; Ji, Yanqing; Zhao, Ruifeng; Zhang, Wenhui; Jin, Kai; Song, Jiuzhou; Zhang, Yani; Li, Bichun

    2016-01-01

    The present study established an efficient genome editing approach for the construction of stable transgenic cell lines of the domestic chicken (Gallus gallus domesticus). Our objectives were to facilitate the breeding of high-yield, high-quality chicken strains, and to investigate gene function in chicken stem cells. Three guide RNA (gRNAs) were designed to knockout the C2EIP gene, and knockout efficiency was evaluated in DF-1 chicken fibroblasts and chicken ESCs using the luciferase single-strand annealing (SSA) recombination assay, T7 endonuclease I (T7EI) assay, and TA clone sequencing. In addition, the polyethylenimine-encapsulated Cas9/gRNA plasmid was injected into fresh fertilized eggs. At 4.5 d later, frozen sections of the embryos were prepared, and knockout efficiency was evaluated by the T7EI assay. SSA assay results showed that luciferase activity of the vector expressing gRNA-3 was double that of the control. Results of the T7EI assay and TA clone sequencing indicated that Cas9/gRNA vector-mediated gene knockdown efficiency was approximately 27% in both DF-1 cells and ESCs. The CRISPR/Cas9 vector was also expressed in chicken embryos, resulting in gene knockdown in three of the 20 embryos (gene knockdown efficiency 15%). Taken together, our results indicate that the CRISPR/Cas9 system can mediate stable gene knockdown at the cell and embryo levels in domestic chickens. PMID:27172204

  15. Site-Directed Genome Knockout in Chicken Cell Line and Embryos Can Use CRISPR/Cas Gene Editing Technology.

    PubMed

    Zuo, Qisheng; Wang, Yinjie; Cheng, Shaoze; Lian, Chao; Tang, Beibei; Wang, Fei; Lu, Zhenyu; Ji, Yanqing; Zhao, Ruifeng; Zhang, Wenhui; Jin, Kai; Song, Jiuzhou; Zhang, Yani; Li, Bichun

    2016-01-01

    The present study established an efficient genome editing approach for the construction of stable transgenic cell lines of the domestic chicken (Gallus gallus domesticus). Our objectives were to facilitate the breeding of high-yield, high-quality chicken strains, and to investigate gene function in chicken stem cells. Three guide RNA (gRNAs) were designed to knockout the C2EIP gene, and knockout efficiency was evaluated in DF-1 chicken fibroblasts and chicken ESCs using the luciferase single-strand annealing (SSA) recombination assay, T7 endonuclease I (T7EI) assay, and TA clone sequencing. In addition, the polyethylenimine-encapsulated Cas9/gRNA plasmid was injected into fresh fertilized eggs. At 4.5 d later, frozen sections of the embryos were prepared, and knockout efficiency was evaluated by the T7EI assay. SSA assay results showed that luciferase activity of the vector expressing gRNA-3 was double that of the control. Results of the T7EI assay and TA clone sequencing indicated that Cas9/gRNA vector-mediated gene knockdown efficiency was approximately 27% in both DF-1 cells and ESCs. The CRISPR/Cas9 vector was also expressed in chicken embryos, resulting in gene knockdown in three of the 20 embryos (gene knockdown efficiency 15%). Taken together, our results indicate that the CRISPR/Cas9 system can mediate stable gene knockdown at the cell and embryo levels in domestic chickens. PMID:27172204

  16. 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). PMID:27289014

  17. MHC II gene knockout in tissue engineering may prevent immune rejection of transplants.

    PubMed

    Yang, Miaomiao; Liu, Lei

    2008-01-01

    The repair and reconstruction of tissue defects and organ loss are severe problems, and many patients are eager to find avenues to these matters. Up until now, the number of methods used to repair tissue defects has increased, but all of these have their own advantages and inconveniences, and do not seem to have been optimized. The development of tissue engineering offers new hopes to patients with tissue defects. To regenerate tissues and organs, we first need a source of seed cells. However, the sources of autologous cells are restricted, cell number is small, and xenogenic cells result in immunological rejections. Major histocompatibility complex (MHC) polymorphism is a key factor in tissue grafts. MHC II, in particular, is associated with allogeneic transplantation. We hypothesize that if we knock-out the MHC II gene of mesenchymal stem cells (MSCs) in vitro, these cells would not express MHC II molecules, and rejection problems will be solved. Accordingly, the industrialization of tissue engineering will be feasible, and products of tissue engineering will be utilized widely for any clinical treatments. PMID:17904760

  18. Age-Dependent Defects of Regulatory B Cells in Wiskott-Aldrich Syndrome Gene Knockout Mice

    PubMed Central

    Yokoyama, Tadafumi; Yoshizaki, Ayumi; Simon, Karen L.; Kirby, Martha R.; Anderson, Stacie M.; Candotti, Fabio

    2015-01-01

    The Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency characterized by recurrent infections, thrombocytopenia, eczema, and high incidence of malignancy and autoimmunity. The cellular mechanisms underlying autoimmune complications in WAS have been extensively studied; however, they remain incompletely defined. We investigated the characteristics of IL-10-producing CD19+CD1dhighCD5+ B cells (CD1dhighCD5+ Breg) obtained from Was gene knockout (WKO) mice and found that their numbers were significantly lower in these mice compared to wild type (WT) controls. Moreover, we found a significant age-dependent reduction of the percentage of IL-10-expressing cells in WKO CD1dhighCD5+ Breg cells as compared to age-matched WT control mice. CD1dhighCD5+ Breg cells from older WKO mice did not suppress the in vitro production of inflammatory cytokines from activated CD4+ T cells. Interestingly, CD1dhighCD5+ Breg cells from older WKO mice displayed a basal activated phenotype which may prevent normal cellular responses, among which is the expression of IL-10. These defects may contribute to the susceptibility to autoimmunity with age in patients with WAS. PMID:26448644

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

    PubMed Central

    Ghosal, Abhisek; Lambrecht, Nils; Subramanya, Sandeep B.; Kapadia, Rubina

    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. PMID:23104561

  20. Antibiotic sensitivity profiles determined with an Escherichia coli gene knockout collection: generating an antibiotic bar code.

    PubMed

    Liu, Anne; Tran, Lillian; Becket, Elinne; Lee, Kim; Chinn, Laney; Park, Eunice; Tran, Katherine; Miller, Jeffrey H

    2010-04-01

    We have defined a sensitivity profile for 22 antibiotics by extending previous work testing the entire KEIO collection of close to 4,000 single-gene knockouts in Escherichia coli for increased susceptibility to 1 of 14 different antibiotics (ciprofloxacin, rifampin [rifampicin], vancomycin, ampicillin, sulfamethoxazole, gentamicin, metronidazole, streptomycin, fusidic acid, tetracycline, chloramphenicol, nitrofurantoin, erythromycin, and triclosan). We screened one or more subinhibitory concentrations of each antibiotic, generating more than 80,000 data points and allowing a reduction of the entire collection to a set of 283 strains that display significantly increased sensitivity to at least one of the antibiotics. We used this reduced set of strains to determine a profile for eight additional antibiotics (spectinomycin, cephradine, aztreonem, colistin, neomycin, enoxacin, tobramycin, and cefoxitin). The profiles for the 22 antibiotics represent a growing catalog of sensitivity fingerprints that can be separated into two components, multidrug-resistant mutants and those mutants that confer relatively specific sensitivity to the antibiotic or type of antibiotic tested. The latter group can be represented by a set of 20 to 60 strains that can be used for the rapid typing of antibiotics by generating a virtual bar code readout of the specific sensitivities. Taken together, these data reveal the complexity of intrinsic resistance and provide additional targets for the design of codrugs (or combinations of drugs) that potentiate existing antibiotics. PMID:20065048

  1. 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

  2. Toward a unified model of vertebrate rod phototransduction

    PubMed Central

    HAMER, R.D.; NICHOLAS, S.C.; TRANCHINA, D.; LAMB, T.D.; JARVINEN, J.L.P.

    2006-01-01

    LA circulating current (as in Koutalos et al., 1995) and LA flash sensitivity measured in rods from four species; (4) step responses from newt rods (Forti et al., 1989) over a large dynamic range; (5) dynamic LA responses, such as the step-flash paradigm of Fain et al. (1989), and the two-flash paradigm of Murnick and Lamb (1996); and (6) the salient response features from four knockout rod preparations. The model was able to meet this stringent test, accounting for almost all the salient qualitative, and many quantitative features, of the responses across this broad array of stimulus conditions, including SPR reproducibility. The model promises to be useful in testing hypotheses regarding both normal and abnormal photoreceptor function, and is a good starting point for development of a full-range model of cone phototransduction. Informative limitations of the model are also discussed. PMID:16212700

  3. Acid Sphingomyelinase Gene Knockout Ameliorates Hyperhomocysteinemic Glomerular Injury in Mice Lacking Cystathionine-β-Synthase

    PubMed Central

    Boini, Krishna M.; Xia, Min; Abais, Justine M.; Xu, Ming; Li, Cai-xia; Li, Pin-Lan

    2012-01-01

    Acid sphingomyelinase (ASM) has been implicated in the development of hyperhomocysteinemia (hHcys)-induced glomerular oxidative stress and injury. However, it remains unknown whether genetically engineering of ASM gene produces beneficial or detrimental action on hHcys-induced glomerular injury. The present study generated and characterized the mice lacking cystathionine β-synthase (Cbs) and Asm mouse gene by cross breeding Cbs+/− and Asm+/− mice. Given that the homozygotes of Cbs−/−/Asm−/− mice could not survive for 3 weeks. Cbs+/−/Asm+/+, Cbs+/−/Asm+/− and Cbs+/−/Asm−/− as well as their Cbs wild type littermates were used to study the role of Asm−/− under a background of Cbs+/− with hHcys. HPLC analysis revealed that plasma Hcys level was significantly elevated in Cbs heterozygous (Cbs+/−) mice with different copies of Asm gene compared to Cbs+/+ mice with different Asm gene copies. Cbs+/−/Asm+/+ mice had significantly increased renal Asm activity, ceramide production and O2.− level compared to Cbs+/+/Asm+/+, while Cbs+/−/Asm−/− mice showed significantly reduced renal Asm activity, ceramide production and O2.− level due to increased plasma Hcys levels. Confocal microscopy demonstrated that colocalization of podocin with ceramide was much lower in Cbs+/−/Asm−/− mice compared to Cbs+/−/Asm+/+ mice, which was accompanied by a reduced glomerular damage index, albuminuria and proteinuria in Cbs+/−/Asm−/− mice. Immunofluorescent analyses of the podocin, nephrin and desmin expression also illustrated less podocyte damages in the glomeruli from Cbs+/−/Asm−/− mice compared to Cbs+/−/Asm+/+ mice. In in vitro studies of podocytes, hHcys-enhanced O2.− production, desmin expression, and ceramide production as well as decreases in VEGF level and podocin expression in podocytes were substantially attenuated by prior treatment with amitriptyline, an Asm inhibitor. In conclusion, Asm gene knockout or

  4. 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.

  5. Prediction of tissue-specific effects of gene knockout on apoptosis in different anatomical structures of human brain

    PubMed Central

    2015-01-01

    Background An important issue in the target identification for the drug design is the tissue-specific effect of inhibition of target genes. The task of assessing the tissue-specific effect in suppressing gene activity is especially relevant in the studies of the brain, because a significant variability in gene expression levels among different areas of the brain was well documented. Results A method is proposed for constructing statistical models to predict the potential effect of the knockout of target genes on the expression of genes involved in the regulation of apoptosis in various brain regions. The model connects the expression of the objective group of genes with expression of the target gene by means of machine learning models trained on available expression data. Information about the interactions between target and objective genes is determined by reconstruction of target-centric gene network. STRING and ANDSystem databases are used for the reconstruction of gene networks. The developed models have been used to analyse gene knockout effects of more than 7,500 target genes on the expression of 1,900 objective genes associated with the Gene Ontology category "apoptotic process". The tissue-specific effect was calculated for 12 main anatomical structures of the human brain. Initial values of gene expression in these anatomical structures were taken from the Allen Brain Atlas database. The results of the predictions of the effect of suppressing the activity of target genes on apoptosis, calculated on average for all brain structures, were in good agreement with experimental data on siRNA-inhibition. Conclusions This theoretical paper presents an approach that can be used to assess tissue-specific gene knockout effect on gene expression of the studied biological process in various structures of the brain. Genes that, according to the predictions of the model, have the highest values of tissue-specific effects on the apoptosis network can be considered as

  6. Interleukin-6 gene knockout antagonizes high-fat-induced trabecular bone loss.

    PubMed

    Wang, Chunyu; Tian, Li; Zhang, Kun; Chen, Yaxi; Chen, Xiang; Xie, Ying; Zhao, Qian; Yu, Xijie

    2016-10-01

    The purpose of the study was to determine the roles of interleukin-6 (IL6) in fat and bone communication. Male wild-type (WT) mice and IL6 knockout (IL6(-/-)) mice were fed with either regular diet (RD) or high-fat diet (HFD) for 12 weeks. Bone mass and bone microstructure were evaluated by micro-computed tomography. Gene expression related to lipid and bone metabolisms was assayed with real-time quantitative polymerase chain reaction. Bone marrow cells from both genotypes were induced to differentiate into osteoblasts or osteoclasts, and treated with palmitic acid (PA). HFD increased the body weight and fat pad weight, and impaired lipid metabolism in both WT and IL6(-/-) mice. The dysregulation of lipid metabolism was more serious in IL6(-/-) mice. Trabecular bone volume fraction, trabecular bone number and trabecular bone thickness were significantly downregulated in WT mice after HFD than those in the RD (P < 0.05). However, these bone microstructural parameters were increased by 53%, 34% and 40%, respectively, in IL6(-/-) mice than those in WT mice on the HFD (P < 0.05). IL6(-/-) osteoblasts displayed higher alkaline phosphatase (ALP) activity and higher mRNA levels of Runx2 and Colla1 than those in WT osteoblasts both in the control and PA treatment group (P < 0.05). IL6(-/-) mice showed significantly lower mRNA levels of PPARγ and leptin and higher mRNA levels of adiponectin in comparison with WT mice on HFD. In conclusion, these findings suggested that IL6 gene deficiency antagonized HFD-induced bone loss. IL6 might bridge lipid and bone metabolisms and could be a new potential therapeutic target for lipid metabolism disturbance-related bone loss. PMID:27493246

  7. 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. PMID:26443220

  8. Melanopsin Tristability for Sustained and Broadband Phototransduction

    PubMed Central

    Emanuel, Alan Joseph; Do, Michael Tri Hoang

    2015-01-01

    SUMMARY Mammals rely upon three ocular photoreceptors to sense light: rods, cones, and intrinsically photosensitive retinal ganglion cells (ipRGCs). Rods and cones resolve details in the visual scene. Conversely, ipRGCs integrate over time and space, primarily to support “non-image” vision. The integrative mechanisms of ipRGCs are enigmatic, particularly since these cells use a phototransduction motif that allows invertebrates like Drosophila to parse light with exceptional temporal resolution. Here, we provide evidence for a single mechanism that allows ipRGCs to integrate over both time and wavelength. Light distributes the visual pigment, melanopsin, across three states, two silent and one signaling. Photoequilibration among states maintains pigment availability for sustained signaling, stability of the signaling state permits minutes-long temporal summation, and modest spectral separation of the silent states promotes uniform activation across wavelengths. By broadening the tuning of ipRGCs in both temporal and chromatic domains, melanopsin tristability produces signal integration for physiology and behavior. PMID:25741728

  9. Gene knockout of the intracellular amylase gene by homologous recombination in Streptococcus bovis.

    PubMed

    Brooker, J D; McCarthy, J M

    1997-09-01

    Streptococcus bovis expresses two different amylases, one intracellular and the other secreted. A suicide vector containing part of the intracellular alpha-amylase gene from Streptococcus bovis WI-1 was recombined into the S. bovis WI-1 chromosome to disrupt the endogenous gene. Recombination was demonstrated by Southern blot, and zymogram analysis confirmed the loss of the intracellular amylase. Amylase activity in cell-free extracts of the recombinant grown in the presence of 1% starch was only 7% of wild type. The rate of logarithmic growth of the recombinant was 15-20% of the wild type in medium containing either 1% glucose, starch, or cellobiose. Revertants and non-amylase control recombinants had logarithmic growth rates that were the same as wild type. Plasmid transformants containing multiple copies of the cloned gene expressed up to threefold higher levels of intracellular amylase activity than wild type but did not demonstrate elevated growth rates. These results suggest that a critical level of expression of the intracellular amylase gene may be important for rapid growth of the bacterium. PMID:9236293

  10. Gene knockout of the KCNJ8-encoded Kir6.1 K(ATP) channel imparts fatal susceptibility to endotoxemia.

    PubMed

    Kane, Garvan C; Lam, Chen-Fuh; O'Cochlain, Fearghas; Hodgson, Denice M; Reyes, Santiago; Liu, Xiao-Ke; Miki, Takashi; Seino, Susumu; Katusic, Zvonimir S; Terzic, Andre

    2006-11-01

    Sepsis, the systemic inflammatory response to infection, imposes a high demand for bodily adaptation, with the cardiovascular response a key determinant of outcome. The homeostatic elements that secure cardiac tolerance in the setting of the sepsis syndrome are poorly understood. Here, in a model of acute septic shock induced by endotoxin challenge with Escherichia coli lipopolysaccharide (LPS), knockout of the KCNJ8 gene encoding the vascular Kir6.1 K(ATP) channel pore predisposed to an early and profound survival disadvantage. The exaggerated susceptibility provoked by disruption of this stress-responsive sensor of cellular metabolism was linked to progressive deterioration in cardiac activity, ischemic myocardial damage, and contractile dysfunction. Deletion of KCNJ8 blunted the responsiveness of coronary vessels to cytokine- or metabolic-mediated vasodilation necessary to support myocardial perfusion in the wild-type (WT), creating a deficit in adaptive response in the Kir6.1 knockout. Application of a K(ATP) channel opener drug improved survival in the endotoxic WT but had no effect in the Kir6.1 knockout. Restoration of the dilatory capacity of coronary vessels was required to rescue the Kir6.1 knockout phenotype and reverse survival disadvantage in lethal endotoxemia. Thus, the Kir6.1-containing K(ATP) channel, by coupling vasoreactivity with metabolic demand, provides a vital feedback element for cardiovascular tolerance in endotoxic shock. PMID:17077304

  11. Molecular characterization and development of Sarcocystis speeri sarcocysts in gamma interferon gene knockout mice.

    PubMed

    Dubey, J P; Verma, S K; Dunams, D; Calero-Bernal, R; Rosenthal, B M

    2015-11-01

    The North American opossum (Didelphis virginiana) is the definitive host for at least three named species of Sarcocystis: Sarcocystis falcatula, Sarcocystis neurona and Sarcocystis speeri. The South American opossums (Didelphis albiventris, Didelphis marsupialis and Didelphis aurita) are definitive hosts for S. falcatula and S. lindsayi. The sporocysts of these Sarcocystis species are similar morphologically. They are also not easily distinguished genetically because of the difficulties of DNA extraction from sporocysts and availability of distinguishing genetic markers. Some of these species can be distinguished by bioassay; S. neurona and S. speeri are infective to gamma interferon gene knockout (KO) mice, but not to budgerigars (Melopsittacus undulatus); whereas S. falcatula and S. lindsayi are infective to budgerigars but not to KO mice. The natural intermediate host of S. speeri is unknown. In the present study, development of sarcocysts of S. speeri in the KO mice is described. Sarcocysts were first seen at 12 days post-inoculation (p.i.), and they became macroscopic (up to 4 mm long) by 25 days p.i. The structure of the sarcocyst wall did not change from the time bradyzoites had formed at 50-220 days p.i. Sarcocysts contained unique villar protrusions, 'type 38'. The polymerase chain reaction amplifications and sequences analysis of three nuclear loci (18S rRNA, 28S rRNA and ITS1) and two mitochondrial loci (cox1 and cytb) of S. speeri isolate from an Argentinean opossum (D. albiventris) confirmed its membership among species of Sarcocystis and indicated an especially close relationship to another parasite in this genus that employs opossums as its definitive host, S. neurona. These results should be useful in finding natural intermediate host of S. speeri. PMID:26303093

  12. Different Effects of sgRNA Length on CRISPR-mediated Gene Knockout Efficiency.

    PubMed

    Zhang, Jian-Ping; Li, Xiao-Lan; Neises, Amanda; Chen, Wanqiu; Hu, Lin-Ping; Ji, Guang-Zhen; Yu, Jun-Yao; Xu, Jing; Yuan, Wei-Ping; Cheng, Tao; Zhang, Xiao-Bing

    2016-01-01

    CRISPR-Cas9 is a powerful genome editing technology, yet with off-target effects. Truncated sgRNAs (17nt) have been found to decrease off-target cleavage without affecting on-target disruption in 293T cells. However, the potency of 17nt sgRNAs relative to the full-length 20nt sgRNAs in stem cells, such as human mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs), has not been assessed. Using a GFP reporter system, we found that both 17nt and 20nt sgRNAs expressed by lentiviral vectors induce ~95% knockout (KO) in 293T cells, whereas the KO efficiencies are significantly lower in iPSCs (60-70%) and MSCs (65-75%). Furthermore, we observed a decrease of 10-20 percentage points in KO efficiency with 17nt sgRNAs compared to full-length sgRNAs in both iPSCs and MSCs. Off-target cleavage was observed in 17nt sgRNAs with 1-2nt but not 3-4nt mismatches; whereas 20nt sgRNAs with up to 5nt mismatches can still induce off-target mutations. Of interest, we occasionally observed off-target effects induced by the 17nt but not the 20nt sgRNAs. These results indicate the importance of balancing on-target gene cleavage potency with off-target effects: when efficacy is a major concern such as genome editing in stem cells, the use of 20nt sgRNAs is preferable. PMID:27338021

  13. Different Effects of sgRNA Length on CRISPR-mediated Gene Knockout Efficiency

    PubMed Central

    Zhang, Jian-Ping; Li, Xiao-Lan; Neises, Amanda; Chen, Wanqiu; Hu, Lin-Ping; Ji, Guang-Zhen; Yu, Jun-Yao; Xu, Jing; Yuan, Wei-Ping; Cheng, Tao; Zhang, Xiao-Bing

    2016-01-01

    CRISPR-Cas9 is a powerful genome editing technology, yet with off-target effects. Truncated sgRNAs (17nt) have been found to decrease off-target cleavage without affecting on-target disruption in 293T cells. However, the potency of 17nt sgRNAs relative to the full-length 20nt sgRNAs in stem cells, such as human mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs), has not been assessed. Using a GFP reporter system, we found that both 17nt and 20nt sgRNAs expressed by lentiviral vectors induce ~95% knockout (KO) in 293T cells, whereas the KO efficiencies are significantly lower in iPSCs (60–70%) and MSCs (65–75%). Furthermore, we observed a decrease of 10–20 percentage points in KO efficiency with 17nt sgRNAs compared to full-length sgRNAs in both iPSCs and MSCs. Off-target cleavage was observed in 17nt sgRNAs with 1-2nt but not 3-4nt mismatches; whereas 20nt sgRNAs with up to 5nt mismatches can still induce off-target mutations. Of interest, we occasionally observed off-target effects induced by the 17nt but not the 20nt sgRNAs. These results indicate the importance of balancing on-target gene cleavage potency with off-target effects: when efficacy is a major concern such as genome editing in stem cells, the use of 20nt sgRNAs is preferable. PMID:27338021

  14. True-Breeding Targeted Gene Knock-Out in Barley Using Designer TALE-Nuclease in Haploid Cells

    PubMed Central

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

    2014-01-01

    Transcription activator-like effector nucleases (TALENs) are customizable fusion proteins able to cleave virtually any genomic DNA sequence of choice, and thereby to generate site-directed genetic modifications in a wide range of cells and organisms. In the present study, we expressed TALENs in pollen-derived, regenerable cells to establish the generation of instantly true-breeding mutant plants. A gfp-specific TALEN pair was expressed via Agrobacterium-mediated transformation in embryogenic pollen of transgenic barley harboring a functional copy of gfp. Thanks to the haploid nature of the target cells, knock-out mutations were readily detected, and homozygous primary mutant plants obtained following genome duplication. In all, 22% of the TALEN transgenics proved knocked out with respect to gfp, and the loss of function could be ascribed to the deletions of between four and 36 nucleotides in length. The altered gfp alleles were transmitted normally through meiosis, and the knock-out phenotype was consistently shown by the offspring of two independent mutants. Thus, here we describe the efficient production of TALEN-mediated gene knock-outs in barley that are instantaneously homozygous and non-chimeric in regard to the site-directed mutations induced. This TALEN approach has broad applicability for both elucidating gene function and tailoring the phenotype of barley and other crop species. PMID:24643227

  15. Differential role of PKA catalytic subunits in mediating phenotypes caused by knockout of the Carney complex gene Prkar1a.

    PubMed

    Yin, Zhirong; Pringle, Daphne R; Jones, Georgette N; Kelly, Kimberly M; Kirschner, Lawrence S

    2011-10-01

    The Carney complex is an inherited tumor predisposition caused by activation of the cAMP-dependent protein kinase [protein kinase A (PKA)] resulting from mutation of the PKA-regulatory subunit gene PRKAR1A. Myxomas and tumors in cAMP-responsive tissues are cardinal features of this syndrome, which is unsurprising given the important role played by PKA in modulating cell growth and function. Previous studies demonstrated that cardiac-specific knockout of Prkar1a causes embryonic heart failure and myxomatous degeneration in the heart, whereas limited Schwann cell-specific knockout of the gene causes schwannoma formation. In this study, we sought to determine the role of PKA activation in this phenotype by using genetic means to reduce PKA enzymatic activity. To accomplish this goal, we introduced null alleles of the PKA catalytic subunits Prkaca (Ca) or Prkacb (Cb) into the Prkar1a-cardiac knockout (R1a-CKO) or limited Schwann cell knockout (R1a-TEC3KO) line. Heterozygosity for Prkaca rescued the embryonic lethality of the R1a-CKO, although mice had a shorter than normal lifespan and died from cardiac failure with atrial thrombosis. In contrast, heterozygosity for Prkacb only enabled the mice to survive 1 extra day during embryogenesis. Biochemical analysis indicated that reduction of Ca markedly reduced PKA activity in embryonic hearts, whereas reduction of Cb had minimal effects. In R1a-TEC3KO mice, tumorigenesis was completely suppressed by a heterozygosity for Prkaca, and by more than 80% by heterozygosity for Prkacb. These data suggest that both developmental and tumor phenotypes caused by Prkar1a mutation result from excess PKA activity due to PKA-Ca. PMID:21852354

  16. Novel therapeutic targets in osteoarthritis: Narrative review on knock-out genes involved in disease development in mouse animal models.

    PubMed

    Veronesi, Francesca; Della Bella, Elena; Cepollaro, Simona; Brogini, Silvia; Martini, Lucia; Fini, Milena

    2016-05-01

    Osteoarthritis (OA) can affect every joint, especially the knee. Given the complexity of this pathology, OA is difficult to treat with current therapies, which only relieve pain and inflammation and are not capable of restoring tissues once OA has started. Currently, researchers focus on finding a therapeutic strategy that may help to arrest disease progression. The present narrative review gives an overview of the genes involved in the development and progression of OA, assessing in vivo studies performed in knock-out mice affected by OA, to suggest new therapeutic strategies. The article search was performed on the PubMed database and www.webofknowledge.com website with the following keywords: "knee osteoarthritis" AND "knockout mice". The included studies were in English and published from 2005 to 2015. Additional papers were found within the references of the selected articles. In the 55 analyzed in vivo studies, genes mainly affected chondrocyte homeostasis, inflammatory processes, extracellular matrix and the relationship between obesity and OA. Genes are defined as inducing, preventing and not influencing OA. This review shows that joint homeostasis depends on a variety of genetic factors, and preventing or restoring the loss of a gene encoding for protective proteins, or inhibiting the expression of proteins that induce OA, might be a potential therapeutic approach. However, conclusions cannot be drawn because of the wide variability concerning the technique used for OA induction, the role of the genes, the method for tissue evaluations and the lack of assessments of all joint tissues. PMID:27059198

  17. Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2 (USF2) knockout mice.

    PubMed

    Nicolas, G; Bennoun, M; Devaux, I; Beaumont, C; Grandchamp, B; Kahn, A; Vaulont, S

    2001-07-17

    We previously reported the disruption of the murine gene encoding the transcription factor USF2 and its consequences on glucose-dependent gene regulation in the liver. We report here a peculiar phenotype of Usf2(-/-) mice that progressively develop multivisceral iron overload; plasma iron overcomes transferrin binding capacity, and nontransferrin-bound iron accumulates in various tissues including pancreas and heart. In contrast, the splenic iron content is strikingly lower in knockout animals than in controls. To identify genes that may account for the abnormalities of iron homeostasis in Usf2(-/-) mice, we used suppressive subtractive hybridization between livers from Usf2(-/-) and wild-type mice. We isolated a cDNA encoding a peptide, hepcidin (also referred to as LEAP-1, for liver-expressed antimicrobial peptide), that was very recently purified from human blood ultrafiltrate and from urine as a disulfide-bonded peptide exhibiting antimicrobial activity. Accumulation of iron in the liver has been recently reported to up-regulate hepcidin expression, whereas our data clearly show that a complete defect in hepcidin expression is responsible for progressive tissue iron overload. The striking similarity of the alterations in iron metabolism between HFE knockout mice, a murine model of hereditary hemochromatosis, and the Usf2(-/-) hepcidin-deficient mice suggests that hepcidin may function in the same regulatory pathway as HFE. We propose that hepcidin acts as a signaling molecule that is required in conjunction with HFE to regulate both intestinal iron absorption and iron storage in macrophages. PMID:11447267

  18. Hepatic Gene Expression Profiling in Nrf2 Knockout Mice after Long-Term High-Fat Diet-Induced Obesity

    PubMed Central

    Chartoumpekis, Dionysios V.; Ziros, Panos G.; Zaravinos, Apostolos; Iskrenova, Ralitsa P.; Psyrogiannis, Agathoklis I.; Kyriazopoulou, Venetsana E.; Sykiotis, Gerasimos P.; Habeos, Ioannis G.

    2013-01-01

    Introduction. The transcription factor NFE2-related factor 2 (Nrf2) is a central regulator of antioxidant and detoxification gene expression in response to electrophilic or oxidative stress. Nrf2 has recently been shown to cross-talk with metabolic pathways, and its gene deletion protected mice from high-fat-diet-(HFD-) induced obesity and insulin resistance. This study aimed to identify potential Nrf2-regulated genes of metabolic interest by comparing gene expression profiles of livers of wild-type (WT) versus Nrf2 knockout (Nrf2-KO) mice after a long-term HFD. Methods. WT and Nrf2-KO mice were fed an HFD for 180 days; total RNA was prepared from liver and used for microarray analysis and quantitative real-time RT-PCR (qRT-PCR). Results. The microarray analysis identified 601 genes that were differentially expressed between WT and Nrf2-KO mice after long-term HFD. Selected genes, including ones known to be involved in metabolic regulation, were prioritized for verification by qRT-PCR: Cyp7a1 and Fabp5 were significantly overexpressed in Nrf2-KO mice; in contrast, Car, Cyp2b10, Lipocalin 13, Aquaporin 8, Cbr3, Me1, and Nqo1 were significantly underexpressed in Nrf2-KO mice. Conclusion. Transcriptome profiling after HFD-induced obesity confirms that Nrf2 is implicated in liver metabolic gene networks. The specific genes identified here may provide insights into Nrf2-dependent mechanisms of metabolic regulation. PMID:23710285

  19. Modeling RNA polymerase competition: the effect of σ-subunit knockout and heat shock on gene transcription level

    PubMed Central

    2011-01-01

    Background Modeling of a complex biological process can explain the results of experimental studies and help predict its characteristics. Among such processes is transcription in the presence of competing RNA polymerases. This process involves RNA polymerases collision followed by transcription termination. Results A mathematical and computer simulation model is developed to describe the competition of RNA polymerases during genes transcription on complementary DNA strands. E.g., in the barley Hordeum vulgare the polymerase competition occurs in the locus containing plastome genes psbA, rpl23, rpl2 and four bacterial type promoters. In heat shock experiments on isolated chloroplasts, a twofold decrease of psbA transcripts and even larger increase of rpl23-rpl2 transcripts were observed, which is well reproduced in the model. The model predictions are in good agreement with virtually all relevant experimental data (knockout, heat shock, chromatogram data, etc.). The model allows to hypothesize a mechanism of cell response to knockout and heat shock, as well as a mechanism of gene expression regulation in presence of RNA polymerase competition. The model is implemented for multiprocessor platforms with MPI and supported on Linux and MS Windows. The source code written in C++ is available under the GNU General Public License from the laboratory website. A user-friendly GUI version is also provided at http://lab6.iitp.ru/en/rivals. Conclusions The developed model is in good agreement with virtually all relevant experimental data. The model can be applied to estimate intensities of binding of the holoenzyme and phage type RNA polymerase to their promoters using data on gene transcription levels, as well as to predict characteristics of RNA polymerases and the transcription process that are difficult to measure directly, e.g., the intensity (frequency) of holoenzyme binding to the promoter in correlation to its nucleotide composition and the type of σ-subunit, the

  20. Prion protein (PrP) gene-knockout cell lines: insight into functions of the PrP

    PubMed Central

    Sakudo, Akikazu; Onodera, Takashi

    2015-01-01

    Elucidation of prion protein (PrP) functions is crucial to fully understand prion diseases. A major approach to studying PrP functions is the use of PrP gene-knockout (Prnp−/−) mice. So far, six types of Prnp−/− mice have been generated, demonstrating the promiscuous functions of PrP. Recently, other PrP family members, such as Doppel and Shadoo, have been found. However, information obtained from comparative studies of structural and functional analyses of these PrP family proteins do not fully reveal PrP functions. Recently, varieties of Prnp−/− cell lines established from Prnp−/− mice have contributed to the analysis of PrP functions. In this mini-review, we focus on Prnp−/− cell lines and summarize currently available Prnp−/− cell lines and their characterizations. In addition, we introduce the recent advances in the methodology of cell line generation with knockout or knockdown of the PrP gene. We also discuss how these cell lines have provided valuable insights into PrP functions and show future perspectives. PMID:25642423

  1. Claudin-2 Knockout by TALEN-Mediated Gene Targeting in MDCK Cells: Claudin-2 Independently Determines the Leaky Property of Tight Junctions in MDCK Cells

    PubMed Central

    Tokuda, Shinsaku; Furuse, Mikio

    2015-01-01

    Tight junctions (TJs) regulate the movements of substances through the paracellular pathway, and claudins are major determinants of TJ permeability. Claudin-2 forms high conductive cation pores in TJs. The suppression of claudin-2 expression by RNA interference in Madin-Darby canine kidney (MDCK) II cells (a low-resistance strain of MDCK cells) was shown to induce a three-fold increase in transepithelial electrical resistance (TER), which, however, was still lower than in high-resistance strains of MDCK cells. Because RNA interference-mediated knockdown is not complete and only reduces gene function, we considered the possibility that the remaining claudin-2 expression in the knockdown study caused the lower TER in claudin-2 knockdown cells. Therefore, we investigated the effects of claudin-2 knockout in MDCK II cells by establishing claudin-2 knockout clones using transcription activator-like effector nucleases (TALENs), a recently developed genome editing method for gene knockout. Surprisingly, claudin-2 knockout increased TER by more than 50-fold in MDCK II cells, and TER values in these cells (3000–4000 Ω·cm2) were comparable to those in the high-resistance strains of MDCK cells. Claudin-2 re-expression restored the TER of claudin-2 knockout cells dependent upon claudin-2 protein levels. In addition, we investigated the localization of claudin-1, -2, -3, -4, and -7 at TJs between control MDCK cells and their respective knockout cells using their TALENs. Claudin-2 and -7 were less efficiently localized at TJs between control and their knockout cells. Our results indicate that claudin-2 independently determines the ‘leaky’ property of TJs in MDCK II cells and suggest the importance of knockout analysis in cultured cells. PMID:25781928

  2. Development of pyrF-based gene knockout systems for genome-wide manipulation of the archaea Haloferax mediterranei and Haloarcula hispanica.

    PubMed

    Liu, Hailong; Han, Jing; Liu, Xiaoqing; Zhou, Jian; Xiang, Hua

    2011-06-20

    The haloarchaea Haloferax mediterranei and Haloarcula hispanica are both polyhydroxyalkanoate producers in the domain Archaea, and they are becoming increasingly attractive for research and biotechnology due to their unique genetic and metabolic features. To accelerate their genome-level genetic and metabolic analyses, we have developed specific and highly efficient gene knockout systems for these two haloarchaea. These gene knockout systems consist of a suicide plasmid vector with the pyrF gene as the selection marker and a uracil auxotrophic haloarchaeon (ΔpyrF) as the host. For in-frame deletion of a target gene, the suicide plasmid carrying the flanking region of the target gene was transferred into the corresponding ΔpyrF host. After positive selection of the single-crossover integration recombinants (pop-in) on AS-168SY medium without uracil and counterselection of the double-crossover pyrF-excised recombinants (pop-out) with 5-fluoroorotic acid (5-FOA), the target gene knockout mutants were confirmed by PCR and Southern blot analysis. We have demonstrated the effectiveness of these systems by knocking out the crtB gene which encodes a phytoene synthase in these haloarchaea. In conclusion, these well-developed knockout systems would greatly accelerate the functional genomic research of these halophilic archaea. PMID:21703550

  3. Epithelial-specific knockout of the Rac1 gene leads to enamel defects

    PubMed Central

    Huang, Zhan; Kim, Jieun; Lacruz, Rodrigo; Bringas, Pablo; Kaartinen, Vesa M.; Snead, Malcolm L.

    2015-01-01

    Rac1 encodes a 21kDa GTP-binding protein belonging to the RAS superfamily. RAS members play important roles in controlling focal adhesion complex formation and cytoskeleton contraction; activities with consequences to cell growth, adhesion, migration, and differentiation. To examine the role(s) played by Rac1 protein in cell-to-matrix interaction and in enamel matrix biomineralization we used the Cre/loxP binary recombination system to characterize enamel matrix proteins expression and enamel formation in Rac1 knockout mice. Mating between mice bearing the floxed Rac1 allele with mice bearing a keratin14-Cre transgene generate animals in which Rac1 is absent from epithelial organs. The enamel of Rac1 conditional knockout mouse was characterized by computerized tomography (microCT), light microscopy, histochemistry, and back-scatter electron microscopy. Enamel matrix protein expression was analyzed by Western blotting. Major findings showed that the Tomes’ processes of Rac1−/− ameloblasts loose contact with the forming enamel matrix in un-erupted teeth. The abundance of amelogenin and ameloblastin was reduced in the Rac1−/− ameloblasts. After eruption, the enamel from the Rac1−/− mice displayed severe structural defects with the complete loss of enamel. These results support an essential role for Rac1 function in the dental epithelium involving cell-matrix interaction and matrix biomineralization. PMID:22243243

  4. 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

  5. Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2 (USF2) knockout mice

    PubMed Central

    Nicolas, Gaël; Bennoun, Myriam; Devaux, Isabelle; Beaumont, Carole; Grandchamp, Bernard; Kahn, Axel; Vaulont, Sophie

    2001-01-01

    We previously reported the disruption of the murine gene encoding the transcription factor USF2 and its consequences on glucose-dependent gene regulation in the liver. We report here a peculiar phenotype of Usf2−/− mice that progressively develop multivisceral iron overload; plasma iron overcomes transferrin binding capacity, and nontransferrin-bound iron accumulates in various tissues including pancreas and heart. In contrast, the splenic iron content is strikingly lower in knockout animals than in controls. To identify genes that may account for the abnormalities of iron homeostasis in Usf2−/− mice, we used suppressive subtractive hybridization between livers from Usf2−/− and wild-type mice. We isolated a cDNA encoding a peptide, hepcidin (also referred to as LEAP-1, for liver-expressed antimicrobial peptide), that was very recently purified from human blood ultrafiltrate and from urine as a disulfide-bonded peptide exhibiting antimicrobial activity. Accumulation of iron in the liver has been recently reported to up-regulate hepcidin expression, whereas our data clearly show that a complete defect in hepcidin expression is responsible for progressive tissue iron overload. The striking similarity of the alterations in iron metabolism between HFE knockout mice, a murine model of hereditary hemochromatosis, and the Usf2−/− hepcidin-deficient mice suggests that hepcidin may function in the same regulatory pathway as HFE. We propose that hepcidin acts as a signaling molecule that is required in conjunction with HFE to regulate both intestinal iron absorption and iron storage in macrophages. PMID:11447267

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  8. ZO-1 Knockout by TALEN-Mediated Gene Targeting in MDCK Cells: Involvement of ZO-1 in the Regulation of Cytoskeleton and Cell Shape

    PubMed Central

    Tokuda, Shinsaku; Higashi, Tomohito; Furuse, Mikio

    2014-01-01

    ZO-1, ZO-2 and ZO-3 are tight junction-associated scaffold proteins that bind to transmembrane proteins of tight junctions and the underlying cytoskeleton. ZO-1 is involved in the regulation of cytoskeletal organization, but its detailed molecular mechanism is less well understood. Gene knockout is an ideal method to investigate the functions of proteins that might have redundant functions such as ZO proteins, when compared with methods such as RNA interference-mediated suppression of gene expression. In this study we applied transcription activator-like effector nucleases (TALENs), a recently developed genome editing method for gene knockout, and established ZO-1 knockout clones in Madin-Darby canine kidney (MDCK) cells. ZO-1 knockout induced striking changes in myosin organization at cell–cell contacts and disrupted the localization of tight junction proteins; these findings were previously unseen in studies of ZO-1 knockdown by RNA interference. Rescue experiments revealed that trace ZO-1 expression reversed these changes while excessive ZO-1 expression induced an intensive zigzag shape of cell–cell junctions. These results suggest a role for ZO-1 in the regulation of cytoskeleton and shape of cell–cell junctions in MDCK cells and indicate the advantage of knockout analysis in cultured cells. PMID:25157572

  9. Expression of the functional cone phototransduction cascade in retinoblastoma.

    PubMed Central

    Hurwitz, R L; Bogenmann, E; Font, R L; Holcombe, V; Clark, D

    1990-01-01

    Retinoblastoma is a malignant intraocular tumor that primarily affects small children. These tumors are primitive neuroectodermal malignancies, however some of them show morphologic evidence of differentiation into photoreceptors. Phototransduction cascades are a series of biochemical reactions that convert a photon of light into a neural impulse in rods and cones. The components of these cascades are uniquely expressed in photoreceptors and, although functionally similar, distinct components of these cascades are expressed in rods and cones. Using HPLC anion exchange chromatography, Western blot analysis, and specific monoclonal and polyclonal antibodies, we found that the cone but not the rod cGMP phosphodiesterase is functionally expressed in all six primary retinoblastomas examined and in three continuous retinoblastoma cell lines. Morphologic evidence of differentiation did not correlate with the expression of the enzyme. Furthermore, GTP analogues could activate the phosphodiesterase activity suggesting that an intact phototransduction cascade is present in the tumors. The presence of the cone phototransduction cascade in retinoblastoma confirms that this tumor has biochemically differentiated along the cone cell lineage. Images PMID:2161431

  10. Duodenal nonheme iron content correlates with iron stores in mice, but the relationship is altered by Hfe gene knock-out.

    PubMed

    Simpson, Robert J; Debnam, Edward S; Laftah, Abas H; Solanky, Nita; Beaumont, Nick; Bahram, Seiamak; Schümann, Klaus; Srai, S Kaila S

    2003-04-15

    Hereditary hemochromatosis is a common iron-loading disorder found in populations of European descent. It has been proposed that mutations causing loss of function of HFE gene result in reduced iron incorporation into immature duodenal crypt cells. These cells then overexpress genes for iron absorption, leading to inappropriate cellular iron balance, a persistent iron deficiency of the duodenal mucosa, and increased iron absorption. The objective was to measure duodenal iron content in Hfe knock-out mice to test whether the mutation causes a persistent decrease in enterocyte iron concentration. In both normal and Hfe knock-out mice, duodenal nonheme iron content was found to correlate with liver iron stores (P <.001, r = 0.643 and 0.551, respectively), and this effect did not depend on dietary iron levels. However, duodenal iron content was reduced in Hfe knock-out mice for any given content of liver iron stores (P <.001). PMID:12468424

  11. Generation of a complete single-gene knockout bacterial artificial chromosome library of cowpox virus and identification of its essential genes.

    PubMed

    Xu, Zhiyong; Zikos, Dimitrios; Osterrieder, Nikolaus; Tischer, B Karsten

    2014-01-01

    Cowpox virus (CPXV) belongs to the genus Orthopoxvirus in the Poxviridae family. It infects a broad range of vertebrates and can cause zoonotic infections. CPXV has the largest genome among the orthopoxviruses and is therefore considered to have the most complete set of genes of all members of the genus. Since CPXV has also become a model for studying poxvirus genetics and pathogenesis, we created and characterized a complete set of single gene knockout bacterial artificial chromosome (BAC) clones of the CPXV strain Brighton Red. These mutants allow a systematic assessment of the contribution of single CPXV genes to the outcome of virus infection and replication, as well as to the virus host range. A full-length BAC clone of CPXV strain Brighton Red (pBRF) harboring the gene expressing the enhanced green fluorescent protein under the control of a viral late promoter was modified by introducing the mrfp1 gene encoding the monomeric red fluorescent protein driven by a synthetic early vaccinia virus promoter. Based on the modified BAC (pBRFseR), a library of targeted knockout mutants for each single viral open reading frame (ORF) was generated. Reconstitution of infectious virus was successful for 109 of the 183 mutant BAC clones, indicating that the deleted genes are not essential for virus replication. In contrast, 74 ORFs were identified as essential because no virus progeny was obtained upon transfection of the mutant BAC clones and in the presence of a helper virus. More than 70% of all late CPXV genes belonged to this latter group of essential genes. PMID:24155400

  12. Knockout and functional analysis of two DExD/H-box family helicase genes in Sulfolobus islandicus REY15A.

    PubMed

    Song, Xueguo; Huang, Qihong; Ni, Jinfeng; Yu, Yang; Shen, Yulong

    2016-07-01

    DExD/H-box helicases represent the largest family of helicases. They belong to superfamily 2 helicases and participate in nucleotide metabolism, ribosome biogenesis, and nucleocytoplasmic transport. The biochemical properties and structures of some DExD/H-box helicases in the archaea have been documented, but many of them have not been characterized; and reports on in vivo functional analyses are limited. In this study, we attempted gene knockout of 8 putative DExD/H-box helicases in Sulfolobus islandicus REY15A and obtained two deletion mutants, SiRe_0681 and SiRe_1605. We determined that ΔSiRe_0681 grew faster than wild type cells in the presence of methyl methanesulfonate (MMS). Flow cytometry analysis showed that this strain had fewer G1/S phase cells than the wild type, and the genes coding for cell division proteins were up-regulated. The stain ΔSiRe_1605 was more sensitive to MMS than the wild type cell, and many nucleotide metabolism and DNA repair enzymes were found to be down-regulated. Intriguingly, deletion of either gene led to silencing simultaneously of over 80 genes located at a specific region. This study provides a novel insight into the in vivo functions of predicted DExD/H-box family helicases in the archaea. PMID:27290726

  13. 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. PMID:26278974

  14. A Gene Trap Knockout of the Tiam-1 Protein Results in Malformation of the Early Embryonic Brain

    PubMed Central

    Yoo, Sooyeon; Kim, Yujin; Lee, Haeryung; Park, Sungjeong; Park, Soochul

    2012-01-01

    Tiam-1 has been implicated in the development of the central nervous system. However, the in vivo function of Tiam-1 has not been fully determined in the developing mouse brain. In this study, we generated Tiam-1 knockout mice using a Tiam-1 gene-trapped embryonic stem cell line. Insertion of a gene trap vector into a genomic site downstream of exon 5 resulted in a mutant allele encoding a truncated protein fused with the β-geo LacZ gene. Primary mouse embryonic fibroblasts lacking Tiam-1 revealed a significant decrease in Rac activity and cell proliferation. In addition, whole-mount embryonic LacZ expression analysis demonstrated that Tiam-1 is specifically expressed in regions of the developing brain, such as the caudal telencephalon and rostral diencephalon. More importantly, mouse embryos deficient in Tiam-1 gene expression displayed a severe defect in embryonic brain development, including neural tube closure defects or a dramatic decrease in brain size. These findings suggest that embryonic Tiam-1 expression plays a critical role during early brain development in mice. PMID:22661025

  15. Rapid-Throughput Skeletal Phenotyping of 100 Knockout Mice Identifies 9 New Genes That Determine Bone Strength

    PubMed Central

    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.

    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. PMID:22876197

  16. 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

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

    PubMed

    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

  18. 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. PMID:26741960

  19. Genome-wide Generation and Systematic Phenotyping of Knockout Mice Reveals New Roles for Many Genes

    PubMed Central

    White, Jacqueline K.; Gerdin, Anna-Karin; Karp, Natasha A.; Ryder, Ed; Buljan, Marija; Bussell, James N.; Salisbury, Jennifer; Clare, Simon; Ingham, Neil J.; Podrini, Christine; Houghton, Richard; Estabel, Jeanne; Bottomley, Joanna R.; Melvin, David G.; Sunter, David; Adams, Niels C.; Baker, Lauren; Barnes, Caroline; Beveridge, Ryan; Cambridge, Emma; Carragher, Damian; Chana, Prabhjoat; Clarke, Kay; Hooks, Yvette; Igosheva, Natalia; Ismail, Ozama; Jackson, Hannah; Kane, Leanne; Lacey, Rosalind; Lafont, David Tino; Lucas, Mark; Maguire, Simon; McGill, Katherine; McIntyre, Rebecca E.; Messager, Sophie; Mottram, Lynda; Mulderrig, Lee; Pearson, Selina; Protheroe, Hayley J.; Roberson, Laura-Anne; Salsbury, Grace; Sanderson, Mark; Sanger, Daniel; Shannon, Carl; Thompson, Paul C.; Tuck, Elizabeth; Vancollie, Valerie E.; Brackenbury, Lisa; Bushell, Wendy; Cook, Ross; Dalvi, Priya; Gleeson, Diane; Habib, Bishoy; Hardy, Matt; Liakath-Ali, Kifayathullah; Miklejewska, Evelina; Price, Stacey; Sethi, Debarati; Trenchard, Elizabeth; von Schiller, Dominique; Vyas, Sapna; West, Anthony P.; Woodward, John; Wynn, Elizabeth; Evans, Arthur; Gannon, David; Griffiths, Mark; Holroyd, Simon; Iyer, Vivek; Kipp, Christian; Lewis, Morag; Li, Wei; Oakley, Darren; Richardson, David; Smedley, Damian; Agu, Chukwuma; Bryant, Jackie; Delaney, Liz; Gueorguieva, Nadia I.; Tharagonnet, Helen; Townsend, Anne J.; Biggs, Daniel; Brown, Ellen; Collinson, Adam; Dumeau, Charles-Etienne; Grau, Evelyn; Harrison, Sarah; Harrison, James; Ingle, Catherine E.; Kundi, Helen; Madich, Alla; Mayhew, Danielle; Metcalf, Tom; Newman, Stuart; Pass, Johanna; Pearson, Laila; Reynolds, Helen; Sinclair, Caroline; Wardle-Jones, Hannah; Woods, Michael; Alexander, Liam; Brown, Terry; Flack, Francesca; Frost, Carole; Griggs, Nicola; Hrnciarova, Silvia; Kirton, Andrea; McDermott, Jordan; Rogerson, Claire; White, Gemma; Zielezinski, Pawel; DiTommaso, Tia; Edwards, Andrew; Heath, Emma; Mahajan, Mary Ann; Yalcin, Binnaz; Tannahill, David; Logan, Darren W.; MacArthur, Daniel G.; Flint, Jonathan; Mahajan, Vinit B.; Tsang, Stephen H.; Smyth, Ian; Watt, Fiona M.; Skarnes, William C.; Dougan, Gordon; Adams, David J.; Ramirez-Solis, Ramiro; Bradley, Allan; Steel, Karen P.

    2013-01-01

    Summary Mutations in whole organisms are powerful ways of interrogating gene function in a realistic context. We describe a program, the Sanger Institute Mouse Genetics Project, that provides a step toward the aim of knocking out all genes and screening each line for a broad range of traits. We found that hitherto unpublished genes were as likely to reveal phenotypes as known genes, suggesting that novel genes represent a rich resource for investigating the molecular basis of disease. We found many unexpected phenotypes detected only because we screened for them, emphasizing the value of screening all mutants for a wide range of traits. Haploinsufficiency and pleiotropy were both surprisingly common. Forty-two percent of genes were essential for viability, and these were less likely to have a paralog and more likely to contribute to a protein complex than other genes. Phenotypic data and more than 900 mutants are openly available for further analysis. PaperClip PMID:23870131

  20. Genetic manipulation to analyze pheromone responses: knockouts of multiple receptor genes.

    PubMed

    Ishii, Tomohiro

    2013-01-01

    Gene targeting in the mouse is an essential technique to study gene function in vivo. Multigene families encoding vomeronasal receptor (VR) type 1 and type 2 consist of ~300 intact genes, which are clustered at multiple loci in the mouse genome. To understand the function of VRs and neurons expressing a particular VR in vivo, individual endogenous receptor genes can be manipulated by conventional gene targeting to create loss-of-function mutations or to visualize neurons and their axons expressing the VR. Multiple receptor genes in a cluster can also be deleted simultaneously by chromosome engineering, allowing analysis of function of a particular VR subfamily. Here, we describe protocols for conventional gene targeting and chromosome engineering for deleting a large genomic region in mouse embryonic stem (ES) cells. PMID:24014359

  1. CRISPR-Cas9 mediated gene knockout in primary human airway epithelial cells reveals a pro-inflammatory role for MUC18

    PubMed Central

    Chu, Hong Wei; Rios, Cydney; Huang, Chunjian; Wesolowska-Andersen, Agata; Burchard, Esteban G.; O'Connor, Brian P.; Fingerlin, Tasha E.; Nichols, David; Reynolds, Susan D.; Seibold, Max A.

    2015-01-01

    Targeted knockout of genes in primary human cells using CRISPR-Cas9 mediated genome-editing represents a powerful approach to study gene function and to discern molecular mechanisms underlying complex human diseases. We used lentiviral delivery of CRISPR-Cas9 machinery and conditional reprogramming culture methods to knockout the MUC18 gene in human primary nasal airway epithelial cells (AECs). Massively parallel sequencing technology was used to confirm that the genome of essentially all cells in the edited AEC populations contained coding region insertions and deletions (indels). Correspondingly, we found mRNA expression of MUC18 was greatly reduced and protein expression was absent. Characterization of MUC18 knockout cell populations stimulated with TLR2, 3 and 4 agonists revealed that IL-8 (a pro-inflammatory chemokine) responses of AECs were greatly reduced in the absence of functional MUC18 protein. Our results show the feasibility of CRISPR-Cas9 mediated gene knockouts in AEC culture (both submerged and polarized), and suggest a pro-inflammatory role for MUC18 in airway epithelial response to bacterial and viral stimuli. PMID:26043872

  2. Model-aided atpE gene knockout strategy in Escherichia coli for enhanced succinic acid production from glycerol.

    PubMed

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

    2016-08-01

    Succinic acid is an important platform chemical with a variety of applications. Model-guided metabolic engineering strategies in Escherichia coli for strain improvement to increase succinic acid production using glucose and glycerol remain largely unexplored. Herein, we report what are, to our knowledge, the first metabolic knockout of the atpE gene to have increased succinic acid production using both glucose and alternative glycerol carbon sources in E. coli. Guided by a genome-scale metabolic model, we engineered the E. coli host to enhance anaerobic production of succinic acid by deleting the atpE gene, thereby generating additional reducing equivalents by blocking H(+) conduction across the mutant cell membrane. This strategy produced 1.58 and .49 g l(-1) of succinic acid from glycerol and glucose substrate, respectively. This work further elucidates a model-guided and/or system-based metabolic engineering, involving only a single-gene deletion strategy for enhanced succinic acid production in E. coli. PMID:26513379

  3. Model-guided metabolic gene knockout of gnd for enhanced succinate production in Escherichia coli from glucose and glycerol substrates.

    PubMed

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

    2016-04-01

    The metabolic role of 6-phosphogluconate dehydrogenase (gnd) under anaerobic conditions with respect to succinate production in Escherichia coli remained largely unspecified. Herein we report what are to our knowledge the first metabolic gene knockout of gnd to have increased succinic acid production using both glucose and glycerol substrates in E. coli. Guided by a genome scale metabolic model, we engineered the E. coli host metabolism to enhance anaerobic production of succinic acid by deleting the gnd gene, considering its location in the boundary of oxidative and non-oxidative pentose phosphate pathway. This strategy induced either the activation of malic enzyme, causing up-regulation of phosphoenolpyruvate carboxylase (ppc) and down regulation of phosphoenolpyruvate carboxykinase (ppck) and/or prevents the decarboxylation of 6 phosphogluconate to increase the pool of glyceraldehyde-3-phosphate (GAP) that is required for the formation of phosphoenolpyruvate (PEP). This approach produced a mutant strain BMS2 with succinic acid production titers of 0.35gl(-1) and 1.40gl(-1) from glucose and glycerol substrates respectively. This work further clearly elucidates and informs other studies that the gnd gene, is a novel deletion target for increasing succinate production in E. coli under anaerobic condition using glucose and glycerol carbon sources. The knowledge gained in this study would help in E. coli and other microbial strains development for increasing succinate production and/or other industrial chemicals. PMID:26878126

  4. Targeted Chromosomal Translocations and Essential Gene Knockout Using CRISPR/Cas9 Technology in Caenorhabditis elegans.

    PubMed

    Chen, Xiangyang; Li, Mu; Feng, Xuezhu; Guang, Shouhong

    2015-12-01

    Many genes play essential roles in development and fertility; their disruption leads to growth arrest or sterility. Genetic balancers have been widely used to study essential genes in many organisms. However, it is technically challenging and laborious to generate and maintain the loss-of-function mutations of essential genes. The CRISPR/Cas9 technology has been successfully applied for gene editing and chromosome engineering. Here, we have developed a method to induce chromosomal translocations and produce genetic balancers using the CRISPR/Cas9 technology and have applied this approach to edit essential genes in Caenorhabditis elegans. The co-injection of dual small guide RNA targeting genes on different chromosomes resulted in reciprocal translocation between nonhomologous chromosomes. These animals with chromosomal translocations were subsequently crossed with animals that contain normal sets of chromosomes. The F1 progeny were subjected to a second round of Cas9-mediated gene editing. Through this method, we successfully produced nematode strains with specified chromosomal translocations and generated a number of loss-of-function alleles of two essential genes (csr-1 and mes-6). Therefore, our method provides an easy and efficient approach to generate and maintain loss-of-function alleles of essential genes with detailed genetic background information. PMID:26482793

  5. A meta-analysis study of gene expression datasets in mouse liver under PPARα knockout.

    PubMed

    He, Kan; Wang, Zhen; Wang, Qishan; Pan, Yuchun

    2013-06-01

    Gene expression profiling of peroxisome-proliferator-activated receptor α (PPARα) has been used in several studies, but there were no consistent results on gene expression patterns involved in PPARα activation in genome-wide due to different sample sizes or platforms. Here, we employed two published microarray datasets both PPARα dependent in mouse liver and applied meta-analysis on them to increase the power of the identification of differentially expressed genes and significantly enriched pathways. As a result, we have improved the concordance in identifying many biological mechanisms involved in PPARα activation. We suggest that our analysis not only leads to more identified genes by combining datasets from different resources together, but also provides some novel hepatic tissue-specific marker genes related to PPARα according to our re-analysis. PMID:23938112

  6. Development of a transformation system for gene knock-out in the flavinogenic yeast Pichia guilliermondii.

    PubMed

    Boretsky, Yuriy R; Pynyaha, Yuriy V; Boretsky, Volodymyr Y; Kutsyaba, Vasyl I; Protchenko, Olga V; Philpott, Caroline C; Sibirny, Andriy A

    2007-07-01

    Pichia guilliermondii is a representative of a yeast species, all of which over-synthesize riboflavin in response to iron deprivation. Molecular genetic studies in this yeast species have been hampered by a lack of strain-specific tools for gene manipulation. Stable P. guilliermondii ura3 mutants were selected on the basis of 5'-fluoroorotic acid resistance. Plasmid carrying Saccharomyces cerevisiae URA3 gene transformed the mutant strains to prototrophy with a low efficiency. Substitution of a single leucine codon CUG by another leucine codon CUC in the URA3 gene increased the efficiency of transformation 100 fold. Deletion cassettes for the RIB1 and RIB7 genes, coding for GTP cyclohydrolase and riboflavin synthase, respectively, were constructed using the modified URA3 gene and subsequently introduced into a P. guilliermondii ura3 strain. Site-specific integrants were identified by selection for the Rib(-) Ura(+) phenotype and confirmed by PCR analysis. Transformation of the P. guilliermondii ura3 strain was performed using electroporation, spheroplasting or lithium acetate treatment. Only the lithium acetate transformation procedure provided selection of uracil prototrophic, riboflavin deficient recombinant strains. Depending on the type of cassette, efficiency of site-specific integration was 0.1% and 3-12% in the case of the RIB1 and RIB7 genes, respectively. We suggest that the presence of the ARS element adjacent to the 3' end of the RIB1 gene significantly reduced the frequency of homologous recombination. Efficient gene deletion in P. guilliermondii can be achieved using the modified URA3 gene of S. cerevisiae flanked by 0.8-0.9 kb sequences homologous to the target gene. PMID:17467833

  7. Knockout of mouse Cyp3a gene enhances synthesis of cholesterol and bile acid in the liver[S

    PubMed Central

    Hashimoto, Mari; Kobayashi, Kaoru; Watanabe, Mio; Kazuki, Yasuhiro; Takehara, Shoko; Inaba, Asumi; Nitta, Shin-ichiro; Senda, Naoto; Oshimura, Mitsuo; Chiba, Kan

    2013-01-01

    Here, we studied the effects of cytochrome P450 (CYP)3A deficiency on the mRNA expression of genes encoding regulators of hepatic cholesterol levels using Cyp3a-knockout (Cyp3a−/−) mice. The mRNA expression levels of genes encoding enzymes involved in cholesterol biosynthesis in the livers of Cyp3a−/− mice were higher than those of wild-type (WT) mice. Nuclear levels of sterol regulatory element-binding protein-2 (SREBP-2), which enhances cholesterol biosynthesis, were also higher in the livers of Cyp3a−/− mice. Binding of SREBP-2 to the Hmgcs1 gene promoter was more abundant in the livers of Cyp3a−/− mice. These results suggest that deficiency of CYP3A enzymes enhances transcription of genes encoding enzymes involved in cholesterol biosynthesis via activation of SREBP-2. On the other hand, hepatic cholesterol levels in Cyp3a−/− mice were 20% lower than those in WT mice. The mRNA expression levels of genes encoding enzymes involved in bile acid synthesis, plasma levels of 7α-hydroxy-4-cholesten-3-one and hepatic levels of total bile acid were significantly higher in Cyp3a−/− mice than in WT mice. These findings suggest that reduction of hepatic total cholesterol in Cyp3a−/− mice would be the consequence of enhanced bile acid synthesis. Therefore, CYP3A enzymes appear to play roles in the synthesis of cholesterol and bile acid in vivo. PMID:23709690

  8. 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

  9. Oral Carcinogenesis Induced by 4-Nitroquinoline 1-Oxide in Lecithin:retinol Acyltransferase Gene Knockout Mice

    PubMed Central

    Liu, Limin; Tang, Xiao-Han; Scognamiglio, Theresa; Gudas, Lorraine J.

    2010-01-01

    Lecithin:retinol acyltransferase (LRAT) regulates retinol (vitamin A) metabolism by esterifying retinol. LRAT expression is decreased in cultured human head and neck squamous cell carcinoma (SCCHN) relative to normal epithelial cells. We investigated whether the carcinogen 4-nitroquinoline 1-oxide (4-NQO) induced a higher incidence of oral cancer in LRAT knockout than wild type (Wt) mice. We also investigated retinol deprivation during 4-NQO treatment in LRAT−/− mice as a model for rapid retinol deficiency. We observed higher levels of secreted frizzled-related protein 2 (Sfrp2), an inhibitor of WNT signaling, in tongue tumors in LRAT−/− versus Wt. LRAT−/− embryonic stem cells also expressed higher Sfrp2 transcripts, indicating an interaction between retinol and WNT signaling. Cox-2, Cyclin D1, p21, Trop2, and RARβ2 were not differentially expressed in Wt versus LRAT−/− tongue tumors. Wt and LRAT−/− mice fed a retinol sufficient diet showed the same oral tumor incidence after 4-NQO. In contrast, tongue tumors developed in 60% of Wt and in 100% of LRAT−/− mice fed a retinol deficient diet during 4-NQO treatment (p=0.22); moreover, the BrdU labeling index was 21.0±2.4% in LRAT−/− normal tongue epithelium as compared to 9.9±0.8% in Wt (p<0.001). Thus, partial retinol deficiency during carcinogen treatment (achieved in LRAT−/−) resulted in more proliferating cells in tongue epithelia from LRAT−/− mice and ultimately a greater probability of carcinogenesis. PMID:19954945

  10. PARP1 Gene Knock-Out Increases Resistance to Retinal Degeneration without Affecting Retinal Function

    PubMed Central

    Sahaboglu, Ayse; Tanimoto, Naoyuki; Kaur, Jasvir; Sancho-Pelluz, Javier; Huber, Gesine; Fahl, Edda; Arango-Gonzalez, Blanca; Zrenner, Eberhart; Ekström, Per; Löwenheim, Hubert; Seeliger, Mathias; Paquet-Durand, François

    2010-01-01

    Retinitis pigmentosa (RP) is a group of inherited neurodegenerative diseases affecting photoreceptors and causing blindness in humans. Previously, excessive activation of enzymes belonging to the poly-ADP-ribose polymerase (PARP) group was shown to be involved in photoreceptor degeneration in the human homologous rd1 mouse model for RP. Since there are at least 16 different PARP isoforms, we investigated the exact relevance of the predominant isoform - PARP1 - for photoreceptor cell death using PARP1 knock-out (KO) mice. In vivo and ex vivo morphological analysis using optic coherence tomography (OCT) and conventional histology revealed no major alterations of retinal phenotype when compared to wild-type (wt). Likewise, retinal function as assessed by electroretinography (ERG) was normal in PARP1 KO animals. We then used retinal explant cultures derived from wt, rd1, and PARP1 KO animals to test their susceptibility to chemically induced photoreceptor degeneration. Since photoreceptor degeneration in the rd1 retina is triggered by a loss-of-function in phosphodiesterase-6 (PDE6), we used selective PDE6 inhibition to emulate the rd1 situation on non-rd1 genotypes. While wt retina subjected to PDE6 inhibition showed massive photoreceptor degeneration comparable to rd1 retina, in the PARP1 KO situation, cell death was robustly reduced. Together, these findings demonstrate that PARP1 activity is in principle dispensable for normal retinal function, but is of major importance for photoreceptor degeneration under pathological conditions. Moreover, our results suggest that PARP dependent cell death or PARthanatos may play a major role in retinal degeneration and highlight the possibility to use specific PARP inhibitors for the treatment of RP. PMID:21124852

  11. 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

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

    PubMed

    Chen, Yanyan; Xu, Yuanyuan; Zheng, Hongzhi; Fu, Jingqi; Hou, Yongyong; Wang, Huihui; Zhang, Qiang; Yamamoto, Masayuki; Pi, Jingbo

    2016-09-01

    Nuclear factor E2-related factor 2 (NRF2) and uncoupling protein 2 (UCP2) are indicated to protect from oxidative stress. They also play roles in the homeostasis of glutathione. However, the detailed mechanisms are not well understood. In the present study, we found Nrf2-knockout (Nrf2-KO) mice exhibited altered glutathione homeostasis and reduced expression of various genes involved in GSH biosynthesis, regeneration, utilization and transport in the liver. Ucp2-knockout (Ucp2-KO) mice exhibited altered glutathione homeostasis in the liver, spleen and blood, as well as increased transcript of cystic fibrosis transmembrane conductance regulator in the liver, a protein capable of mediating glutathione efflux. Nrf2-Ucp2-double knockout (DKO) mice showed characteristics of both Nrf2-KO and Ucp2-KO mice. But no significant difference was observed in DKO mice when compared with Nrf2-KO or Ucp2-KO mice, except in blood glutathione levels. These data suggest that ablation of Nrf2 and Ucp2 leads to disrupted GSH balance, which could result from altered expression of genes involved in GSH metabolism. DKO may not evoke more severe oxidative stress than the single gene knockout. PMID:27453341

  13. malT knockout mutation invokes a stringent type gene-expression profile in Actinobacillus pleuropneumoniae in bronchoalveolar fluid

    PubMed Central

    2009-01-01

    Background Actinobacillus pleuropneumoniae causes contagious pleuropneumonia, an economically important disease of commercially reared pigs throughout the world. To cause this disease, A. pleuropneumoniae must rapidly overcome porcine pulmonary innate immune defenses. Since bronchoalveolar fluid (BALF) contains many of the innate immune and other components found in the lungs, we examined the gene expression of a virulent serovar 1 strain of A. pleuropneumoniae after exposure to concentrated BALF for 30 min. Results In reverse transcription PCR differential display (RT-PCR DD) experiments, A. pleuropneumoniae CM5 exposed to BALF up-regulated, among other genes, a gene predicted to encode LamB, an outer-membrane transport protein of the maltose regulon. To determine the role of the lamB and other genes of the maltose regulon in the pathogenesis of A. pleuropneumoniae, knockout mutations were created in the lamB and malT genes, the latter being the positive transcriptional regulator of the maltose regulon. Relative to the lamB mutant and the wild type, the malT mutant had a significant (P < 0.05) decrease in growth rate and an increased sensitivity to fresh porcine serum and high concentrations (more than 0.5 M) of sodium chloride. In DNA microarray experiments, the BALF-exposed malT mutant exhibited a gene-expression profile resembling that of a stringent type gene-expression profile seen in bacteria facing amino acid or carbon starvation. Genes encoding proteins for protein synthesis, energy metabolism, and DNA replication were down-regulated, while genes involved in stringent response (e.g., relA), amino acid and nucleotide biosynthesis, biofilm formation, DNA transformation, and stress response were up-regulated. Conclusion These results suggest that MalT may be involved in protection against some stressors and in the transport of one or more essential nutrients in BALF. Moreover, if MalT is directly or indirectly linked to the stringent response, an important

  14. Knockout of the TauT Gene Predisposes C57BL/6 Mice to Streptozotocin-Induced Diabetic Nephropathy

    PubMed Central

    Han, Xiaobin; Patters, Andrea B.; Ito, Takashi; Schaffer, Stephen W.; Chesney, Russell W.

    2015-01-01

    Diabetic nephropathy is the leading cause of end stage renal disease in the world. Although tremendous efforts have been made, scientists have yet to identify an ideal animal model that can reproduce the characteristics of human diabetic nephropathy. In this study, we hypothesize that taurine insufficiency is a critical risk factor for development of diabetic nephropathy associated with diabetes mellitus. This hypothesis was tested in vivo in TauT heterozygous (TauT+/-) and homozygous (TauT-/-) knockout in C57BL/6 background mice. We have shown that alteration of the TauT gene (also known as SLC6A6) has a substantial effect on the susceptibility to development of extensive diabetic kidney disease in both TauT+/- and TauT-/-mouse models of diabetes. These animals developed histological changes characteristic of human diabetic nephropathy that included glomerulosclerosis, nodular lesions, arteriosclerosis, arteriolar dilation, and tubulointerstitial fibrosis. Immunohistochemical staining of molecular markers of smooth muscle actin, CD34, Ki67 and collagen IV further confirmed these observations. Our results demonstrated that both homozygous and heterozygous TauT gene deletion predispose C57BL/6 mice to develop end-stage diabetic kidney disease, which closely replicates the pathological features of diabetic nephropathy in human diabetic patients. PMID:25629817

  15. Sub-optimal phenotypes of double-knockout mutants of Escherichia coli depend on the order of gene deletions.

    PubMed

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

    2015-08-01

    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 the aim of investigating the sub-optimal physiology of the mutants and the possible roles of latent reactions. Specifically, we investigated the effects of the 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 when no other latent reactions are concurrently inactivated. 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

  16. 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

  17. Engineering Clostridium beijerinckii with the Cbei_4693 gene knockout for enhanced ferulic acid tolerance.

    PubMed

    Liu, Jun; Guo, Ting; Shen, Xiaoning; Xu, Jiahui; Wang, Junzhi; Wang, Yanyan; Liu, Dong; Niu, Huanqing; Liang, Lei; Ying, Hanjie

    2016-07-10

    A mutant strain of Clostridium beijerinckii NCIMB 8052, C. beijerinckii M11, which exhibited ferulic acid tolerance up to 0.9g/L, was generated using atmospheric pressure glow discharge and high-throughput screening. Comparative genomic analysis revealed that this strain harbored a mutation of the Cbei_4693 gene, which encodes a hypothetical protein suspected to be an NADPH-dependent FMN reductase. After disrupting the Cbei_4693 gene in C. beijerinckii NCIMB 8052 using the ClosTron group II intron-based gene inactivation system, we obtained the Cbei_4693 gene inactivated mutant strain, C. beijerinckii 4693::int. Compared with C. beijerinckii NCIMB 8052, 6.23g/L of butanol was produced in P2 medium containing 0.5g/L of ferulic acid by 4693::int, and the ferulic acid tolerance was also significantly increased up to 0.8g/L. These data showed, for the first time, that the Cbei_4693 gene plays an important role in regulating ferulic acid tolerance in ABE fermentation by C. beijerinckii. PMID:27164255

  18. 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

  19. Systematic screening of glycosylation- and trafficking-associated gene knockouts in Saccharomyces cerevisiae identifies mutants with improved heterologous exocellulase activity and host secretion

    PubMed Central

    2013-01-01

    Background As a strong fermentator, Saccharomyces cerevisiae has the potential to be an excellent host for ethanol production by consolidated bioprocessing. For this purpose, it is necessary to transform cellulose genes into the yeast genome because it contains no cellulose genes. However, heterologous protein expression in S. cerevisiae often suffers from hyper-glycosylation and/or poor secretion. Thus, there is a need to genetically engineer the yeast to reduce its glycosylation strength and to increase its secretion ability. Results Saccharomyces cerevisiae gene-knockout strains were screened for improved extracellular activity of a recombinant exocellulase (PCX) from the cellulose digesting fungus Phanerochaete chrysosporium. Knockout mutants of 47 glycosylation-related genes and 10 protein-trafficking-related genes were transformed with a PCX expression construct and screened for extracellular cellulase activity. Twelve of the screened mutants were found to have a more than 2-fold increase in extracellular PCX activity in comparison with the wild type. The extracellular PCX activities in the glycosylation-related mnn10 and pmt5 null mutants were, respectively, 6 and 4 times higher than that of the wild type; and the extracellular PCX activities in 9 protein-trafficking-related mutants, especially in the chc1, clc1 and vps21 null mutants, were at least 1.5 times higher than the parental strains. Site-directed mutagenesis studies further revealed that the degree of N-glycosylation also plays an important role in heterologous cellulase activity in S. cerevisiae. Conclusions Systematic screening of knockout mutants of glycosylation- and protein trafficking-associated genes in S. cerevisiae revealed that: (1) blocking Golgi-to-endosome transport may force S. cerevisiae to export cellulases; and (2) both over- and under-glycosylation may alter the enzyme activity of cellulases. This systematic gene-knockout screening approach may serve as a convenient means for

  20. Heritable gene knockout in Caenorhabditis elegans by direct injection of Cas9-sgRNA ribonucleoproteins.

    PubMed

    Cho, Seung Woo; Lee, Jihyun; Carroll, Dana; Kim, Jin-Soo; Lee, Junho

    2013-11-01

    We present a novel method of targeted gene disruption that involves direct injection of recombinant Cas9 protein complexed with guide RNA into the gonad of the nematode Caenorhabditis elegans. Biallelic mutants were recovered among the F1 progeny, demonstrating the high efficiency of this method. PMID:23979576

  1. 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. PMID:24969022

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

    PubMed

    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

    2015-07-01

    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

  3. 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

  4. Calbindin Knockout Alters Sex-Specific Regulation of Behavior and Gene Expression in Amygdala and Prefrontal Cortex.

    PubMed

    Harris, Erin P; Abel, Jean M; Tejada, Lucia D; Rissman, Emilie F

    2016-05-01

    Calbindin-D(28K) (Calb1), a high-affinity calcium buffer/sensor, shows abundant expression in neurons and has been associated with a number of neurobehavioral diseases, many of which are sexually dimorphic in incidence. Behavioral and physiological end points are affected by experimental manipulations of calbindin levels, including disruption of spatial learning, hippocampal long-term potentiation, and circadian rhythms. In this study, we investigated novel aspects of calbindin function on social behavior, anxiety-like behavior, and fear conditioning in adult mice of both sexes by comparing wild-type to littermate Calb1 KO mice. Because Calb1 mRNA and protein are sexually dimorphic in some areas of the brain, we hypothesized that sex differences in behavioral responses of these behaviors would be eliminated or revealed in Calb1 KO mice. We also examined gene expression in the amygdala and prefrontal cortex, two areas of the brain intimately connected with limbic system control of the behaviors tested, in response to sex and genotype. Our results demonstrate that fear memory and social behavior are altered in male knockout mice, and Calb1 KO mice of both sexes show less anxiety. Moreover, gene expression studies of the amygdala and prefrontal cortex revealed several significant genotype and sex effects in genes related to brain-derived neurotrophic factor signaling, hormone receptors, histone deacetylases, and γ-aminobutyric acid signaling. Our findings are the first to directly link calbindin with affective and social behaviors in rodents; moreover, the results suggest that sex differences in calbindin protein influence behavior. PMID:27010449

  5. Type II Toxoplasma gondii KU80 knockout strains enable functional analysis of genes required for cyst development and latent infection.

    PubMed

    Fox, Barbara A; Falla, Alejandra; Rommereim, Leah M; Tomita, Tadakimi; Gigley, Jason P; Mercier, Corinne; Cesbron-Delauw, Marie-France; Weiss, Louis M; Bzik, David J

    2011-09-01

    Type II Toxoplasma gondii KU80 knockouts (Δku80) deficient in nonhomologous end joining were developed to delete the dominant pathway mediating random integration of targeting episomes. Gene targeting frequency in the type II Δku80 Δhxgprt strain measured at the orotate (OPRT) and the uracil (UPRT) phosphoribosyltransferase loci was highly efficient. To assess the potential of the type II Δku80 Δhxgprt strain to examine gene function affecting cyst biology and latent stages of infection, we targeted the deletion of four parasite antigen genes (GRA4, GRA6, ROP7, and tgd057) that encode characterized CD8(+) T cell epitopes that elicit corresponding antigen-specific CD8(+) T cell populations associated with control of infection. Cyst development in these type II mutant strains was not found to be strictly dependent on antigen-specific CD8(+) T cell host responses. In contrast, a significant biological role was revealed for the dense granule proteins GRA4 and GRA6 in cyst development since brain tissue cyst burdens were drastically reduced specifically in mutant strains with GRA4 and/or GRA6 deleted. Complementation of the Δgra4 and Δgra6 mutant strains using a functional allele of the deleted GRA coding region placed under the control of the endogenous UPRT locus was found to significantly restore brain cyst burdens. These results reveal that GRA proteins play a functional role in establishing cyst burdens and latent infection. Collectively, our results suggest that a type II Δku80 Δhxgprt genetic background enables a higher-throughput functional analysis of the parasite genome to reveal fundamental aspects of parasite biology controlling virulence, pathogenesis, and transmission. PMID:21531875

  6. The effect of pyruvate decarboxylase gene knockout in Saccharomyces cerevisiae on L-lactic acid production.

    PubMed

    Ishida, Nobuhiro; Saitoh, Satoshi; Onishi, Toru; Tokuhiro, Kenro; Nagamori, Eiji; Kitamoto, Katsuhiko; Takahashi, Haruo

    2006-05-01

    A plant- and crop-based renewable plastic, poly-lactic acid (PLA), is receiving attention as a new material for a sustainable society in place of petroleum-based plastics. We constructed a metabolically engineered Saccharomyces cerevisiae that has both pyruvate decarboxylase genes (PDC1 and PDC5) disrupted in the genetic background to express two copies of the bovine L-lactate dehydrogenase (LDH) gene. With this recombinant, the yield of lactate was 82.3 g/liter, up to 81.5% of the glucose being transformed into lactic acid on neutralizing cultivation, although pdc1 pdc5 double disruption led to ineffective decreases in cell growth and fermentation speed. This strain showed lactate productivity improvement as much as 1.5 times higher than the previous strain. This production yield is the highest value for a lactic acid-producing yeast yet reported. PMID:16717415

  7. 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. PMID:22841313

  8. UVA Phototransduction Drives Early Melanin Synthesis in Human Melanocytes

    PubMed Central

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

    2013-01-01

    Summary Exposure of human skin to solar ultraviolet radiation (UVR), a powerful carcinogen [1] comprising ~95% UVA and ~5% 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 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 five-fold after 24 hours. 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. PMID:22055294

  9. Regulation of Mammalian Cone Phototransduction by Recoverin and Rhodopsin Kinase*

    PubMed Central

    Sakurai, Keisuke; Chen, Jeannie; Khani, Shahrokh C.; Kefalov, Vladimir J.

    2015-01-01

    Cone photoreceptors function under daylight conditions and are essential for color perception and vision with high temporal and spatial resolution. A remarkable feature of cones is that, unlike rods, they remain responsive in bright light. In rods, light triggers a decline in intracellular calcium, which exerts a well studied negative feedback on phototransduction that includes calcium-dependent inhibition of rhodopsin kinase (GRK1) by recoverin. Rods and cones share the same isoforms of recoverin and GRK1, and photoactivation also triggers a calcium decline in cones. However, the molecular mechanisms by which calcium exerts negative feedback on cone phototransduction through recoverin and GRK1 are not well understood. Here, we examined this question using mice expressing various levels of GRK1 or lacking recoverin. We show that although GRK1 is required for the timely inactivation of mouse cone photoresponse, gradually increasing its expression progressively delays the cone response recovery. This surprising result is in contrast with the known effect of increasing GRK1 expression in rods. Notably, the kinetics of cone responses converge and become independent of GRK1 levels for flashes activating more than ∼1% of cone pigment. Thus, mouse cone response recovery in bright light is independent of pigment phosphorylation and likely reflects the spontaneous decay of photoactivated visual pigment. We also find that recoverin potentiates the sensitivity of cones in dim light conditions but does not contribute to their capacity to function in bright light. PMID:25673692

  10. 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

  11. 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

  12. Heavy-ion-induced mutations in the gpt delta transgenic mouse: effect of p53 gene knockout.

    PubMed

    Yatagai, Fumio; Kurobe, Toshihiro; Nohmi, Takehiko; Masumura, Ken-ichi; Tsukada, Teruyo; Yamaguchi, Hirotake; Kasai-Eguchi, Kiyomi; Fukunishi, Nobuhisa

    2002-01-01

    The influence of the loss of p53 gene on heavy-ion-induced mutations was examined by constructing a new line of transgenic mice, p53 knockout (p53(-/-)) gpt delta. In this mouse model, deletions in lambda DNA integrated into the mouse genome are preferentially selected as Spi(-) phages, which can then be subjected to molecular analysis. Mice were exposed to 10 Gy of whole-body carbon-ion irradiation. The carbon ions were accelerated to 135 MeV/u by the RIKEN Ring Cyclotron. The p53 defect markedly enhanced the Spi(-) mutant frequency (MF) in the kidneys of mice exposed to C-ion irradiation: the Spi(-) MF increased 4.4- and 2.8-fold over the background level after irradiation in p53(-/-) and p53(+/+) mice, respectively. There was no significant difference in the background Spi(-) MF between p53(-/-) and p53(+/+) mice. Sequence analysis of the Spi(-) mutants indicated that the enhancement of kidney Spi(-) MF in p53(-/-) mice was primarily due to an increase in complex or rearranged-type deletions. In contrast to the kidney, the p53 defect had no effect on the Spi(-) MF in liver: Spi(-) MF increased 3.0- and 2.7-fold after the irradiation in p53(-/-) and p53(+/+) mice, respectively. Our results suggest that p53 suppresses deletion mutations induced by heavy-ion irradiation in an organ-specific manner. PMID:12355556

  13. Antibiotic Sensitivity Profiles Determined with an Escherichia coli Gene Knockout Collection: Generating an Antibiotic Bar Code ▿ †

    PubMed Central

    Liu, Anne; Tran, Lillian; Becket, Elinne; Lee, Kim; Chinn, Laney; Park, Eunice; Tran, Katherine; Miller, Jeffrey H.

    2010-01-01

    We have defined a sensitivity profile for 22 antibiotics by extending previous work testing the entire KEIO collection of close to 4,000 single-gene knockouts in Escherichia coli for increased susceptibility to 1 of 14 different antibiotics (ciprofloxacin, rifampin [rifampicin], vancomycin, ampicillin, sulfamethoxazole, gentamicin, metronidazole, streptomycin, fusidic acid, tetracycline, chloramphenicol, nitrofurantoin, erythromycin, and triclosan). We screened one or more subinhibitory concentrations of each antibiotic, generating more than 80,000 data points and allowing a reduction of the entire collection to a set of 283 strains that display significantly increased sensitivity to at least one of the antibiotics. We used this reduced set of strains to determine a profile for eight additional antibiotics (spectinomycin, cephradine, aztreonem, colistin, neomycin, enoxacin, tobramycin, and cefoxitin). The profiles for the 22 antibiotics represent a growing catalog of sensitivity fingerprints that can be separated into two components, multidrug-resistant mutants and those mutants that confer relatively specific sensitivity to the antibiotic or type of antibiotic tested. The latter group can be represented by a set of 20 to 60 strains that can be used for the rapid typing of antibiotics by generating a virtual bar code readout of the specific sensitivities. Taken together, these data reveal the complexity of intrinsic resistance and provide additional targets for the design of codrugs (or combinations of drugs) that potentiate existing antibiotics. PMID:20065048

  14. Recovery of NV Knockout Infectious Hematopoietic Necrosis Virus Expressing Foreign Genes

    PubMed Central

    Biacchesi, Stéphane; Thoulouze, Maria-Isabel; Béarzotti, Monique; Yu, Yan-Xing; Brémont, Michel

    2000-01-01

    Infectious hematopoietic necrosis virus (IHNV) is a Novirhabdovirus and is the causative agent of a devastating acute, lethal disease in wild and farmed rainbow trout. The virus is enzootic throughout western North America and has spread to Asia and Europe. A full-length cDNA of the IHNV antigenome (pIHNV-Pst) was assembled from subgenomic overlapping cDNA fragments and cloned in a transcription plasmid between the T7 RNA polymerase promoter and the autocatalytic hepatitis delta virus ribozyme. Recombinant IHNV (rIHNV) was recovered from fish cells at 14°C, following infection with a recombinant vaccinia virus expressing the T7 RNA polymerase (vTF7-3) and cotransfection of pIHNV-Pst together with plasmids encoding the nucleoprotein N (pT7-N), the phosphoprotein P (pT7-P), the RNA polymerase L (pT7-L), and the nonvirion protein NV (pT7-NV). When pT7-N and pT7-NV were omitted, rIHNV was also recovered, although less efficiently. Incidental mutations introduced in pIHNV-Pst were all present in the rIHNV genome; however, a targeted mutation located in the L gene was eliminated from the recombinant genome by homologous recombination with the added pT7-L expression plasmid. To investigate the role of NV protein in virus replication, the pIHNV-Pst construct was engineered such that the entire NV open reading frame was deleted and replaced by the genes encoding green fluorescent protein or chloramphenicol acetyltransferase. The successful recovery of recombinant virus expressing foreign genes instead of the NV gene demonstrated that the NV protein was not absolutely required for viral replication in cell cultures, although its presence greatly improves virus growth. The ability to generate rIHNV from cDNA provides the basis to manipulate the genome in order to engineer new live viral vaccine strains. PMID:11070023

  15. Identification of gene knockout strategies using a hybrid of an ant colony optimization algorithm and flux balance analysis to optimize microbial strains.

    PubMed

    Lu, Shi Jing; Salleh, Abdul Hakim Mohamed; Mohamad, Mohd Saberi; Deris, Safaai; Omatu, Sigeru; Yoshioka, Michifumi

    2014-09-28

    Reconstructions of genome-scale metabolic networks from different organisms have become popular in recent years. Metabolic engineering can simulate the reconstruction process to obtain desirable phenotypes. In previous studies, optimization algorithms have been implemented to identify the near-optimal sets of knockout genes for improving metabolite production. However, previous works contained premature convergence and the stop criteria were not clear for each case. Therefore, this study proposes an algorithm that is a hybrid of the ant colony optimization algorithm and flux balance analysis (ACOFBA) to predict near optimal sets of gene knockouts in an effort to maximize growth rates and the production of certain metabolites. Here, we present a case study that uses Baker's yeast, also known as Saccharomyces cerevisiae, as the model organism and target the rate of vanillin production for optimization. The results of this study are the growth rate of the model organism after gene deletion and a list of knockout genes. The ACOFBA algorithm was found to improve the yield of vanillin in terms of growth rate and production compared with the previous algorithms. PMID:25462325

  16. Lycopene production in recombinant strains of Escherichia coli is improved by knockout of the central carbon metabolism gene coding for glucose-6-phosphate dehydrogenase.

    PubMed

    Zhou, Yan; Nambou, Komi; Wei, Liujing; Cao, Jingjing; Imanaka, Tadayuki; Hua, Qiang

    2013-12-01

    Genetic manipulation was undertaken in order to understand the mechanism involved in the heterologous synthesis of lycopene in Escherichia coli. Knockout of the central carbon metabolic gene zwf (glucose-6-phosphate dehydrogenase) resulted in the enhancement of lycopene production (above 130 % relative to control). The amplification and overexpression of rate-limiting steps encoded by idi (isopentenyl diphosphate isomerase), dxs (1-deoxyxylulose-5-phosphate synthase) and ispDF (4-diphosphocytidyl-2C-methyl-D-erythritol synthase and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase) genes improved lycopene synthesis from 0.89 to 5.39 mg g(-1) DCW. The combination of central metabolic genes knockout with the amplification of MEP pathway genes yielded best amounts of lycopene (6.85-7.55 mg g(-1) DCW). Transcript profiling revealed that idi and dxs were up-regulated in the zwf knock-out strain, providing a plausible explanation for the increase in lycopene yield observed in this strain. An increase in precursor availability might also have contributed to the improved lycopene production. PMID:24062132

  17. The kiss/kissr Systems Are Dispensable for Zebrafish Reproduction: Evidence From Gene Knockout Studies

    PubMed Central

    Tang, Haipei; Liu, Yun; Luo, Daji; Ogawa, Satoshi; Yin, Yike; Li, Shuisheng; Zhang, Yong; Hu, Wei; Parhar, Ishwar S.; Lin, Haoran

    2015-01-01

    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. PMID:25406015

  18. Identification of α1L-adrenoceptor in mice and its abolition by α1A-adrenoceptor gene knockout

    PubMed Central

    Muramatsu, I; Morishima, S; Suzuki, F; Yoshiki, H; Anisuzzaman, A S M; Tanaka, T; Rodrigo, M C; Myagmar, B E; Simpson, P C

    2008-01-01

    Background and purpose: The α1L-adrenoceptor has pharmacological properties that distinguish it from three classical α1-adrenoceptors (α1A, α1B and α1D). The purpose of this was to identify α1L-adrenoceptors in mice and to examine their relationship to classical α1-adrenoceptors. Experimental approach: Radioligand binding and functional bioassay experiments were performed on the cerebral cortex, vas deferens and prostate of wild-type (WT) and α1A-, α1B- and α1D-adrenoceptor gene knockout (AKO, BKO and DKO) mice. Key results: The radioligand [3H]-silodosin bound to intact segments of the cerebral cortex, vas deferens and prostate of WT, BKO and DKO but not of AKO mice. The binding sites were composed of two components with high and low affinities for prazosin or RS-17053, indicating the pharmacological profiles of α1A-adrenoceptors and α1L-adrenoceptors. In membrane preparations of WT mouse cortex, however, [3H]-silodosin bound to a single population of prazosin high-affinity sites, suggesting the presence of α1A-adrenoceptors alone. In contrast, [3H]-prazosin bound to two components having α1A-adrenoceptor and α1B-adrenoceptor profiles in intact segments of WT and DKO mouse cortices, but AKO mice lacked α1A-adrenoceptor profiles and BKO mice lacked α1B-adrenoceptor profiles. Noradrenaline produced contractions through α1L-adrenoceptors with low affinity for prazosin in the vas deferens and prostate of WT, BKO and DKO mice. However, the contractions were abolished or markedly attenuated in AKO mice. Conclusions and implications: α1L-Adrenoceptors were identified as binding and functional entities in WT, BKO and DKO mice but not in AKO mice, suggesting that the α1L-adrenoceptor is one phenotype derived from the α1A-adrenoceptor gene. PMID:18806813

  19. Experimental evolution and gene knockout studies reveal AcrA-mediated isobutanol tolerance in Ralstonia eutropha.

    PubMed

    Bernardi, Amanda C; Gai, Claudia S; Lu, Jingnan; Sinskey, Anthony J; Brigham, Christopher J

    2016-07-01

    Isobutanol (IBT) has attracted much attention from researchers as a next generation drop-in biofuel. Ralstonia eutropha is a gram-negative bacterium which naturally produces polyhydroxybutyrate (PHB), and has been reported to produce IBT after metabolic engineering. Similar to other microbes, R. eutropha experiences toxicity from branched-chain alcohols and is unable to grow in the presence of IBT concentrations higher than 0.5% (v v(-1)). Such low tolerance greatly limits the ability of R. eutropha to grow and produce IBT. In order to study toxicity to the cells, IBT-tolerant strains were developed by experimental evolution, revealing that two genes, previously described as being related to IBT tolerance in Escherichia coli (acrA and acrA6), also presented mutations in R. eutropha evolved strains. The effect on the physiology of the cells of in-frame deletions of each of these genes was assessed in wild type and engineered IBT-producing strains in an attempt to reproduce a tolerant phenotype. The mutant strains' ability to tolerate, consume, and produce IBT were also analyzed. Although deletions of acrA6 and acrA did not significantly improve R. eutropha growth in the presence of IBT, these deletions improved cell survival in the presence of high concentrations of IBT in the extracellular milieu. Moreover, an in-frame acrA deletion in an engineered IBT-producing R. eutropha enhanced the strain's ability to produce IBT, which could potentially be associated with enhanced survival at high IBT concentrations. PMID:26811221

  20. The ability to solve elementary logic tasks in mice with the knockout of sodium-calcium exchanger gene 2 (NCX2).

    PubMed

    Poletaeva, I I; Perepelkina, O V; Boyarshinova, O S; Golibrodo, V A; Lilp, I G; Lipp, H-P; Shin, Hee Sup

    2016-07-01

    Mice with a knockout of the sodium-calcium exchanger 2 (NCX2) gene were statistically significantly more successful than wild-type controls in the solution of two cognitive tasks, the test for the capacity to extrapolate the direction of the stimulus movement and the "puzzle-box" test for the capacity to find a hidden route to safe environment, which were based on food and aversive motivations, respectively. In both tests, the success of task solution was based on the animal's ability to use the object's "permanence" rule (according to J. Piaget). The data confirm that the knockout of this gene, which is accompanied by modulation of the temporal pattern of calcium membrane flux, also induces changes in mouse CNS plasticity. PMID:27595821

  1. Systematic Analysis of Zn2Cys6 Transcription Factors Required for Development and Pathogenicity by High-Throughput Gene Knockout in the Rice Blast Fungus

    PubMed Central

    Huang, Pengyun; Lin, Fucheng

    2014-01-01

    Because of great challenges and workload in deleting genes on a large scale, the functions of most genes in pathogenic fungi are still unclear. In this study, we developed a high-throughput gene knockout system using a novel yeast-Escherichia-Agrobacterium shuttle vector, pKO1B, in the rice blast fungus Magnaporthe oryzae. Using this method, we deleted 104 fungal-specific Zn2Cys6 transcription factor (TF) genes in M. oryzae. We then analyzed the phenotypes of these mutants with regard to growth, asexual and infection-related development, pathogenesis, and 9 abiotic stresses. The resulting data provide new insights into how this rice pathogen of global significance regulates important traits in the infection cycle through Zn2Cys6TF genes. A large variation in biological functions of Zn2Cys6TF genes was observed under the conditions tested. Sixty-one of 104 Zn2Cys6 TF genes were found to be required for fungal development. In-depth analysis of TF genes revealed that TF genes involved in pathogenicity frequently tend to function in multiple development stages, and disclosed many highly conserved but unidentified functional TF genes of importance in the fungal kingdom. We further found that the virulence-required TF genes GPF1 and CNF2 have similar regulation mechanisms in the gene expression involved in pathogenicity. These experimental validations clearly demonstrated the value of a high-throughput gene knockout system in understanding the biological functions of genes on a genome scale in fungi, and provided a solid foundation for elucidating the gene expression network that regulates the development and pathogenicity of M. oryzae. PMID:25299517

  2. 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. PMID:24841241

  3. Knockout of ERK1 enhances cocaine-evoked immediate early gene expression and behavioral plasticity.

    PubMed

    Ferguson, Susan M; Fasano, Stefania; Yang, Pengwei; Brambilla, Riccardo; Robinson, Terry E

    2006-12-01

    The ability of cocaine to produce lasting neural adaptations in mesocorticolimbic brain regions is thought to promote drug seeking and facilitate addiction in humans. The Ras-controlled Raf-MEK-ERK protein kinase signaling cascade has been implicated in the behavioral and neurobiological actions of cocaine in animals. However, these pharmacological studies have not been able to determine the specific role of the two predominant isoforms of ERK (ERK1 and ERK2) in these processes. We report here that deletion of the ERK1 isoform, which leads to increased ERK2 stimulus-dependent signaling, facilitates the development of cocaine-induced psychomotor sensitization and the acquisition of a cocaine conditioned place preference. Conversely, pharmacological blockade of ERK signaling attenuates the development of psychomotor sensitization to cocaine. Finally, cocaine-evoked gene expression in mesocorticolimbic brain regions is potentiated in ERK1-deficient mice. Thus, alterations in ERK signaling influence both the neurobiological impact of cocaine and its ability to produce enduring forms of drug experience-dependent behavioral plasticity. Our results suggest that enhanced ERK2 signaling following repeated drug exposure may facilitate the development of forms of cocaine-induced plasticity that contribute to addiction. PMID:16407894

  4. Systematic analysis of palatal transcriptome to identify cleft palate genes within TGFβ3-knockout mice alleles: RNA-Seq analysis of TGFβ3 Mice

    PubMed Central

    2013-01-01

    Background In humans, cleft palate (CP) accounts for one of the largest number of birth defects with a complex genetic and environmental etiology. TGFβ3 has been established as an important regulator of palatal fusion in mice and it has been shown that TGFβ3-null mice exhibit CP without any other major deformities. However, the genes that regulate cellular decisions and molecular mechanisms maintained by the TGFβ3 pathway throughout palatogenesis are predominantly unexplored. Our objective in this study was to analyze global transcriptome changes within the palate during different gestational ages within TGFβ3 knockout mice to identify TGFβ3-associated genes previously unknown to be associated with the development of cleft palate. We used deep sequencing technology, RNA-Seq, to analyze the transcriptome of TGFβ3 knockout mice at crucial stages of palatogenesis, including palatal growth (E14.5), adhesion (E15.5), and fusion (E16.5). Results The overall transcriptome analysis of TGFβ3 wildtype mice (C57BL/6) reveals that almost 6000 genes were upregulated during the transition from E14.5 to E15.5 and more than 2000 were downregulated from E15.5 to E16.5. Using bioinformatics tools and databases, we identified the most comprehensive list of CP genes (n = 322) in which mutations cause CP either in humans or mice, and analyzed their expression patterns. The expression motifs of CP genes between TGFβ3+/− and TGFβ3−/− were not significantly different from each other, and the expression of the majority of CP genes remained unchanged from E14.5 to E16.5. Using these patterns, we identified 8 unique genes within TGFβ3−/− mice (Chrng, Foxc2, H19, Kcnj13, Lhx8, Meox2, Shh, and Six3), which may function as the primary contributors to the development of cleft palate in TGFβ3−/− mice. When the significantly altered CP genes were overlaid with TGFβ signaling, all of these genes followed the Smad-dependent pathway. Conclusions Our study represents the

  5. Small molecule antagonists of melanopsin-mediated phototransduction

    PubMed Central

    Jones, Kenneth A.; Hatori, Megumi; Mure, Ludovic S.; Bramley, Jayne R.; Artymyshyn, Roman; Hong, Sang-Phyo; Marzabadi, Mohammad; Zhong, Huailing; Sprouse, Jeffrey; Zhu, Quansheng; Hartwick, Andrew T.E.; Sollars, Patricia J.; Pickard, Gary E.; Panda, Satchidananda

    2013-01-01

    Melanopsin, expressed in a subset of retinal ganglion cells, mediates behavioral adaptation to ambient light and other non-image forming photic responses. This has raised the possibility that pharmacological manipulation of melanopsin can modulate several CNS responses including photophobia, sleep, circadian rhythms and neuroendocrine function. Here we describe the identification of a potent synthetic melanopsin antagonist with in vivo activity. Novel sulfonamide compounds inhibiting melanopsin (opsinamides) compete with retinal binding to melanopsin and inhibit its function without affecting rod/cone mediated responses. In vivo administration of opsinamides to mice specifically and reversibly modified melanopsin-dependent light responses including the pupillary light reflex and light aversion. The discovery of opsinamides raises the prospect of therapeutic control of the melanopsin phototransduction system to regulate light-dependent behavior and remediate pathological conditions. PMID:23974117

  6. [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. PMID:27349115

  7. Reduction of Inflammatory Bowel Disease-induced Tumor Development in IL-10 Knockout Mice with Soluble Epoxide Hydrolase Gene Deficiency

    PubMed Central

    Zhang, Wanying; Liao, Jie; Li, Haonan; Dong, Hua; Bai, Han; Yang, Allison; Hammock, Bruce D.; Yang, Guang-Yu

    2012-01-01

    Soluble epoxide hydrolase (sEH) quickly inactivates anti-inflammatory epoxyeicosatrienoic acids (EETs) by converting them to dihydroxyeicosatrienoic acids (DHETs). Inhibition of sEH has shown effects against inflammation, but little is studied about the role of sEH in inflammatory bowel disease (IBD) and its induced carcinogenesis. In the present study, the effect of sEH gene deficiency on the development of IBD-induced tumor development was determined in IL-10 knockout mice combined with sEH gene deficiency. Tumor development in the bowel was examined at the age of 25 weeks for male mice and 35 weeks for female mice. Compared to IL-10(−/−) mice, sEH (−/−)/IL-10 (−/−) mice exhibited a significant decrease of tumor multiplicity (2 ± 0.9 vs. 1 ± 0.3 tumors/mouse) and tumor size (344.55±71.73 vs. 126.94±23.18 mm3), as well as a marked decrease of precancerous dysplasia. The significantly lower inflammatory scores were further observed in the bowel in sEH(−/−)/IL-10(−/−) mice as compared to IL-10(−/−) mice, including parameters of inflammation-involved area (0.70±0.16 vs 1.4±0.18), inflammation cell infiltration (1.55±0.35 vs 2.15±0.18), and epithelial hyperplasia (0.95±0.21 vs 1.45±0.18), as well as larger ulcer formation. qPCR and western blotting assays demonstrated a significant down-regulation of cytokines/chemokines (TNFα, MCP1, and IL-12, 17 and 23) and NF-kB signals. Eicosanoid acid metabolic profiling revealed a significant increase of ratios of EETs to DHETs and EpOMEs to DiOMEs. These results indicate that sEH plays an important role in IBD and its-induced carcinogenesis and could serve as a highly potential target of chemoprevention and treatment for IBD. PMID:22517541

  8. Zinc finger nuclease-mediated gene knockout results in loss of transport activity for P-glycoprotein, BCRP, and MRP2 in Caco-2 cells.

    PubMed

    Sampson, Kathleen E; Brinker, Amanda; Pratt, Jennifer; Venkatraman, Neetu; Xiao, Yongling; Blasberg, Jim; Steiner, Toni; Bourner, Maureen; Thompson, David C

    2015-02-01

    Membrane transporters P-glycoprotein [P-gp; multidrug resistance 1 (MDR1)], multidrug resistance-associated protein (MRP) 2, and breast cancer resistance protein (BCRP) affect drug absorption and disposition and can also mediate drug-drug interactions leading to safety/toxicity concerns in the clinic. Challenges arise with interpreting cell-based transporter assays when substrates or inhibitors affect more than one actively expressed transporter and when endogenous or residual transporter activity remains following overexpression or knockdown of a given transporter. The objective of this study was to selectively knock out three drug efflux transporter genes (MDR1, MRP2, and BCRP), both individually as well as in combination, in a subclone of Caco-2 cells (C2BBe1) using zinc finger nuclease technology. The wild-type parent and knockout cell lines were tested for transporter function in Transwell bidirectional assays using probe substrates at 5 or 10 μM for 2 hours at 37°C. P-gp substrates digoxin and erythromycin, BCRP substrates estrone 3-sulfate and nitrofurantoin, and MRP2 substrate 5-(and-6)-carboxy-2',7'-dichlorofluorescein each showed a loss of asymmetric transport in the MDR1, BCRP, and MRP2 knockout cell lines, respectively. Furthermore, transporter interactions were deduced for cimetidine, ranitidine, fexofenadine, and colchicine. Compared with the knockout cell lines, standard transporter inhibitors showed substrate-specific variation in reducing the efflux ratios of the test compounds. These data confirm the generation of a panel of stable Caco-2 cell lines with single or double knockout of human efflux transporter genes and a complete loss of specific transport activity. These cell lines may prove useful in clarifying complex drug-transporter interactions without some of the limitations of current chemical or genetic knockdown approaches. PMID:25388687

  9. Modeling and simulation of the main metabolism in Escherichia coli and its several single-gene knockout mutants with experimental verification

    PubMed Central

    2010-01-01

    Background It is quite important to simulate the metabolic changes of a cell in response to the change in culture environment and/or specific gene knockouts particularly for the purpose of application in industry. If this could be done, the cell design can be made without conducting exhaustive experiments, and one can screen out the promising candidates, proceeded by experimental verification of a select few of particular interest. Although several models have so far been proposed, most of them focus on the specific metabolic pathways. It is preferred to model the whole of the main metabolic pathways in Escherichia coli, allowing for the estimation of energy generation and cell synthesis, based on intracellular fluxes and that may be used to characterize phenotypic growth. Results In the present study, we considered the simulation of the main metabolic pathways such as glycolysis, TCA cycle, pentose phosphate (PP) pathway, and the anapleorotic pathways using enzymatic reaction models of E. coli. Once intracellular fluxes were computed by this model, the specific ATP production rate, the specific CO2 production rate, and the specific NADPH production rate could be estimated. The specific ATP production rate thus computed was used for the estimation of the specific growth rate. The CO2 production rate could be used to estimate cell yield, and the specific NADPH production rate could be used to determine the flux of the oxidative PP pathway. The batch and continuous cultivations were simulated where the changing patterns of extracellular and intra-cellular metabolite concentrations were compared with experimental data. Moreover, the effects of the knockout of such pathways as Ppc, Pck and Pyk on the metabolism were simulated. It was shown to be difficult for the cell to grow in Ppc mutant due to low concentration of OAA, while Pck mutant does not necessarily show this phenomenon. The slower growth rate of the Ppc mutant was properly estimated by taking into account

  10. Identification of transcription factors and single nucleotide polymorphisms of Lrh1 and its homologous genes in Lrh1-knockout pancreas of mice

    PubMed Central

    2014-01-01

    Background To identify transcription factors (TFs) and single nucleotide polymorphisms (SNPs) of Lrh1 (also named Nr5a2) and its homologous genes in Lrh1-knockout pancreas of mice. Methods The RNA-Seq data GSE34030 were downloaded from Gene Expression Omnibus (GEO) database, including 2 Lrh1 pancreas knockout samples and 2 wild type samples. All reads were processed through TopHat and Cufflinks package to calculate gene-expression level. Then, the differentially expressed genes (DEGs) were identified via non-parametric algorithm (NOISeq) methods in R package, of which the homology genes of Lrh1 were identified via BLASTN analysis. Furthermore, the TFs of Lrh1 and its homologous genes were selected based on TRANSFAC database. Additionally, the SNPs were analyzed via SAM tool to record the locations of mutant sites. Results Total 15683 DEGs were identified, of which 23 was Lrh1 homology genes (3 up-regulated and 20 down-regulated). Fetoprotein TF (FTF) was the only TF of Lrh1 identified and the promoter-binding factor of FTF was CYP7A. The SNP annotations of Lrh1 homologous genes showed that 92% of the mutation sites were occurred in intron and upstream. Three SNPs of Lrh1 were located in intron, while 1819 SNPs of Phkb were located in intron and 1343 SNPs were located in the upstream region. Conclusion FTF combined with CYP7A might play an important role in Lrh1 regulated pancreas-specific transcriptional network. Furthermore, the SNPs analysis of Lrh1 and its homology genes provided the candidate mutant sites that might affect the Lrh1-related production and secretion of pancreatic fluid. PMID:24735206

  11. Plastid casein kinase 2 knockout reduces abscisic acid (ABA) sensitivity, thermotolerance, and expression of ABA- and heat-stress-responsive nuclear genes.

    PubMed

    Wang, Yu; Chang, Hongping; Hu, Shuai; Lu, Xiutao; Yuan, Congying; Zhang, Chen; Wang, Ping; Xiao, Wenjun; Xiao, Langtao; Xue, Gang-Ping; Guo, Xinhong

    2014-08-01

    Plastid casein kinase 2 (CK2) is a major Ser/Thr-specific enzyme for protein phosphorylation in the chloroplast stroma and its kinase activity is regulated by redox signals. To understand the role of CK2 phosphorylation of chloroplast proteins in abiotic stress signalling, an Arabidopsis plastid CK2 (CKA4) knockout mutant was investigated in terms of the plant response to abscisic acid (ABA) and heat stress. CKA4 expression was upregulated by ABA and heat treatment. The cka4 mutant showed reduced sensitivity to ABA during seed germination and seedling growth, and increased stomatal aperture and leaf water loss with a slightly reduced leaf ABA level. The cka4 mutant was more sensitive to heat stress than the wild-type Columbia-0. The expression levels of a number of genes in the ABA regulatory network were reduced in the cka4 mutant. Many heat-upregulated genes (heat-shock factors and heat-shock proteins) were also reduced in the cka4 mutant. The cka4 mutant showed reduced expression levels of plastid-encoded RNA polymerase target genes (atpB and psbA). CKA4 knockout mutation also resulted in a reduction in expression of some critical genes (PTM, ABI4, and PRS1) involved in retrograde signalling from the chloroplast to the nucleus. Similar results were observed in mutant plants with the knockout mutation in both CKA4 and CKA3, which encodes a nuclear CK2 α3 subunit. CKA3 expression was not responsive to ABA and heat stress. These results suggest that CKA4 is an enhancing factor in abiotic stress signalling through modulating the expression of some molecular players in retrograde signalling. PMID:24803505

  12. Plastid casein kinase 2 knockout reduces abscisic acid (ABA) sensitivity, thermotolerance, and expression of ABA- and heat-stress-responsive nuclear genes

    PubMed Central

    Wang, Yu; Chang, Hongping; Hu, Shuai; Lu, Xiutao; Yuan, Congying; Zhang, Chen; Wang, Ping; Xiao, Wenjun; Xiao, Langtao; Xue, Gang-Ping; Guo, Xinhong

    2014-01-01

    Plastid casein kinase 2 (CK2) is a major Ser/Thr-specific enzyme for protein phosphorylation in the chloroplast stroma and its kinase activity is regulated by redox signals. To understand the role of CK2 phosphorylation of chloroplast proteins in abiotic stress signalling, an Arabidopsis plastid CK2 (CKA4) knockout mutant was investigated in terms of the plant response to abscisic acid (ABA) and heat stress. CKA4 expression was upregulated by ABA and heat treatment. The cka4 mutant showed reduced sensitivity to ABA during seed germination and seedling growth, and increased stomatal aperture and leaf water loss with a slightly reduced leaf ABA level. The cka4 mutant was more sensitive to heat stress than the wild-type Columbia-0. The expression levels of a number of genes in the ABA regulatory network were reduced in the cka4 mutant. Many heat-upregulated genes (heat-shock factors and heat-shock proteins) were also reduced in the cka4 mutant. The cka4 mutant showed reduced expression levels of plastid-encoded RNA polymerase target genes (atpB and psbA). CKA4 knockout mutation also resulted in a reduction in expression of some critical genes (PTM, ABI4, and PRS1) involved in retrograde signalling from the chloroplast to the nucleus. Similar results were observed in mutant plants with the knockout mutation in both CKA4 and CKA3, which encodes a nuclear CK2 α3 subunit. CKA3 expression was not responsive to ABA and heat stress. These results suggest that CKA4 is an enhancing factor in abiotic stress signalling through modulating the expression of some molecular players in retrograde signalling. PMID:24803505

  13. Rapid construction of multiple sgRNA vectors and knockout of the Arabidopsis IAA2 gene using the CRISPR/Cas9 genomic editing technology.

    PubMed

    Dingyuan, Liu; Ting, Qiu; Xiaohui, Ding; Miaomiao, Li; Muyuan, Zhu; Junhui, Wang

    2016-08-01

    IAA2 is a member of the Aux/IAA auxin responsive gene family in Arabidopsis thaliana. No iaa2 mutant has been reported until now, thus hindering its further mechanistic investigations. The normal genomic editing technology of CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) uses only a single guide RNA (sgRNA) to target one site in a specific gene, and the gene knockout efficiency is not high. Instead, multiple sgRNAs can target multiple sites; therefore, the efficiency may be improved. In the present investigation, we used the golden-gate cloning strategy and two rounds of PCR reactions to combine three sgRNAs in the same entry vector. The final expression vector was obtained by LR reactions with the destination vector containing the Cas9 expression cassette. Four out of the six sgRNAs were effective, and we also obtained a lot of insertion and deletion mutations. Compared with one sgRNA approach, multiple sgRNAs displayed higher gene-knockout efficiency and produced more germ-line mutants. Thus, we established a more rapid and efficient method and generated five mutants for further studies of IAA2 functions. PMID:27531614

  14. Modeling the Role of Incisures in Vertebrate Phototransduction

    PubMed Central

    Caruso, Giovanni; Bisegna, Paolo; Shen, Lixin; Andreucci, Daniele; Hamm, Heidi E.; DiBenedetto, Emmanuele

    2006-01-01

    Phototransduction is mediated by a G-protein-coupled receptor-mediated cascade, activated by light and localized to rod outer segment (ROS) disk membranes, which, in turn, drives a diffusion process of the second messengers cGMP and Ca2+ in the ROS cytosol. This process is hindered by disks—which, however, bear physical cracks, known as incisures, believed to favor the longitudinal diffusion of cGMP and Ca2+. This article is aimed at highlighting the biophysical functional role and significance of incisures, and their effect on the local and global response of the photocurrent. Previous work on this topic regarded the ROS as well stirred in the radial variables, lumped the diffusion mechanism on the longitudinal axis of the ROS, and replaced the cytosolic diffusion coefficients by effective ones, accounting for incisures through their total patent area only. The fully spatially resolved model recently published by our group is a natural tool to take into account other significant details of incisures, including their geometry and distribution. Using mathematical theories of homogenization and concentrated capacity, it is shown here that the complex diffusion process undergone by the second messengers cGMP and Ca2+ in the ROS bearing incisures can be modeled by a family of two-dimensional diffusion processes on the ROS cross sections, glued together by other two-dimensional diffusion processes, accounting for diffusion in the ROS outer shell and in the bladelike regions comprised by the stack of incisures. Based on this mathematical model, a code has been written, capable of incorporating an arbitrary number of incisures and activation sites, with any given arbitrary distribution within the ROS. The code is aimed at being an operational tool to perform numerical experiments of phototransduction, in rods with incisures of different geometry and structure, under a wide spectrum of operating conditions. The simulation results show that incisures have a dual

  15. [Phototransduction mediated by melanopsin in intrinsically photosensitive retinal ganglion cells].

    PubMed

    Domínguez-Solís, Carlos Augusto; Pérez-León, Jorge Alberto

    2015-01-01

    Melanopsin is the most recent photopigment described. As all the other opsins, it attaches in the retina as chromophore. Its amino acid sequence resembles more invertebrate opsins than those of vertebrates. The signal transduction pathway of opsins in vertebrates is based on the coupling to the G protein transducin, triggering a signaling cascade that results in the hyperpolarization of the plasma membrane. On the contrary, the photoreceptors of invertebrates activate the Gq protein pathway, which leads to depolarizing responses. Phototransduction mediated by melanopsin leads to the depolarization of those cells where it is expressed, the intrinsically photosensitive retinal ganglion cells; the cellular messengers and the ion channel type(s) responsible for the cells´ response is still unclear. Studies to elucidate the signaling cascade of melanopsin in heterologous expression systems, in retina and isolated/cultured intrinsically photosensitive retinal ganglion cells, have provided evidence for the involvement of protein Gq and phospholipase C together with the likely participation of an ion channel member of the transient receptor potential-canonical family, a transduction pathway similar to invertebrate photopigments, particularly Drosophila melanogaster. The intrinsically photosensitive retinal ganglion cells are the sole source of retinal inferences to the suprachiasmatic nucleus; thus, clarifying completely the melanopsin signaling pathway will impact the chronobiology field, including the clinical aspects. PMID:26581535

  16. Characterisation of light responses in the retina of mice lacking principle components of rod, cone and melanopsin phototransduction signalling pathways.

    PubMed

    Hughes, Steven; Rodgers, Jessica; Hickey, Doron; Foster, Russell G; Peirson, Stuart N; Hankins, Mark W

    2016-01-01

    Gnat(-/-), Cnga3(-/-), Opn4(-/-) triple knockout (TKO) mice lack essential components of phototransduction signalling pathways present in rods, cones and photosensitive retinal ganglion cells (pRGCs), and are therefore expected to lack all sensitivity to light. However, a number of studies have shown that light responses persist in these mice. In this study we use multielectrode array (MEA) recordings and light-induced c-fos expression to further characterise the light responses of the TKO retina. Small, but robust electroretinogram type responses are routinely detected during MEA recordings, with properties consistent with rod driven responses. Furthermore, a distinctive pattern of light-induced c-fos expression is evident in the TKO retina, with c-fos expression largely restricted to a small subset of amacrine cells that express disabled-1 (Dab1) but lack expression of glycine transporter-1 (GlyT-1). Collectively these data are consistent with the persistence of a novel light sensing pathway in the TKO retina that originates in rod photoreceptors, potentially a rare subset of rods with distinct functional properties, and which is propagated to an atypical subtype of AII amacrine cells. Furthermore, the minimal responses observed following UV light stimulation suggest only a limited role for the non-visual opsin OPN5 in driving excitatory light responses within the mouse retina. PMID:27301998

  17. Characterisation of light responses in the retina of mice lacking principle components of rod, cone and melanopsin phototransduction signalling pathways

    PubMed Central

    Hughes, Steven; Rodgers, Jessica; Hickey, Doron; Foster, Russell G.; Peirson, Stuart N.; Hankins, Mark W.

    2016-01-01

    Gnat−/−, Cnga3−/−, Opn4−/− triple knockout (TKO) mice lack essential components of phototransduction signalling pathways present in rods, cones and photosensitive retinal ganglion cells (pRGCs), and are therefore expected to lack all sensitivity to light. However, a number of studies have shown that light responses persist in these mice. In this study we use multielectrode array (MEA) recordings and light-induced c-fos expression to further characterise the light responses of the TKO retina. Small, but robust electroretinogram type responses are routinely detected during MEA recordings, with properties consistent with rod driven responses. Furthermore, a distinctive pattern of light-induced c-fos expression is evident in the TKO retina, with c-fos expression largely restricted to a small subset of amacrine cells that express disabled-1 (Dab1) but lack expression of glycine transporter-1 (GlyT-1). Collectively these data are consistent with the persistence of a novel light sensing pathway in the TKO retina that originates in rod photoreceptors, potentially a rare subset of rods with distinct functional properties, and which is propagated to an atypical subtype of AII amacrine cells. Furthermore, the minimal responses observed following UV light stimulation suggest only a limited role for the non-visual opsin OPN5 in driving excitatory light responses within the mouse retina. PMID:27301998

  18. Toll-like receptor 4 (Tlr4) knockout rats produced by transcriptional activator-like effector nuclease- (TALEN)-mediated gene inactivation

    PubMed Central

    Ferguson, Carolyn; McKay, Matthew; Harris, R. Adron; Homanics, Gregg E.

    2013-01-01

    Genetically engineered mice are a valuable resource for studies of the behavioral effects of ethanol. However, for some behavioral tests of ethanol action, the rat is a superior model organism. Production of genetically engineered rats has been severely hampered due to technical limitations. Here we utilized a promising new technique for efficient site-specific gene modification to create a novel gene knockout rat line. This approach is based on Transcriptional Activator-Like Effector Nucleases (TALENs). TALENs function in pairs and bind DNA in a sequence-specific manner. Upon binding to the target sequence, a functional nuclease is reconstituted that creates double-stranded breaks in the DNA that are efficiently repaired by non-homologous end joining. This error-prone process often results in deletions of varying lengths at the targeted locus. The toll-like receptor 4 (Tlr4) gene was selected for TALEN-mediated gene inactivation. Tlr4 has been implicated in ethanol-induced neuroinflammation and neurodegeneration, as well as multiple ethanol-induced behavioral effects. To generate Tlr4 knockout rats, a pair of TALEN constructs was created that specifically target Exon 1 immediately downstream of the start of translation. TALEN mRNAs were microinjected into the cytoplasm of one-cell Wistar rat embryos. Of 13 live-born pups that resulted, one harbored a mutation in Exon 1 of Tlr4. The mutated allele consisted of a 13 base-pair deletion that was predicted to create a frameshift mutation after amino acid 25. This founder rat successfully transmitted the mutation to F1 offspring. Heterozygous F1 offspring were interbred to produce homozygous F2 animals. Homozygous mutants expressed the 13-bp deletion in Tlr4 mRNA. In contrast to control rats that produced a robust increase in plasma tumor necrosis factor alpha in response to a lipopolysaccharide challenge, homozygous rats had a markedly attenuated response. Thus, the mutant Tlr4 allele generated by TALEN-mediated gene

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

    PubMed

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

    2016-07-01

    In the cortex of subjects with schizophrenia, expression of glutamic acid decarboxylase 67 (GAD67), the enzyme primarily responsible for cortical GABA synthesis, is reduced in the subset of GABA neurons that express parvalbumin (PV). This GAD67 deficit is accompanied by lower cortical levels of other GABA-associated transcripts, including GABA transporter-1, PV, brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinase B, somatostatin, GABAA receptor α1 subunit, and KCNS3 potassium channel subunit mRNAs. In contrast, messenger RNA (mRNA) levels for glutamic acid decarboxylase 65 (GAD65), another enzyme for GABA synthesis, are not altered. We tested the hypothesis that this pattern of GABA-associated transcript levels is secondary to the GAD67 deficit in PV neurons by analyzing cortical levels of these GABA-associated mRNAs in mice with a PV neuron-specific GAD67 knockout. Using in situ hybridization, we found that none of the examined GABA-associated transcripts had lower cortical expression in the knockout mice. In contrast, PV, BDNF, KCNS3, and GAD65 mRNA levels were higher in the homozygous mice. In addition, our behavioral test battery failed to detect a change in sensorimotor gating or working memory, although the homozygous mice exhibited increased spontaneous activities. These findings suggest that reduced GAD67 expression in PV neurons is not an upstream cause of the lower levels of GABA-associated transcripts, or of the characteristic behaviors, in schizophrenia. In PV neuron-specific GAD67 knockout mice, increased levels of PV, BDNF, and KCNS3 mRNAs might be the consequence of increased neuronal activity secondary to lower GABA synthesis, whereas increased GAD65 mRNA might represent a compensatory response to increase GABA synthesis. PMID:26980143

  20. Alternative splicing directs two IL-20R2 isoforms and is responsible for the incomplete gene knockout via the exon I ablation.

    PubMed

    Zhou, H; Liu, X; Yu, R; Long, T; Zhao, R; Liu, H; Xu, Y; Liang, J G; Liang, P

    2016-06-01

    Two heterodimeric receptors consisting of interleukin (IL)-20R2 are shared by three of the IL-20 family of cytokines, IL-19, IL-20 and IL-24. Along with IL-22, these cytokines are downstream effectors of IL-23 and have been implicated in keratinocyte functions and the pathogenesis of psoriasis. Surprisingly, whereas knocking out either the IL-23 or IL-22 gene abolished imiquimod-induced psoriatic phenotypes in mice, similar attempt for IL-20R2 had little effect. Here, we report that the apparent disparity may result from a new IL-20R2 isoform encoded by an alternatively spliced transcript which survived the previous attempt for IL-20R2 gene knockout via the exon I deletion. PMID:27009487

  1. 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

  2. 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

  3. 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

  4. Comparison of hematologic, biochemical, and coagulation parameters in α1,3-galactosyltransferase gene-knockout pigs, wild-type pigs, and 4 primate species

    PubMed Central

    Ekser, Burcin; Bianchi, John; Ball, Suyapa; Iwase, Hayato; Walters, Anneke; Ezzelarab, Mohamed; Veroux, Massimiliano; Gridelli, Bruno; Wagner, Robert; Ayares, David; Cooper, David K.C.

    2012-01-01

    Background The increasing availability of genetically-engineered pigs is steadily improving the results of pig organ and cell transplantation in nonhuman primates (NHPs). Current techniques offer knock-out of pig genes and/or knock-in of human genes. Knowledge of normal values of hematologic, biochemical, coagulation, and other parameters in healthy genetically-engineered pigs and NHPs is important, particularly following pig organ transplantation in NHPs. Furthermore, information on parameters in various NHP species may prove important in selecting the optimal NHP model for specific studies. Methods We have collected hematologic, biochemical, and coagulation data on 71 α1,3-galactosyltransferase gene-knockout (GTKO) pigs, 18 GTKO pigs additionally transgenic for human CD46 (GTKO.hCD46), 4 GTKO.hCD46 pigs additionally transgenic for human CD55 (GTKO.hCD46.hCD55), and 2 GTKO.hCD46 pigs additionally transgenic for human thrombomodulin (GTKO.hCD46.hTBM). Results We report these data and compare them with similar data from wild-type pigs, and the 3 major NHP species commonly used in biomedical research (baboons, cynomolgus, and rhesus monkeys) and humans, largely from previously published reports. Conclusions Genetic modification of the pig (e.g., deletion of the Gal antigen and/or the addition of a human transgene) (i) does not result in abnormalities in hematologic, biochemical, or coagulation parameters that might impact animal welfare, (ii) seems not to alter metabolic function of vital organs, though this needs to be confirmed after their xenotransplantation, and (iii) possibly (though by no means certainly) modifies the hematologic, biochemical, and coagulation parameters closer to human values. The present study may provide a good reference for those working with genetically-engineered pigs in xenotransplantation research and eventually in clinical xenotransplantation. PMID:23145497

  5. Knockout of REST/NRSF shows that the protein is a potent repressor of neuronally expressed genes in non-neural tissues.

    PubMed

    Jones, F S; Meech, R

    1999-05-01

    The protein repressor element 1 silencing transcription factor/neuron restrictive silencer factor (REST/NRSF) is a negative regulator of neuronal genes that contain a particular DNA sequence, the neuron restrictive silencer element (NRSE). REST is expressed ubiquitously in non-neural tissues but is down-regulated in neural precursors and turned off in postmitotic neurons, suggesting that it can act both to prevent extraneural expression of certain genes and to delay the differentiation of neuronal subtypes. In a recent paper, Chen et al.(1) describe the production of a null mutant for REST in mice and the mosaic inactivation of REST function in chicken embryos. Knockout of REST led to malformations in several non-neural tissues, as well as apoptosis and embryonic lethality in mice. In addition, the expression of several REST target genes was derepressed in non-neural tissues and in neural progenitors in both mouse and chicken embryos. These studies clearly demonstrate that active repression of tissue-specific genes is required for proper tissue differentiation during embryonic development. PMID:10376008

  6. Knockout of the Bcmo1 gene results in an inflammatory response in female lung, which is suppressed by dietary beta-carotene

    PubMed Central

    van Helden, Yvonne G. J.; Heil, Sandra G.; van Schooten, Frederik J.; Kramer, Evelien; Hessel, Susanne; Amengual, Jaume; Ribot, Joan; Teerds, Katja; Wyss, Adrian; Lietz, Georg; Bonet, M. Luisa; von Lintig, Johannes; Godschalk, Roger W. L.

    2010-01-01

    Beta-carotene 15,15′-monooxygenase 1 knockout (Bcmo1−/−) mice accumulate beta-carotene (BC) similarly to humans, whereas wild-type (Bcmo1+/+) mice efficiently cleave BC. Bcmo1−/− mice are therefore suitable to investigate BC-induced alterations in gene expression in lung, assessed by microarray analysis. Bcmo1−/− mice receiving control diet had increased expression of inflammatory genes as compared to BC-supplemented Bcmo1−/− mice and Bcmo1+/+ mice that received either control or BC-supplemented diets. Differential gene expression in Bcmo1−/− mice was confirmed by real-time quantitative PCR. Histochemical analysis indeed showed an increase in inflammatory cells in lungs of control Bcmo1−/− mice. Supported by metabolite and gene-expression data, we hypothesize that the increased inflammatory response is due to an altered BC metabolism, resulting in an increased vitamin A requirement in Bcmo1−/− mice. This suggests that effects of BC may depend on inter-individual variations in BC-metabolizing enzymes, such as the frequently occurring human polymorphisms in BCMO1. PMID:20372966

  7. Knockout of the prion protein (PrP)-like Sprn gene does not produce embryonic lethality in combination with PrP(C)-deficiency.

    PubMed

    Daude, Nathalie; Wohlgemuth, Serene; Brown, Rebecca; Pitstick, Rose; Gapeshina, Hristina; Yang, Jing; Carlson, George A; Westaway, David

    2012-06-01

    The Sprn gene encodes Shadoo (Sho), a glycoprotein with biochemical properties similar to the unstructured region of cellular prion protein (PrP(C)). Sho has been considered a candidate for the hypothetical π protein that supplies a PrP(C)-like function to maintain the viability of Prnp(0/0) mice lacking the PrP(C) protein. To understand these relationships more clearly we probed the cell biology of Sho and created knockout mice. Besides full-length and a "C1" C-terminal fragment, we describe a 6-kDa N-terminal Sho neuropeptide, "N1," which is present in membrane-enriched subcellular fractions of wild-type mice. Sprn null alleles were produced that delete all protein coding sequences yet spare the Mtg1 gene transcription unit that overlaps the Sprn 3' UTR; the resulting mice bred to homozygosity were viable and fertile, although Sprn(0/0) mice maintained in two genetic backgrounds weighed less than wild-type mice. Lack of Sho protein did not affect prion incubation time. Contrasting with lethality reported for knockdown of expression in Prnp(0/0) embryos using lentiviruses targeted against the Sprn 3' UTR, we established that double-knockout mice deficient in both Sho and PrP(C) are fertile and viable up to 690 d of age. Our data reduce the impetus for equating Sho with the notional π protein and are not readily reconciled with hypotheses wherein expression of PrP(C) and Sho are both required for completion of embryogenesis. Alternatively, and in accord with some reports for PrP(C), we infer that Sho's activity will prove germane to the maintenance of neuronal viability in postnatal life. PMID:22619325

  8. Recombinant adeno-associated virus type 2-mediated gene delivery into the Rpe65-/- knockout mouse eye results in limited rescue

    PubMed Central

    Lai, Chooi-May; Yu, Meaghan JT; Brankov, Meliha; Barnett, Nigel L; Zhou, Xiaohuai; Redmond, T Michael; Narfstrom, Kristina; Rakoczy, P Elizabeth

    2004-01-01

    Background Leber's congenital amaurosis (LCA) is a severe form of retinal dystrophy. Mutations in the RPE65 gene, which is abundantly expressed in retinal pigment epithelial (RPE) cells, account for approximately 10–15% of LCA cases. In this study we used the high turnover, and rapid breeding and maturation time of the Rpe65-/- knockout mice to assess the efficacy of using rAAV-mediated gene therapy to replace the disrupted RPE65 gene. The potential for rAAV-mediated gene treatment of LCA was then analyzed by determining the pattern of RPE65 expression, the physiological and histological effects that it produced, and any improvement in visual function. Methods rAAV.RPE65 was injected into the subretinal space of Rpe65-/- knockout mice and control mice. Histological and immunohistological analyses were performed to evaluate any rescue of photoreceptors and to determine longevity and pattern of transgene expression. Electron microscopy was used to examine ultrastructural changes, and electroretinography was used to measure changes in visual function following rAAV.RPE65 injection. Results rAAV-mediated RPE65 expression was detected for up to 18 months post injection. The delivery of rAAV.RPE65 to Rpe65-/- mouse retinas resulted in a transient improvement in the maximum b-wave amplitude under both scotopic and photopic conditions (76% and 59% increase above uninjected controls, respectively) but no changes were observed in a-wave amplitude. However, this increase in b-wave amplitude was not accompanied by any slow down in photoreceptor degeneration or apoptotic cell death. Delivery of rAAV.RPE65 also resulted in a decrease in retinyl ester lipid droplets and an increase in short wavelength cone opsin-positive cells, suggesting that the recovery of RPE65 expression has long-term benefits for retinal health. Conclusion This work demonstrated the potential benefits of using the Rpe65-/- mice to study the effects and mechanism of rAAV.RPE65-mediated gene delivery into

  9. cAMP controls rod photoreceptor sensitivity via multiple targets in the phototransduction cascade

    PubMed Central

    Astakhova, Luba A.; Samoiliuk, Evgeniia V.; Govardovskii, Victor I.

    2012-01-01

    In early studies, both cyclic AMP (cAMP) and cGMP were considered as potential secondary messengers regulating the conductivity of the vertebrate photoreceptor plasma membrane. Later discovery of the cGMP specificity of cyclic nucleotide–gated channels has shifted attention to cGMP as the only secondary messenger in the phototransduction cascade, and cAMP is not considered in modern schemes of phototransduction. Here, we report evidence that cAMP may also be involved in regulation of the phototransduction cascade. Using a suction pipette technique, we recorded light responses of isolated solitary rods from the frog retina in normal solution and in the medium containing 2 µM of adenylate cyclase activator forskolin. Under forskolin action, flash sensitivity rose more than twofold because of a retarded photoresponse turn-off. The same concentration of forskolin lead to a 2.5-fold increase in the rod outer segment cAMP, which is close to earlier reported natural day/night cAMP variations. Detailed analysis of cAMP action on the phototransduction cascade suggests that several targets are affected by cAMP increase: (a) basal dark phosphodiesterase (PDE) activity decreases; (b) at the same intensity of light background, steady background-induced PDE activity increases; (c) at light backgrounds, guanylate cyclase activity at a given fraction of open channels is reduced; and (d) the magnitude of the Ca2+ exchanger current rises 1.6-fold, which would correspond to a 1.6-fold elevation of [Ca2+]in. Analysis by a complete model of rod phototransduction suggests that an increase of [Ca2+]in might also explain effects (b) and (c). The mechanism(s) by which cAMP could regulate [Ca2+]in and PDE basal activity is unclear. We suggest that these regulations may have adaptive significance and improve the performance of the visual system when it switches between day and night light conditions. PMID:23008435

  10. 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

  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

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

    2009-05-01

    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)-beta-D-Galp-(1[alpha-D-Glcp-(1-->6)]-->4)-beta-D-ManpNAc-(1-->] repeating units that is linked by an adaptor of -[GroA-2-->OPO2-->4-beta-D-ManpNAc-(1-->4)]-->3)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->3)-beta-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. 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

  13. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection.

    PubMed

    Baba, Tomoya; Ara, Takeshi; Hasegawa, Miki; Takai, Yuki; Okumura, Yoshiko; Baba, Miki; Datsenko, Kirill A; Tomita, Masaru; Wanner, Barry L; Mori, Hirotada

    2006-01-01

    We have systematically made a set of precisely defined, single-gene deletions of all nonessential genes in Escherichia coli K-12. Open-reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one-step method for inactivation of chromosomal genes and primers designed to create in-frame deletions upon excision of the resistance cassette. Of 4288 genes targeted, mutants were obtained for 3985. To alleviate problems encountered in high-throughput studies, two independent mutants were saved for every deleted gene. These mutants-the 'Keio collection'-provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome-wide testing of mutational effects in a common strain background, E. coli K-12 BW25113. We were unable to disrupt 303 genes, including 37 of unknown function, which are candidates for essential genes. Distribution is being handled via GenoBase (http://ecoli.aist-nara.ac.jp/). PMID:16738554

  14. Genetic knockout of the α7 nicotinic acetylcholine receptor gene alters hippocampal long-term potentiation in a background strain-dependent manner.

    PubMed

    Freund, Ronald K; Graw, Sharon; Choo, Kevin S; Stevens, Karen E; Leonard, Sherry; Dell'Acqua, Mark L

    2016-08-01

    Reduced α7 nicotinic acetylcholine receptor (nAChR) function is linked to impaired hippocampal-dependent sensory processing and learning and memory in schizophrenia. While knockout of the Chrna7 gene encoding the α7nAChR on a C57/Bl6 background results in changes in cognitive measures, prior studies found little impact on hippocampal synaptic plasticity in these mice. However, schizophrenia is a multi-genic disorder where complex interactions between specific genetic mutations and overall genetic background may play a prominent role in determining phenotypic penetrance. Thus, we compared the consequences of knocking out the α7nAChR on synaptic plasticity in C57/Bl6 and C3H mice, which differ in their basal α7nAChR expression levels. Homozygous α7 deletion in C3H mice, which normally express higher α7nAChR levels, resulted in impaired long-term potentiation (LTP) at hippocampal CA1 synapses, while C3H α7 heterozygous mice maintained robust LTP. In contrast, homozygous α7 deletion in C57 mice, which normally express lower α7nAChR levels, did not alter LTP, as had been previously reported for this strain. Thus, the threshold of Chrna7 expression required for LTP may be different in the two strains. Measurements of auditory gating, a hippocampal-dependent behavioral paradigm used to identify schizophrenia-associated sensory processing deficits, was abnormal in C3H α7 knockout mice confirming that auditory gating also requires α7nAChR expression. Our studies highlight the importance of genetic background on the regulation of synaptic plasticity and could be relevant for understanding genetic and cognitive heterogeneity in human studies of α7nAChR dysfunction in mental disorders. PMID:27233215

  15. 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. PMID:20472720

  16. Comparing Gene Expression during Cadmium Uptake and Distribution: Untreated versus Oral Cd-Treated Wild-Type and ZIP14 Knockout Mice

    PubMed Central

    Jorge-Nebert, Lucia F.; Gálvez-Peralta, Marina; Landero Figueroa, Julio; Somarathna, Maheshika; Hojyo, Shintaro; Fukada, Toshiyuki; Nebert, Daniel W.

    2015-01-01

    The nonessential metal cadmium (Cd) is toxic only after entering the cell. Proteins possibly relevant to intracellular Cd accumulation include the divalent metal transporter-1 (DMT1) and all 14 zinc-like iron-like protein (ZIP) importers, 10 zinc transporter (ZnT) exporters, and metallothionein chaperones MT1 and MT2. Comparing oral Cd-treated ZIP14 knockout (KO) with wild-type (WT) mice, we predicted Cd uptake and distribution would be diminished in the KO—because ZIP14 is very highly expressed in GI tract and liver; this was indeed observed for Cd content in liver. However, the reverse was found in kidney and lung from 6 or 12 h through 10 days of Cd exposure; at these times, Cd accumulation was unexpectedly greater in KO than WT mice; mRNA levels of the 27 above-mentioned genes were thus examined in proximal small intestine (PSI) versus kidney to see if these paradoxical effects could be explained by substantial alterations in any of the other 26 genes. PSI genes highly expressed in untreated WT animals included seven ZIP and five ZnT transporters, DMT1, MT1, and MT2; kidney genes included 11 ZIP and 7 ZnT transporters, DMT1, MT1, and MT2. Over 10 days of oral Cd, a bimodal response was seen for Cd content in PSI and for various mRNAs; initially, acute effects caused by the toxic metal; subsequently, the up- or down-regulation of important genes presumably to combat the sustained adversity. These data underscore the complex interplay between the gastrointestinal tract and renal proteins that might be relevant to Cd uptake and distribution in animals exposed to oral Cd. PMID:25294218

  17. 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

    2016-01-01

    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. 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

  19. Gal knockout and beyond.

    PubMed

    Zhong, R

    2007-01-01

    Recently, Galalpha1-3Galbeta1-4GlcNAc (Gal) knockout (k/o) pigs have been developed using genetic cloning technologies. This remarkable achievement has generated great enthusiasm in xenotransplantation studies. This review summarizes the current status of nonhuman primate experiments using Gal k/o pig organs. Briefly, when Gal k/o pig organs are transplanted into primates, hyperacute rejection does not occur. Although graft survival has been prolonged up to a few months in some cases, the overall results were not better than those using Gal-positive pig organs with human complement regulatory protein transgenes. Gal k/o pig kidneys rapidly developed rejection which was associated with increased anti-non-Gal antibodies. Although the precise mechanisms of Gal k/o pig organ rejection are not clear, it could result from incomplete deletion of Gal, up-regulation of new antigen (non-Gal antigen) and/or production of non-Gal antibodies. Future work in xenotransplantation should place emphasis on further modification of donors, such as combining human complement regulatory genes with Gal k/o, deleting non-Gal antigens and adding protective/surviving genes or a gene that inhibits coagulation. Induction of donor-specific T- and B-cell tolerance and promotion of accommodation are also warranted. PMID:17227553

  20. Knockout Zbtb33 gene results in an increased locomotion, exploration and pre-pulse inhibition in mice.

    PubMed

    Kulikov, Alexander V; Korostina, Valeria S; Kulikova, Elizabeth A; Fursenko, Dariya V; Akulov, Andrey E; Moshkin, Mikhail P; Prokhortchouk, Egor B

    2016-01-15

    The Zbtb33 gene encodes the Kaiso protein-a bimodal transcriptional repressor. Here, the effects of Zbtb33 gene disruption on the brain and behaviour of the Kaiso-deficient (KO) and C57BL/6 (WT) male mice were investigated. Behaviour was studied using the open field, novel object, elevated plus maze and acoustic startle reflex tests. Brain morphology was investigated with magnetic resonance imaging. Biogenic amine levels and gene expression in the brain were measured with high-performance liquid chromatography and quantitative real-time RT-PCR, respectively. Zbtb33 gene mRNA was not detected in the brain of KO mice. KO mice exhibited increased locomotion, exploration in the open field, novel object and elevated plus-maze test. At the same time, Zbtb33 gene disruption did not alter anxiety-related behaviour in the elevated plus-maze test. KO mice showed elevated amplitudes and pre-pulse inhibitions of the acoustic startle reflex. These behavioural alterations were accompanied by significant reductions in the volumes of the lateral ventricles without significant alterations in the volumes of the hippocampus, striatum, thalamus and corpus callosum. Norepinephrine concentration was reduced in the hypothalami and hippocampi in KO mice, while the levels of serotonin, dopamine, their metabolites as well as mRNA of the gene coding brain-derived neurotrophic factor were not altered in the brain of KO mice compared to WT mice. This study is the first to reveal the involvement of the Zbtb33 gene in the regulation of behaviour and the central nervous system. PMID:26454239

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

    PubMed

    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

  2. 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

  3. 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. PMID:24615917

  4. Generation of conditional knockout mice.

    PubMed

    Sakamoto, Kazuhito; Gurumurthy, Channabasavaiah B; Wagner, Kay-Uwe

    2014-01-01

    Conditional knockout mouse models are powerful tools to examine the biological and molecular function(s) of genes in specific tissues. The general procedure to generate such genetically engineered mouse models consists of three main steps. The first step is to find the appropriate genomic clone of the gene of interest and to design the cloning and Southern blot strategies. The second step is the cloning of the gene-targeting vector with all its essential components including positive and negative selection cassettes and the insertion of LoxP sites. Although conventional methods are still being widely used for DNA cloning, we describe in this book chapter the use of λ Red phage-based homologous recombination in Escherichia coli to capture the genomic DNA of the gene of interest and to assemble the gene-targeting vector. This new method provides several advantages as it does not require the presence of restriction sites within the gene of interest to insert LoxP-flanked DNA fragments. In the final step, the gene-targeting vector is transferred into embryonic stem (ES) cells, and successfully targeted ES cell clones are injected into mouse blastocysts to generate conditional knockout mice. PMID:25064096

  5. Development of a Gene Knockout System Using Mobile Group II Introns (Targetron) and Genetic Disruption of Acid Production Pathways in Clostridium beijerinckii

    PubMed Central

    Wang, Yi; Li, Xiangzhen; Milne, Caroline B.; Janssen, Holger; Lin, Weiyin; Phan, Gloria; Hu, Huiying; Jin, Yong-Su; Price, Nathan D.

    2013-01-01

    Clostridium beijerinckii is a well-known solvent-producing microorganism with great potential for biofuel and biochemical production. To better understand and improve the biochemical pathway to solvents, the development of genetic tools for engineering C. beijerinckii is highly desired. Based on mobile group II intron technology, a targetron gene knockout system was developed for C. beijerinckii in this study. This system was successfully employed to disrupt acid production pathways in C. beijerinckii, leading to pta (encoding phosphotransacetylase)- and buk (encoding butyrate kinase)-negative mutants. In addition to experimental characterization, the mutant phenotypes were analyzed in the context of our C. beijerinckii genome-scale model. Compared to those of the parental strain (C. beijerinckii 8052), acetate production in the pta mutant was substantially reduced and butyrate production was remarkably increased, while solvent production was dependent on the growth medium. The pta mutant also produced much higher levels of lactate, suggesting that disrupting pta influenced the energy generation and electron flow pathways. In contrast, acetate and butyrate production in the buk mutant was generally similar to that of the wild type, but solvent production was consistently 20 to 30% higher and glucose consumption was more rapid and complete. Our results suggest that the acid and solvent production of C. beijerinckii can be effectively altered by disrupting the acid production pathways. As the gene disruption method developed in this study does not leave any antibiotic marker in a disrupted allele, multiple and high-throughput gene disruption is feasible for elucidating genotype and phenotype relationships in C. beijerinckii. PMID:23872562

  6. Type II Toxoplasma gondii KU80 Knockout Strains Enable Functional Analysis of Genes Required for Cyst Development and Latent Infection ▿ †

    PubMed Central

    Fox, Barbara A.; Falla, Alejandra; Rommereim, Leah M.; Tomita, Tadakimi; Gigley, Jason P.; Mercier, Corinne; Cesbron-Delauw, Marie-France; Weiss, Louis M.; Bzik, David J.

    2011-01-01

    Type II Toxoplasma gondii KU80 knockouts (Δku80) deficient in nonhomologous end joining were developed to delete the dominant pathway mediating random integration of targeting episomes. Gene targeting frequency in the type II Δku80 Δhxgprt strain measured at the orotate (OPRT) and the uracil (UPRT) phosphoribosyltransferase loci was highly efficient. To assess the potential of the type II Δku80 Δhxgprt strain to examine gene function affecting cyst biology and latent stages of infection, we targeted the deletion of four parasite antigen genes (GRA4, GRA6, ROP7, and tgd057) that encode characterized CD8+ T cell epitopes that elicit corresponding antigen-specific CD8+ T cell populations associated with control of infection. Cyst development in these type II mutant strains was not found to be strictly dependent on antigen-specific CD8+ T cell host responses. In contrast, a significant biological role was revealed for the dense granule proteins GRA4 and GRA6 in cyst development since brain tissue cyst burdens were drastically reduced specifically in mutant strains with GRA4 and/or GRA6 deleted. Complementation of the Δgra4 and Δgra6 mutant strains using a functional allele of the deleted GRA coding region placed under the control of the endogenous UPRT locus was found to significantly restore brain cyst burdens. These results reveal that GRA proteins play a functional role in establishing cyst burdens and latent infection. Collectively, our results suggest that a type II Δku80 Δhxgprt genetic background enables a higher-throughput functional analysis of the parasite genome to reveal fundamental aspects of parasite biology controlling virulence, pathogenesis, and transmission. PMID:21531875

  7. 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. PMID:25599829

  8. 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

  9. Piper betle induces phase I & II genes through Nrf2/ARE signaling pathway in mouse embryonic fibroblasts derived from wild type and Nrf2 knockout cells

    PubMed Central

    2014-01-01

    Background Nuclear factor-erythroid 2 p45 related factor 2 (Nrf2) is a primary transcription factor, protecting cells from oxidative stress by regulating a number of antioxidants and phase II detoxifying enzymes. Dietary components such as sulforaphane in broccoli and quercetin in onions have been shown to be inducers of Nrf2. Piper betle (PB) grows well in tropical climate and the leaves are used in a number of traditional remedies for the treatment of stomach ailments and infections among Asians. The aim of this study was to elucidate the effect of Piper betle (PB) leaves extract in Nrf2 signaling pathway by using 2 types of cells; mouse embryonic fibroblasts (MEFs) derived from wild-type (WT) and Nrf2 knockout (N0) mice. Methods WT and N0 cells were treated with 5 and 10 μg/ml of PB for 10 and 12-h for the determination of nuclear translocation of Nrf2 protein. Luciferase reporter gene activity was performed to evaluate the antioxidant response element (ARE)-induction by PB. Real-time PCR and Western blot were conducted on both WT and N0 cells after PB treatment for the determination of antioxidant enzymes [superoxide dismutase (SOD1) and heme-oxygenase (HO-1)], phase I oxidoreductase enzymes [NAD(P)H: quinone oxidoreductase (NQO1)] and phase II detoxifying enzyme [glutathione S-transferase (GST)]. Results Nuclear translocation of Nrf2 by PB in WT cells was better after 10 h incubation compared to 12 h. Real time PCR and Western blot analysis showed increased expressions of Nrf2, NQO1 and GSTA1 genes with corresponding increases in glutathione, NQO1 and HO-1 proteins in WT cells. Reporter gene ARE was stimulated by PB as shown by ARE/luciferase assay. Interestingly, PB induced SOD1 gene and protein expressions in N0 cells but not in WT cells. Conclusion The results of this study confirmed that PB activated Nrf2-ARE signaling pathway which subsequently induced some phase I oxidoreductase, phase II detoxifying and antioxidant genes expression via ARE reporter

  10. Formation of acellular cementum-like layers, with and without extrinsic fiber insertion, along inert bone surfaces of aging c-Src gene knockout mice.

    PubMed

    Baba, Otto; Miyata, Atsushi; Abe, Tatsuhiko; Shibata, Shunichi; Nakano, Yukiko; Terashima, Tatsuo; Oda, Tsuyoshi; Kudo, Akira; Takano, Yoshiro

    2006-12-01

    To investigate the long-term effects of c-src deficiency on skeletal and dental tissues, we examined the lower jaws and long bones of c-src gene knockout (c-src KO) mice by histological and histochemical methods. Numerous multinucleated osteoclasts were distributed throughout the mandible in 5-wk-old c-src KO mice, but by 14 wk they had almost completely disappeared from the alveolar bone, leaving tartrate-resistant acid phosphatase (TRAP)-positive layers along the bone surface. Deposition of osteopontin-positive mineralized tissue, reminiscent of acellular afibrillar cementum (AAC), was confirmed along the TRAP-positive bone surface at 14 wk. The layer progressively thickened up to 21 months. A comparable mineralized layer was noted along the trabeculae of long bones as thickened cement lines. In the periostin-rich areas of jaw bones, but not in the long bones, portions of AAC-like mineralized layers were often replaced with and/or covered by acellular extrinsic fiber cementum (AEFC)-like tissue. These data suggest that the deposition of AAC-like mineralized tissue is a general phenomenon that may occur along inert or slowly remodeling bone surfaces under conditions characterized by reduced bone-resorbing activity, whereas the induction of AEFC-like tissue seems to be associated with the expression of certain molecules that are particularly abundant in the microenvironment of the periodontal ligament. PMID:17184236

  11. 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

  12. Rejection of Cardiac Xenografts Transplanted from α 1,3-Galactosyltransferase Gene-Knockout (GalT-KO) Pigs to Baboons

    PubMed Central

    Hisashi, Y.; Yamada, K.; Kuwaki, K.; Tseng, Y.-L; Dor, F. J. M. F.; Houser, S. L; Robson, S. C.; Schuurman, H.-J.; Cooper, D. K. C.; Sachs, D. H.; Colvin, R. B.; Shimizu, A.

    2010-01-01

    The use of α 1,3-galactosyltransferase gene-knockout (GalT-KO) swine donors in discordant xenotransplantation has extended the survival of cardiac xenografts in baboons following transplantation. Eight baboons received heterotopic cardiac xenografts from GalT-KO swine and were treated with a chronic immunosuppressive regimen. The pathologic features of acute humoral xenograft rejection (AHXR), acute cellular xenograft rejection (ACXR) and chronic rejection were assessed in the grafts. No hyperacute rejection developed and one graft survived up to 6 months after transplantation. However, all GalT-KO heart grafts underwent graft failure with AHXR, ACXR and/or chronic rejection. AHXR was characterized by interstitial hemorrhage and multiple thrombi in vessels of various sizes. ACXR was characterized by TUNEL+ graft cell injury with the infiltration of T cells (including CD3 and TIA-1+ cytotoxic T cells), CD4+ cells, CD8+ cells, macrophages and a small number of B and NK cells. Chronic xenograft vasculopathy, a manifestation of chronic rejection, was characterized by arterial intimal thickening with TUNEL+ dead cells, antibody and complement deposition, and/or cytotoxic T-cell infiltration. In conclusion, despite the absence of the Gal epitope, acute and chronic antibody and cell-mediated rejection developed in grafts, maintained by chronic immunosupression, presumably due to de novo responses to non-Gal antigens. PMID:19032222

  13. 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

  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

    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

    2003-05-01

    1 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. 2 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. 3 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. 4 Relative to UT((+/+)) mouse isolated thoracic aorta, where hU-II was a potent spasmogen (pEC(50)=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. 5 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. The neuronal isoform of constitutive nitric oxide synthase is up-regulated in the macula densa of angiotensinogen gene-knockout mice.

    PubMed

    Kihara, M; Umemura, S; Kadota, T; Yabana, M; Tamura, K; Nyuui, N; Ogawa, N; Murakami, K; Fukamizu, A; Ishii, M

    1997-02-01

    Angiotensinogen gene-knockout (Atg -/-) mice lacking angiotensin II exhibit chronic hypotension and an increase in renal renin gene expression. The present study was designed to provide evidence for the possible involvement of neuronal type nitric oxide synthase (N-NOS) at the macula densa in the increased renin production in Atg -/- mice. The enzyme activity of N-NOS was histochemically detected by NADPH diaphorase (NADPHd) reaction combined with N-NOS immunohistochemistry. N-NOS mRNA expression in the renal cortical tissue was determined using reverse transcription-PCR in a semiquantitative manner. The levels of renal renin mRNA were evaluated by Northern blot analysis. In the kidneys of wild-type (Atg +/+) mice, N-NOS activity was localized to the macula densa as reported previously. On the other hand, N-NOS-positive macula densa cells of Atg -/- mice were distributed beyond the original location of the macula densa. They often occupy the entire cross-sectional profiles of the tubules. In addition, Atg -/- mice showed a stronger signal intensity for the enzyme reaction than Atg +/+ mice. The mean total number of N-NOS-positive cells per 100 glomeruli was 6 times higher in Atg -/- mice than in Atg +/+ mice. Semiquantitative reverse transcription-PCR revealed an increase in the N-NOS mRNA level in renal cortical tissue of Atg -/- mice compared with Atg +/+ mice. Furthermore, the selective inhibition of N-NOS activity by 7-nitrondazole significantly decreased the level of renal renin mRNA in Atg -/- mice. These results suggest that increased N-NOS activity at the macula densa is involved a renal renin overproduction in Atg -/- mice. PMID:9042165

  16. Effects of repeated adolescent stress and serotonin transporter gene partial knockout in mice on behaviors and brain structures relevant to major depression

    PubMed Central

    Spinelli, Simona; Müller, Tanja; Friedel, Miriam; Sigrist, Hannes; Lesch, Klaus-Peter; Henkelman, Mark; Rudin, Markus; Seifritz, Erich; Pryce, Christopher R.

    2013-01-01

    In humans, exposure to stress during development is associated with structural and functional alterations of the prefrontal cortex (PFC), amygdala (AMY), and hippocampus (HC) and their circuits of connectivity, and with an increased risk for developing major depressive disorder particularly in carriers of the short (s) variant of the serotonin transporter (5-HTT) gene-linked polymorphic region (5-HTTLPR). Although changes in these regions are found in carriers of the s allele and/or in depressed patients, evidence for a specific genotype × developmental stress effect on brain structure and function is limited. Here, we investigated the effect of repeated stress exposure during adolescence in mice with partial knockout of the 5-HTT gene (HET) vs. wildtype (WT) on early-adulthood behavioral measures and brain structure [using magnetic resonance imaging (MRI)] relevant to human major depression. Behaviorally, adolescent stress (AS) increased anxiety and decreased activity and did so to a similar degree in HET and WT. In a probabilistic reversal learning task, HET-AS mice achieved fewer reversals than did HET-No-AS mice. 5-HTT genotype and AS were without effect on corticosterone stress response. In terms of structural brain differences, AS reduced the volume of two long-range white matter tracts, the optic tract (OT) and the cerebral peduncle (CP), in WT mice specifically. In a region-of-interest analysis, AS was associated with increased HC volume and HET genotype with a decreased frontal lobe volume. In conclusion, we found that 5-HTT and AS genotype exerted long-term effects on behavior and development of brain regions relevant to human depression. PMID:24427124

  17. Conditional Knockout of Tumor Overexpressed Gene in Mouse Neurons Affects RNA Granule Assembly, Granule Translation, LTP and Short Term Habituation

    PubMed Central

    Barbarese, Elisa; Ifrim, Marius F.; Hsieh, Lawrence; Guo, Caiying; Tatavarty, Vedakumar; Maggipinto, Michael J.; Korza, George; Tutolo, Jessica W.; Giampetruzzi, Anthony; Le, Hien; Ma, Xin-Ming; Levine, Eric; Bishop, Brian; Kim, Duck O.; Kuwada, Shigeyuki; Carson, John H.

    2013-01-01

    In neurons, specific RNAs are assembled into granules, which are translated in dendrites, however the functional consequences of granule assembly are not known. Tumor overexpressed gene (TOG) is a granule-associated protein containing multiple binding sites for heterogeneous nuclear ribonucleoprotein (hnRNP) A2, another granule component that recognizes cis-acting sequences called hnRNP A2 response elements (A2REs) present in several granule RNAs. Translation in granules is sporadic, which is believed to reflect monosomal translation, with occasional bursts, which are believed to reflect polysomal translation. In this study, TOG expression was conditionally knocked out (TOG cKO) in mouse hippocampal neurons using cre/lox technology. In TOG cKO cultured neurons granule assembly and bursty translation of activity-regulated cytoskeletal associated (ARC) mRNA, an A2RE RNA, are disrupted. In TOG cKO brain slices synaptic sensitivity and long term potentiation (LTP) are reduced. TOG cKO mice exhibit hyperactivity, perseveration and impaired short term habituation. These results suggest that in hippocampal neurons TOG is required for granule assembly, granule translation and synaptic plasticity, and affects behavior. PMID:23936366

  18. Acinar cell-specific knockout of the PTHrP gene decreases the proinflammatory and profibrotic responses in pancreatitis

    PubMed Central

    Bhatia, Vandanajay; Rastellini, Cristiana; Han, Song; Aronson, Judith F.; Greeley, George H.

    2014-01-01

    Pancreatitis is a necroinflammatory disease with acute and chronic manifestations. Accumulated damage incurred during repeated bouts of acute pancreatitis (AP) can lead to chronic pancreatitis (CP). Pancreatic parathyroid hormone-related protein (PTHrP) levels are elevated in a mouse model of cerulein-induced AP. Here, we show elevated PTHrP levels in mouse models of pancreatitis induced by chronic cerulein administration and pancreatic duct ligation. Because acinar cells play a major role in the pathophysiology of pancreatitis, mice with acinar cell-specific targeted disruption of the Pthrp gene (PTHrPΔacinar) were generated to assess the role of acinar cell-secreted PTHrP in pancreatitis. These mice were generated using Cre-LoxP technology and the acinar cell-specific elastase promoter. PTHrPΔacinar exerted protective effects in cerulein and pancreatic duct ligation models, evident as decreased edema, histological damage, amylase secretion, pancreatic stellate cell (PSC) activation, and extracellular matrix deposition. Treating acinar cells in vitro with cerulein increased IL-6 expression and NF-κB activity; these effects were attenuated in PTHrPΔacinar cells, as were the cerulein- and carbachol-induced elevations in amylase secretion. The cerulein-induced upregulation of procollagen I expression was lost in PSCs from PTHrPΔacinar mice. PTHrP immunostaining was elevated in human CP sections. The cerulein-induced upregulation of IL-6 and ICAM-1 (human acinar cells) and procollagen I (human PSCs) was suppressed by pretreatment with the PTH1R antagonist, PTHrP (7–34). These findings establish PTHrP as a novel mediator of inflammation and fibrosis associated with CP. Acinar cell-secreted PTHrP modulates acinar cell function via its effects on proinflammatory cytokine release and functions via a paracrine pathway to activate PSCs. PMID:25035110

  19. AAV-Mediated Gene Therapy in the Guanylate Cyclase (RetGC1/RetGC2) Double Knockout Mouse Model of Leber Congenital Amaurosis

    PubMed Central

    Boye, Sanford L.; Peshenko, Igor V.; Huang, Wei Chieh; Min, Seok Hong; McDoom, Issam; Kay, Christine N.; Liu, Xuan; Dyka, Frank M.; Foster, Thomas C.; Umino, Yumiko; Karan, Sukanya; Jacobson, Samuel G.; Baehr, Wolfgang; Dizhoor, Alexander; Hauswirth, William W.

    2013-01-01

    Mutations in GUCY2D are associated with recessive Leber congenital amaurosis-1 (LCA1). GUCY2D encodes photoreceptor-specific, retinal guanylate cyclase-1 (RetGC1). Reports of retinal degeneration in LCA1 are conflicting; some describe no obvious degeneration and others report loss of both rods and cones. Proof of concept studies in models representing the spectrum of phenotypes is warranted. We have previously demonstrated adeno-associated virus (AAV)-mediated RetGC1 is therapeutic in GC1ko mice, a model exhibiting loss of cones only. The purpose of this study was to characterize AAV-mediated gene therapy in the RetGC1/RetGC2 double knockout (GCdko) mouse, a model lacking rod and cone function and exhibiting progressive loss of both photoreceptor subclasses. Use of this model also allowed for the evaluation of the functional efficiency of transgenic RetGC1 isozyme. Subretinal delivery of AAV8(Y733F) vector containing the human rhodopsin kinase (hGRK1) promoter driving murine Gucy2e was performed in GCdko mice at various postnatal time points. Treatment resulted in restoration of rod and cone function at all treatment ages and preservation of retinal structure in GCdko mice treated as late as 7 weeks of age. Functional gains and structural preservation were stable for at least 1 year. Treatment also conferred cortical- and subcortical-based visually-guided behavior. Functional efficiency of transgenic RetGC1 was indistinguishable from that of endogenous isozyme in congenic wild-type (WT) mice. This study clearly demonstrates AAV-mediated RetGC1 expression restores function to and preserves structure of rod and cone photoreceptors in a degenerative model of retinal guanylate cyclase deficiency, further supporting development of an AAV-based vector for treatment of LCA1. PMID:23210611

  20. Knockout of the Host Resistance Gene Pkr Fully Restores Replication of Murine Cytomegalovirus m142 and m143 Mutants In Vivo

    PubMed Central

    Ostermann, Eleonore; Warnecke, Gabriele; Waibler, Zoe

    2015-01-01

    Murine cytomegalovirus (MCMV) proteins m142 and m143 are essential for viral replication. They bind double-stranded RNA and prevent protein kinase R-induced protein synthesis shutoff. Whether the two viral proteins have additional functions such as their homologs in human cytomegalovirus do remained unknown. We show that MCMV m142 and m143 knockout mutants attain organ titers equivalent to those attained by wild-type MCMV in Pkr knockout mice, suggesting that these viral proteins do not encode additional PKR-independent functions relevant for pathogenesis in vivo. PMID:26512090

  1. Gene-specific alterations of hepatic nuclear receptor regulated gene expression by ligand activation or hepatocyte-selective knockout inhibition of RXRα signaling during inflammation

    PubMed Central

    Kosters, Astrid; Tian, Feng; Wan, Yvonne Yu-Jie; Karpen, Saul J.

    2013-01-01

    Background Inflammation leads to transcriptional downregulation of many hepatic genes, particulary those activated by RXRα-heterodimers. Inflammation-mediated reduction of nuclear RXRα levels is a main factor in reduced nuclear receptor (NR)–regulated hepatic gene expression, eventually leading to cholestasis and liver damage. Aim To investigate roles for RXRα in hepatic gene expression during inflammation, using two complementary mouse models: ligand–activation of RXRα, and in mice expressing hepatocyte-specific expression of RXRα missing its DNA-binding-domain (DBD; hs-RxrαΔex4−/−) Methods To activate RXRα, mice were gavage-fed with LG268 or vehicle for 5 days. To inhibit RXRα function, hs-RxrαΔex4−/− were used. All mice were IP-injected with LPS or saline for 16 hrs prior to analysis of hepatic RNA, protein and NR-DNA binding. Results LG268-treatment attenuated the LPS-mediated reductions of several RXRα-regulated genes, coinciding with maintained RXRα occupancy in both Bsep and Ostβ promoters. Lacking full hepatocyte-RXRα function (hs-RxrαΔex4−/− mice) led to enhancement of LPS-mediated changes in gene expression, but surprisingly, maintenance of RNA levels of some RXRα-regulated genes. Investigations revealed that Hs-Rxrα−/− hepatocytes expressed an internally-truncated, ~44 kDa, RXRα-form. DNA-binding capacity of NR-heterodimers was equivalent in wt and hs-RxrαΔex4−/− livers, but reduced by LPS in both. ChIP-QPCR revealed reduced RXRα occupancy to the Bsep RXRα:FXR site was reduced, but not absent, in hs-RxrαΔex4−/− livers. Conclusions There are differential regulatory roles for hepatic RXRα, both in basal and inflammatory states, suggesting new and complex multi-domain roles for RXRα in regulating hepatic gene expression. Moreover, there is an unexpected non-obligate role for the DBD of RXRα. PMID:22098603

  2. Mechanisms in Knockout Reactions

    NASA Astrophysics Data System (ADS)

    Bazin, D.; Charity, R. J.; de Souza, R. T.; Famiano, M. A.; Gade, A.; Henzl, V.; Henzlova, D.; Hudan, S.; Lee, J.; Lukyanov, S.; Lynch, W. G.; McDaniel, S.; Mocko, M.; Obertelli, A.; Rogers, A. M.; Sobotka, L. G.; Terry, J. R.; Tostevin, J. A.; Tsang, M. B.; Wallace, M. S.

    2009-06-01

    We report the first detailed study of the relative importance of the stripping and diffraction mechanisms involved in nucleon knockout reactions, by the use of a coincidence measurement of the residue and fast proton following one-proton knockout reactions. The measurements used the S800 spectrograph in combination with the HiRA detector array at the NSCL. Results for the reactions Be9(C9,B8+X)Y and Be9(B8,Be7+X)Y are presented and compared with theoretical predictions for the two reaction mechanisms calculated using the eikonal model. The data show a clear distinction between the stripping and diffraction mechanisms and the measured relative proportions are very well reproduced by the reaction theory. This agreement adds support to the results of knockout reaction analyses and their applications to the spectroscopy of rare isotopes.

  3. The Na(+)/Ca(2+), K(+) exchanger 2 modulates mammalian cone phototransduction.

    PubMed

    Sakurai, Keisuke; Vinberg, Frans; Wang, Tian; Chen, Jeannie; Kefalov, Vladimir J

    2016-01-01

    Calcium ions (Ca(2+)) modulate the phototransduction cascade of vertebrate cone photoreceptors to tune gain, inactivation, and light adaptation. In darkness, the continuous current entering the cone outer segment through cGMP-gated (CNG) channels is carried in part by Ca(2+), which is then extruded back to the extracellular space. The mechanism of Ca(2+) extrusion from mammalian cones is not understood. The dominant view has been that the cone-specific isoform of the Na(+)/Ca(2+), K(+) exchanger, NCKX2, is responsible for removing Ca(2+) from their outer segments. However, indirect evaluation of cone function in NCKX2-deficient (Nckx2(-/-)) mice by electroretinogram recordings revealed normal photopic b-wave responses. This unexpected result suggested that NCKX2 may not be involved in the Ca(2+) homeostasis of mammalian cones. To address this controversy, we examined the expression of NCKX2 in mouse cones and performed transretinal recordings from Nckx2(-/-) mice to determine the effect of NCKX2 deletion on cone function directly. We found that Nckx2(-/-) cones exhibit compromised phototransduction inactivation, slower response recovery and delayed background adaptation. We conclude that NCKX2 is required for the maintenance of efficient Ca(2+) extrusion from mouse cones. However, surprisingly, Nckx2(-/-) cones adapted normally in steady background light, indicating the existence of additional Ca(2+)-extruding mechanisms in mammalian cones. PMID:27580676

  4. The Na+/Ca2+, K+ exchanger 2 modulates mammalian cone phototransduction

    PubMed Central

    Sakurai, Keisuke; Vinberg, Frans; Wang, Tian; Chen, Jeannie; Kefalov, Vladimir J.

    2016-01-01

    Calcium ions (Ca2+) modulate the phototransduction cascade of vertebrate cone photoreceptors to tune gain, inactivation, and light adaptation. In darkness, the continuous current entering the cone outer segment through cGMP-gated (CNG) channels is carried in part by Ca2+, which is then extruded back to the extracellular space. The mechanism of Ca2+ extrusion from mammalian cones is not understood. The dominant view has been that the cone-specific isoform of the Na+/Ca2+, K+ exchanger, NCKX2, is responsible for removing Ca2+ from their outer segments. However, indirect evaluation of cone function in NCKX2-deficient (Nckx2−/−) mice by electroretinogram recordings revealed normal photopic b-wave responses. This unexpected result suggested that NCKX2 may not be involved in the Ca2+ homeostasis of mammalian cones. To address this controversy, we examined the expression of NCKX2 in mouse cones and performed transretinal recordings from Nckx2−/− mice to determine the effect of NCKX2 deletion on cone function directly. We found that Nckx2−/− cones exhibit compromised phototransduction inactivation, slower response recovery and delayed background adaptation. We conclude that NCKX2 is required for the maintenance of efficient Ca2+ extrusion from mouse cones. However, surprisingly, Nckx2−/− cones adapted normally in steady background light, indicating the existence of additional Ca2+-extruding mechanisms in mammalian cones. PMID:27580676

  5. 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

  6. 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. PMID:26551692

  7. A review of current large-scale mouse knockout efforts.

    PubMed

    Guan, Chunmei; Ye, Chao; Yang, Xiaomei; Gao, Jiangang

    2010-02-01

    After the successful completion of the human genome project (HGP), biological research in the postgenome era urgently needs an efficient approach for functional analysis of genes. Utilization of knockout mouse models has been powerful for elucidating the function of genes as well as finding new therapeutic interventions for human diseases. Gene trapping and gene targeting are two independent techniques for making knockout mice from embryonic stem (ES) cells. Gene trapping is high-throughput, random, and sequence-tagged while gene targeting enables the knockout of specific genes. It has been about 20 years since the first gene targeting and gene trapping mice were generated. In recent years, new tools have emerged for both gene targeting and gene trapping, and organizations have been formed to knock out genes in the mouse genome using either of the two methods. The knockout mouse project (KOMP) and the international gene trap consortium (IGTC) were initiated to create convenient resources for scientific research worldwide and knock out all the mouse genes. Organizers of KOMP regard it as important as the HGP. Gene targeting methods have changed from conventional gene targeting to high-throughput conditional gene targeting. The combined advantages of trapping and targeting elements are improving the gene trapping spectrum and gene targeting efficiency. As a newly-developed insertional mutation system, transposons have some advantages over retrovirus in trapping genes. Emergence of the international knockout mouse consortium (IKMP) is the beginning of a global collaboration to systematically knock out all the genes in the mouse genome for functional genomic research. PMID:20095055

  8. Mechanisms in knockout reactions

    NASA Astrophysics Data System (ADS)

    Bazin, D.; Charity, R. J.; de Souza, R. T.; Famiano, M. A.; Gade, A.; Henzl, V.; Henzlova, D.; Hudan, S.; Lee, J.; Lukyanov, S.; Lynch, W. G.; McDaniel, S.; Mocko, M.; Obertelli, A.; Rogers, A. M.; Sobotka, L. G.; Terry, J. R.; Tostevin, J. A.; Tsang, M. B.; Wallace, M. S.

    2009-10-01

    We report on the first detailed study of the mechanisms involved in knockout reactions, via a coincidence measurement of the residue and fast proton in one-proton knockout reactions, using the S800 spectrograph in combination with the HiRA detector array at the NSCL. Results on the reactions ^9Be(^9C,^8B+X)Y and ^9Be(^8B,^7Be+X)Y are presented. They are compared with theoretical predictions for both the diffraction (elastic breakup) and stripping (inelastic breakup) reaction mechanisms, as calculated in the eikonal model. The data shows a clear distinction between the two reaction mechanisms, and the observed respective proportions are very well reproduced by the reaction theory. This agreement supports the results of knockout reaction analyses and their applications to the spectroscopy of rare isotopes. In particular, this add considerable support to the use of the eikonal model as a quantitative tool, able, for example, to determine single-particle spectroscopic strengths in rare isotopes.

  9. Effect of 11-Cis 13-Demethylretinal on Phototransduction in Bleach-Adapted Rod and Cone Photoreceptors

    PubMed Central

    Corson, D.Wesley; Kefalov, Vladimir J.; Cornwall, M. Carter; Crouch, Rosalie K.

    2000-01-01

    We used 11-cis 13-demethylretinal to examine the physiological consequences of retinal's noncovalent interaction with opsin in intact rod and cone photoreceptors during visual pigment regeneration. 11-Cis 13-demethylretinal is an analog of 11-cis retinal in which the 13 position methyl group has been removed. Biochemical experiments have shown that it is capable of binding in the chromophore pocket of opsin, forming a Schiff-base linkage with the protein to produce a pigment, but at a much slower rate than the native 11-cis retinal (Nelson, R., J. Kim deReil, and A. Kropf. 1970. Proc. Nat. Acad. Sci. USA. 66:531–538). Experimentally, this slow rate of pigment formation should allow separate physiological examination of the effects of the initial binding of retinal in the pocket and the subsequent formation of the protonated Schiff-base linkage. Currents from solitary rods and cones from the tiger salamander were recorded in darkness before and after bleaching and then after exposure to 11-cis 13-demethylretinal. In bleach-adapted rods, 11-cis 13-demethylretinal caused transient activation of phototransduction, as evidenced by a decrease of the dark current and sensitivity, acceleration of the dim flash responses, and activation of cGMP phosphodiesterase and guanylyl cyclase. The steady state of phototransduction activity was still higher than that of the bleach-adapted rod. In contrast, exposure of bleach-adapted cones to 11-cis 13-demethylretinal resulted in an immediate deactivation of transduction as measured by the same parameters. These results extend the validity of a model for the effects of the noncovalent binding of a retinoid in the chromophore pockets of rod and cone opsins to analogs capable of forming a Schiff-base and imply that the noncovalent binding by itself may play a role for the dark adaptation of photoreceptors. PMID:10919871

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

    PubMed Central

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

    2016-01-01

    ABSTRACT 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. PMID:27387532

  11. 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

    2016-01-01

    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. PMID:27387532

  12. Expression pattern of matrix metalloproteinase and TIMP genes in fibroblasts derived from Ets-1 knock-out mice compared to wild-type mouse fibroblasts.

    PubMed

    Hahne, Jens Claus; Fuchs, Tanja; El Mustapha, Haddouti; Okuducu, Ali Fuat; Bories, Jean Christophe; Wernert, Nicolas

    2006-07-01

    Matrix-degrading proteases play a key role in normal development, wound healing, many diseases such as rheumatoid arthritis and, in particular, tumour invasion. In invasive tumours, these enzymes are expressed by fibroblasts of the tumour stroma. Their expression and activity are tightly regulated at several levels, an important one being transcription. Previous in vitro and in vivo findings pointed to a major role of the Ets-1 transcription factor for this level of regulation. In the present study, we tried to prove this role in fibroblasts. We stimulated wild-type mouse fibroblasts with physiological doses of basic fibroblast growth factor (bFGF, known to induce different proteases and expressed by tumour cells) and compared the results to those obtained in Ets-1 -/- fibroblasts derived from Ets-1 knock-out mice. We found that basal Ets-1 levels are necessary not only for a fast induction of MMPs 2, 3 and 13 by bFGF but also for maintenance of the bFGF-induced expression of tissue inhibitors of metalloproteinases (TIMPs) 1, 2 and 3, which are known not only to inhibit but also participate as activators of certain pro-MMPs. PMID:16786167

  13. Increased Expression of Chitinase 3-Like 1 in Aorta of Patients with Atherosclerosis and Suppression of Atherosclerosis in Apolipoprotein E-Knockout Mice by Chitinase 3-Like 1 Gene Silencing

    PubMed Central

    Gong, Zushun; Xing, Shanshan; Zheng, Fei; Xing, Qichong

    2014-01-01

    Introduction. The purpose of this study was to investigate the changes of chitinase 3-like 1 (CHI3L1) in the aorta of patients with coronary atherosclerosis and to determine whether inhibition of CHI3L1 by lentivirus-mediated RNA interference could stabilize atherosclerotic plaques in apolipoprotein E-knockout (ApoE−/−) mice. Methods. We collected discarded aortic specimens from patients undergoing coronary artery bypass graft surgery and renal arterial tissues from kidney donors. A lentivirus carrying small interfering RNA targeting the expression of CHI3L1 was constructed. Fifty ApoE−/− mice were divided into control group and CHI3L1 gene silenced group. A constrictive collar was placed around carotid artery to induce plaques formation. Then lentivirus was transfected into carotid plaques. Results. We found that CHI3L1 was overexpressed in aorta of patients with atherosclerosis and its expression was correlated with the atherosclerotic risk factors. After lentivirus transduction, mRNA and protein expression of CHI3L1 were attenuated in carotid plaques, leading to reduced plaque content of lipids and macrophages, and increased plaque content of collagen and smooth muscle cells. Moreover, CHI3L1 gene silencing downregulated the expression of local proinflammatory mediators. Conclusions. CHI3L1 is overexpressed in aorta from patients with atherosclerosis and the lentivirus-mediated CHI3L1 gene silencing could represent a new strategy to inhibit plaques progression. PMID:24729664

  14. Skeletal muscle gene expression after myostatin knockout in mature mice Address for reprint requests and other correspondence: S. Welle, Univ. of Rochester Medical Center, 601 Elmwood Ave., Box 693, Rochester, NY 14642 (e-mail: stephen_welle@urmc.rochester.edu).

    PubMed Central

    Welle, Stephen; Cardillo, Andrew; Zanche, Michelle; Tawil, Rabi

    2009-01-01

    There is much interest in developing anti-myostatin agents to reverse or prevent muscle atrophy in adults, so it is important to characterize the effects of reducing myostatin activity after normal muscle development. For assessment of the effect of loss of myostatin signaling on gene expression in muscle, RNA from mice with postdevelopmental myostatin knockout was analyzed with oligonucleotide microarrays. Myostatin was undetectable in muscle within 2 wk after Cre recombinase activation in 4-month-old male mice with floxed myostatin genes. Three months after myostatin depletion, muscle mass had increased 26% (vs. 2% after induction of Cre activity in mice with normal myostatin genes), at which time the expression of several hundred genes differed in knockout and control mice at nominal P < 0.01. In contrast to previously reported effects of constitutive myostatin knockout, postdevelopmental knockout did not downregulate expression of genes encoding slow isoforms of contractile proteins or genes encoding proteins involved in energy metabolism. Several collagen genes were expressed at 20–50% lower levels in the myostatin-deficient muscles, which had ∼25% less collagen than normal muscles as reflected by hydroxyproline content. Most of the other genes affected by myostatin depletion have not been previously linked to myostatin signaling. Gene set enrichment analysis suggested that Smads are not the only transcription factors with reduced activity after myostatin depletion. These data reinforce other evidence that myostatin regulates collagen production in muscle and demonstrate that many of the previously reported effects of constitutive myostatin deficiency do not occur when myostatin is knocked out in mature muscles. PMID:19509079

  15. Knockout beyond the dripline

    SciTech Connect

    Bonaccorso, A.; Charity, R. J.; Kumar, R.; Salvioni, G.

    2015-02-24

    In this contribution, we will describe neutron and proton removal from {sup 9}C and {sup 7}Be which are two particularly interesting nuclei entering the nucleo-synthesis pp-chain [1, 2]. Neutron and proton removal reactions have been used in the past twenty years to probe the single-particle structure of exotic nuclei. The core parallel-momentum distribution can give information on the angular momentum and spin of the nucleon initial state while the total removal cross section is sensitive to the asymptotic part of the initial wave function and also to the reaction mechanism. Because knockout is a peripheral reaction from which the Asymptotic Normalization Constant (ANC) of the single-particle wave function can be extracted, it has been used as an indirect method to obtain the rate of reactions like {sup 8}B(p,γ){sup 9}C or {sup 7}Be(p,γ){sup 8}B. Nucleon removal has recently been applied by the HiRA collaboration [3] to situations in which the remaining “core” is beyond the drip line, such as {sup 8}C and {sup 6}Be, unbound by one or more protons, and whose excitation-energy spectrum can be obtained by the invariant-mass method. By gating on the ground-state peak, “core” parallel-momentum distributions and total knockout cross sections have been obtained similar to previous studies with well-bound “cores”. In addition for each projectile, knock out to final bound states has also been obtained in several cases. We will report on the theoretical description and comparison to this experimental data for a few cases for which advances in the accuracy of the transfer-to-the continuum model [4, 5] have been made [6]. These include the use, when available, of “ab-initio” overlaps for the initial state [7] and in particular their ANC values [8]. Also, the construction of a nucleus-target folding potential for the treatment of the core-target S-matrix [9] using for the cores “ab-initio” densities [10] and state-of-the-art n−{sup 9}Be optical

  16. DNA methylation in cystathionine-γ-lyase (CSE) gene promoter induced by ox-LDL in macrophages and in apoE knockout mice.

    PubMed

    Du, Hua-Ping; Li, Jiaojiao; You, Shou-Jiang; Wang, Ya-Li; Wang, Fen; Cao, Yong-Jun; Hu, Li-Fang; Liu, Chun-Feng

    2016-01-15

    Recent studies suggest that epigenetic alterations such as DNA methylation control many aspects of monocytes/macrophages and participate in the pathogenesis of atherosclerosis, a lipid-driven inflammatory disorder. Our and other groups demonstrated that dysregulation of cystathionine γ-lyase (CSE) -hydrogen sulfide (H2S) pathway was involved in monocyte/macrophages-mediated inflammation and atherosclerosis. However, it remains unknown whether altered cse methylation in macrophages may play a role in linking CSE-H2S dysregulation and atherosclerosis. In the present study, we showed that plasma H2S and H2S production in the peritoneal macrophages of apolipoprotein knockout (apoE(-/-)) mice gradually decreased with ages, and were also lower than that in control mice at 12 weeks older. Moreover, CSE mRNA expressions decreased while DNA methyltransferase (DNMT) expressions increased in the peritoneal macrophages isolated from apoE(-/-) mice, compared to age-matched wildtype mice. Similar observations were obtained in an in vitro study. In oxidized low-density lipoprotein (ox-LDL)-treated raw264.7 macrophages, cse transcription was down-regulated while the expression and activity of DNMT was up-regulated, associated with enhanced DNA methylation in cse promoter. Suppression of DNMT with its inhibitor or siRNA reversed the decrease of CSE mRNA. Therefore, our data suggest that DNA hypermethylation of CpG rich region in cse promoter might contribute to the decrease of cse transcription and H2S production in macrophages, and thus contribute to atherosclerosis development. PMID:26692478

  17. Cathepsin L Plays a Major Role in Cholecystokinin Production in Mouse Brain Cortex and in Pituitary AtT-20 Cells: Protease Gene Knockout and Inhibitor Studies

    PubMed Central

    Beinfeld, Margery C.; Funkelstein, Lydiane; Foulon, Thierry; Cadel, Sandrine; Kitagawa, Kouki; Toneff, Thomas; Reinheckel, Thomas; Peters, Christoph; Hook, Vivian

    2009-01-01

    Cholecystokinin (CCK) is a peptide neurotransmitter whose production requires proteolytic processing of the proCCK precursor to generate active CCK8 neuropeptide in brain. This study demonstrates the significant role of the cysteine protease cathepsin L for CCK8 production. In cathepsin L knockout (KO) mice, CCK8 levels were substantially reduced in brain cortex by an average of 75%. To evaluate the role of cathepsin L in producing CCK in the regulated secretory pathway of neuroendocrine cells, pituitary AtT-20 cells that stably produce CCK were treated with the specific cathepsin L inhibitor, CLIK-148. CLIK-148 inhibitor treatment resulted in decreased amounts of CCK secreted from the regulated secretory pathway of AtT-20 cells. CLIK-148 also reduced cellular levels of CCK9 (Arg-CCK8), consistent with CCK9 as an intermediate product of cathepsin L, shown by the decreased ratio of CCK9/CCK8. The decreased CCK0/CCK8 ratio also suggests a shift in the production to CCK8 over CCK9 during inhibition of cathepsin L. During reduction of the PC1/3 processing enzyme by siRNA, the ratio of CCK9/CCK8 was increased, suggesting a shift to the cathepsin L pathway for production of CCK9. The changes in ratios of CCK9 compared to CCK8 are consistent with dual roles of the cathepsin L protease pathway that includes aminopeptidase B to remove NH2-terminal Arg or Lys, and the PC1/3 protease pathway. These results suggest that cathepsin L functions as a major protease responsible for CCK8 production in mouse brain cortex, and participates with PC1/3 for CCK8 production in pituitary cells. PMID:19589362

  18. Understanding the progression of atherosclerosis through gene profiling and co-expression network analysis in Apob(tm2Sgy)Ldlr(tm1Her) double knockout mice.

    PubMed

    Deshpande, Vrushali; Sharma, Ankit; Mukhopadhyay, Rupak; Thota, Lakshmi Narasimha Rao; Ghatge, Madankumar; Vangala, Rajani Kanth; Kakkar, Vijay V; Mundkur, Lakshmi

    2016-06-01

    The objective of the study was to gain molecular insights into the progression of atherosclerosis in Apob(tm2Sgy)Ldlr(tm1Her) mice, using transcriptome profiles. Weighted gene co network analysis (WGCNA) and time course analysis using limma were used to study disease progression from 0 to 20weeks. Five co-expression modules were identified by WGCNA using the expression values of 2153 genes. Genes associated with autophagy, endoplasmic reticulum stress, inflammation and lipid metabolism were differentially expressed at early stages of atherosclerosis. Time course analysis highlighted activation of inflammatory gene signaling at 4weeks, cell proliferation and calcification at 8weeks, amyloid like structures and oxidative stress at 14weeks and enhanced production of inflammatory cytokines at 20weeks. Our results suggest that maximum gene perturbations occur during early atherosclerosis which could be the danger signals associated with subclinical disease. Understanding these genes and associated pathways can help in improvement of diagnostic and therapeutic targets for atherosclerosis. PMID:27133569

  19. Alterations of gene expression of sodium channels in dorsal root ganglion neurons of estrogen receptor knockout (ERKO) mice induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).

    PubMed

    Ding, Haixia; Wang, Qiang; Liu, Jingli; Qian, Wenyi; Wang, Wenjuan; Wang, Jun; Gao, Rong; Xiao, Hang

    2012-08-01

    Estrogen receptors (ERα and ERβ) mediate the neuroprotection of estrogens against MPTP-induced striatal dopamine (DA) depletion. Pain is an important and distressing symptom in Parkinson's disease (PD). Voltage-gated sodium channels in sensory neurons are involved in the development of neuropathic pain. In this study, MPTP caused changes in nociception and alterations of gene expression of voltage-gated sodium channels in dorsal root ganglion (DRG) neurons in ER knockout (ERKO) mice were investigated. We found that administration of MPTP (11 mg/kg) to WT mice led to an extensive depletion of DA and its two metabolites, αERKO mice were observed to be more susceptible to MPTP toxicity than βERKO or WT mice. In addition, we found that the mRNA levels of TTX-S and TTX-R sodium channel subtypes were differentially affected in MPTP-treated WT animals. The MPTP-induced up-regulation of Nav1.1 and Nav1.9, down-regulation of Nav1.6 in DRG neurons may be through ERβ, up-regulation of Nav1.7 and down-regulation of Nav1.8 are dependent on both ERα and ERβ. Therefore, the MPTP-induced alterations of gene expression of sodium channels in DRG neurons could be an important mechanism to affect excitability and nociceptive thresholds, and the ERs appear to play a role in nociception in PD. PMID:22371119

  20. Speed and sensitivity of phototransduction in Drosophila depend on degree of saturation of membrane phospholipids.

    PubMed

    Randall, Alex S; Liu, Che-Hsiung; Chu, Brian; Zhang, Qifeng; Dongre, Sidharta A; Juusola, Mikko; Franze, Kristian; Wakelam, Michael J O; Hardie, Roger C

    2015-02-11

    Drosophila phototransduction is mediated via a G-protein-coupled PLC cascade. Recent evidence, including the demonstration that light evokes rapid contractions of the photoreceptors, suggested that the light-sensitive channels (TRP and TRPL) may be mechanically gated, together with protons released by PLC-mediated PIP2 hydrolysis. If mechanical gating is involved we predicted that the response to light should be influenced by altering the physical properties of the membrane. To achieve this, we used diet to manipulate the degree of saturation of membrane phospholipids. In flies reared on a yeast diet, lacking polyunsaturated fatty acids (PUFAs), mass spectrometry showed that the proportion of polyunsaturated phospholipids was sevenfold reduced (from 38 to ∼5%) but rescued by adding a single species of PUFA (linolenic or linoleic acid) to the diet. Photoreceptors from yeast-reared flies showed a 2- to 3-fold increase in latency and time to peak of the light response, without affecting quantum bump waveform. In the absence of Ca(2+) influx or in trp mutants expressing only TRPL channels, sensitivity to light was reduced up to ∼10-fold by the yeast diet, and essentially abolished in hypomorphic G-protein mutants (Gαq). PLC activity appeared little affected by the yeast diet; however, light-induced contractions measured by atomic force microscopy or the activation of ectopic mechanosensitive gramicidin channels were also slowed ∼2-fold. The results are consistent with mechanosensitive gating and provide a striking example of how dietary fatty acids can profoundly influence sensory performance in a classical G-protein-coupled signaling cascade. PMID:25673862

  1. Speed and Sensitivity of Phototransduction in Drosophila Depend on Degree of Saturation of Membrane Phospholipids

    PubMed Central

    Randall, Alex S.; Liu, Che-Hsiung; Chu, Brian; Zhang, Qifeng; Dongre, Sidharta A.; Juusola, Mikko; Franze, Kristian; Wakelam, Michael J.O.

    2015-01-01

    Drosophila phototransduction is mediated via a G-protein-coupled PLC cascade. Recent evidence, including the demonstration that light evokes rapid contractions of the photoreceptors, suggested that the light-sensitive channels (TRP and TRPL) may be mechanically gated, together with protons released by PLC-mediated PIP2 hydrolysis. If mechanical gating is involved we predicted that the response to light should be influenced by altering the physical properties of the membrane. To achieve this, we used diet to manipulate the degree of saturation of membrane phospholipids. In flies reared on a yeast diet, lacking polyunsaturated fatty acids (PUFAs), mass spectrometry showed that the proportion of polyunsaturated phospholipids was sevenfold reduced (from 38 to ∼5%) but rescued by adding a single species of PUFA (linolenic or linoleic acid) to the diet. Photoreceptors from yeast-reared flies showed a 2- to 3-fold increase in latency and time to peak of the light response, without affecting quantum bump waveform. In the absence of Ca2+ influx or in trp mutants expressing only TRPL channels, sensitivity to light was reduced up to ∼10-fold by the yeast diet, and essentially abolished in hypomorphic G-protein mutants (Gαq). PLC activity appeared little affected by the yeast diet; however, light-induced contractions measured by atomic force microscopy or the activation of ectopic mechanosensitive gramicidin channels were also slowed ∼2-fold. The results are consistent with mechanosensitive gating and provide a striking example of how dietary fatty acids can profoundly influence sensory performance in a classical G-protein-coupled signaling cascade. PMID:25673862

  2. Prolongation of Actions of Ca2+ Early in Phototransduction by 9-Demethylretinal

    PubMed Central

    Matthews, Hugh R.; Cornwall, M.C.; Crouch, R.K.

    2001-01-01

    During adaptation Ca2+ acts on a step early in phototransduction, which is normally available for only a brief period after excitation. To investigate the identity of this step, we studied the effect of the light-induced decline in intracellular Ca2+ concentration on the response to a bright flash in normal rods, and in rods bleached and regenerated with 11-cis 9-demethylretinal, which forms a photopigment with a prolonged photoactivated lifetime. Changes in cytoplasmic Ca2+ were opposed by rapid superfusion of the outer segment with a 0Na+/0Ca2+ solution designed to minimize Ca2+ fluxes across the surface membrane. After regeneration of a bleached rod with 9-demethlyretinal, the response in Ringer's to a 440-nm bright flash was prolonged in comparison with the unbleached control, and the response remained in saturation for 10–15s. If the dynamic fall in Ca2+i induced by the flash was delayed by stepping the outer segment to 0Na+/0Ca2+ solution just before the flash and returning it to Ringer's shortly before recovery, then the response saturation was prolonged further, increasing linearly by 0.41 ± 0.01 of the time spent in this solution. In contrast, even long exposures to 0Na+/0Ca2+ solution of rods containing native photopigment evoked only a modest response prolongation on the return to Ringer's. Furthermore, if the rod was preexposed to steady subsaturating light, thereby reducing the cytoplasmic calcium concentration, then the prolongation of the bright flash response evoked by 0Na+/0Ca2+ solution was reduced in a graded manner with increasing background intensity. These results indicate that altering the chromophore of rhodopsin prolongs the time course of the Ca2+-dependent step early in the transduction cascade so that it dominates response recovery, and suggest that it is associated with photopigment quenching by phosphorylation. PMID:11585850

  3. Activation and quenching of the phototransduction cascade in retinal cones as inferred from electrophysiology and mathematical modeling

    PubMed Central

    Astakhova, Luba; Firsov, Michael

    2015-01-01

    Purpose To experimentally identify and quantify factors responsible for the lower sensitivity of retinal cones compared to rods. Methods Electrical responses of frog rods and fish (Carassius) cones to short flashes of light were recorded using the suction pipette technique. A fast solution changer was used to apply a solution that fixed intracellular Ca2+ concentration at the prestimulus level, thereby disabling Ca2+ feedback, to the outer segment (OS). The results were analyzed with a specially designed mathematical model of phototransduction. The model included all basic processes of activation and quenching of the phototransduction cascade but omitted unnecessary mechanistic details of each step. Results Judging from the response versus intensity curves, Carassius cones were two to three orders of magnitude less sensitive than frog rods. There was a large scatter in sensitivity among individual cones, with red-sensitive cones being on average approximately two times less sensitive than green-sensitive ones. The scatter was mostly due to different signal amplification, since the kinetic parameters of the responses among cones were far less variable than sensitivity. We argue that the generally accepted definition of the biochemical amplification in phototransduction cannot be used for comparing amplification in rods and cones, since it depends on an irrelevant factor, that is, the cell’s volume. We also show that the routinely used simplified parabolic curve fitting to an initial phase of the response leads to a few-fold underestimate of the amplification. We suggest a new definition of the amplification that only includes molecular parameters of the cascade activation, and show how it can be derived from experimental data. We found that the mathematical model with unrestrained parameters can yield an excellent fit to experimental responses. However, the fits with wildly different sets of parameters can be virtually indistinguishable, and therefore cannot

  4. Construction of Deletion-knockout Mutant Fowlpox Virus (FWPV)

    PubMed Central

    Laidlaw, Stephen M.; Skinner, Michael A.

    2016-01-01

    The construction of deletion-knockout poxviruses is a useful approach to determining the function of specific virus genes. This protocol is an adaptation of the transient dominant knockout selection protocol published by Falkner and Moss (1990) for use with vaccinia virus. The protocol makes use of the dominant selectable marker Escherichia coli guanine phosphoribosyltransferase (gpt) gene (Mulligan and Berg, 1981), under the control of an early/late poxvirus promoter. The deletion viruses that are produced no longer contain a selectable marker, which may be preferable for the production of vaccines.

  5. CRISPR/Cas9-mediated knockout of factors in non-homologous end joining pathway enhances gene targeting in silkworm cells

    PubMed Central

    Zhu, Li; Mon, Hiroaki; Xu, Jian; Lee, Jae Man; Kusakabe, Takahiro

    2015-01-01

    Gene targeting can be achieved by precise genetic modifications through homology-directed repair (HDR) after DNA breaks introduced by genome editing tools such as CRISPR/Cas9 system. The most common form of HDR is homologous recombination (HR). Binding to the DNA breaks by HR factors is thought to compete with non-homologous end joining (NHEJ), an alternative DNA repair pathway. Here, we knocked out the factors in NHEJ by CRISPR/Cas9 system in silkworm cells, so that increased the activities of HR up to 7-fold. Also efficient HR-mediated genome editing events occurred between the chromosomal BmTUDOR-SN gene and donor DNA sequences with an EGFP gene in the middle of two homologous arms for the target gene. Utilizing the NHEJ-deficient silkworm cells, we found that homologous arms as short as 100 bp in donor DNA could be designed to perform precise genome editing. These studies should greatly accelerate investigations into genome editing of silkworm. PMID:26657947

  6. Clues to VIP function from knockout mice.

    PubMed

    Hamidi, S A; Szema, A M; Lyubsky, S; Dickman, K G; Degene, A; Mathew, S M; Waschek, J A; Said, S I

    2006-07-01

    We have taken advantage of the availability of vasoactive intestinal polypeptide (VIP) knockout (KO) mice to examine the possible influence of deletion of the VIP gene on: (a) airway reactivity and airway inflammation, as indicators of bronchial asthma; (b) mortality from endotoxemia, a model of septic shock; and (c) the pulmonary circulation. VIP KO mice showed: (a) airway hyperresponsiveness to the cholinergic agonist methacholine, as well as peribronchial and perivascular inflammation; (b) a greater susceptibility to death from endotoxemia; and (c) evidence suggestive of pulmonary hypertension. PMID:16888146

  7. Improved Survival and Reduced Phenotypic Severity Following AAV9/MECP2 Gene Transfer to Neonatal and Juvenile Male Mecp2 Knockout Mice

    PubMed Central

    Gadalla, Kamal KE; Bailey, Mark ES; Spike, Rosemary C; Ross, Paul D; Woodard, Kenton T; Kalburgi, Sahana Nagabhushan; Bachaboina, Lavanya; Deng, Jie V; West, Anne E; Samulski, R Jude; Gray, Steven J; Cobb, Stuart R

    2013-01-01

    Typical Rett syndrome (RTT) is a pediatric disorder caused by loss-of-function mutations in the methyl-CpG binding protein 2 (MECP2) gene. The demonstrated reversibility of RTT-like phenotypes in mice suggests that MECP2 gene replacement is a potential therapeutic option in patients. We report improvements in survival and phenotypic severity in Mecp2-null male mice after neonatal intracranial delivery of a single-stranded (ss) AAV9/chicken β-actin (CBA)-MECP2 vector. Median survival was 16.6 weeks for MECP2-treated versus 9.3 weeks for green fluorescent protein (GFP)-treated mice. ssAAV9/CBA-MECP2–treated mice also showed significant improvement in the phenotype severity score, in locomotor function, and in exploratory activity, as well as a normalization of neuronal nuclear volume in transduced cells. Wild-type (WT) mice receiving neonatal injections of the same ssAAV9/CBA-MECP2 vector did not show any significant deficits, suggesting a tolerance for modest MeCP2 overexpression. To test a MECP2 gene replacement approach in a manner more relevant for human translation, a self-complementary (sc) adeno-associated virus (AAV) vector designed to drive MeCP2 expression from a fragment of the Mecp2 promoter was injected intravenously (IV) into juvenile (4–5 weeks old) Mecp2-null mice. While the brain transduction efficiency in juvenile mice was low (~2–4% of neurons), modest improvements in survival were still observed. These results support the concept of MECP2 gene therapy for RTT. PMID:23011033

  8. Elimination of Manganese(II,III) Oxidation in Pseudomonas putida GB-1 by a Double Knockout of Two Putative Multicopper Oxidase Genes

    PubMed Central

    McCarthy, James K.; Tebo, Bradley M.

    2013-01-01

    Bacterial manganese(II) oxidation impacts the redox cycling of Mn, other elements, and compounds in the environment; therefore, it is important to understand the mechanisms of and enzymes responsible for Mn(II) oxidation. In several Mn(II)-oxidizing organisms, the identified Mn(II) oxidase belongs to either the multicopper oxidase (MCO) or the heme peroxidase family of proteins. However, the identity of the oxidase in Pseudomonas putida GB-1 has long remained unknown. To identify the P. putida GB-1 oxidase, we searched its genome and found several homologues of known or suspected Mn(II) oxidase-encoding genes (mnxG, mofA, moxA, and mopA). To narrow this list, we assumed that the Mn(II) oxidase gene would be conserved among Mn(II)-oxidizing pseudomonads but not in nonoxidizers and performed a genome comparison to 11 Pseudomonas species. We further assumed that the oxidase gene would be regulated by MnxR, a transcription factor required for Mn(II) oxidation. Two loci met all these criteria: PputGB1_2447, which encodes an MCO homologous to MnxG, and PputGB1_2665, which encodes an MCO with very low homology to MofA. In-frame deletions of each locus resulted in strains that retained some ability to oxidize Mn(II) or Mn(III); loss of oxidation was attained only upon deletion of both genes. These results suggest that PputGB1_2447 and PputGB1_2665 encode two MCOs that are independently capable of oxidizing both Mn(II) and Mn(III). The purpose of this redundancy is unclear; however, differences in oxidation phenotype for the single mutants suggest specialization in function for the two enzymes. PMID:23124227

  9. Knockout of the dhfr-ts Gene in Trypanosoma cruzi Generates Attenuated Parasites Able to Confer Protection against a Virulent Challenge

    PubMed Central

    Perez Brandan, Cecilia; Padilla, Angel M.; Xu, Dan; Tarleton, Rick L.; Basombrio, Miguel A.

    2011-01-01

    Background Trypanosoma cruzi is a protozoan parasite that causes severe disease in millions of habitants of developing countries. Currently there is no vaccine to prevent this disease and the available drugs have the consequences of side effects. Live vaccines are likely to be more effective in inducing protection than recombinant proteins or DNA vaccines; however, safety problems associated to their use have been pointed out. In recent years, increasing knowledge on the molecular genetics of Trypanosomes has allowed the identification and elimination of genes that may be necessary for parasite infectivity and survival. In this sense, targeted deletion or disruption of specific genes in the parasite genome may protect against such reversion to virulent genotypes. Methods and Findings By targeted gene disruption we generated monoallelic mutant parasites for the dhfr-ts gene in a T. cruzi strain that has been shown to be naturally attenuated. In comparison to T. cruzi wild type epimastigotes, impairment in growth of dhfr-ts+/− mutant parasites was observed and mutant clones displayed decreased virulence in mice. Also, a lower number of T. cruzi-specific CD8+ T cells, in comparison to those induced by wild type parasites, was detected in mice infected with mutant parasites. However, no remarkable differences in the protective effect of TCC wild type versus TCC mutant parasites were observed. Mice challenged with virulent parasites a year after the original infection with the mutant parasites still displayed a significant control over the secondary infection. Conclusion This study indicates that it is possible to generate genetically attenuated T. cruzi parasites able to confer protection against further T. cruzi infections. PMID:22180798

  10. Establishment of Functional Genomics Pipeline in Epiblast-Like Tissue by Combining Transcriptomic Analysis and Gene Knockdown/Knockin/Knockout, Using RNA Interference and CRISPR/Cas9.

    PubMed

    Takata, Nozomu; Sakakura, Eriko; Kasukawa, Takeya; Sakuma, Tetsushi; Yamamoto, Takashi; Sasai, Yoshiki

    2016-06-01

    The epiblast (foremost embryonic ectoderm) generates all three germ layers and therefore has crucial roles in the formation of all mammalian body cells. However, regulation of epiblast gene expression is poorly understood because of the difficulty of manipulating epiblast tissues in vivo. In the present study, using the self-organizing properties of embryonic stem cell (ESC), we generated and characterized epiblast-like tissue in three-dimensional culture. We identified significant genome-wide gene expression changes in this epiblast-like tissue by transcriptomic analysis. In addition, we identified the particular significance of the Erk/Mapk and integrin-linked kinase pathways, and genes related to ectoderm/epithelial formation, using the bioinformatics resources IPA and DAVID. Here, we focused on Fgf5, which ranked in the top 10 among the discovered genes. To develop a functional analysis of Fgf5, we created an efficient method combining CRISPR/Cas9-mediated genome engineering and RNA interference (RNAi). Notably, we show one-step generation of various Fgf5 reporter lines including heterozygous and homozygous knockins (the GET method). For time- and dose-dependent depletion of fgf5 over the course of development, we generated an ESC line harboring Tol2 transposon-mediated integration of an inducible short hairpin RNA interference system (pdiRNAi). Our findings raised the possibility that Fgf/Erk signaling and apicobasal epithelial integrity are important factors in epiblast development. In addition, our methods provide a framework for a broad array of applications in the areas of mammalian genetics and molecular biology to understand development and to improve future therapeutics. PMID:26839115

  11. Knockout of the p-Coumarate Decarboxylase Gene from Lactobacillus plantarum Reveals the Existence of Two Other Inducible Enzymatic Activities Involved in Phenolic Acid Metabolism

    PubMed Central

    Barthelmebs, Lise; Divies, Charles; Cavin, Jean-François

    2000-01-01

    Lactobacillus plantarum NC8 contains a pdc gene coding for p-coumaric acid decarboxylase activity (PDC). A food grade mutant, designated LPD1, in which the chromosomal pdc gene was replaced with the deleted pdc gene copy, was obtained by a two-step homologous recombination process using an unstable replicative vector. The LPD1 mutant strain remained able to weakly metabolize p-coumaric and ferulic acids into vinyl derivatives or into substituted phenyl propionic acids. We have shown that L. plantarum has a second acid phenol decarboxylase enzyme, better induced with ferulic acid than with p-coumaric acid, which also displays inducible acid phenol reductase activity that is mostly active when glucose is added. Those two enzymatic activities are in competition for p-coumaric and ferulic acid degradation, and the ratio of the corresponding derivatives depends on induction conditions. Moreover, PDC appeared to decarboxylate ferulic acid in vitro with a specific activity of about 10 nmol · min−1 · mg−1 in the presence of ammonium sulfate. Finally, PDC activity was shown to confer a selective advantage on LPNC8 grown in acidic media supplemented with p-coumaric acid, compared to the LPD1 mutant devoid of PDC activity. PMID:10919793

  12. 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. PMID:27318252

  13. 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. PMID:23375195

  14. 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

  15. Rod phototransduction modulated by bicarbonate in the frog retina: roles of carbonic anhydrase and bicarbonate exchange.

    PubMed Central

    Donner, K; Hemilä, S; Kalamkarov, G; Koskelainen, A; Shevchenko, T

    1990-01-01

    1. Effects on rod phototransduction following manipulation of retinal CO2-HCO3- and H+ fluxes were studied in dark-adapted retinas of the frog and the tiger salamander. 2. Rod photoresponses to brief flashes of light were recorded from the isolated sensory retina as electroretinogram mass receptor potentials and from isolated rods by the suction-pipette technique. The experimental treatments were: (1) varying [CO2] + [HCO3-] in the perfusion fluid: (2) applying acetazolamide (AAA), which inhibits the enzyme carbonic anhydrase (CA); and (3) applying 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) which blocks exchange mechanisms transporting HCO3- across cell membranes. 3. The concentration of the internal transmitter of the rods, cyclic GMP, was biochemically determined from the rod outer segment layer of retinas that had been incubated in the same solutions as were used for perfusion in the electrophysiological experiments. 4. The introduction of 6 mM-sodium bicarbonate to replace half the buffer of a nominally CO2-HCO3(-)-free (12 mM-phosphate or HEPES, [Na+] constant) Ringer solution doubled the cyclic GMP concentration in the rod outer segment layer and increased the saturating response amplitude and the relative sensitivity of rods in the intact retina. 5. The introduction of 0.5 mM-AAA into bicarbonate-containing Ringer solution accelerated the growth of saturated responses and sensitivity. Incubation of the retina in AAA-bicarbonate Ringer solution elevated the concentration of cyclic GMP ninefold compared with the phosphate control. 6. No effects of switching to bicarbonate-AAA Ringer solution were observed in the photocurrent of isolated rods drawn into suction pipettes with only the outer segment protruding into the perfusion fluid. The target of AAA is probably the CA-containing Müller cell. 7. The introduction of DIDS into the perfusate (at normal pH 7.5) set off a continuous decay of photoresponses which finally abolished light sensitivity

  16. Knockout of a starch synthase gene OsSSIIIa/Flo5 causes white-core floury endosperm in rice (Oryza sativa L.).

    PubMed

    Ryoo, Nayeon; Yu, Chul; Park, Cheon-Seok; Baik, Moo-Yeol; Park, In Myoung; Cho, Man-Ho; Bhoo, Seong Hee; An, Gynheung; Hahn, Tae-Ryong; Jeon, Jong-Seong

    2007-07-01

    To elucidate the role of SSIIIa during starch synthesis in rice (Oryza sativa L.) endosperm, we characterized null mutants of this gene, generated by T-DNA insertions. Scanning electron microscope (SEM) analysis revealed that the starch granules in these mutants are smaller and rounder compared with the wild type controls, and that the mutant endosperm is characterized by a loosely packed central portion exhibiting a floury-like phenotype. Hence, the OsSSIIIa (Oryza sativa SSIIIa) mutations are referred to as white-core floury endosperm 5-1 (flo5-1) and flo5-2. Based upon their X-ray diffraction patterns, the crystallinity of the starch in the flo5 mutant endosperm is decreased compared with wild type. Through determination of the chain-length distribution of the mutant endosperm starch, we found that flo5-1 and flo5-2 mutants have reduced the content of long chains with degree of polymerization (DP) 30 or greater compared with the controls. This suggests that OsSSIIIa/Flo5 plays an important role in generating relatively long chains in rice endosperm. In addition, DP 6 to 8 and DP 16 to 20 appeared to be reduced in endosperm starch of flo5-1 and flo5-2, whereas DP 9 to 15 and DP 22 to 29 were increased in these mutants. By the use of differential scanning calorimetry (DSC), the gelatinization temperatures of endosperm starch were found to be 1-5 degrees C lower than those of the control. We propose a distinct role for OsSSIIIa/Flo5 and the coordinated action of other SS isoforms during starch synthesis in the seed endosperm of rice. PMID:17297616

  17. Live Attenuated Leishmania donovani p27 Gene Knockout Parasites Are Non-pathogenic and Elicit Long Term Protective Immunity in BALB/c Mice

    PubMed Central

    Dey, Ranadhir; Dagur, Pradeep K.; Selvapandiyan, Angamuthu; McCoy, J. Philip; Salotra, Poonam; Duncan, Robert; Nakhasi, Hira L.

    2013-01-01

    Leishmaniasis causes significant morbidity and mortality worldwide and there are no vaccines available against this disease. Previously, we had shown that the amastigote specific protein p27 (Ldp27) is a component of an active cytochrome c oxidase complex in L. donovani and upon deletion of its gene the parasite had reduced virulence in vivo. In this study, we have shown that Ldp27−/− parasites do not survive beyond 20 weeks in BALB/c mice, hence are safe as an immunogen. Upon virulent challenge, 12 weeks post-immunized mice showed significantly lower parasite burden in liver and spleen. When mice were challenged 20 weeks post immunization, there was still a significant reduction in parasite burden suggesting long term protection by Ldp27−/− immunization. Immunization with Ldp27−/− induced both pro- and anti- inflammatory cytokine responses and activated splenocytes for enhanced leishmaniacidal activity in association with NO production. Protection in both short and long term immunized mice after challenge with the wild type parasite correlated with the stimulation of multifunctional Th1 type CD4 and CD8 T cells. Adoptive transfer of T cells from long term immunized mice conferred protection against virulent challenge in naïve recipient mice suggesting involvement of memory T cell response in the protection against Leishmania infection. Immunization of mice with Ldp27−/− also demonstrated cross-protection against the Leishmania major and Leishmania braziliensis infection. Our data show that genetically modified live attenuated Ldp27−/− parasites are safe, induce protective immunity even in the absence of parasites and can provide protection against homologous and heterologous Leishmania species. PMID:23338240

  18. 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. PMID:26209783

  19. 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...

  20. Murine mentors: transgenic and knockout models of surgical disease.

    PubMed Central

    Arbeit, J M; Hirose, R

    1999-01-01

    OBJECTIVE: Transgenic and knockout technologies have emerged from the "molecular biology revolution" as unprecedented techniques for manipulating gene function in intact mice. The goals of this review are to outline the techniques of creating transgenic and knockout mice, and to demonstrate their use in elucidation of the molecular mechanisms underlying common surgical diseases. SUMMARY BACKGROUND DATA: Gain of gene function is created by transgenic technology, whereas gene function is ablated using gene knockouts. Each technique has distinctive applications and drawbacks. A unique feature of genetically manipulated mice is that combinatorial genetic experiments can be executed that precisely define the functional contribution of a gene to disease progression. Transgenic and knockout mouse models of wound healing, cardiovascular disease, transplant immunology, gut motility and inflammatory bowel disease, and oncology are beginning to illuminate the precise molecular regulation of these diseases. Transgenic technology has also been extended to larger mammals such as pigs, with the goal of using genetic manipulation of the xenogenic immune response to increase the availability of transplant organs. Continual refinements in gene manipulation technology in mice offer the opportunity to turn genes on or off at precise time intervals and in particular tissues, according to the needs of the investigator. Ultimately, investigation of disease development and progression in genetically manipulated mammals may delineate new molecular targets for drug discovery and provide novel platforms for drug efficacy screens. CONCLUSIONS: Emulation of human disease and therapy using genetically manipulated mammals fulfills a promise of molecular medicine: fusion of molecular biochemistry with "classical" biology and physiology. Surgeons have unique skills spanning both worlds that can facilitate their success in this expanding arena. PMID:9923797

  1. Designer Nuclease-Mediated Generation of Knockout THP1 Cells.

    PubMed

    Schmidt, Tobias; Schmid-Burgk, Jonathan L; Ebert, Thomas S; Gaidt, Moritz M; Hornung, Veit

    2016-01-01

    Recent developments in the field of designer nucleases allow the efficient and specific manipulation of genomic architectures in eukaryotic cell lines. To this end, it has become possible to introduce DNA double strand breaks (DSBs) at user-defined genomic loci. If located in critical coding regions of genes, thus induced DSBs can lead to insertions or deletions (indels) that result in frameshift mutations and thereby the knockout of the target gene. In this chapter, we describe a step-by-step workflow for establishing knockout cell clones of the difficult-to-transfect suspension cell line THP1. The here described protocol encompasses electroporation, cell cloning, and a deep sequencing-based genotyping step that allows the in-parallel analysis of 96 cell clones per gene of interest. Furthermore, we describe the use of the analysis tool OutKnocker that allows rapid identification of cell clones with all-allelic frameshift mutations. PMID:26443227

  2. Rare complete knockouts in humans: population distribution and significant role in autism spectrum disorders

    PubMed Central

    Lim, Elaine T.; Raychaudhuri, Soumya; Sanders, Stephan J.; Stevens, Christine; Sabo, Aniko; MacArthur, Daniel G.; Neale, Benjamin M.; Kirby, Andrew; Ruderfer, Douglas M.; Fromer, Menachem; Lek, Monkol; Liu, Li; Flannick, Jason; Ripke, Stephan; Nagaswamy, Uma; Muzny, Donna; Reid, Jeffrey G.; Hawes, Alicia; Newsham, Irene; Wu, Yuanqing; Lewis, Lora; Dinh, Huyen; Gross, Shannon; Wang, Li-San; Lin, Chiao-Feng; Valladares, Otto; Gabriel, Stacey B.; dePristo, Mark; Altshuler, David M.; Purcell, Shaun M.; State, Matthew W.; Boerwinkle, Eric; Buxbaum, Joseph D.; Cook, Edwin H.; Gibbs, Richard A.; Schellenberg, Gerard D.; Sutcliffe, James S.; Devlin, Bernie; Roeder, Kathryn; Daly, Mark J.

    2013-01-01

    SUMMARY To characterize the role of rare complete human knockouts in autism spectrum disorders (ASD), we identify genes with homozygous or compound heterozygous loss-of-function (LoF) variants (defined as nonsense and essential splice sites) from exome sequencing of 933 cases and 869 controls. We identify a two-fold increase in complete knockouts of autosomal genes with low rates of LoF variation (≤5% frequency) in cases and estimate a 3% contribution to ASD risk by these events, confirming this observation in an independent set of 563 probands and 4,605 controls. Outside the pseudo-autosomal regions on the X-chromosome, we similarly observe a significant 1.5-fold increase in rare hemizygous knockouts in males, contributing to another 2% of ASDs in males. Taken together these results provide compelling evidence that rare autosomal and X-chromosome complete gene knockouts are important inherited risk factors for ASD. PMID:23352160

  3. Assignment of the phosducin (PDC) gene to human chromosome 1q25-1q32. 1 by somatic cell hybridization and in situ hybridization

    SciTech Connect

    Sparkes, R.S.; Kojis, T.; Klisak, I.; Heinzmann, C.; Bateman, J.B. ); Lee, R.H. ); Shinohara, T. ); Craft, C.M. )

    1993-11-01

    Phosducin is a soluble photoreceptor phosphoprotein that probably modulates phototransduction in the retina and thus qualifies as a potential candidate gene for retinitis pigmentosa. Using both human/mouse somatic cell hybrids and in situ hybridization to human metaphase chromosomes, the authors have mapped this gene to chromosome 1q25-1q32.1. 18 refs., 2 figs.

  4. The evolution of thymic lymphomas in p53 knockout mice.

    PubMed

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

    2014-12-01

    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

  5. 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

  6. 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

  7. Knockout of Foxp2 disrupts vocal development in mice.

    PubMed

    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

  8. Knock-out models reveal new aquaporin functions.

    PubMed

    Verkman, Alan S

    2009-01-01

    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

  9. Conditional knockouts generated by engineered CRISPR-Cas9 endonuclease reveal the roles of coronin in C. elegans neural development.

    PubMed

    Shen, Zhongfu; Zhang, Xianliang; Chai, Yongping; Zhu, Zhiwen; Yi, Peishan; Feng, Guoxin; Li, Wei; Ou, Guangshuo

    2014-09-01

    Conditional gene knockout animals are valuable tools for studying the mechanisms underlying cell and developmental biology. We developed a conditional knockout strategy by spatiotemporally manipulating the expression of an RNA-guided DNA endonuclease, CRISPR-Cas9, in Caenorhabditis elegans somatic cell lineages. We showed that this somatic CRISPR-Cas9 technology provides a quick and efficient approach to generate conditional knockouts in various cell types at different developmental stages. Furthermore, we demonstrated that this method outperforms our recently developed somatic TALEN technique and enables the one-step generation of multiple conditional knockouts. By combining these techniques with live-cell imaging, we showed that an essential embryonic gene, Coronin, which is associated with human neurobehavioral dysfunction, regulates actin organization and cell morphology during C. elegans postembryonic neuroblast migration and neuritogenesis. We propose that the somatic CRISPR-Cas9 platform is uniquely suited for conditional gene editing-based biomedical research. PMID:25155554

  10. 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

  11. Studying TGF-beta superfamily signaling by knockouts and knockins.

    PubMed

    Chang, H; Lau, A L; Matzuk, M M

    2001-06-30

    The transforming growth factor beta (TGF-beta) superfamily has profound effects on many aspects of animal development. In the last decade, our laboratory and others have performed in vivo functional studies on multiple components of the TGF-beta superfamily signal transduction pathway, including upstream ligands, transmembrane receptors, receptor-associated proteins and downstream Smad proteins. We have taken gene knockout approaches to generate null alleles of the genes of interest, as well as a gene knockin approach to replace the mature region of one TGF-beta superfamily ligand with another. We found that activin betaB, expressed in the spatiotemporal pattern of activin betaA, can function as a hypomorphic allele of activin betaA and rescue the craniofacial defects and neonatal lethal phenotype of activin betaA-deficient mice. With the knockout approach, we have shown that the expression pattern of a component in the TGF-beta superfamily signal transduction cascade does not necessarily predict its in vivo function. Two liver-specific activins, activin betaC and activin betaE are dispensable for liver development, regeneration and function, whereas ubiquitously expressed Smad5 has specific roles in the development of multiple embryonic and extraembryonic tissues. PMID:11451570

  12. Apolipoprotein E knockout as the basis for mouse models of dyslipidemia-induced neuropathy.

    PubMed

    Hinder, Lucy M; Vincent, Andrea M; Hayes, John M; McLean, Lisa L; Feldman, Eva L

    2013-01-01

    Dyslipidemia has been identified as an important pathogenic risk factor for diabetic neuropathy, but current animal models do not adequately reproduce the lipid profile observed in human diabetics (increased triglycerides with an elevated LDL-cholesterol and reduced HDL-cholesterol). High fat feeding of mice produces hyperlipidemia, but mice are resistant to increases in the LDL to HDL ratio, reducing the potential for peripheral lipid deposits to impact neuropathy, as is postulated to occur in human subjects. Genetic manipulations provide an alternative approach to reproducing a neuropathic plasma lipid profile. Based on findings from the atherosclerosis literature, we began with knockout of ApoE. Since knockout of ApoE alone only partially mimics the human diabetic lipid profile, we examined the impact of its combination with a well-characterized model of type 2 diabetes exhibiting neuropathy, the db/db mouse. We added further gene manipulations to increase hyperlipidemia by using mice with both ApoE and ApoB48 knockout on the ob/+ (leptin mutation) mice. In all of these models, we found that either the db/db or ob/ob genotypes had increased body weight, hyperlipidemia, hyperglycemia, and evidence of neuropathy compared with the control groups (db/+ or ob/+, respectively). We found that ApoE knockout combined with leptin receptor knockout produced a lipid profile most closely modeling human dyslipidemia that promotes neuropathy. ApoE knockout combined with additional ApoB48 and leptin knockout produced similar changes of smaller magnitude, but, notably, an increase in HDL-cholesterol. Our data suggest that the overall effects of ApoE knockout, either directly upon nerve structure and function or indirectly on lipid metabolism, are insufficient to significantly alter the course of diabetic neuropathy. Although these models ultimately do not deliver optimal lipid profiles for translational diabetic neuropathy research, they do present glycemic and lipid profile

  13. 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. PMID

  14. Creation and preliminary characterization of a Tp53 knockout rat

    PubMed Central

    McCoy, Aaron; Besch-Williford, Cynthia L.; Franklin, Craig L.; Weinstein, Edward J.; Cui, Xiaoxia

    2013-01-01

    SUMMARY The tumor suppressor TP53 plays a crucial role in cancer biology, and the TP53 gene is the most mutated gene in human cancer. Trp53 knockout mouse models have been widely used in cancer etiology studies and in search for a cure of cancer with some limitations that other model organisms might help overcome. Via pronuclear microinjection of zinc finger nucleases (ZFNs), we created a Tp53 knockout rat that contains an 11-bp deletion in exon 3, resulting in a frameshift and premature terminations in the open reading frame. In cohorts of 25 homozygous (Tp53Δ11/Δ11), 37 heterozygous (Tp53Δ11/+) and 30 wild-type rats, the Tp53Δ11/Δ11 rats lived an average of 126 days before death or removal from study because of clinical signs of abnormality or formation of tumors. Half of Tp53Δ11/+ were removed from study by 1 year of age because of tumor formation. Both Tp53Δ11/+ and Tp53Δ11/Δ11 rats developed a wide spectrum of tumors, most commonly sarcomas. Interestingly, there was a strikingly high incidence of brain lesions, especially in Tp53Δ11/Δ11 animals. We believe that this mutant rat line will be useful in studying cancer types rarely observed in mice and in carcinogenicity assays for drug development. PMID:22917926

  15. 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…

  16. 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.

  17. Phototransduction Motifs and Variations

    PubMed Central

    Yau, King-Wai; Hardie, Roger C.

    2010-01-01

    Seeing begins in the photoreceptors, where light is absorbed and signaled to the nervous system. Throughout the animal kingdom, photoreceptors are diverse in design and purpose. Nonetheless, phototransduction—the mechanism by which absorbed photons are converted into an electrical response—is highly conserved and based almost exclusively on a single class of photoproteins, the opsins. In this Review, we survey the G protein-coupled signaling cascades downstream from opsins in photoreceptors across vertebrate and invertebrate species, noting their similarities as well as differences. PMID:19837030

  18. Activation of NADPH-recycling systems in leaves and roots of Arabidopsis thaliana under arsenic-induced stress conditions is accelerated by knock-out of Nudix hydrolase 19 (AtNUDX19) gene.

    PubMed

    Corpas, Francisco J; Aguayo-Trinidad, Simeón; Ogawa, Takahisa; Yoshimura, Kazuya; Shigeoka, Shigeru

    2016-03-15

    NADPH is an important cofactor in cell growth, proliferation and detoxification. Arabidopsis thaliana Nudix hydrolase 19 (AtNUDX19) belongs to a family of proteins defined by the conserved amino-acid sequence GX5-EX7REUXEEXGU which has the capacity to hydrolyze NADPH as a physiological substrate in vivo. Given the importance of NADPH in the cellular redox homeostasis of plants, the present study compares the responses of the main NADPH-recycling systems including NADP-isocitrate dehydrogenase (ICDH), glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH) and NADP-malic enzyme (ME) in the leaves and roots of Arabidopsis wild-type (Wt) and knock-out (KO) AtNUDX19 mutant (Atnudx19) plants under physiological and arsenic-induced stress conditions. Two major features were observed in the behavior of the main NADPH-recycling systems: (i) under optimal conditions in both organs, the levels of these activities were higher in nudx19 mutants than in Wt plants; and, (ii) under 500μM AsV conditions, these activities increase, especially in nudx19 mutant plants. Moreover, G6PDH activity in roots was the most affected enzyme in both Wt and nudx19 mutant plants, with a 4.6-fold and 5.0-fold increase, respectively. In summary, the data reveals a connection between the absence of chloroplastic AtNUDX19 and the rise in all NADP-dehydrogenase activities under physiological and arsenic-induced stress conditions, particularly in roots. This suggests that AtNUDX19 could be a key factor in modulating the NADPH pool in plants and consequently in redox homeostasis. PMID:26878367

  19. Neuregulin 3 Knockout Mice Exhibit Behaviors Consistent with Psychotic Disorders.

    PubMed

    Hayes, Lindsay N; Shevelkin, Alexey; Zeledon, Mariela; Steel, Gary; Chen, Pei-Lung; Obie, Cassandra; Pulver, Ann; Avramopoulos, Dimitrios; Valle, David; Sawa, Akira; Pletnikov, Mikhail V

    2016-07-01

    Neuregulin 3 (NRG3) is a paralog of NRG1. Genetic studies in schizophrenia demonstrate that risk variants in NRG3 are associated with cognitive and psychotic symptom severity, and several intronic single nucleotide polymorphisms in NRG3 are associated with delusions in patients with schizophrenia. In order to gain insights into the biological function of the gene, we generated a novel Nrg3 knockout (KO) mouse model and tested for neurobehavioral phenotypes relevant to psychotic disorders. KO mice displayed novelty-induced hyperactivity, impaired prepulse inhibition of the acoustic startle response, and deficient fear conditioning. No gross cytoarchitectonic or layer abnormalities were noted in the brain of KO mice. Our findings suggest that deletion of the Nrg3 gene leads to alterations consistent with aspects of schizophrenia. We propose that KO mice will provide a valuable animal model to determine the role of the NRG3 in the molecular pathogenesis of schizophrenia and other psychotic disorders. PMID:27606322

  20. 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. PMID:27071940

  1. Phosphorylation of the Drosophila Transient Receptor Potential Ion Channel Is Regulated by the Phototransduction Cascade and Involves Several Protein Kinases and Phosphatases

    PubMed Central

    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. PMID:24040070

  2. Microarray analysis of E9.5 reduced folate carrier (RFC1; Slc19a1) knockout embryos reveals altered expression of genes in the cubilin-megalin multiligand endocytic receptor complex

    PubMed Central

    Gelineau-van Waes, Janee; Maddox, Joyce R; Smith, Lynette M; van Waes, Michael; Wilberding, Justin; Eudy, James D; Bauer, Linda K; Finnell, Richard H

    2008-01-01

    Background The reduced folate carrier (RFC1) is an integral membrane protein and facilitative anion exchanger that mediates delivery of 5-methyltetrahydrofolate into mammalian cells. Adequate maternal-fetal transport of folate is necessary for normal embryogenesis. Targeted inactivation of the murine RFC1 gene results in post-implantation embryolethality, but daily folic acid supplementation of pregnant dams prolongs survival of homozygous embryos until mid-gestation. At E10.5 RFC1-/- embryos are developmentally delayed relative to wildtype littermates, have multiple malformations, including neural tube defects, and die due to failure of chorioallantoic fusion. The mesoderm is sparse and disorganized, and there is a marked absence of erythrocytes in yolk sac blood islands. The identification of alterations in gene expression and signaling pathways involved in the observed dysmorphology following inactivation of RFC1-mediated folate transport are the focus of this investigation. Results Affymetrix microarray analysis of the relative gene expression profiles in whole E9.5 RFC1-/- vs. RFC1+/+ embryos identified 200 known genes that were differentially expressed. Major ontology groups included transcription factors (13.04%), and genes involved in transport functions (ion, lipid, carbohydrate) (11.37%). Genes that code for receptors, ligands and interacting proteins in the cubilin-megalin multiligand endocytic receptor complex accounted for 9.36% of the total, followed closely by several genes involved in hematopoiesis (8.03%). The most highly significant gene network identified by Ingenuity™ Pathway analysis included 12 genes in the cubilin-megalin multiligand endocytic receptor complex. Altered expression of these genes was validated by quantitative RT-PCR, and immunohistochemical analysis demonstrated that megalin protein expression disappeared from the visceral yolk sac of RFC1-/- embryos, while cubilin protein was widely misexpressed. Conclusion Inactivation of

  3. Comparison of Parallel High-Throughput RNA Sequencing Between Knockout of TDP-43 and Its Overexpression Reveals Primarily Nonreciprocal and Nonoverlapping Gene Expression Changes in the Central Nervous System of Drosophila

    PubMed Central

    Hazelett, Dennis J.; Chang, Jer-Cherng; Lakeland, Daniel L.; Morton, David B.

    2012-01-01

    The human Tar-DNA binding protein, TDP-43, is associated with amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. TDP-43 contains two conserved RNA-binding motifs and has documented roles in RNA metabolism, including pre-mRNA splicing and repression of transcription. Here, using Drosophila melanogaster as a model, we generated loss-of-function and overexpression genotypes of Tar-DNA binding protein homolog (TBPH) to study their effect on the transcriptome of the central nervous system (CNS). By using massively parallel sequencing methods (RNA-seq) to profile the CNS, we find that loss of TBPH results in widespread gene activation and altered splicing, much of which are reversed by rescue of TBPH expression. Conversely, TBPH overexpression results in decreased gene expression. Although previous studies implicated both absence and mis-expression of TDP-43 in ALS, our data exhibit little overlap in the gene expression between them, suggesting that the bulk of genes affected by TBPH loss-of-function and overexpression are different. In combination with computational approaches to identify likely TBPH targets and orthologs of previously identified vertebrate TDP-43 targets, we provide a comprehensive analysis of enriched gene ontologies. Our data suggest that TDP-43 plays a role in synaptic transmission, synaptic release, and endocytosis. We also uncovered a potential novel regulation of the Wnt and BMP pathways, many of whose targets appear to be conserved. PMID:22870402

  4. 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

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

    PubMed

    Wu, Xudong; Indzhykulian, Artur A; 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

  6. 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...

  7. 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

  8. 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

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

    2015-10-01

    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

  9. Mup-knockout mice generated through CRISPR/Cas9-mediated deletion for use in urinary protein analysis.

    PubMed

    Yang, Haixia; Zhang, Wei; Lu, Shan; Lu, Guangqing; Zhang, Hongjuan; Zhuang, Yinghua; Wang, Yue; Dong, Mengqiu; Zhang, Yu; Zhou, Xingang; Wang, Peng; Yu, Lei; Wang, Fengchao; Chen, Liang

    2016-05-01

    Major urinary proteins (MUPs) are the most abundant protein species in mouse urine, accounting for more than 90% of total protein content. Twenty-one Mup genes and 21 pseudogenes are clustered in a region of around 2 megabase pairs (Mbp) on chromosome 4. A Mup-knockout mouse model would greatly facilitate researches in the field of proteomic analysis of mouse urine. Here, we report the successful knockout of the Mup gene cluster of 2.2 Mbp using the CRISPR/Cas9 system. Homozygous Mup-knockout mice survived to adulthood and exhibited no obvious defects. The patterns of the proteomes of non-MUP urinary proteins in homozygous Mup-knockout mice were similar to those of wild-type mice judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The sensitivity of enzyme-linked immunosorbent assay to detect non-MUP urinary protein was significantly enhanced in Mup-knockout mice. In short, we have developed a Mup-knockout mouse model. This mouse model will be useful for the research of urinary biomarker testing that may have relevance for humans. PMID:26851484

  10. 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

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

    2013-10-01

    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

  11. 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

  12. Transcriptomic profiling comparison of YAP over-expression and conditional knockout mouse tooth germs

    PubMed Central

    Liu, Ming; Wang, Xiu-Ping

    2015-01-01

    To identify the downstream target genes of YAP, we used RNA-Seq technology to compare the transcriptomic profilings of Yap conditional knockout (Yap CKO) and YAP over-expression mouse tooth germs. Our results showed that some Hox, Wnt and Laminin family genes had concurrent changes with YAP transcripts, indicating that the expression of these genes may be regulated by YAP. Here, we provide the detailed experimental procedure for the transcriptomic profiling results (NCBI GEO accession number GSE65524). The associated study on the regulation of Hoxa1 and Hoxc13 genes by YAP was published in Molecular Cellular Biology in 2015 [Liu et al., 2015]. PMID:26484260

  13. Behavior training reverses asymmetry in hippocampal transcriptome of the cav3.2 knockout mice.

    PubMed

    Chung, Ni-Chun; Huang, Ying-Hsueh; Chang, Chuan-Hsiung; Liao, James C; Yang, Chih-Hsien; Chen, Chien-Chang; Liu, Ingrid Y

    2015-01-01

    Homozygous Cav3.2 knockout mice, which are defective in the pore-forming subunit of a low voltage activated T-type calcium channel, have been documented to show impaired maintenance of late-phase long-term potentiation (L-LTP) and defective retrieval of context-associated fear memory. To investigate the role of Cav3.2 in global gene expression, we performed a microarray transcriptome study on the hippocampi of the Cav3.2-/- mice and their wild-type littermates, either naïve (untrained) or trace fear conditioned. We found a significant left-right asymmetric effect on the hippocampal transcriptome caused by the Cav3.2 knockout. Between the naive Cav3.2-/- and the naive wild-type mice, 3522 differentially expressed genes (DEGs) were found in the left hippocampus, but only 4 DEGs were found in the right hippocampus. Remarkably, the effect of Cav3.2 knockout was partially reversed by trace fear conditioning. The number of DEGs in the left hippocampus was reduced to 6 in the Cav3.2 knockout mice after trace fear conditioning, compared with the wild-type naïve mice. To our knowledge, these results demonstrate for the first time the asymmetric effects of the Cav3.2 and its partial reversal by behavior training on the hippocampal transcriptome. PMID:25768289

  14. A baculovirus alkaline nuclease knockout construct produces fragmented DNA and aberrant capsids

    SciTech Connect

    Okano, Kazuhiro; Vanarsdall, Adam L.; Rohrmann, George F. . E-mail: rohrmanng@orst.edu

    2007-03-01

    DNA replication of bacmid-derived constructs of the Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) was analyzed by field inversion gel electrophoresis (FIGE) in combination with digestion at a unique Eco81I restriction enzyme site. Three constructs were characterized: a parental bacmid, a bacmid deleted for the alkaline nuclease gene, and a bacmid from which the gp64 gene had been deleted. The latter was employed as a control for comparison with the alkaline nuclease knockout because neither yields infectious virus and their replication is limited to the initially transfected cells. The major difference between DNA replicated by the different constructs was the presence in the alkaline nuclease knockout of high concentrations of relatively small, subgenome length DNA in preparations not treated with Eco81I. Furthermore, upon Eco81I digestion, the alkaline nuclease knockout bacmid also yielded substantially more subgenome size DNA than the other constructs. Electron microscopic examination of cells transfected with the alkaline nuclease knockout indicated that, in addition to a limited number of normal-appearing electron-dense nucleocapsids, numerous aberrant capsid-like structures were observed indicating a defect in nucleocapsid maturation or in a DNA processing step that is necessary for encapsidation. Because of the documented role of the baculovirus alkaline nuclease and its homologs from other viruses in homologous recombination, these data suggest that DNA recombination may play a major role in the production of baculovirus genomes.

  15. 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. PMID:26948338

  16. 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.

  17. Adaptation of the myoglobin knockout mouse to hypoxic stress.

    PubMed

    Schlieper, Georg; Kim, Jie-Hoon; Molojavyi, Andrei; Jacoby, Christoph; Laussmann, Tim; Flögel, Ulrich; Gödecke, Axel; Schrader, Jürgen

    2004-04-01

    Myoglobin knockout (myo-/-) mice were previously reported to show no obvious phenotype but revealed several compensatory mechanisms that include increases in cardiac capillary density, coronary flow, and hemoglobin. The aim of this study was to investigate whether severe hypoxic stress can exhaust these compensatory mechanisms and whether this can be monitored on the gene and protein level. Myo-/- and wild-type (WT) mice we e exposed to hypoxia (10% O(2)) fo 2 wk. Thereafter hemodynamic parameters were investigated by invasive measurement combined with magnetic resonance imaging. Cardiac gene and protein expression were analyzed using cDNA arrays and two-dimensional gel electrophoresis plus mass spectrometry, respectively. Hematocrit levels increased from 44% (WT) and 48% (myo-/-) to 72% in both groups. Similar to WT controls, hypoxic myo-/- animals maintained stable cardiovascular function (mean arterial blood pressure 82.4 mmHg, ejection fraction 72.5%). Cardiac gene expression of hypoxic myo-/- mice differed significantly from WT controls in 17 genes (e.g., keratinocyte lipid binding protein +202%, cytochrome c oxidase Vb +41%). Interestingly, hypoxia inducible factor-1alpha remained unchanged in both groups. Proteome analysis revealed reduced levels of heart fatty acid-binding protein and heat shock protein 27 both in hypoxic myo-/- and WT mice. Our data thus demonstrate that myo-/- mice do not decompensate du ing hypoxic st ess but a e surprisingly well adapted. Changes in ene gy metabolism of fatty acids may contribute to the robustness of myoglobin-deficient mice. PMID:14656764

  18. Modeling fragile X syndrome in the Fmr1 knockout mouse

    PubMed Central

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

    2014-01-01

    Summary 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. PMID:25606362

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

    PubMed Central

    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. PMID:26580070

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

    PubMed

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

    2016-08-01

    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 Ca(2+) entry on single AP stimulation, particularly in the axon proximal segment, reduced AP duration and reduced firing frequency to a 400 ms stimulation in α2δ-1 knockout neurons, consistent with the expected role of voltage-gated calcium channels in these events. Furthermore, lower intracellular Ca(2+) buffering also resulted in reduced AP duration, and a lower frequency of AP firing in WT neurons, mimicking the effect of α2δ-1 knockout. By contrast, we did not obtain any consistent evidence for the involvement of Ca(2+)-activation of large conductance calcium-activated potassium (BK) and small conductance calcium-activated potassium (SK) channels in these events. In conclusion, the reduced Ca(2+) elevation as a result of single AP stimulation is likely to result from the reduced duration of the AP in α2δ-1 knockout sensory neurons.This article is part of the themed issue 'Evolution brings Ca(2+) and ATP together to control life and death'. PMID:27377724

  1. 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

  2. Modeling leucine's metabolic pathway and knockout prediction improving the production of surfactin, a biosurfactant from Bacillus subtilis.

    PubMed

    Coutte, François; Niehren, Joachim; Dhali, Debarun; John, Mathias; Versari, Cristian; Jacques, Philippe

    2015-08-01

    A Bacillus subtilis mutant strain overexpressing surfactin biosynthetic genes was previously constructed. In order to further increase the production of this biosurfactant, our hypothesis is that the surfactin precursors, especially leucine, must be overproduced. We present a three step approach for leucine overproduction directed by methods from computational biology. Firstly, we develop a new algorithm for gene knockout prediction based on abstract interpretation, which applies to a recent modeling language for reaction networks with partial kinetic information. Secondly, we model the leucine metabolic pathway as a reaction network in this language, and apply the knockout prediction algorithm with the target of leucine overproduction. Out of the 21 reactions corresponding to potential gene knockouts, the prediction algorithm selects 12 reactions. Six knockouts were introduced in B. subtilis 168 derivatives strains to verify their effects on surfactin production. For all generated mutants, the specific surfactin production is increased from 1.6- to 20.9-fold during the exponential growth phase, depending on the medium composition. These results show the effectiveness of the knockout prediction approach based on formal models for metabolic reaction networks with partial kinetic information, and confirms our hypothesis that precursors supply is one of the main parameters to optimize surfactin overproduction. PMID:26220295

  3. Identification of essential genes and synthetic lethal gene combinations in Escherichia coli K-12.

    PubMed

    Mori, Hirotada; Baba, Tomoya; Yokoyama, Katsushi; Takeuchi, Rikiya; Nomura, Wataru; Makishi, Kazuichi; Otsuka, Yuta; Dose, Hitomi; Wanner, Barry L

    2015-01-01

    Here we describe the systematic identification of single genes and gene pairs, whose knockout causes lethality in Escherichia coli K-12. During construction of precise single-gene knockout library of E. coli K-12, we identified 328 essential gene candidates for growth in complex (LB) medium. Upon establishment of the Keio single-gene deletion library, we undertook the development of the ASKA single-gene deletion library carrying a different antibiotic resistance. In addition, we developed tools for identification of synthetic lethal gene combinations by systematic construction of double-gene knockout mutants. We introduce these methods herein. PMID:25636612

  4. Distinct mixtures of muscarinic receptor subtypes mediate inhibition of noradrenaline release in different mouse peripheral tissues, as studied with receptor knockout mice

    PubMed Central

    Trendelenburg, Anne-Ulrike; Meyer, Angelika; Wess, Jürgen; Starke, Klaus

    2005-01-01

    The muscarinic heteroreceptors modulating noradrenaline release in atria, urinary bladder and vas deferens were previously studied in mice in which the M2 or the M4 muscarinic receptor genes had been disrupted. These experiments showed that these tissues possessed both M2 and non-M2 heteroreceptors. The analysis was now extended to mice in which either the M3, both the M2 and the M3, or both the M2 and the M4 genes had been disrupted (M3-knockout, M2/3-knockout and M2/4-knockout). Tissues were preincubated with 3H-noradrenaline and then stimulated electrically (20 pulses per 50 Hz). In wild-type atria, carbachol (0.01–100 μM) decreased the electrically evoked tritium overflow by maximally 60–78%. The maximum inhibition of carbachol was reduced to 57% in M3-knockout and to 23% in M2/4-knockout atria. Strikingly, the effect of carbachol was abolished in M2/3-knockout atria. In wild-type bladder, carbachol (0.01–100 μM) reduced the evoked tritium overflow by maximally 57–71%. This effect remained unchanged in the M3-knockout, but was abolished in the M2/4-knockout bladder. In wild-type vas deferens, carbachol (0.01–100 μM) reduced the evoked tritium overflow by maximally 34–48%. The maximum inhibition of carbachol was reduced to 40% in the M3-knockout and to 18% in the M2/4-knockout vas deferens. We conclude that the postganglionic sympathetic axons of mouse atria possess M2 and M3, those of the urinary bladder M2 and M4, and those of the vas deferens M2, M3 and M4 release-inhibiting muscarinic receptors. PMID:15965496

  5. 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

  6. Altered behavioral development in Nrf2 knockout mice following early postnatal exposure to valproic acid

    PubMed Central

    Furnari, Melody A.; Saw, Constance Lay-Lay; Kong, Ah-Ng; Wagner, George C

    2015-01-01

    Early exposure to valproic acid results in autism-like neural and behavioral deficits in humans and other animals through oxidative stress-induced neural damage. In the present study, valproic acid was administered to genetically altered mice lacking the Nrf2 (nuclear factor-erythroid 2 related factor 2) gene on postnatal day 14 (P14). Nrf2 is a transcription factor that induces genes that protect against oxidative stress. It was found that valproic acid-treated Nrf2 knockout mice were less active in open field activity chambers, less successful on the rotorod, and had deficits in learning and memory in the Morris water maze compared to the valproic acid-treated wild type mice. Given these results, it appears that Nrf2 knockout mice were more sensitive to the neural damage caused by valproic acid administered during early development. PMID:25454122

  7. 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.

  8. Ancestral multipartite units in light-responsive plant promoters have structural features correlating with specific phototransduction pathways.

    PubMed Central

    Argüello-Astorga, G R; Herrera-Estrella, L R

    1996-01-01

    Regulation of plant gene transcription by light is mediated by multipartite cis-regulatory units. Previous attempts to identify structural features that are common to all light-responsive elements (LREs) have been unsuccessful. To address the question of what is needed to confer photoresponsiveness to a promoter, the upstream sequences from more than 110 light-regulated plant genes were analyzed by a new, phylogenetic-structural method. As a result, 30 distinct conserved DNA module arrays (CMAs) associated with light-responsive promoter regions were identified. Several of these CMAs have remained invariant throughout the evolutionary radiation of angiosperms and are conserved between homologous genes as well as between members of different gene families. The identified CMAs share a gene superfamily-specific core that correlates with the particular phytochrome-dependent transduction pathway that controls their expression, i.e. ACCTA(A/C)C(A/C) for the cGMP-dependent phenylpropanoid metabolism-associated genes, and GATA(A/T)GR for the Ca2+/calmodulin-dependent photosynthesis-associated nuclear genes. In addition to suggesting a general model for the functional and structural organization of LREs, the data obtained in this study indicate that angiosperm LREs probably evolved from complex cis-acting elements involved in regulatory processes other than photoregulation in gymnosperms. PMID:8938415

  9. 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

  10. 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.

  11. 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.

    PubMed

    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; Williams, David E; Baird, William M

    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 8h post-initiation. A significant reduction in levels of Cyp 1a1, Nqo1 at 8h 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 4h 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 8h 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. PMID:26049101

  12. 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.

  13. 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

  14. Proteomic Analysis of Loricrin Knockout Mouse Epidermis.

    PubMed

    Rice, Robert H; Durbin-Johnson, Blythe P; Ishitsuka, Yosuke; Salemi, Michelle; Phinney, Brett S; Rocke, David M; Roop, Dennis R

    2016-08-01

    The crosslinked envelope of the mammalian epidermal corneocyte serves as a scaffold for assembly of the lipid barrier of the epidermis. Thus, deficient envelope crosslinking by keratinocyte transglutaminase (TGM1) is a major cause of the human autosomal recessive congenital ichthyoses characterized by barrier defects. Expectations that loss of some envelope protein components would also confer an ichthyosis phenotype have been difficult to demonstrate. To help rationalize this observation, the protein profile of epidermis from loricrin knockout mice has been compared to that of wild type. Despite the mild phenotype of the knockout, some 40 proteins were incorporated into envelope material to significantly different extents compared to those of wild type. Nearly half were also incorporated to similarly altered extents into the disulfide bonded keratin network of the corneocyte. The results suggest that loss of loricrin alters their incorporation into envelopes as a consequence of protein-protein interactions during cell maturation. Mass spectrometric protein profiling revealed that keratin 1, keratin 10, and loricrin are prominent envelope components and that dozens of other proteins are also components. This finding helps rationalize the potential formation of functional envelopes, despite loss of a single component, due to the availability of many alternative transglutaminase substrates. PMID:27418529

  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. 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

  17. Hesr1 knockout mice exhibit behavioral alterations through the dopaminergic nervous system.

    PubMed

    Fuke, Satoshi; Minami, Natsumi; Kokubo, Hiroki; Yoshikawa, Ayumu; Yasumatsu, Hiroshi; Sasagawa, Noboru; Saga, Yumiko; Tsukahara, Toshifumi; Ishiura, Shoichi

    2006-11-15

    The basic helix-loop-helix (bHLH) transcriptional factor Hesr1 gene (hairy and enhancer of split-related 1, also called Hey1/HRT1/CHF2/HERP2) has been identified and characterized as a member of the subfamily of hairy/Enhancer of split, and shown to be involved in cardiovascular and neural development. We report that HESR1 binds directly to a part of the 3' non-coding region of the human dopamine transporter (DAT1) gene and represses the endogenous DAT1 gene in HEK293 cells. To investigate functions of the HESR1 gene in the dopaminergic nervous system in vivo, we analyzed the expressions of dopamine-related genes in the postnatal day 0 whole brains of Hesr1 knockout mice by real-time RT-PCR analysis. Several dopamine-related genes, such as DAT, dopamine receptors D1, D2, D4, and D5, were significantly upregulated. Moreover, young adults of Hesr1 knockout mice showed a decrease in spontaneous locomotor activity and a reduction in exploratory behavior or behavioral responses to novelty in the open-field, and elevated plus-maze tests. These results indicate that the HESR1 gene is related to neuropsychiatric disorders and behavioral traits through the dopaminergic nervous system. PMID:16998899

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

    PubMed

    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

    2013-12-10

    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. Technical knockout, a Drosophila model of mitochondrial deafness.

    PubMed Central

    Toivonen, J M; O'Dell, K M; Petit, N; Irvine, S C; Knight, G K; Lehtonen, M; Longmuir, M; Luoto, K; Touraille, S; Wang, Z; Alziari, S; Shah, Z H; Jacobs, H T

    2001-01-01

    Mutations in mtDNA-encoded components of the mitochondrial translational apparatus are associated with diverse pathological states in humans, notably sensorineural deafness. To develop animal models of such disorders, we have manipulated the nuclear gene for mitochondrial ribosomal protein S12 in Drosophila (technical knockout, tko). The prototypic mutant tko(25t) exhibits developmental delay, bang sensitivity, impaired male courtship, and defective response to sound. On the basis of a transgenic reversion test, these phenotypes are attributable to a single substitution (L85H) at a conserved residue of the tko protein. The mutant is hypersensitive to doxycyclin, an antibiotic that selectively inhibits mitochondrial protein synthesis, and mutant larvae have greatly diminished activities of mitochondrial redox enzymes and decreased levels of mitochondrial small-subunit rRNA. A second mutation in the tko gene, Q116K, which is predicted to impair the accuracy of mitochondrial translation, results in the completely different phenotype of recessive female sterility, based on three independent transgenic insertions. We infer that the tko(25t) mutant provides a model of mitochondrial hearing impairment resulting from a quantitative deficiency of mitochondrial translational capacity. PMID:11560901

  20. Methylphenidate improves the behavioral and cognitive deficits of neurogranin knockout mice

    PubMed Central

    Huang, Freesia L.; Huang, Kuo-Ping

    2012-01-01

    Neurogranin (Ng), a brain-specific calmodulin-binding protein, is expressed highly in hippocampus, and is important for cognitive function. Deletion of the Ng gene from mice caused attenuation of signal reaction cascade in hippocampus, impairments in learning and memory and high frequency stimulation-induced long-term potentiation. Environmental enrichment alone failed to improve cognitive function. In the present study, behavioral testing revealed that Ng knockout mice were both hyperactive and socially withdrawn. Methylphenidate (MPH) was given to mice while they were also kept under an enrichment condition. MPH treatment reduced the hyperactivity of Ng knockout mice tested in both the open field and forced swim chamber. MPH improved their social abilities such that mice recognized and interacted better with novel subjects. The cognitive memories of MPH-treated mutants were improved in both water maze and contextual fear conditioning tests. High frequency stimulation-induced long-term potentiation of Ng knockout mice was also improved by MPH. The present treatment regimen, however, did not fully reverse the deficits of the mutant mice. In contrast, MPH exerted only a minimal effect on the wild type mice. At the cellular level, MPH increased the number of glial fibrillary acidic protein-positive cells in hippocampus, particularly within the dentate gyrus of Ng knockout mice. Therefore it will be of interest to determine the nature of MPH-mediated astrocyte activation and how it may modulate behavior in future studies. Taken together these Ng knockout mice may be useful for the development of better drug treatment to improve cognitive and behavioral impairments. PMID:22809330

  1. Changes in the expression of neurotransmitter receptors in Parkin and DJ-1 knockout mice--A quantitative multireceptor study.

    PubMed

    Cremer, J N; Amunts, K; Schleicher, A; Palomero-Gallagher, N; Piel, M; Rösch, F; Zilles, K

    2015-12-17

    Parkinson's disease (PD) is a well-characterized neurological disorder with regard to its neuropathological and symptomatic appearance. At the genetic level, mutations of particular genes, e.g. Parkin and DJ-1, were found in human hereditary PD with early onset. Neurotransmitter receptors constitute decisive elements in neural signal transduction. Furthermore, since they are often altered in neurological and psychiatric diseases, receptors have been successful targets for pharmacological agents. However, the consequences of PD-associated gene mutations on the expression of transmitter receptors are largely unknown. Therefore, we studied the expression of 16 different receptor binding sites of the neurotransmitters glutamate, GABA, acetylcholine, adrenaline, serotonin, dopamine and adenosine by means of quantitative receptor autoradiography in Parkin and DJ-1 knockout mice. These knockout mice exhibit electrophysiological and behavioral deficits, but do not show the typical dopaminergic cell loss. We demonstrated differential changes of binding site densities in eleven brain regions. Most prominently, we found an up-regulation of GABA(B) and kainate receptor densities in numerous cortical areas of Parkin and DJ-1 knockout mice, as well as increased NMDA but decreased AMPA receptor densities in different brain regions of the Parkin knockout mice. The alterations of three different glutamate receptor types may indicate the potential relevance of the glutamatergic system in the pathogenesis of PD. Furthermore, the cholinergic M1, M2 and nicotinic receptors as well as the adrenergic α2 and the adenosine A(2A) receptors showed differentially increased densities in Parkin and DJ-1 knockout mice. Taken together, knockout of the PD-associated genes Parkin or DJ-1 results in differential changes of neurotransmitter receptor densities, highlighting a possible role of altered non-dopaminergic, and in particular of glutamatergic neurotransmission in PD pathogenesis. PMID

  2. Rapid Knockout and Reporter Mouse Line Generation and Breeding Colony Establishment Using EUCOMM Conditional-Ready Embryonic Stem Cells: A Case Study

    PubMed Central

    Coleman, James L. J.; Brennan, Karen; Ngo, Tony; Balaji, Poornima; Graham, Robert M.; Smith, Nicola J.

    2015-01-01

    As little as a decade ago, generation of a single knockout mouse line was an expensive and time-consuming undertaking available to relatively few researchers. The International Knockout Mouse Consortium, established in 2007, has revolutionized the use of such models by creating an open-access repository of embryonic stem (ES) cells that, through sequential breeding with first FLP1 recombinase and then Cre recombinase transgenic mice, facilitates germline global or conditional deletion of almost every gene in the mouse genome. In this Case Study, we describe our experience using the repository to create mouse lines for a variety of experimental purposes. Specifically, we discuss the process of obtaining germline transmission of two European Conditional Mouse Mutagenesis Program (EUCOMM) “knockout-first” gene targeted constructs and the advantages and pitfalls of using this system. We then outline our breeding strategy and the outcomes of our efforts to generate global and conditional knockouts and reporter mice for the genes of interest. Line maintenance, removal of recombinase transgenes, and cryopreservation are also considered. Our approach led to the generation of heterozygous knockout mice within 6 months of commencing breeding to the founder mice. By describing our experiences with the EUCOMM ES cells and subsequent breeding steps, we hope to assist other researchers with the application of this valuable approach to generating versatile knockout mouse lines. PMID:26175717

  3. Universal statistics of the knockout tournament

    PubMed Central

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

    2013-01-01

    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. PMID:24217406

  4. Ppp2ca knockout in mice spermatogenesis.

    PubMed

    Pan, Xiaoyun; Chen, Xia; Tong, Xin; Tang, Chao; Li, Jianmin

    2015-04-01

    Protein phosphatase 2A (PP2A) is a ubiquitous serine/threonine phosphatase involved in meiosis, mitosis, sperm capacitation, and apoptosis. Abberant activity of PP2A has been associated with a number of diseases. The homolog PPP2CA and PPP2CB can each function as the phosphatase catalytic subunit generally referred to as PP2AC. We generated a Ppp2ca conditional knockout (CKO) in C57BL/6J mice. Exon 2 of Ppp2ca was knocked out in a spatial or temporal-specific manner in primordial germ cells at E12.5. This Ppp2ca-null mutation caused infertility in male C57BL/6J mice. These CKO mice provide a powerful tool to study the mechanisms of Ppp2ca in development and disease. PMID:25628439

  5. 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. PMID:25064116

  6. 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.

  7. Knockout Mice Challenge our Concepts of Glucose Homeostasis and the Pathogenesis of Diabetes

    PubMed Central

    2003-01-01

    A central component of type 2 diabetes and the metabolic syndrome is insulin resistance. Insulin exerts a multifaceted and highly integrated series of actions via its intracellular signaling systems. Generation of mice carrying null mutations of the genes encoding proteins in the insulin signaling pathway provides a unique approach to determining the role of individual proteins in the molecular mechanism of insulin action and the pathogenesis of insulin resistance and diabetes. The role of the four major insulin receptor substrates (IRS1-4) in insulin and IGF-1 signaling have been examined by creating mice with targeted gene knockouts. Each produces a unique phenotype, indicating the complementary role of these signaling components. Combined heterozygous defects often produce synergistic or epistatic effects, although the final severity of the phenotype depends on the genetic background of the mice. Conditional knockouts of the insulin receptor have also been created using the Cre-lox system. These tissue specific knockouts have provide unique insights into the control of glucose homeostasis and the pathogenesis of type 2 diabetes, and have led to development of new hypotheses about the nature of the insulin action and development of diabetes. PMID:15061645

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

    PubMed

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

    2016-01-01

    Previously, we demonstrated the association between autophagy and gossypol-induced growth inhibition of mutant BRAF melanoma cells. Here, we investigate the role of autophagy in ATG5 knockout cell lines generated by the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas-mediated genome editing. The MTT assay revealed that the inhibitory effect of gossypol was weaker on ATG5 knockout cells than that on the wild type (WT) cells. The conversion of non-autophagic LC3-I to autophagic LC3-II and RT-PCR confirmed the functional gene knockout. However, Cyto-ID autophagy assay revealed that gossypol induced ATG5- and LC3-independent autophagy in ATG5 knockout cells. Moreover, gossypol acts as an autophagy inducer in ATG5 knockout cells while blocking the later stages of the autophagy process in WT cells, which was determined by measuring autophagic flux after co-treatment of gossypol with chloroquine (late-stage autophagy inhibitor). On the other hand, inhibition of autophagy with 3-MA or Beclin-1 siRNA caused a partial increase in the sensitivity to gossypol in ATG5 knockout cells, but not in the WT cells. Together, our findings suggest that the resistance to gossypol in ATG5 knockout cells is associated with increased cytoprotective autophagy, independent of ATG5. PMID:26476415

  9. Protein Production with a Pichia pastoris OCH1 Knockout Strain in Fed-Batch Mode.

    PubMed

    Gmeiner, Christoph; Spadiut, Oliver

    2015-01-01

    The methylotrophic yeast Pichia pastoris is a widely used host organism for recombinant protein production in biotechnology and pharmaceutical industry. However, if the target product describes a glycoprotein, an α-1,6-mannosyltransferase located in the Golgi apparatus of P. pastoris, called OCH1, triggers hypermannosylation of the recombinant protein which significantly impedes following unit operations and hampers biopharmaceutical product applications. A knockout of the och1 gene allows the production of less-glycosylated proteins-however, morphology and physiology of P. pastoris also change, complicating the upstream process. Here, we describe a controlled and efficient bioprocess based on the specific substrate uptake rate (q s) for a recombinant P. pastoris OCH1 knockout strain expressing a peroxidase as model protein. PMID:26082217

  10. [A comparison of the knockout efficiencies of two codon-optimized Cas9 coding sequences in zebrafish embryos].

    PubMed

    Fenghua, Zhang; Houpeng, Wang; Siyu, Huang; Feng, Xiong; Zuoyan, Zhu; Yonghua, Sun

    2016-02-01

    Recent years have witnessed the rapid development of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein(CRISPR/Cas9)system. In order to realize gene knockout with high efficiency and specificity in zebrafish, several labs have synthesized distinct Cas9 cDNA sequences which were cloned into different vectors. In this study, we chose two commonly used zebrafish-codon-optimized Cas9 coding sequences (zCas9_bz, zCas9_wc) from two different labs, and utilized them to knockout seven genes in zebrafish embryos, including the exogenous egfp and six endogenous genes (chd, hbegfa, th, eef1a1b, tyr and tcf7l1a). We compared the knockout efficiencies resulting from the two zCas9 coding sequences, by direct sequencing of PCR products, colony sequencing and phenotypic analysis. The results showed that the knockout efficiency of zCas9_wc was higher than that of zCas9_bz in all conditions. PMID:26907778

  11. Autism-related behavioral abnormalities in synapsin knockout mice

    PubMed Central

    Greco, Barbara; Managò, Francesca; Tucci, Valter; Kao, Hung-Teh; Valtorta, Flavia; Benfenati, Fabio

    2013-01-01

    Several synaptic genes predisposing to autism-spectrum disorder (ASD) have been identified. Nonsense and missense mutations in the SYN1 gene encoding for Synapsin I have been identified in families segregating for idiopathic epilepsy and ASD and genetic mapping analyses have identified variations in the SYN2 gene as significantly contributing to epilepsy predisposition. Synapsins (Syn I/II/III) are a multigene family of synaptic vesicle-associated phosphoproteins playing multiple roles in synaptic development, transmission and plasticity. Lack of SynI and/or SynII triggers a strong epileptic phenotype in mice associated with mild cognitive impairments that are also present in the non-epileptic SynIII−/− mice. SynII−/− and SynIII−/− mice also display schizophrenia-like traits, suggesting that Syns could be involved in the regulation of social behavior. Here, we studied social interaction and novelty, social recognition and social dominance, social transmission of food preference and social memory in groups of male SynI−/−, SynII−/− and SynIII−/− mice before and after the appearance of the epileptic phenotype and compared their performances with control mice. We found that deletion of Syn isoforms widely impairs social behaviors and repetitive behaviors, resulting in ASD-related phenotypes. SynI or SynIII deletion altered social behavior, whereas SynII deletion extensively impaired various aspects of social behavior and memory, altered exploration of a novel environment and increased self-grooming. Social impairments of SynI−/− and SynII−/− mice were evident also before the onset of seizures. The results demonstrate an involvement of Syns in generation of the behavioral traits of ASD and identify Syn knockout mice as a useful experimental model of ASD and epilepsy. PMID:23280234

  12. Social dominance in male vasopressin 1b receptor knockout mice.

    PubMed

    Caldwell, Heather K; Dike, Obianuju E; Stevenson, Erica L; Storck, Kathryn; Young, W Scott

    2010-07-01

    We have previously reported that mice with a targeted disruption of their vasopressin 1b receptor gene, Avpr1b, have mild impairments in social recognition and reduced aggression. The reductions in aggression are limited to social forms of aggression, i.e., maternal and inter-male aggression, while predatory aggression remains unaffected. To further clarify the role of the Avpr1b in the regulation of social behavior we first examined anxiety-like and depression-like behaviors in Avpr1b knockout (Avpr1b -/-) mice. We then went on to test the ability of Avpr1b -/- mice to form dominance hierarchies. No major differences were found between Avpr1b -/- and wildtype mice in anxiety-like behaviors, as measured using an elevated plus maze and an open field test, or depression-like behaviors, as measured using a forced swim test. In the social dominance study we found that Avpr1b -/- mice are able to form dominance hierarchies, though in early hierarchy formation dominant Avpr1b -/- mice display significantly more mounting behavior on Day 1 of testing compared to wildtype controls. Further, non-socially dominant Avpr1b -/- mice spend less time engaged in attack behavior than wildtype controls. These findings suggest that while Avpr1b -/- mice may be able to form dominance hierarchies they appear to employ alternate strategies. PMID:20298692

  13. 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.

  14. Bone Growth and Turnover in Progesterone Receptor Knockout Mice

    PubMed Central

    Rickard, David J.; Iwaniec, Urszula T.; Evans, Glenda; Hefferan, Theresa E.; Hunter, Jamie C.; Waters, Katrina M.; Lydon, John P.; O’Malley, Bert W.; Khosla, Sundeep; Spelsberg, Thomas C.; Turner, Russell T.

    2008-01-01

    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 microcomputed tomography analyses of bone from homozygous female PR knockout (PRKO) mice at 6, 12, and 26 wk 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 were normal in PRKO mice. In contrast, total, cancellous, and cortical bone mass were increased in the humerus of 12-wk-old PRKO mice, whereas cortical and cancellous bone mass in the tibia was normal. At 26 wk of age, cancellous bone area in the proximal tibia metaphysis of PRKO mice was 153% greater than age matched wild-type mice. The improved cancellous bone balance in 6-month-old 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 is not essential for bone growth and turnover. However, at some skeletal sites, PR signaling attenuates the accumulation of cortical and cancellous bone mass during adolescence. PMID:18276762

  15. 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

  16. Theoretical knock-outs on biological networks.

    PubMed

    Miranda, Pedro J; de S Pinto, Sandro E; Baptista, Murilo S; La Guardia, Giuliano G

    2016-08-21

    In this work we redefine the concept of biological importance and how to compute it, based on a model of complex networks and random walk. We call this new procedure, theoretical knock-out (KO). The proposed method generalizes the procedure presented in a recent study about Oral Tolerance. To devise this method, we make two approaches: algebraically and algorithmically. In both cases we compute a vector on an asymptotic state, called flux vector. The flux is given by a random walk on a directed graph that represents a biological phenomenon. This vector gives us the information about the relative flux of walkers on a vertex which represents a biological agent. With two vector of this kind, we can calculate the relative mean error between them by averaging over its coefficients. This quantity allows us to assess the degree of importance of each vertex of a complex network that evolves in time and has experimental background. We find out that this procedure can be applied in any sort of biological phenomena in which we can know the role and interrelationships of its agents. These results also provide experimental biologists to predict the order of importance of biological agents on a mounted complex network. PMID:27188251

  17. Characterization of Heterogeneous Prostate Tumors in Targeted Pten Knockout Mice

    PubMed Central

    Korsten, Hanneke; Ziel-van der Made, Angelique C. J.; van Weerden, Wytske M.; van der Kwast, Theo; Trapman, Jan; Van Duijn, Petra W.

    2016-01-01

    Previously, we generated a preclinical mouse prostate tumor model based on PSA-Cre driven inactivation of Pten. In this model homogeneous hyperplastic prostates (4-5m) developed at older age (>10m) into tumors. Here, we describe the molecular and histological characterization of the tumors in order to better understand the processes that are associated with prostate tumorigenesis in this targeted mouse Pten knockout model. The morphologies of the tumors that developed were very heterogeneous. Different histopathological growth patterns could be identified, including intraductal carcinoma (IDC), adenocarcinoma and undifferentiated carcinoma, all strongly positive for the epithelial cell marker Cytokeratin (CK), and carcinosarcomas, which were negative for CK. IDC pattern was already detected in prostates of 7–8 month old mice, indicating that it could be a precursor stage. At more than 10 months IDC and carcinosarcoma were most frequently observed. Gene expression profiling discriminated essentially two molecular subtypes, denoted tumor class 1 (TC1) and tumor class 2 (TC2). TC1 tumors were characterized by high expression of epithelial markers like Cytokeratin 8 and E-Cadherin whereas TC2 tumors showed high expression of mesenchyme/stroma markers such as Snail and Fibronectin. These molecular subtypes corresponded with histological growth patterns: where TC1 tumors mainly represented adenocarcinoma / intraductal carcinoma, in TC2 tumors carcinosarcoma was the dominant growth pattern. Further molecular characterization of the prostate tumors revealed an increased expression of genes associated with the inflammatory response. Moreover, functional markers for senescence, proliferation, angiogenesis and apoptosis were higher expressed in tumors compared to hyperplasia. The highest expression of proliferation and angiogenesis markers was detected in TC2 tumors. Our data clearly showed that in the genetically well-defined PSA-Cre;Pten-loxP/loxP prostate tumor model

  18. 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

  19. Gomafu lncRNA knockout mice exhibit mild hyperactivity with enhanced responsiveness to the psychostimulant methamphetamine.

    PubMed

    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

  20. Protein Degradation and Quality Control in Cells from Laforin and Malin Knockout Mice*

    PubMed Central

    Garyali, Punitee; Segvich, Dyann M.; DePaoli-Roach, Anna A.; Roach, Peter J.

    2014-01-01

    Lafora disease is a progressive myoclonus epilepsy caused by mutations in the EPM2A or EPM2B genes that encode a glycogen phosphatase, laforin, and an E3 ubiquitin ligase, malin, respectively. Lafora disease is characterized by accumulation of insoluble, poorly branched, hyperphosphorylated glycogen in brain, muscle, heart, and liver. The laforin-malin complex has been proposed to play a role in the regulation of glycogen metabolism and protein quality control. We evaluated three arms of the protein degradation/quality control process (the autophago-lysosomal pathway, the ubiquitin-proteasomal pathway, and the endoplasmic reticulum (ER) stress response) in mouse embryonic fibroblasts from Epm2a−/−, Epm2b−/−, and Epm2a−/− Epm2b−/− mice. The levels of LC3-II, a marker of autophagy, were decreased in all knock-out cells as compared with wild type even though they still showed a slight response to starvation and rapamycin. Furthermore, ribosomal protein S6 kinase and S6 phosphorylation were increased. Under basal conditions there was no effect on the levels of ubiquitinated proteins in the knock-out cells, but ubiquitinated protein degradation was decreased during starvation or stress. Lack of malin (Epm2b−/− and Epm2a−/− Epm2b−/− cells) but not laforin (Epm2a−/− cells) decreased LAMP1, a lysosomal marker. CHOP expression was similar in wild type and knock-out cells under basal conditions or with ER stress-inducing agents. In conclusion, both laforin and malin knock-out cells display mTOR-dependent autophagy defects and reduced proteasomal activity but no defects in the ER stress response. We speculate that these defects may be secondary to glycogen overaccumulation. This study also suggests a malin function independent of laforin, possibly in lysosomal biogenesis and/or lysosomal glycogen disposal. PMID:24914213

  1. Establishment and phenotypic analysis of an Mstn knockout rat.

    PubMed

    Gu, Hao; Cao, Yong; Qiu, Bin; Zhou, Zhiqiang; Deng, Ran; Chen, Zhuang; Li, Rongfeng; Li, Xueling; Wei, Qiang; Xia, Xianzhu; Yong, Weidong

    2016-08-12

    Myostatin (Mstn) is an inhibitor of myogenesis, regulating the number and size of skeletal myocytes. In addition to its myogenic regulatory function, Mstn plays important roles in the development of adipose tissues and in metabolism. In the present study, an Mstn knockout rat model was generated using the zinc finger nuclease (ZFN) technique in order to further investigate the function and mechanism of Mstn in metabolism. The knockout possesses a frame shift mutation resulting in an early termination codon and a truncated peptide of 109 amino acids rather than the full 376 amino acids. The absence of detectable mRNA confirmed successful knockout of Mstn. Relative to wild-type (WT) littermates, Knockout (KO) rats exhibited significantly greater body weight, body circumference, and muscle mass. However, no significant differences in grip force was observed, indicating that Mstn deletion results in greater muscle mass but not greater muscle fiber strength. Additionally, KO rats were found to possess less body fat relative to WT littermates, which is consistent with previous studies in mice and cattle. The aforementioned results indicate that Mstn knockout increases muscle mass while decreasing fat content, leading to observed increases in body weight and body circumference. The Mstn knockout rat model provides a novel means to study the role of Mstn in metabolism and Mstn-related muscle hypertrophy. PMID:27289021

  2. Impaired neurogenesis in embryonic spinal cord of Phgdh knockout mice, a serine deficiency disorder model.

    PubMed

    Kawakami, Yuriko; Yoshida, Kazuyuki; Yang, Jung Hoon; Suzuki, Takeshi; Azuma, Norihiro; Sakai, Kazuhisa; Hashikawa, Tsutomu; Watanabe, Masahiko; Yasuda, Kaori; Kuhara, Satoru; Hirabayashi, Yoshio; Furuya, Shigeki

    2009-03-01

    Mutations in the d-3-phosphoglycerate dehydrogenase (PHGDH; EC 1.1.1.95) gene, which encodes an enzyme involved in de novol-serine biosynthesis, are shown to cause human serine deficiency disorder. This disorder has been characterized by severe neurological symptoms including congenital microcephaly and psychomotor retardation. Our previous work demonstrated that targeted disruption of mouse Phgdh leads to a marked decrease in serine and glycine, severe growth retardation of the central nervous system, and lethality after embryonic day 13.5. To clarify how a serine deficiency causes neurodevelopmental defects, we characterized changes in metabolites, gene expression and morphological alterations in the spinal cord of Phgdh knockout mice. BeadChip microarray analysis revealed significant dysregulation of genes involved in the cell cycle. Ingenuity Pathway Analysis also revealed a significant perturbation of regulatory networks that operate in the cell cycle progression. Moreover, morphological examinations of the knockout spinal cord demonstrated a marked deficit in dorsal horn neurons. Radial glia cells, native neural stem/progenitor cells, accumulated in the dorsal ventricular zone, but they did not proceed to a G(0)-like quiescent state. The present integrative study provides in vivo evidence that normal cell cycle progression and subsequent neurogenesis of radial glia cells are severely impaired by serine deficiency. PMID:19114063

  3. 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

  4. P2X6 Knockout Mice Exhibit Normal Electrolyte Homeostasis

    PubMed Central

    Viering, Daan H. H. M.; Bos, Caro; Bindels, René J. M.; Hoenderop, Joost G. J.

    2016-01-01

    ATP-mediated signaling is an important regulator of electrolyte transport in the kidney. The purinergic cation channel P2X6 has been previously localized to the distal convoluted tubule (DCT), a nephron segment important for Mg2+ and Na+ reabsorption, but its role in ion transport remains unknown. In this study, P2x6 knockout (P2x6-/-) mice were generated to investigate the role of P2X6 in renal electrolyte transport. The P2x6-/- animals displayed a normal phenotype and did not differ physiologically from wild type mice. Differences in serum concentration and 24-hrs urine excretion of Na+, K+, Mg2+ and Ca2+ were not detected between P2x6+/+, P2x6+/- and P2x6-/- mice. Quantitative PCR was applied to examine potential compensatory changes in renal expression levels of other P2x subunits and electrolyte transporters, including P2x1-5, P2x7, Trpm6, Ncc, Egf, Cldn16, Scnn1, Slc12a3, Slc41a1, Slc41a3, Cnnm2, Kcnj10 and Fxyd2. Additionally, protein levels of P2X2 and P2X4 were assessed in P2x6+/+ and P2x6-/- mouse kidneys. However, significant changes in expression were not detected. Furthermore, no compensatory changes in gene expression could be demonstrated in heart material isolated from P2x6-/- mice. Except for a significant (P<0.05) upregulation of P2x2 in the heart of P2x6-/- mice compared to the P2x6+/+ mice. Thus, our data suggests that purinergic signaling via P2X6 is not significantly involved in the regulation of renal electrolyte handling under normal physiological conditions. PMID:27254077

  5. Targeting cancer using KAT inhibitors to mimic lethal knockouts.

    PubMed

    Brown, James A L; Bourke, Emer; Eriksson, Leif A; Kerin, Michael J

    2016-08-15

    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

  6. Impaired conditioned taste aversion learning in spinophilin knockout mice.

    PubMed

    Stafstrom-Davis, C A; Ouimet, C C; Feng, J; Allen, P B; Greengard, P; Houpt, T A

    2001-01-01

    Plasticity in dendritic spines may underlie learning and memory. Spinophilin, a protein enriched in dendritic spines, has the properties of a scaffolding protein and is believed to regulate actin cytoskeletal dynamics affecting dendritic spine morphology. It also binds protein phosphatase-1 (PP-1), an enzyme that regulates dendritic spine physiology. In this study, we tested the role of spinophilin in conditioned taste aversion learning (CTA) using transgenic spinophilin knockout mice. CTA is a form of associative learning in which an animal rejects a food that has been paired previously with a toxic effect (e.g., a sucrose solution paired with a malaise-inducing injection of lithium chloride). Acquisition and extinction of CTA was tested in spinophilin knockout and wild-type mice using taste solutions (sucrose or sodium chloride) or flavors (Kool-Aid) paired with moderate or high doses of LiCl (0.15 M, 20 or 40 mL/kg). When sucrose or NaCl solutions were paired with a moderate dose of LiCl, spinophilin knockout mice were unable to learn a CTA. At the higher dose, knockout mice acquired a CTA but extinguished more rapidly than wild-type mice. A more salient flavor stimulus (taste plus odor) revealed similar CTA learning at both doses of LiCl in both knockouts and wild types. Sensory processing in the knockouts appeared normal because knockout mice and wild-type mice expressed identical unconditioned taste preferences in two-bottle tests, and identical lying-on-belly responses to acute LiCl. We conclude that spinophilin is a candidate molecule required for normal CTA learning. PMID:11584074

  7. Knockout of the abundant Trichomonas vaginalis hydrogenosomal membrane protein TvHMP23 increases hydrogenosome size but induces no compensatory up-regulation of paralogous copies.

    PubMed

    Brás, Xavier Pereira; Zimorski, Verena; Bolte, Kathrin; Maier, Uwe-G; Martin, William F; Gould, Sven B

    2013-05-01

    The Trichomonas vaginalis genome encodes up to 60000 genes, many of which stem from genome duplication events. Paralogous copies thus accompany most T. vaginalis genes, a phenomenon that limits genetic manipulation. We characterized one of the parasite's most abundant hydrogenosomal membrane proteins, TvHMP23, which is phylogenetically distinct from canonical metabolite carriers, and which localizes to the inner hydrogenosomal membrane as shown through sub-organellar fractionation and protease protection assays. Knockout of Tvhmp23 through insertion of the selectable neomycin marker led to a size increase of hydrogenosomes, the first knockout-induced phenotypes reported for Trichomonas, but no growth impairment. The transcriptional response of its four paralogous copies then analyzed revealed that they are not up-regulated, and hence do not compensate for the Tvhmp23 knockout. PMID:23499435

  8. 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

  9. Metabolomic Characterization of Knockout Mutants in Arabidopsis: Development of a Metabolite Profiling Database for Knockout Mutants in Arabidopsis1[W][OPEN

    PubMed Central

    Fukushima, Atsushi; Kusano, Miyako; Mejia, Ramon Francisco; Iwasa, Mami; Kobayashi, Makoto; Hayashi, Naomi; Watanabe-Takahashi, Akiko; Narisawa, Tomoko; Tohge, Takayuki; Hur, Manhoi; Wurtele, Eve Syrkin; Nikolau, Basil J.; Saito, Kazuki

    2014-01-01

    Despite recent intensive research efforts in functional genomics, the functions of only a limited number of Arabidopsis (Arabidopsis thaliana) genes have been determined experimentally, and improving gene annotation remains a major challenge in plant science. As metabolite profiling can characterize the metabolomic phenotype of a genetic perturbation in the plant metabolism, it provides clues to the function(s) of genes of interest. We chose 50 Arabidopsis mutants, including a set of characterized and uncharacterized mutants, that resemble wild-type plants. We performed metabolite profiling of the plants using gas chromatography-mass spectrometry. To make the data set available as an efficient public functional genomics tool for hypothesis generation, we developed the Metabolite Profiling Database for Knock-Out Mutants in Arabidopsis (MeKO). It allows the evaluation of whether a mutation affects metabolism during normal plant growth and contains images of mutants, data on differences in metabolite accumulation, and interactive analysis tools. Nonprocessed data, including chromatograms, mass spectra, and experimental metadata, follow the guidelines set by the Metabolomics Standards Initiative and are freely downloadable. Proof-of-concept analysis suggests that MeKO is highly useful for the generation of hypotheses for genes of interest and for improving gene annotation. MeKO is publicly available at http://prime.psc.riken.jp/meko/. PMID:24828308

  10. 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.

  11. Cathepsin K knockout alleviates aging-induced cardiac dysfunction

    PubMed Central

    Hua, Yinan; Robinson, Timothy J; Cao, Yongtao; Shi, Guo-Ping; Ren, Jun; Nair, Sreejayan

    2015-01-01

    Aging is a major risk factor for cardiovascular disease. It has previously been shown that protein levels of cathepsin K, a lysosomal cysteine protease, are elevated in the failing heart and that genetic ablation of cathepsin K protects against pressure overload-induced cardiac hypertrophy and contractile dysfunction. Here we test the hypothesis that cathepsin K knockout alleviates age-dependent decline in cardiac function. Cardiac geometry, contractile function, intracellular Ca2+ properties, and cardiomyocyte apoptosis were evaluated using echocardiography, fura-2 technique, immunohistochemistry, Western blot and TUNEL staining, respectively. Aged (24-month-old) mice exhibited significant cardiac remodeling (enlarged chamber size, wall thickness, myocyte cross-sectional area, and fibrosis), decreased cardiac contractility, prolonged relengthening along with compromised intracellular Ca2+ release compared to young (6-month-old) mice, which were attenuated in the cathepsin K knockout mice. Cellular markers of senescence, including cardiac lipofuscin, p21 and p16, were lower in the aged-cathepsin K knockout mice compared to their wild-type counterpart. Mechanistically, cathepsin K knockout mice attenuated an age-induced increase in cardiomyocyte apoptosis and nuclear translocation of mitochondrial apoptosis-inducing factor (AIF). In cultured H9c2 cells, doxorubicin stimulated premature senescence and apoptosis. Silencing of cathepsin K blocked the doxorubicin-induced translocation of AIF from the mitochondria to the nuclei. Collectively, these results suggest that cathepsin K knockout attenuates age-related decline in cardiac function via suppressing caspase-dependent and caspase-independent apoptosis. PMID:25692548

  12. Roles of hepatic glucokinase in intertissue metabolic communication: Examination of novel liver-specific glucokinase knockout mice.

    PubMed

    Hayashi, Hirofumi; Sato, Yoshifumi; Li, Zhenghua; Yamamura, Ken-ichi; Yoshizawa, Tatsuya; Yamagata, Kazuya

    2015-05-01

    Glucokinase is expressed principally in pancreatic β-cells and hepatocytes, and catalyzes the phosphorylation of glucose to glucose-6-phosphate, a rate-limiting step of glycolysis. To better understand the roles of hepatic glucokinase, we generated Gck knockout mice by ablating liver-specific exon 1b. The knockout mice exhibited impaired glucose tolerance, decreased hepatic glycogen content, and reduced Pklr and Fas gene expression in the liver, indicating that hepatic glucokinase plays important roles in glucose metabolism. It has also been reported that hepatic glucokinase regulates the expression of thermogenesis-related genes in brown adipose tissue (BAT) and insulin secretion in response to glucose. However, the liver-specific Gck knockout mice displayed neither altered expression of thermogenesis-related genes in BAT nor impaired insulin secretion by β-cells under a normal chow diet. These results suggest that chronic suppression of hepatic glucokinase has a small influence on intertissue (liver-to-BAT as well as liver-to-β-cell) metabolic communication. PMID:25817793

  13. 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. PMID:24430555

  14. Knockout silkworms reveal a dispensable role for juvenile hormones in holometabolous life cycle.

    PubMed

    Daimon, Takaaki; Uchibori, Miwa; Nakao, Hajime; Sezutsu, Hideki; Shinoda, Tetsuro

    2015-08-01

    Insect juvenile hormones (JHs) prevent precocious metamorphosis and allow larvae to undergo multiple rounds of status quo molts. However, the roles of JHs during the embryonic and very early larval stages have not been fully understood. We generated and characterized knockout silkworms (Bombyx mori) with null mutations in JH biosynthesis or JH receptor genes using genome-editing tools. We found that embryonic growth and morphogenesis are largely independent of JHs in Bombyx and that, even in the absence of JHs or JH signaling, pupal characters are not formed in first- or second-instar larvae, and precocious metamorphosis is induced after the second instar at the earliest. We also show by mosaic analysis that a pupal specifier gene broad, which is dramatically up-regulated in the late stage of the last larval instar, is essential for pupal commitment in the epidermis. Importantly, the mRNA expression level of broad, which is thought to be repressed by JHs, remained at very low basal levels during the early larval instars of JH-deficient or JH signaling-deficient knockouts. Therefore, our study suggests that the long-accepted paradigm that JHs maintain the juvenile status throughout larval life should be revised because the larval status can be maintained by a JH-independent mechanism in very early larval instars. We propose that the lack of competence for metamorphosis during the early larval stages may result from the absence of an unidentified broad-inducing factor, i.e., a competence factor. PMID:26195792

  15. 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

    2016-01-01

    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

  16. 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

  17. 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

    2016-01-01

    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

  18. 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

  19. Knockout silkworms reveal a dispensable role for juvenile hormones in holometabolous life cycle

    PubMed Central

    Daimon, Takaaki; Uchibori, Miwa; Nakao, Hajime; Sezutsu, Hideki; Shinoda, Tetsuro

    2015-01-01

    Insect juvenile hormones (JHs) prevent precocious metamorphosis and allow larvae to undergo multiple rounds of status quo molts. However, the roles of JHs during the embryonic and very early larval stages have not been fully understood. We generated and characterized knockout silkworms (Bombyx mori) with null mutations in JH biosynthesis or JH receptor genes using genome-editing tools. We found that embryonic growth and morphogenesis are largely independent of JHs in Bombyx and that, even in the absence of JHs or JH signaling, pupal characters are not formed in first- or second-instar larvae, and precocious metamorphosis is induced after the second instar at the earliest. We also show by mosaic analysis that a pupal specifier gene broad, which is dramatically up-regulated in the late stage of the last larval instar, is essential for pupal commitment in the epidermis. Importantly, the mRNA expression level of broad, which is thought to be repressed by JHs, remained at very low basal levels during the early larval instars of JH-deficient or JH signaling-deficient knockouts. Therefore, our study suggests that the long-accepted paradigm that JHs maintain the juvenile status throughout larval life should be revised because the larval status can be maintained by a JH-independent mechanism in very early larval instars. We propose that the lack of competence for metamorphosis during the early larval stages may result from the absence of an unidentified broad-inducing factor, i.e., a competence factor. PMID:26195792

  20. 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

  1. 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. PMID:25468940

  2. 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

  3. 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...

  4. 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. PMID:22697126

  5. Rapidly generating knockout mice from H19-Igf2 engineered androgenetic haploid embryonic stem cells

    PubMed Central

    Zhang, Meili; Liu, Yufang; Liu, Guang; Li, Xin; Jia, Yuyan; Sun, Lihong; Wang, Liu; Zhou, Qi; Huang, Yue

    2015-01-01

    Haploid mammalian embryonic stem cells (ESCs) hold great promise for functional genetic studies and assisted reproduction. Recently, rodent androgenetic haploid ESCs (AG-haESCs) were generated from androgenetic blastocysts and functioned like sperm to produce viable offspring via the intracytoplasmic AG-haESCs injection into oocytes. However, the efficiency of this reproduction was very low. Most pups were growth-retarded and died shortly after birth, which is not practical for producing knockout animals. Further investigation suggested a possible link between the low birthrate and aberrant expression of imprinted genes. Here, we report the high-frequency generation of healthy, fertile mice from H19-Igf2 imprinting-locus modified AG-haESCs, which maintained normal paternal imprinting and pluripotency. Moreover, it is feasible to perform further genetic manipulations in these AG-haESCs. Our study provides a reliable and efficient tool to rapidly produce gene-modified mouse models and will benefit reproductive medicine in the future.

  6. Animal models for the study of liver fibrosis: new insights from knockout mouse models

    PubMed Central

    Hayashi, Hiromitsu

    2011-01-01

    Fibrosis arises as part of a would-healing response that maintains organ structure and integrity following tissue damage but also contributes to a variety of human pathologies such as liver fibrosis. Liver fibrosis is an abnormal response of the liver to persistent injury with the excessive accumulation of collagenous extracellular matrices. Currently there is no effective treatment, and many patients end up with a progressive form of the disease, eventually requiring a liver transplant. The clarification of mechanisms underlying pathogenesis of liver fibrosis and the development of effective therapy are of clinical importance. Experimental animal models, in particular targeted gene knockouts (loss of function) in mice, have become a powerful resource to address the molecular mechanisms or significance of the targeted gene in hepatic functions and diseases. This review will focus on the recent advances in knowledge obtained from genetically engineered mice that provide novel insights into the pathophysiology of liver fibrosis. PMID:21350186

  7. CAF1-knockout mice are more susceptive to lipopolysaccharide-induced acute lung injury

    PubMed Central

    Shi, Jia-Xin; Li, Jia-Shu; Hu, Rong; Li, Xiao-Min; Wang, Hong

    2016-01-01

    The carbon catabolite repressor protein 4 (CCR4)–negative on TATA (NOT) complex includes multiple subunits and is conserved in the eukaryotic cells. The CCR4–NOT complex can regulate gene expression at different levels. Two subunits of the CCR4–NOT complex, CCR4 and CCR4-associated factor 1 (CAF1), possess deadenylase activity. In yeast, the deadenylase activity is mainly provided by the CCR4 subunit; however, the deadenylase activity is provided by both CCR4 and CAF1 in other eukaryotes. A previous study reported that CAF1 but not CCR4 is required for the decay of a reporter mRNA with AU-rich elements. Our previous study showed that CAF1 is involved in the regulation of intercellular adhesion molecule-1 (ICAM-1) and interleukin-8 (IL-8) expression. Both ICAM-1 and IL-8 play crucial roles in acute lung injury. In the present study, we examined the effects of CAF1 deficiency on IL-8 and ICAM-1 expression and acute lung injury in mice. Here we showed that there were no differences between the wild-type and CAF1-knockout mice on phenotypes. The lung histology and protein and mRNA levels of IL-8 and ICAM-1 in unstimulated wild-type mice were comparable to those in unstimulated CAF1-knockout mice. However, lipopolysaccharide stimulation led to more severe lung histological injury and greatly higher IL-8 and ICAM-1 expression in CAF1-knockout mice compared to the wild-type mice. These results, together with our previous study, suggest that CAF1 is involved in the regulation of lipopolysaccharide-stimulated IL-8 and ICAM-1 expression in vivo and affects the progression of acute lung injury. PMID:27358572

  8. Spatiotemporal Alterations in Primary Odorant Representations in Olfactory Marker Protein Knockout Mice

    PubMed Central

    Kass, Marley D.; Moberly, Andrew H.; McGann, John P.

    2013-01-01

    Olfactory marker protein (OMP) is highly and selectively expressed in primary olfactory sensory neurons (OSNs) across species, but its physiological function remains unclear. Previous studies in the olfactory epithelium suggest that it accelerates the neural response to odorants and may modulate the odorant-selectivity of OSNs. Here we used a line of gene-targeted mice that express the fluorescent exocytosis indicator synaptopHluorin in place of OMP to compare spatiotemporal patterns of odorant-evoked neurotransmitter release from OSNs in adult mice that were heterozygous for OMP or OMP-null. We found that these patterns, which constitute the primary neural representation of each odorant, developed more slowly during the odorant presentation in OMP knockout mice but eventually reached the same magnitude as in heterozygous mice. In the olfactory bulb, each glomerulus receives synaptic input from a subpopulation of OSNs that all express the same odor receptor and thus typically respond to a specific subset of odorants. We observed that in OMP knockout mice, OSNs innervating a given glomerulus typically responded to a broader range of odorants than in OMP heterozygous mice and thus each odorant evoked synaptic input to a larger number of glomeruli. In an olfactory habituation task, OMP knockout mice behaved differently than wild-type mice, exhibiting a delay in their onset to investigate an odor stimulus during its first presentation and less habituation to that stimulus over repeated presentations. These results suggest that the actions of OMP in olfactory transduction carry through to the primary sensory representations of olfactory stimuli in adult mice in vivo. PMID:23630588

  9. Cardiomyocyte-specific conditional knockout of the histone chaperone HIRA in mice results in hypertrophy, sarcolemmal damage and focal replacement fibrosis.

    PubMed

    Valenzuela, Nicolas; Fan, Qiying; Fa'ak, Faisal; Soibam, Benjamin; Nagandla, Harika; Liu, Yu; Schwartz, Robert J; McConnell, Bradley K; Stewart, M David

    2016-03-01

    HIRA is the histone chaperone responsible for replication-independent incorporation of histone variant H3.3 within gene bodies and regulatory regions of actively transcribed genes, and within the bivalent promoter regions of developmentally regulated genes. The HIRA gene lies within the 22q11.2 deletion syndrome critical region; individuals with this syndrome have multiple congenital heart defects. Because terminally differentiated cardiomyocytes have exited the cell cycle, histone variants should be utilized for the bulk of chromatin remodeling. Thus, HIRA is likely to play an important role in epigenetically defining the cardiac gene expression program. In this study, we determined the consequence of HIRA deficiency in cardiomyocytes in vivo by studying the phenotype of cardiomyocyte-specific Hira conditional-knockout mice. Loss of HIRA did not perturb heart development, but instead resulted in cardiomyocyte hypertrophy and susceptibility to sarcolemmal damage. Cardiomyocyte degeneration gave way to focal replacement fibrosis and impaired cardiac function. Gene expression was widely altered in Hira conditional-knockout hearts. Significantly affected pathways included responses to cellular stress, DNA repair and transcription. Consistent with heart failure, fetal cardiac genes were re-expressed in the Hira conditional knockout. Our results suggest that transcriptional regulation by HIRA is crucial for cardiomyocyte homeostasis. PMID:26935106

  10. Cardiomyocyte-specific conditional knockout of the histone chaperone HIRA in mice results in hypertrophy, sarcolemmal damage and focal replacement fibrosis

    PubMed Central

    Valenzuela, Nicolas; Fan, Qiying; Fa'ak, Faisal; Soibam, Benjamin; Nagandla, Harika; Liu, Yu; Schwartz, Robert J.; McConnell, Bradley K.; Stewart, M. David

    2016-01-01

    ABSTRACT HIRA is the histone chaperone responsible for replication-independent incorporation of histone variant H3.3 within gene bodies and regulatory regions of actively transcribed genes, and within the bivalent promoter regions of developmentally regulated genes. The HIRA gene lies within the 22q11.2 deletion syndrome critical region; individuals with this syndrome have multiple congenital heart defects. Because terminally differentiated cardiomyocytes have exited the cell cycle, histone variants should be utilized for the bulk of chromatin remodeling. Thus, HIRA is likely to play an important role in epigenetically defining the cardiac gene expression program. In this study, we determined the consequence of HIRA deficiency in cardiomyocytes in vivo by studying the phenotype of cardiomyocyte-specific Hira conditional-knockout mice. Loss of HIRA did not perturb heart development, but instead resulted in cardiomyocyte hypertrophy and susceptibility to sarcolemmal damage. Cardiomyocyte degeneration gave way to focal replacement fibrosis and impaired cardiac function. Gene expression was widely altered in Hira conditional-knockout hearts. Significantly affected pathways included responses to cellular stress, DNA repair and transcription. Consistent with heart failure, fetal cardiac genes were re-expressed in the Hira conditional knockout. Our results suggest that transcriptional regulation by HIRA is crucial for cardiomyocyte homeostasis. PMID:26935106

  11. Impaired extinction of learned contextual fear memory in early growth response 1 knockout mice.

    PubMed

    Han, Seungrie; Hong, Soontaek; Mo, Jiwon; Lee, Dongmin; Choi, Eunju; Choi, June-seek; Sun, Woong; Lee, Hyun Woo; Kim, Hyun

    2014-01-01

    Inductive expression of early growth response 1 (Egr-1) in neurons is associated with many forms of neuronal activity. However, only a few Egr-1 target genes are known in the brain. The results of this study demonstrate that Egr-1 knockout (KO) mice display impaired contextual extinction learning and normal fear acquisition relative to wild-type (WT) control animals. Genome-wide microarray experiments revealed 368 differentially expressed genes in the hippocampus of Egr-1 WT exposed to different phases of a fear conditioning paradigm compared to gene expression profiles in the hippocampus of KO mice. Some of genes, such as serotonin receptor 2C (Htr2c), neuropeptide B (Npb), neuronal PAS domain protein 4 (Npas4), NPY receptor Y1 (Npy1r), fatty acid binding protein 7 (Fabp7), and neuropeptide Y (Npy) are known to regulate processing of fearful memories, and promoter analyses demonstrated that several of these genes contained Egr-1 binding sites. This study provides a useful list of potential Egr-1 target genes which may be regulated during fear memory processing. PMID:24552706

  12. Knockout of Extracytoplasmic Function Sigma Factor ECF-10 Affects Stress Resistance and Biofilm Formation in Pseudomonas putida KT2440

    PubMed Central

    Tettmann, Beatrix; Dötsch, Andreas; Armant, Olivier; Fjell, Christopher D.

    2014-01-01

    Pseudomonas putida is a Gram-negative soil bacterium which is well-known for its versatile lifestyle, controlled by a large repertoire of transcriptional regulators. Besides one- and two-component regulatory systems, the genome of P. putida reveals 19 extracytoplasmic function (ECF) sigma factors involved in the adaptation to changing environmental conditions. In this study, we demonstrate that knockout of extracytoplasmic function sigma factor ECF-10, encoded by open reading frame PP4553, resulted in 2- to 4-fold increased antibiotic resistance to quinolone, β-lactam, sulfonamide, and chloramphenicol antibiotics. In addition, the ECF-10 mutant exhibited enhanced formation of biofilms after 24 h of incubation. Transcriptome analysis using Illumina sequencing technology resulted in the detection of 12 genes differentially expressed (>2-fold) in the ECF-10 knockout mutant strain compared to their levels of expression in wild-type cells. Among the upregulated genes were ttgA, ttgB, and ttgC, which code for the major multidrug efflux pump TtgABC in P. putida KT2440. Investigation of an ECF-10 and ttgA double-knockout strain and a ttgABC-overexpressing strain demonstrated the involvement of efflux pump TtgABC in the stress resistance and biofilm formation phenotypes of the ECF-10 mutant strain, indicating a new role for this efflux pump beyond simple antibiotic resistance in P. putida KT2440. PMID:24907323

  13. Knockout confirmation for Hurries: rapid genotype identification of Trypanosoma cruzi transfectants by polymerase chain reaction directly from liquid culture.

    PubMed

    Alcantara, Monica Visnieski; Fragoso, Stenio Perdigão; Picchi, Gisele Fernanda Assine

    2014-07-01

    Gene knockout is a widely used approach to evaluate loss-of-function phenotypes and it can be facilitated by the incorporation of a DNA cassette having a drug-selectable marker. Confirmation of the correct knockout cassette insertion is an important step in gene removal validation and has generally been performed by polymerase chain reaction (PCR) assays following a time-consuming DNA extraction step. Here, we show a rapid procedure for the identification of Trypanosoma cruzi transfectants by PCR directly from liquid culture - without prior DNA extraction. This simple approach enabled us to generate PCR amplifications from different cultures varying from 106-108 cells/mL. We also show that it is possible to combine different primer pairs in a multiplex detection reaction and even to achieve knockout confirmation with an extremely simple interpretation of a real-time PCR result. Using the "culture PCR" approach, we show for the first time that we can assess different DNA sequence combinations by PCR directly from liquid culture, saving time in several tasks for T. cruzi genotype interrogation. PMID:24936912

  14. 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.

  15. 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

  16. 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. PMID:25762293

  17. 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

  18. Dentin Dysplasia in Notum Knockout Mice.

    PubMed

    Vogel, P; Read, R W; Hansen, G M; Powell, D R; Kantaputra, P N; Zambrowicz, B; Brommage, R

    2016-07-01

    Secreted WNT proteins control cell differentiation and proliferation in many tissues, and NOTUM is a secreted enzyme that modulates WNT morphogens by removing a palmitoleoylate moiety that is essential for their activity. To better understand the role this enzyme in development, the authors produced NOTUM-deficient mice by targeted insertional disruption of the Notum gene. The authors discovered a critical role for NOTUM in dentin morphogenesis suggesting that increased WNT activity can disrupt odontoblast differentiation and orientation in both incisor and molar teeth. Although molars in Notum(-/-) mice had normal-shaped crowns and normal mantle dentin, the defective crown dentin resulted in enamel prone to fracture during mastication and made teeth more susceptible to endodontal inflammation and necrosis. The dentin dysplasia and short roots contributed to tooth hypermobility and to the spread of periodontal inflammation, which often progressed to periapical abscess formation. The additional incidental finding of renal agenesis in some Notum (-/-) mice indicated that NOTUM also has a role in kidney development, with undiagnosed bilateral renal agenesis most likely responsible for the observed decreased perinatal viability of Notum(-/-) mice. The findings support a significant role for NOTUM in modulating WNT signaling pathways that have pleiotropic effects on tooth and kidney development. PMID:26926082

  19. Diacylglycerol Lipase α Knockout Mice Demonstrate Metabolic and Behavioral Phenotypes Similar to Those of Cannabinoid Receptor 1 Knockout Mice

    PubMed Central

    Powell, David R.; Gay, Jason P.; Wilganowski, Nathaniel; Doree, Deon; Savelieva, Katerina V.; Lanthorn, Thomas H.; Read, Robert; Vogel, Peter; Hansen, Gwenn M.; Brommage, Robert; Ding, Zhi-Ming; Desai, Urvi; Zambrowicz, Brian

    2015-01-01

    After creating >4,650 knockouts (KOs) of independent mouse genes, we screened them by high-throughput phenotyping and found that cannabinoid receptor 1 (Cnr1) KO mice had the same lean phenotype published by others. We asked if our KOs of DAG lipase α or β (Dagla or Daglb), which catalyze biosynthesis of the endocannabinoid (EC) 2-arachidonoylglycerol (2-AG), or Napepld, which catalyzes biosynthesis of the EC anandamide, shared the lean phenotype of Cnr1 KO mice. We found that Dagla KO mice, but not Daglb or Napepld KO mice, were among the leanest of 3651 chow-fed KO lines screened. In confirmatory studies, chow- or high fat diet-fed Dagla and Cnr1 KO mice were leaner than wild-type (WT) littermates; when data from multiple cohorts of adult mice were combined, body fat was 47 and 45% lower in Dagla and Cnr1 KO mice, respectively, relative to WT values. By contrast, neither Daglb nor Napepld KO mice were lean. Weanling Dagla KO mice ate less than WT mice and had body weight (BW) similar to pair-fed WT mice, and adult Dagla KO mice had normal activity and VO2 levels, similar to Cnr1 KO mice. Our Dagla and Cnr1 KO mice also had low fasting insulin, triglyceride, and total cholesterol levels, and after glucose challenge had normal glucose but very low insulin levels. Dagla and Cnr1 KO mice also showed similar responses to a battery of behavioral tests. These data suggest: (1) the lean phenotype of young Dagla and Cnr1 KO mice is mainly due to hypophagia; (2) in pathways where ECs signal through Cnr1 to regulate food intake and other metabolic and behavioral phenotypes observed in Cnr1 KO mice, Dagla alone provides the 2-AG that serves as the EC signal; and (3) small molecule Dagla inhibitors with a pharmacokinetic profile similar to that of Cnr1 inverse agonists are likely to mirror the ability of these Cnr1 inverse agonists to lower BW and improve glycemic control in obese patients with type 2 diabetes, but may also induce undesirable neuropsychiatric side

  20. Diacylglycerol Lipase α Knockout Mice Demonstrate Metabolic and Behavioral Phenotypes Similar to Those of Cannabinoid Receptor 1 Knockout Mice.

    PubMed

    Powell, David R; Gay, Jason P; Wilganowski, Nathaniel; Doree, Deon; Savelieva, Katerina V; Lanthorn, Thomas H; Read, Robert; Vogel, Peter; Hansen, Gwenn M; Brommage, Robert; Ding, Zhi-Ming; Desai, Urvi; Zambrowicz, Brian

    2015-01-01

    After creating >4,650 knockouts (KOs) of independent mouse genes, we screened them by high-throughput phenotyping and found that cannabinoid receptor 1 (Cnr1) KO mice had the same lean phenotype published by others. We asked if our KOs of DAG lipase α or β (Dagla or Daglb), which catalyze biosynthesis of the endocannabinoid (EC) 2-arachidonoylglycerol (2-AG), or Napepld, which catalyzes biosynthesis of the EC anandamide, shared the lean phenotype of Cnr1 KO mice. We found that Dagla KO mice, but not Daglb or Napepld KO mice, were among the leanest of 3651 chow-fed KO lines screened. In confirmatory studies, chow- or high fat diet-fed Dagla and Cnr1 KO mice were leaner than wild-type (WT) littermates; when data from multiple cohorts of adult mice were combined, body fat was 47 and 45% lower in Dagla and Cnr1 KO mice, respectively, relative to WT values. By contrast, neither Daglb nor Napepld KO mice were lean. Weanling Dagla KO mice ate less than WT mice and had body weight (BW) similar to pair-fed WT mice, and adult Dagla KO mice had normal activity and VO2 levels, similar to Cnr1 KO mice. Our Dagla and Cnr1 KO mice also had low fasting insulin, triglyceride, and total cholesterol levels, and after glucose challenge had normal glucose but very low insulin levels. Dagla and Cnr1 KO mice also showed similar responses to a battery of behavioral tests. These data suggest: (1) the lean phenotype of young Dagla and Cnr1 KO mice is mainly due to hypophagia; (2) in pathways where ECs signal through Cnr1 to regulate food intake and other metabolic and behavioral phenotypes observed in Cnr1 KO mice, Dagla alone provides the 2-AG that serves as the EC signal; and (3) small molecule Dagla inhibitors with a pharmacokinetic profile similar to that of Cnr1 inverse agonists are likely to mirror the ability of these Cnr1 inverse agonists to lower BW and improve glycemic control in obese patients with type 2 diabetes, but may also induce undesirable neuropsychiatric side

  1. Depdc5 knockout rat: A novel model of mTORopathy.

    PubMed

    Marsan, Elise; Ishida, Saeko; Schramm, Adrien; Weckhuysen, Sarah; Muraca, Giuseppe; Lecas, Sarah; Liang, Ning; Treins, Caroline; Pende, Mario; Roussel, Delphine; Le Van Quyen, Michel; Mashimo, Tomoji; Kaneko, Takehito; Yamamoto, Takashi; Sakuma, Tetsushi; Mahon, Séverine; Miles, Richard; Leguern, Eric; Charpier, Stéphane; Baulac, Stéphanie

    2016-05-01

    DEP-domain containing 5 (DEPDC5), encoding a repressor of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway, has recently emerged as a major gene mutated in familial focal epilepsies and focal cortical dysplasia. Here we established a global knockout rat using TALEN technology to investigate in vivo the impact of Depdc5-deficiency. Homozygous Depdc5(-/-) embryos died from embryonic day 14.5 due to a global growth delay. Constitutive mTORC1 hyperactivation was evidenced in the brains and in cultured fibroblasts of Depdc5(-/-) embryos, as reflected by enhanced phosphorylation of its downstream effectors S6K1 and rpS6. Consistently, prenatal treatment with mTORC1 inhibitor rapamycin rescued the phenotype of Depdc5(-/-) embryos. Heterozygous Depdc5(+/-) rats developed normally and exhibited no spontaneous electroclinical seizures, but had altered cortical neuron excitability and firing patterns. Depdc5(+/-) rats displayed cortical cytomegalic dysmorphic neurons and balloon-like cells strongly expressing phosphorylated rpS6, indicative of mTORC1 upregulation, and not observed after prenatal rapamycin treatment. These neuropathological abnormalities are reminiscent of the hallmark brain pathology of human focal cortical dysplasia. Altogether, Depdc5 knockout rats exhibit multiple features of rodent models of mTORopathies, and thus, stand as a relevant model to study their underlying pathogenic mechanisms. PMID:26873552

  2. Generation of knockout alleles by RFLP based BAC targeting of polymorphic embryonic stem cells.

    PubMed

    Barakat, Tahsin Stefan; Gribnau, Joost

    2015-01-01

    The isolation of germ line competent mouse Embryonic Stem (ES) cells and the ability to modify the genome by homologous recombination has revolutionized life science research. Since its initial discovery, several approaches have been introduced to increase the efficiency of homologous recombination, including the use of isogenic DNA for the generation of targeting constructs, and the use of Bacterial Artificial Chromosomes (BACs). BACs have the advantage of combining long stretches of homologous DNA, thereby increasing targeting efficiencies, with the possibilities delivered by BAC recombineering approaches, which provide the researcher with almost unlimited possibilities to efficiently edit the genome in a controlled fashion. Despite these advantages of BAC targeting approaches, a widespread use has been hampered, mainly because of the difficulties in identifying BAC-targeted knockout alleles by conventional methods like Southern Blotting. Recently, we introduced a novel BAC targeting strategy, in which Restriction Fragment Length Polymorphisms (RFLPs) are targeted in polymorphic mouse ES cells, enabling an efficient and easy PCR-based readout to identify properly targeted alleles. Here we provide a detailed protocol for the generation of targeting constructs, targeting of ES cells, and convenient PCR-based analysis of targeted clones, which enable the user to generate knockout ES cells of almost every gene in the mouse genome within a 2-month period. PMID:25239745

  3. 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

  4. Optimization of transcription factor binding map accuracy utilizing knockout-mouse models

    PubMed Central

    Krebs, Wolfgang; Schmidt, Susanne V.; Goren, Alon; De Nardo, Dominic; Labzin, Larisa; Bovier, Anton; Ulas, Thomas; Theis, Heidi; Kraut, Michael; Latz, Eicke; Beyer, Marc; Schultze, Joachim L.

    2014-01-01

    Genome-wide assessment of protein–DNA interaction by chromatin immunoprecipitation followed by massive parallel sequencing (ChIP-seq) is a key technology for studying transcription factor (TF) localization and regulation of gene expression. Signal-to-noise-ratio and signal specificity in ChIP-seq studies depend on many variables, including antibody affinity and specificity. Thus far, efforts to improve antibody reagents for ChIP-seq experiments have focused mainly on generating higher quality antibodies. Here we introduce KOIN (knockout implemented normalization) as a novel strategy to increase signal specificity and reduce noise by using TF knockout mice as a critical control for ChIP-seq data experiments. Additionally, KOIN can identify ‘hyper ChIPable regions’ as another source of false-positive signals. As the use of the KOIN algorithm reduces false-positive results and thereby prevents misinterpretation of ChIP-seq data, it should be considered as the gold standard for future ChIP-seq analyses, particularly when developing ChIP-assays with novel antibody reagents. PMID:25378309

  5. Myeloid Deletion of α1AMPK Exacerbates Atherosclerosis in LDL Receptor Knockout (LDLRKO) Mice.

    PubMed

    Cao, Qiang; Cui, Xin; Wu, Rui; Zha, Lin; Wang, Xianfeng; Parks, John S; Yu, Liqing; Shi, Hang; Xue, Bingzhong

    2016-06-01

    Macrophage inflammation marks all stages of atherogenesis, and AMPK is a regulator of macrophage inflammation. We therefore generated myeloid α1AMPK knockout (MAKO) mice on the LDL receptor knockout (LDLRKO) background to investigate whether myeloid deletion of α1AMPK exacerbates atherosclerosis. When fed an atherogenic diet, MAKO/LDLRKO mice displayed exacerbated atherosclerosis compared with LDLRKO mice. To determine the underlying pathophysiological pathways, we characterized macrophage inflammation/chemotaxis and lipid/cholesterol metabolism in MAKO/LDLRKO mice. Myeloid deletion of α1AMPK increased macrophage inflammatory gene expression and enhanced macrophage migration and adhesion to endothelial cells. Remarkably, MAKO/LDLRKO mice also displayed higher composition of circulating chemotaxically active Ly-6C(high) monocytes, enhanced atherosclerotic plaque chemokine expression, and monocyte recruitment into plaques, leading to increased atherosclerotic plaque macrophage content and inflammation. MAKO/LDLRKO mice also exhibited higher plasma LDL and VLDL cholesterol content, increased circulating apolipoprotein B (apoB) levels, and higher liver apoB expression. We conclude that macrophage α1AMPK deficiency promotes atherogenesis in LDLRKO mice and is associated with enhanced macrophage inflammation and hypercholesterolemia and that macrophage α1AMPK may serve as a therapeutic target for prevention and treatment of atherosclerosis. PMID:26822081

  6. Idebenone and Resveratrol Extend Lifespan and Improve Motor Function of HtrA2 Knockout Mice

    PubMed Central

    Szegő, Éva M.; Moisoi, Nicoleta; Martins, L. Miguel; Outeiro, Tiago F.; Kermer, Pawel

    2011-01-01

    Heterozygous loss-of-function mutation of the human gene for the mitochondrial protease HtrA2 has been associated with increased risk to develop mitochondrial dysfunction, a process known to contribute to neurodegenerative disorders such as Huntington's disease (HD) and Parkinson's disease (PD). Knockout of HtrA2 in mice also leads to mitochondrial dysfunction and to phenotypes that resemble those found in neurodegenerative disorders and, ultimately, lead to death of animals around postnatal day 30. Here, we show that Idebenone, a synthetic antioxidant of the coenzyme Q family, and Resveratrol, a bioactive compound extracted from grapes, are both able to ameliorate this phenotype. Feeding HtrA2 knockout mice with either compound extends lifespan and delays worsening of the motor phenotype. Experiments conducted in cell culture and on brain tissue of mice revealed that each compound has a different mechanism of action. While Idebenone acts by downregulating the integrated stress response, Resveratrol acts by attenuating apoptosis at the level of Bax. These activities can account for the delay in neuronal degeneration in the striata of these mice and illustrate the potential of these compounds as effective therapeutic approaches against neurodegenerative disorders such as HD or PD. PMID:22205977

  7. Necroptotic Cell Death Signaling and Execution Pathway: Lessons from Knockout Mice

    PubMed Central

    Belizário, José; Vieira-Cordeiro, Luiz; Enns, Sylvia

    2015-01-01

    Under stress conditions, cells in living tissue die by apoptosis or necrosis depending on the activation of the key molecules within a dying cell that either transduce cell survival or death signals that actively destroy the sentenced cell. Multiple extracellular (pH, heat, oxidants, and detergents) or intracellular (DNA damage and Ca2+ overload) stress conditions trigger various types of the nuclear, endoplasmic reticulum (ER), cytoplasmatic, and mitochondrion-centered signaling events that allow cells to preserve the DNA integrity, protein folding, energetic, ionic and redox homeostasis, thus escaping from injury. Along the transition from reversible to irreversible injury, death signaling is highly heterogeneous and damaged cells may engage autophagy, apoptotic, or necrotic cell death programs. Studies on multiple double- and triple- knockout mice identified caspase-8, flip, and fadd genes as key regulators of embryonic lethality and inflammation. Caspase-8 has a critical role in pro- and antinecrotic signaling pathways leading to the activation of receptor interacting protein kinase 1 (RIPK1), RIPK3, and the mixed kinase domain-like (MLKL) for a convergent execution pathway of necroptosis or regulated necrosis. Here we outline the recent discoveries into how the necrotic cell death execution pathway is engaged in many physiological and pathological outcome based on genetic analysis of knockout mice. PMID:26491219

  8. Exacerbated neuronal ceroid lipofuscinosis phenotype in Cln1/5 double-knockout mice

    PubMed Central

    Blom, Tea; Schmiedt, Mia-Lisa; Wong, Andrew M.; Kyttälä, Aija; Soronen, Jarkko; Jauhiainen, Matti; Tyynelä, Jaana; Cooper, Jonathan D.; Jalanko, Anu

    2013-01-01

    SUMMARY Both CLN1 and CLN5 deficiencies lead to severe neurodegenerative diseases of childhood, known as neuronal ceroid lipofuscinoses (NCLs). The broadly similar phenotypes of NCL mouse models, and the potential for interactions between NCL proteins, raise the possibility of shared or convergent disease mechanisms. To begin addressing these issues, we have developed a new mouse model lacking both Cln1 and Cln5 genes. These double-knockout (Cln1/5 dko) mice were fertile, showing a slight decrease in expected Mendelian breeding ratios, as well as impaired embryoid body formation by induced pluripotent stem cells derived from Cln1/5 dko fibroblasts. Typical disease manifestations of the NCLs, i.e. seizures and motor dysfunction, were detected at the age of 3 months, earlier than in either single knockout mouse. Pathological analyses revealed a similar exacerbation and earlier onset of disease in Cln1/5 dko mice, which exhibited a pronounced accumulation of autofluorescent storage material. Cortical demyelination and more pronounced glial activation in cortical and thalamic regions was followed by cortical neuron loss. Alterations in lipid metabolism in Cln1/5 dko showed a specific increase in plasma phospholipid transfer protein (PLTP) activity. Finally, gene expression profiling of Cln1/5 dko cortex revealed defects in myelination and immune response pathways, with a prominent downregulation of α-synuclein in Cln1/5 dko mouse brains. The simultaneous loss of both Cln1 and Cln5 genes might enhance the typical pathological phenotypes of these mice by disrupting or downregulating shared or convergent pathogenic pathways, which could potentially include interactions of CLN1 and CLN5. PMID:23065637

  9. Common mechanisms for calorie restriction and adenylyl cyclase type 5 knockout models of longevity.

    PubMed

    Yan, Lin; Park, Ji Yeon; Dillinger, Jean-Guillaume; De Lorenzo, Mariana S; Yuan, Chujun; Lai, Lo; Wang, Chunbo; Ho, David; Tian, Bin; Stanley, William C; Auwerx, Johan; Vatner, Dorothy E; Vatner, Stephen F

    2012-12-01

    Adenylyl cyclase type 5 knockout mice (AC5 KO) live longer and are stress resistant, similar to calorie restriction (CR). AC5 KO mice eat more, but actually weigh less and accumulate less fat compared with WT mice. CR applied to AC5 KO results in rapid decrease in body weight, metabolic deterioration, and death. These data suggest that despite restricted food intake in CR, but augmented food intake in AC5 KO, the two models affect longevity and metabolism similarly. To determine shared molecular mechanisms, mRNA expression was examined genome-wide for brain, heart, skeletal muscle, and liver. Significantly more genes were regulated commonly rather than oppositely in all the tissues in both models, indicating commonality between AC5 KO and CR. Gene ontology analysis identified many significantly regulated, tissue-specific pathways shared by the two models, including sensory perception in heart and brain, muscle function in skeletal muscle, and lipid metabolism in liver. Moreover, when comparing gene expression changes in the heart under stress, the glutathione regulatory pathway was consistently upregulated in the longevity models but downregulated with stress. In addition, AC5 and CR shared changes in genes and proteins involved in the regulation of longevity and stress resistance, including Sirt1, ApoD, and olfactory receptors in both young- and intermediate-age mice. Thus, the similarly regulated genes and pathways in AC5 KO and CR mice, particularly related to the metabolic phenotype, suggest a unified theory for longevity and stress resistance. PMID:23020244

  10. Changes in gene expression associated with retinal degeneration in the rd3 mouse

    PubMed Central

    Cheng, Christiana L.

    2013-01-01

    Purpose To identify and characterize changes in gene expression associated with photoreceptor degeneration in the rd3 mouse model of Leber congenital amaurosis (LCA) type 12. Methods Global genome expression profiling using microarray technology was performed on total RNA extracts from rd3 and wild-type control mouse retinas at postnatal day 21. Quantitative PCR analysis of selected transcripts was performed to validate the microarray results. Results Functional annotation of differentially regulated genes in the rd3 mouse defined key canonical pathways, including phototransduction, glycerophospholipid metabolism, tumor necrosis factor receptor 1 signaling, and endothelin signaling. Overall, 1,140 of approximately 55,800 transcripts were differentially represented. In particular, a large percentage of the upregulated transcripts encode proteins involved in the immune response; whereas the downregulated transcripts encode proteins involved in phototransduction and lipid metabolism. Conclusions This analysis has elucidated several candidate genes and pathways, thus providing insight into the pathogenic mechanisms underlying the photoreceptor degeneration in the rd3 mouse retina and indicating directions for future studies. PMID:23687432

  11. [From alcohol to liquid ecstasy (GHB)--a survey of old and modern knockout agents. Part 1: historic and classic knockout agents].

    PubMed

    Schütz, Harald; Jansen, Malin; Dettmeyer, Reinhard; Verhoff, Marcel A

    2011-01-01

    Alcohol has been the most important knockout drug in history and literature and continues to play an essential role up to now. Blunt force to the head in the form of a knockout punch is another mechanism leading to a transient loss of consciousness. Diethyl ether and chloroform are among the classical knockout substances. Although they have meanwhile been replaced by modern sedatives and hypnotics, their use is still observed in isolated cases. PMID:22039694

  12. Pauli blocking and medium effects in nucleon knockout reactions

    SciTech Connect

    Bertulani, C. A.; De Conti, C.

    2010-06-15

    We study medium modifications of the nucleon-nucleon (NN) cross sections and their influence on the nucleon knockout reactions. Using the eikonal approximation, we compare the results obtained with free NN cross sections with those obtained with a purely geometrical treatment of Pauli blocking and with NN obtained with more elaborated Dirac-Bruecker methods. The medium effects are parametrized in terms of the baryon density. We focus on symmetric nuclear matter, although the geometrical Pauli blocking also allows for the treatment of asymmetric nuclear matter. It is shown that medium effects can change the nucleon knockout cross sections and momentum distributions up to 10% in the energy range E{sub lab}=50-300 MeV/nucleon. The effect is more evident in reactions involving halo nuclei.

  13. 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.

  14. Creation and Preliminary Characterization of a Leptin Knockout Rat

    PubMed Central

    Vaira, Sergio; Yang, Chang; McCoy, Aaron; Keys, Kelly; Xue, Shurong; Weinstein, Edward J.; Novack, Deborah V.

    2012-01-01

    Leptin, a cytokine-like hormone secreted mainly by adipocytes, regulates various pathways centered on food intake and energy expenditure, including insulin sensitivity, fertility, immune system, and bone metabolism. Here, using zinc finger nuclease technology, we created the first leptin knockout rat. Homozygous leptin null rats are obese with significantly higher serum cholesterol, triglyceride, and insulin levels than wild-type controls. Neither gender produced offspring despite of repeated attempts. The leptin knockout rats also have depressed immune system. In addition, examination by microcomputed tomography of the femurs of the leptin null rats shows a significant increase in both trabecular bone mineral density and bone volume of the femur compared with wild-type littermates. Our model should be useful for many different fields of studies, such as obesity, diabetes, and bone metabolism-related illnesses. PMID:22948215

  15. Protection of Cftr knockout mice from acute lung infection by a helper-dependent adenoviral vector expressing Cftr in airway epithelia

    PubMed Central

    Koehler, David R.; Sajjan, Umadevi; Chow, Yu-Hua; Martin, Bernard; Kent, Geraldine; Tanswell, A. Keith; McKerlie, Colin; Forstner, Janet F.; Hu, Jim

    2003-01-01

    We developed a helper-dependent adenoviral vector for cystic fibrosis lung gene therapy. The vector expresses cystic fibrosis transmembrane conductance regulator (Cftr) using control elements from cytokeratin 18. The vector expressed properly localized CFTR in cultured cells and in the airway epithelia of mice. Cftr RNA and protein were present in whole lung and bronchioles, respectively, for 28 days after a vector dose. Acute inflammation was minimal to moderate. To test the therapeutic potential of the vector, we challenged mice with a clinical strain of Burkholderia cepacia complex (Bcc). Cftr knockout mice (but not Cftr+/+ littermates) challenged with Bcc developed severe lung histopathology and had high lung bacteria counts. Cftr knockout mice receiving gene therapy 7 days before Bcc challenge had less severe histopathology, and the number of lung bacteria was reduced to the level seen in Cftr+/+ littermates. These data suggest that gene therapy could benefit cystic fibrosis patients by reducing susceptibility to opportunistic pathogens. PMID:14673110

  16. Protection of Cftr knockout mice from acute lung infection by a helper-dependent adenoviral vector expressing Cftr in airway epithelia.

    PubMed

    Koehler, David R; Sajjan, Umadevi; Chow, Yu-Hua; Martin, Bernard; Kent, Geraldine; Tanswell, A Keith; McKerlie, Colin; Forstner, Janet F; Hu, Jim

    2003-12-23

    We developed a helper-dependent adenoviral vector for cystic fibrosis lung gene therapy. The vector expresses cystic fibrosis transmembrane conductance regulator (Cftr) using control elements from cytokeratin 18. The vector expressed properly localized CFTR in cultured cells and in the airway epithelia of mice. Cftr RNA and protein were present in whole lung and bronchioles, respectively, for 28 days after a vector dose. Acute inflammation was minimal to moderate. To test the therapeutic potential of the vector, we challenged mice with a clinical strain of Burkholderia cepacia complex (Bcc). Cftr knockout mice (but not Cftr+/+ littermates) challenged with Bcc developed severe lung histopathology and had high lung bacteria counts. Cftr knockout mice receiving gene therapy 7 days before Bcc challenge had less severe histopathology, and the number of lung bacteria was reduced to the level seen in Cftr+/+ littermates. These data suggest that gene therapy could benefit cystic fibrosis patients by reducing susceptibility to opportunistic pathogens. PMID:14673110

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

    PubMed Central

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

    2016-01-01

    The SLAM family receptors contribute to diverse aspects of lymphocyte biology and signal via the small adaptor molecule SAP. Mutations affecting SAP lead to X-linked lymphoproliferative syndrome Type 1, a severe immunodysregulation characterized by fulminant mononucleosis, dysgammaglobulinemia, and lymphoproliferation/lymphomas. Patients and mice having mutations affecting SAP also lack germinal centers due to a defect in T:B cell interactions and are devoid of invariant NKT (iNKT) cells. However, which and how SLAM family members contribute to these phenotypes remains uncertain. Three SLAM family members: SLAMF1, SLAMF5 and SLAMF6, are highly expressed on T follicular helper cells and germinal center B cells. SLAMF1 and SLAMF6 are also implicated in iNKT development. Although individual receptor knockout mice have limited iNKT and germinal center phenotypes compared to SAP knockout mice, the generation of multi-receptor knockout mice has been challenging, due to the genomic linkage of the genes encoding SLAM family members. Here, we used Cas9/CRISPR-based mutagenesis to generate mutations simultaneously in Slamf1, Slamf5 and Slamf6. Genetic disruption of all three receptors in triple-knockout mice (TKO) did not grossly affect conventional T or B cell development and led to mild defects in germinal center formation post-immunization. However, the TKO worsened defects in iNKT cells development seen in SLAMF6 single gene-targeted mice, supporting data on positive signaling and potential redundancy between these receptors. PMID:27258160

  18. LRRK2 knockout mice have an intact dopaminergic system but display alterations in exploratory and motor co-ordination behaviors

    PubMed Central

    2012-01-01

    Mutations in the LRRK2 gene are the most common cause of genetic Parkinson’s disease. Although the mechanisms behind the pathogenic effects of LRRK2 mutations are still not clear, data emerging from in vitro and in vivo models suggests roles in regulating neuronal polarity, neurotransmission, membrane and cytoskeletal dynamics and protein degradation. We created mice lacking exon 41 that encodes the activation hinge of the kinase domain of LRRK2. We have performed a comprehensive analysis of these mice up to 20 months of age, including evaluation of dopamine storage, release, uptake and synthesis, behavioral testing, dendritic spine and proliferation/neurogenesis analysis. Our results show that the dopaminergic system was not functionally comprised in LRRK2 knockout mice. However, LRRK2 knockout mice displayed abnormal exploratory activity in the open-field test. Moreover, LRRK2 knockout mice stayed longer than their wild type littermates on the accelerated rod during rotarod testing. Finally, we confirm that loss of LRRK2 caused degeneration in the kidney, accompanied by a progressive enhancement of autophagic activity and accumulation of autofluorescent material, but without evidence of biphasic changes. PMID:22647713

  19. Versatile strategy for isolating transcription activator-like effector nuclease-mediated knockout mutants in Caenorhabditis elegans.

    PubMed

    Sugi, Takuma; Sakuma, Tetsushi; Ohtani, Yasuko; Yamamoto, Takashi

    2014-01-01

    Targeted genome editing using transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems has recently emerged as a potentially powerful method for creating locus-specific mutations in Caenorhabditis elegans. Due to the low mutation frequencies, one of the crucial steps in using these technologies is screening animals that harbor a targeted mutation. In previous studies, identifying targeted mutations in C. elegans usually depended on observations of fluorescent markers such as a green fluorescent protein or visible phenotypes such as dumpy and uncoordinated phenotypes. However, this strategy is limited in practice because the phenotypes caused by targeted mutations such as defects in sensory behaviors are often apparently invisible. Here, we describe a versatile strategy for isolating C. elegans knockout mutants by TALEN-mediated genome editing and a heteroduplex mobility assay. We applied TALENs to engineer the locus of the neural gene glr-1, which is a C. elegans AMPA-type receptor orthologue that is known to have crucial roles in various sensory behaviors. Knockout mutations in the glr-1 locus, which caused defective mechanosensory behaviors, were efficiently identified by the heteroduplex mobility assay. Thus, we demonstrated the utility of a TALEN-based knockout strategy for creating C. elegans with mutations that cause invisible phenotypes. PMID:24409999

  20. Neural stem cells from protein tyrosine phosphatase sigma knockout mice generate an altered neuronal phenotype in culture

    PubMed Central

    Kirkham, David L; Pacey, Laura KK; Axford, Michelle M; Siu, Roberta; Rotin, Daniela; Doering, Laurie C

    2006-01-01

    Background The LAR family Protein Tyrosine Phosphatase sigma (PTPσ) has been implicated in neuroendocrine and neuronal development, and shows strong expression in specific regions within the CNS, including the subventricular zone (SVZ). We established neural stem cell cultures, grown as neurospheres, from the SVZ of PTPσ knockout mice and sibling controls to determine if PTPσ influences the generation and the phenotype of the neuronal, astrocyte and oligodendrocyte cell lineages. Results The neurospheres from the knockout mice acquired heterogeneous developmental characteristics and they showed similar morphological characteristics to the age matched siblings. Although Ptprs expression decreases as a function of developmental age in vivo, it remains high with the continual renewal and passage of the neurospheres. Stem cells, progenitors and differentiated neurons, astrocytes and oligodendrocytes all express the gene. While no apparent differences were observed in developing neurospheres or in the astrocytes and oligodendrocytes from the PTPσ knockout mice, the neuronal migration patterns and neurites were altered when studied in culture. In particular, neurons migrated farther from the neurosphere centers and the neurite outgrowth exceeded the length of the neuronal processes from age matched sibling controls. Conclusion Our results imply a specific role for PTPσ in the neuronal lineage, particularly in the form of inhibitory influences on neurite outgrowth, and demonstrate a role for tyrosine phosphatases in neuronal stem cell differentiation. PMID:16784531

  1. Phenotypic and Molecular Alterations in the Mammary Tissue of R-Spondin1 Knock-Out Mice during Pregnancy.

    PubMed

    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

  2. Impairment of social and emotional behaviors in Cadm1-knockout mice.

    PubMed

    Takayanagi, Yuki; Fujita, Eriko; Yu, Zhiling; Yamagata, Takanori; Momoi, Mariko Y; Momoi, Takashi; Onaka, Tatsushi

    2010-06-01

    Cell adhesion molecule 1 (CADM1), a member of the immunoglobulin superfamily, mediates synaptic cell adhesion. Missense mutations in the CADM1 gene have been identified in autism spectrum disorder (ASD) patients. In the present study, we examined emotional behaviors, social behaviors and motor performances in Cadm1-knockout (KO) mice. Cadm1-KO mice showed increased anxiety-related behavior in open-field and light-dark transition tests. Social behaviors of Cadm1-KO mice were impaired in social interaction, resident-intruder and social memory/recognition tests. Furthermore, motor coordination and gait of Cadm1-KO mice were impaired in rotarod and footprint tests. Our study demonstrates that CADM1 plays roles in regulating emotional behaviors, social behaviors and motor performances, and that CADM1 has important implications for psychiatric disorders with disruptions in social behavior, such as autism. PMID:20450890

  3. Generation of Rag1-knockout immunodeficient rats and mice using engineered meganucleases.

    PubMed

    Ménoret, Séverine; Fontanière, Sandra; Jantz, Derek; Tesson, Laurent; Thinard, Reynald; Rémy, Séverine; Usal, Claire; Ouisse, Laure-Hélène; Fraichard, Alexandre; Anegon, Ignacio

    2013-02-01

    Despite the recent availability of gene-specific nucleases, such as zinc-finger nucleases (ZFNs) and transcription activator-like nucleases (TALENs), there is still a need for new tools to modify the genome of different species in an efficient, rapid, and less costly manner. One aim of this study was to apply, for the first time, engineered meganucleases to mutate an endogenous gene in animal zygotes. The second aim was to target the mouse and rat recombination activating gene 1 (Rag1) to describe, for the first time, Rag1 knockout immunodeficient rats. We microinjected a plasmid encoding a meganuclease for Rag1 into the pronucleus of mouse and rat zygotes. Mutant animals were detected by PCR sequencing of the targeted sequence. A homozygous RAG1-deficient rat line was generated and immunophenotyped. Meganucleases were efficient, because 3.4 and 0.6% of mouse and rat microinjected zygotes, respectively, generated mutated animals. RAG1-deficient rats showed significantly decreased proportions and numbers of immature and mature T and B lymphocytes and normal NK cells vs. littermate wild-type controls. In summary, we describe the use of engineered meganucleases to inactivate an endogenous gene with efficiencies comparable to those of ZFNs and TALENs. Moreover, we generated an immunodeficient rat line useful for studies in which there is a need for biological parameters to be analyzed in the absence of immune responses. PMID:23150522

  4. Effect of chronic glutathione deficiency on the behavioral phenotype of Gclm-/- knockout mice.

    PubMed

    Chen, Ying; Curran, Christine P; Nebert, Daniel W; Patel, Krishna V; Williams, Michael T; Vorhees, Charles V

    2012-07-01

    Enhanced oxidative stress or deficient oxidative stress response in the brain is associated with neurodegenerative disorders and behavioral abnormalities. Previously we generated a knockout mouse line lacking the gene encoding glutamate-cysteine ligase modifier subunit (GCLM). Gclm(-/-) knockout (KO) mice are viable and fertile, yet exhibit only 9-35% of wild-type levels of reduced glutathione (GSH) in tissues, making them a useful model for chronic GSH depletion. Having the global absence of this gene, KO mice--from the time of conception and throughout postnatal life--experience chronic oxidative stress in all tissues, including brain. Between postnatal day (P) 60 and P100, we carried out behavioral phenotyping tests in adults, comparing male and female Gclm(-/-) with Gclm(-/-) wild-type (WT) littermates. Compared with WT, KO mice exhibited: subnormal anxiety in the elevated zero maze; normal overall exploratory open-field activity, but slightly more activity in the peripheral zones; normal acoustic startle and prepulse inhibition reactions; normal novel object recognition with increased time attending to the stimulus objects; slightly reduced latencies to reach a random marked platform in the Morris water maze; normal spatial learning and memory in multiple phases of the Morris water maze; and significantly greater hyperactivity in response to methamphetamine in the open field. These findings are generally in agreement with two prior studies on these mice and suggest that the brain is remarkably resilient to lowered GSH levels, implying significant reserve capacity to regulate reactive oxygen species-but with regional differences such that anxiety and stimulated locomotor control brain regions might be more vulnerable. PMID:22580179

  5. 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

    2016-01-01

    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 (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. PMID:27362799

  6. Effect of chronic glutathione deficiency on the behavioral phenotype of Gclm(−/−) knockout mice

    PubMed Central

    Chen, Ying; Curran, Christine P.; Nebert, Daniel W.; Patel, Krishna V.; Williams, Michael T.; Vorhees, Charles V.

    2012-01-01

    Enhanced oxidative stress or deficient oxidative stress response in the brain is associated with neurodegenerative disorders and behavioral abnormalities. Previously we generated a knockout mouse line lacking the gene encoding γ-glutamylcysteine ligase modifier subunit (GCLM). Gclm(−/−) knockout (KO) mice are viable and fertile, yet exhibit only 9–35% of wild-type levels of reduced glutathione (GSH) in tissues, making them a useful model for chronic GSH depletion. Having the global absence of this gene, KO mice—from the time of conception and throughout postnatal life—experience chronic oxidative stress in all tissues, including brain. Between postnatal day (P) 60 and P100, we carried out behavioral phenotyping tests in adults, comparing male and female Gclm(−/−) with Gclm(+/+) wild-type (WT) littermates. Compared with WT, KO mice exhibited: subnormal anxiety in the elevated zero maze; normal overall exploratory open-field activity, but slightly more activity in the peripheral zones; normal acoustic startle and prepulse inhibition reactions; normal novel object recognition with increased time attending to the stimulus objects; slightly reduced latencies to reach a random marked platform in the Morris water maze; normal spatial learning and memory in multiple phases of the Morris water maze; and significantly greater hyperactivity in response to methamphetamine in the open field. These findings are in general agreement with two prior studies on these mice and suggest that the brain is remarkably resilient to lowered GSH levels, implying significant reserve capacity to regulate reactivity oxygen species—but with regional differences such that anxiety and stimulated locomotor control brain regions might be more vulnerable. PMID:22580179

  7. 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…

  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. PMID:26496859

  9. Chronic minocycline treatment improves social recognition memory in adult male Fmr1 knockout mice.

    PubMed

    Yau, Suk Yu; Chiu, Christine; Vetrici, Mariana; Christie, Brian R

    2016-10-01

    Fragile X syndrome (FXS) is caused by a mutation in the Fmr1 gene that leads to silencing of the gene and a loss of its gene product, Fragile X mental retardation protein (FMRP). Some of the key behavioral phenotypes for FXS include abnormal social anxiety and sociability. Here we show that Fmr1 knock-out (KO) mice exhibit impaired social recognition when presented with a novel mouse, and they display normal social interactions in other sociability tests. Administering minocycline to Fmr1 KO mice throughout critical stages of neural development improved social recognition memory in the novel mouse recognition task. To determine if synaptic changes in the prefrontal cortex (PFC) could have played a role in this improvement, we examined PSD-95, a member of the membrane-associated guanylate kinase family, and signaling molecules (ERK1/2, and Akt) linked to synaptic plasticity in the PFC. Our analyses indicated that while minocycline treatment can enhance behavioral performance, it does not enhance expression of PSD-95, ERK1/2 or Akt in the PFC. PMID:27291517

  10. Utilising the resources of the International Knockout Mouse Consortium: the Australian experience.

    PubMed

    Cotton, Leanne M; Meilak, Michelle L; Templeton, Tanya; Gonzales, Jose G; Nenci, Arianna; Cooney, Melissa; Truman, Dirk; Rodda, Fleur; Lynas, Alyce; Viney, Elizabeth; Rosenthal, Nadia; Bianco, Deborah M; O'Bryan, Moira K; Smyth, Ian M

    2015-04-01

    Mouse models play a key role in the understanding gene function, human development and disease. In 2007, the Australian Government provided funding to establish the Monash University embryonic stem cell-to-mouse (ES2M) facility. This was part of the broader Australian Phenomics Network, a national infrastructure initiative aimed at maximising access to global resources for understanding gene function in the mouse. The remit of the ES2M facility is to provide subsidised access for Australian biomedical researchers to the ES cell resources available from the International Knockout Mouse Consortium (IKMC). The stated aim of the IKMC is to generate a genetically modified mouse ES cell line for all of the ~23,000 genes in the mouse genome. The principal function of the Monash University ES2M service is to import genetically modified ES cells into Australia and to convert them into live mice with the potential to study human disease. Through advantages of economy of scale and established relationships with ES cell repositories worldwide, we have created over 110 germline mouse strains sourced from all of the major ES providers worldwide. We comment on our experience in generating these mouse lines; providing a snapshot of a "clients" perspective of using the IKMC resource and one which we hope will serve as a guide to other institutions or organisations contemplating establishing a similar centralised service. PMID:25645994

  11. Knockout of Drosophila RNase ZL impairs mitochondrial transcript processing, respiration and cell cycle progression.

    PubMed

    Xie, Xie; Dubrovsky, Edward B

    2015-12-01

    RNase Z(L) is a highly conserved tRNA 3'-end processing endoribonuclease. Similar to its mammalian counterpart, Drosophila RNase Z(L) (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 Z(L) 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

  12. CRISPR/Cas9-mediated GJA8 knockout in rabbits recapitulates human congenital cataracts.

    PubMed

    Yuan, Lin; Sui, Tingting; Chen, Mao; Deng, Jichao; Huang, Yongye; Zeng, Jian; Lv, Qingyan; Song, Yuning; Li, Zhanjun; Lai, Liangxue

    2016-01-01

    Cataracts are the leading cause of vision loss in the world, although surgical treatment can restore vision in cataract patients. Until now, there have been no adequate animal models for in vivo studies of artificial lens safety and drug interactions. Genetic studies have demonstrated that GJA8 is involved in maintaining lens opacity and proper lens development. In this study, a cataract model with GJA8 gene knockout was developed via co-injection of Cas9/sgRNA mRNA into rabbit zygotes. Our results showed that gene mutation efficiency in the GJA8 locus reached 98.7% in embryos and 100% in pups, demonstrating that the Cas9/sgRNA system is a highly efficient tool for gene editing in rabbits. In agreement with other studies, our genetic and histology results showed that impaired GJA8 function caused microphthalmia, small lens size and cataracts. In summary, our novel rabbit model of cataracts will be an important drug-screening tool for cataract prevention and treatment. PMID:26912477

  13. Comprehensive reanalysis of transcription factor knockout expression data in Saccharomyces cerevisiae reveals many new targets.

    PubMed

    Reimand, Jüri; Vaquerizas, Juan M; Todd, Annabel E; Vilo, Jaak; Luscombe, Nicholas M

    2010-08-01

    Transcription factor (TF) perturbation experiments give valuable insights into gene regulation. Genome-scale evidence from microarray measurements may be used to identify regulatory interactions between TFs and targets. Recently, Hu and colleagues published a comprehensive study covering 269 TF knockout mutants for the yeast Saccharomyces cerevisiae. However, the information that can be extracted from this valuable dataset is limited by the method employed to process the microarray data. Here, we present a reanalysis of the original data using improved statistical techniques freely available from the BioConductor project. We identify over 100,000 differentially expressed genes-nine times the total reported by Hu et al. We validate the biological significance of these genes by assessing their functions, the occurrence of upstream TF-binding sites, and the prevalence of protein-protein interactions. The reanalysed dataset outperforms the original across all measures, indicating that we have uncovered a vastly expanded list of relevant targets. In summary, this work presents a high-quality reanalysis that maximizes the information contained in the Hu et al. compendium. The dataset is available from ArrayExpress (accession: E-MTAB-109) and it will be invaluable to any scientist interested in the yeast transcriptional regulatory system. PMID:20385592

  14. Hippocampal dysfunction in the Euchromatin histone methyltransferase 1 heterozygous knockout mouse model for Kleefstra syndrome.

    PubMed

    Balemans, Monique C M; Kasri, Nael Nadif; Kopanitsa, Maksym V; Afinowi, Nurudeen O; Ramakers, Ger; Peters, Theo A; Beynon, Andy J; Janssen, Sanne M; van Summeren, Rik C J; Eeftens, Jorine M; Eikelenboom, Nathalie; Benevento, Marco; Tachibana, Makoto; Shinkai, Yoichi; Kleefstra, Tjitske; van Bokhoven, Hans; Van der Zee, Catharina E E M

    2013-03-01

    Euchromatin histone methyltransferase 1 (EHMT1) is a highly conserved protein that catalyzes mono- and dimethylation of histone H3 lysine 9, thereby epigenetically regulating transcription. Kleefstra syndrome (KS), is caused by haploinsufficiency of the EHMT1 gene, and is an example of an emerging group of intellectual disability (ID) disorders caused by genes encoding epigenetic regulators of neuronal gene activity. Little is known about the mechanisms underlying this disorder, prompting us to study the Euchromatin histone methyltransferase 1 heterozygous knockout (Ehmt1(+/-)) mice as a model for KS. In agreement with the cognitive disturbances observed in patients with KS, we detected deficits in fear extinction learning and both novel and spatial object recognition in Ehmt1(+/-) mice. These learning and memory deficits were associated with a significant reduction in dendritic arborization and the number of mature spines in hippocampal CA1 pyramidal neurons of Ehmt1(+/-) mice. In-depth analysis of the electrophysiological properties of CA3-CA1 synapses revealed no differences in basal synaptic transmission or theta-burst induced long-term potentiation (LTP). However, paired-pulse facilitation (PPF) was significantly increased in Ehmt1(+/-) neurons, pointing to a potential deficiency in presynaptic neurotransmitter release. Accordingly, a reduction in the frequency of miniature excitatory post-synaptic currents (mEPSCs) was observed in Ehmt1(+/-) neurons. These data demonstrate that Ehmt1 haploinsufficiency in mice leads to learning deficits and synaptic dysfunction, providing a possible mechanism for the ID phenotype in patients with KS. PMID:23175442

  15. THERAPEUTIC ISSUES IN THE TREATMENT OF VASCULARIZED XENOTRANSPLANTS USING GAL-KNOCKOUT DONORS IN NONHUMAN PRIMATES

    PubMed Central

    Ekser, Burcin; Kumar, Goutham; Veroux, Massimiliano; Cooper, David K.C.

    2011-01-01

    Purpose of review Solid organ xenotransplantation could be the future of transplantation, but improved outcomes are required in experimental models before clinical trials are justified. This review summarizes recent advances in solid organ xenotransplantation using organs from α1,3-galactosyltransferase gene-knockout (GTKO) pigs (with or without other genetic modifications) and novel therapeutic approaches. Recent findings Work on the development of genetically-engineered pigs has been considerable during the past few years, with many research institutes reporting the outcomes of research. Multiple gene modifications on a GTKO background have been reported, and the results of transplantation using organs from these pigs have been published. Progress, however, has been variable, and several obstacles, e.g., coagulation dysregulation, have been identified. Heterotopic pig heart xenotransplantation has been associated with graft survival exceeding 8 months, but kidney graft survival has not improved significantly. Summary The availability of GTKO pigs with additional genetic modifications aimed towards expression of multiple complement-regulatory proteins and/or human thromboregulatory genes, combined with novel immunosuppressive regimens, e.g., the inclusion of B cell-depleting agents, should improve pig organ survival in the near future. PMID:21415825

  16. 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

  17. A Sertoli Cell-Specific Knockout of Connexin43 Prevents Initiation of Spermatogenesis

    PubMed Central

    Brehm, Ralph; Zeiler, Martina; Rüttinger, Christina; Herde, Katja; Kibschull, Mark; Winterhager, Elke; Willecke, Klaus; Guillou, Florian; Lécureuil, Charlotte; Steger, Klaus; Konrad, Lutz; Biermann, Katharina; Failing, Klaus; Bergmann, Martin

    2007-01-01

    The predominant testicular gap junctional protein connexin43 (cx43) is located between neighboring Sertoli cells (SCs) and between SCs and germ cells. It is assumed to be involved in testicular development, cell differentiation, initiation, and maintenance of spermatogenesis with alterations of its expression being correlated with various testicular disorders. Because total disruption of the cx43 gene leads to perinatal death, we generated a conditional cx43 knockout (KO) mouse using the Cre/loxP recombination system, which lacks the cx43 gene solely in SCs (SCCx43KO), to evaluate the SC-specific functions of cx43 on spermatogenesis in vivo. Adult SCCx43KO−/− mice showed normal testis descent and development of the urogenital tract, but testis size and weight were drastically lower compared with heterozygous and wild-type littermates. Histological analysis and quantitation of mRNA expression of germ cell-specific marker genes revealed a significant reduction in the number of spermatogonia but increased SC numbers/tubule with only a few tubules left showing normal spermatogenesis. Thus, SC-specific deletion of cx43 mostly resulted in an arrest of spermatogenesis at the level of spermatogonia or SC-only syndrome and in intratubular SC clusters. Our data demonstrate for the first time that cx43 expression in SCs is an absolute requirement for normal testicular development and spermatogenesis. PMID:17591950

  18. CRISPR/Cas9-mediated GJA8 knockout in rabbits recapitulates human congenital cataracts

    PubMed Central

    Yuan, Lin; Sui, Tingting; Chen, Mao; Deng, Jichao; Huang, Yongye; Zeng, Jian; Lv, Qingyan; Song, Yuning; Li, Zhanjun; Lai, Liangxue

    2016-01-01

    Cataracts are the leading cause of vision loss in the world, although surgical treatment can restore vision in cataract patients. Until now, there have been no adequate animal models for in vivo studies of artificial lens safety and drug interactions. Genetic studies have demonstrated that GJA8 is involved in maintaining lens opacity and proper lens development. In this study, a cataract model with GJA8 gene knockout was developed via co-injection of Cas9/sgRNA mRNA into rabbit zygotes. Our results showed that gene mutation efficiency in the GJA8 locus reached 98.7% in embryos and 100% in pups, demonstrating that the Cas9/sgRNA system is a highly efficient tool for gene editing in rabbits. In agreement with other studies, our genetic and histology results showed that impaired GJA8 function caused microphthalmia, small lens size and cataracts. In summary, our novel rabbit model of cataracts will be an important drug-screening tool for cataract prevention and treatment. PMID:26912477

  19. Nrf2 Knockout Attenuates the Anti-Inflammatory Effects of Phenethyl Isothiocyanate and Curcumin

    PubMed Central

    2015-01-01

    The role of phytochemicals in preventive and therapeutic medicine is a major area of scientific research. Several studies have illustrated the mechanistic roles of phytochemicals in Nrf2 transcriptional activation. The present study aims to examine the importance of the transcription factor Nrf2 by treating peritoneal macrophages from Nrf2+/+ and Nrf2–/– mice ex vivo with phenethyl isothiocyanate (PEITC) and curcumin (CUR). The peritoneal macrophages were pretreated with the drugs and challenged with lipopolysaccharides (LPSs) alone and in combination with PEITC or CUR to assess their anti-inflammatory and antioxidative effects based on gene and protein expression in the treated cells. LPS treatment resulted in an increase in the expression of inflammatory markers such as cycloxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in both Nrf2+/+ and Nrf2–/– macrophages, detected by quantitative polymerase chain reaction (qPCR). Nrf2+/+ macrophages treated with PEITC and CUR exhibited a significant decrease in the expression of these anti-inflammatory genes along with an increase in the expression of hemeoxygenase-1 (HO-1), which is an antioxidative stress gene downstream of the Nrf2 transcription factor battery. Although there was no significant decrease in the expression of the anti-inflammatory genes or an increase in HO-1 expression in Nrf2–/– macrophages treated with either PEITC or CUR, there was a significant decrease in the protein expression of COX-2 and an increase in the expression of HO-1 in Nrf2+/+ macrophages treated with PEITC compared to that with CUR treatment. No significant changes were observed in the macrophages from knockout animals. Additionally, there was a significant decrease in LPS-induced IL-6 and TNF-α production following PEITC treatment compared with that following CUR in Nrf2+/+ macrophages, whereas no change was observed in the macrophages from knockout

  20. 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.

  1. A knockout mutation of a constitutive GPCR in Tetrahymena decreases both G-protein activity and chemoattraction.

    PubMed

    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

  2. Thyrotoropin receptor knockout changes monoaminergic neuronal system and produces methylphenidate-sensitive emotional and cognitive dysfunction.

    PubMed

    Mouri, Akihiro; Hoshino, Yuta; Narusawa, Shiho; Ikegami, Keisuke; Mizoguchi, Hiroyuki; Murata, Yoshiharu; Yoshimura, Takashi; Nabeshima, Toshitaka

    2014-10-01

    Attention deficit/hyperactivity disorder (ADHD) has been reported in association with resistance to thyroid hormone, a disease caused by a mutation in the thyroid hormone receptor β (TRβ) gene. TRβ is a key protein mediating down-regulation of thyrotropin (TSH) expression by 3,3',5-tri-iodothyronine (T3), an active form of thyroid hormone. Dysregulation of TSH and its receptor (TSHR) is implicated in the pathophysiology of ADHD but the role of TSHR remains elusive. Here, we clarified a novel role for TSHR in emotional and cognitive functions related to monoaminergic nervous systems. TSHR knockout mice showed phenotypes of ADHD such as hyperactivity, impulsiveness, a decrease in sociality and increase in aggression, and an impairment of short-term memory and object recognition memory. Administration of methylphenidate (1, 5 and 10mg/kg) reversed impulsiveness, aggression and object recognition memory impairment. In the knockout mice, monoaminergic changes including decrease in the ratio of 3-methoxy-4-hydroxyphenylglycol/noradrenaline and increase in the ratio of homovanillic acid/dopamine were observed in some brain regions, accompanied by increase in the expression of noradrenaline transporter in the frontal cortex. When TSH was completely suppressed by the supraphysiological administration of T3 to the adult mice, some behavioral and neurological changes in TSHR KO mice were also observed, suggesting that these changes were not due to developmental hypothyroidism induced by the inactivation of TSHR but to the loss of the TSH-TSHR pathway itself. Taken together, the present findings suggest a novel role for TSHR in behavioral and neurological phenotypes of ADHD. PMID:25016105

  3. Masticatory HyperMuscularity is not related to Reduced Cranial Volume in Myostatin-Knockout Mice

    PubMed Central

    Cray, James; Kneib, Jared; Vecchione, Lisa; Byron, Craig; Cooper, Gregory M.; Losee, Joseph E.; Siegel, Michael I.; Hamrick, Mark W.; Sciote, James J.; Mooney, Mark P.

    2013-01-01

    It has been suggested recently that masticatory muscle size reduction in humans resulted in greater encephalization through decreased compressive forces on the cranial vault. Following this logic, if masticatory muscle size were increased, then a reduction in brain growth should also occur. The present study was designed to test this hypothesis using a myostatin (GDF-8) knockout mouse model. Myostatin is a negative regulator of skeletal muscle growth, and individuals lacking this gene show significant hypermuscularity. Sixty-two (32 wild-type and 30 GDF-8 −/− knockout), 1, 28, 56 and 180 day old CD-1 mice were used. Body and masseter muscle weights were collected following dissection and standardized lateral and dorsoventral cephalographs were obtained. Cephalometric landmarks were identified on the radiographs and cranial volume was calculated. Mean differences were assessed using a two-way ANOVA. KO mice had significantly greater body and masseter weights beginning at 28 days compared to WT controls. No significant differences in cranial volumes were noted between KO and WT. Muscle weight was not significantly correlated with cranial volume in 1, 28, or 180 day old mice. Muscle weights exhibited a positive correlation with cranial volume at 56 days. Results demonstrate that masticatory hypermuscularity is not associated with reduced cranial volume. In contrast, there is abundant data demonstrating the opposite, brain growth determines cranial vault growth and masticatory apparatus only affects ectocranial morphology. The results presented here do not support the hypothesis that a reduction in masticatory musculature relaxed compressive forces on the cranial vault allowing for greater encephalization. PMID:21618442

  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. 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.

  6. Substrate uptake and metabolism are preserved in hypertrophic caveolin-3 knockout hearts

    PubMed Central

    Augustus, Ayanna S.; Buchanan, Jonathan; Addya, Sankar; Rengo, Giuseppe; Pestell, Richard G.; Fortina, Paolo; Koch, Walter J.; Bensadoun, Andre; Abel, E. Dale; Lisanti, Michael P.

    2008-01-01

    Caveolin-3 (Cav3), the primary protein component of caveolae in muscle cells, regulates numerous signaling pathways including insulin receptor signaling and facilitates free fatty acid (FA) uptake by interacting with several FA transport proteins. We previously reported that Cav3 knockout mice (Cav3KO) develop cardiac hypertrophy with diminished contractile function; however, the effects of Cav3 gene ablation on cardiac substrate utilization are unknown. The present study revealed that the uptake and oxidation of FAs and glucose were normal in hypertrophic Cav3KO hearts. Real-time PCR analysis revealed normal expression of lipid metabolism genes including FA translocase (CD36) and carnitine palmitoyl transferase-1 in Cav3KO hearts. Interestingly, myocardial cAMP content was significantly increased by 42%; however, this had no effect on PKA activity in Cav3KO hearts. Microarray expression analysis revealed a marked increase in the expression of genes involved in receptor trafficking to the plasma membrane, including Rab4a and the expression of WD repeat/FYVE domain containing proteins. We observed a fourfold increase in the expression of cellular retinol binding protein-III and a 3.5-fold increase in 17β-hydroxysteroid dehydrogenase type 11, a member of the short-chain dehydrogenase/reductase family involved in the biosynthesis and inactivation of steroid hormones. In summary, a loss of Cav3 in the heart leads to cardiac hypertrophy with normal substrate utilization. Moreover, a loss of Cav3 mRNA altered the expression of several genes not previously linked to cardiac growth and function. Thus we have identified a number of new target genes associated with the pathogenesis of cardiac hypertrophy. PMID:18552160

  7. Screening methods to identify TALEN-mediated knockout mice.

    PubMed

    Nakagawa, Yoshiko; Yamamoto, Takashi; Suzuki, Ken-Ichi; Araki, Kimi; Takeda, Naoki; Ohmuraya, Masaki; Sakuma, Tetsushi

    2014-01-01

    Genome editing with site-specific nucleases, such as zinc-finger nucleases or transcription activator-like effector nucleases (TALENs), and RNA-guided nucleases, such as the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system, is becoming the new standard for targeted genome modification in various organisms. Application of these techniques to the manufacture of knockout mice would be greatly aided by simple and easy methods for genotyping of mutant and wild-type pups among litters. However, there are no detailed or comparative reports concerning the identification of mutant mice generated using genome editing technologies. Here, we genotyped TALEN-derived enhanced green fluorescent protein (eGFP) knockout mice using a combination of approaches, including fluorescence observation, heteroduplex mobility assay, restriction fragment length polymorphism analysis and DNA sequencing. The detection sensitivities for TALEN-induced mutations differed among these methods, and we therefore concluded that combinatorial testing is necessary for the screening and determination of mutant genotypes. Since the analytical methods tested can be carried out without specialized equipment, costly reagents and/or sophisticated protocols, our report should be of interest to a broad range of researchers who are considering the application of genome editing technologies in various organisms. PMID:24521866

  8. BDNF restricted knockout mice as an animal model for aggression

    PubMed Central

    Ito, Wataru; Chehab, Mahmoud; Thakur, Siddarth; Li, Jiayang; Morozov, Alexei

    2011-01-01

    Mice with global deletion of one BDNF allele, or with forebrain-restricted deletion of both alleles show elevated aggression, but this phenotype is accompanied by other behavioral changes, including increases in anxiety and deficits in cognition. Here, we performed behavioral characterization of conditional BDNF knockout mice generated using a Cre recombinase driver line, KA1-Cre, which expresses Cre in few areas of brain: highly at hippocampal area CA3, moderately in dentate gyrus, cerebellum and facial nerve nucleus. The mutant animals exhibited elevated conspecific aggression and social dominance, but did not show changes in anxiety-like behaviors assessed using the elevated plus maze and open field test. There were no changes in depression like behaviors tested in the forced swim test, but small increase in immobility in the tail suspension test. In cognitive tasks, mutants showed normal social recognition and normal spatial and fear memory, but exhibited a deficit in object recognition. Thus, this knockout can serve as a robust model of BDNF-dependent aggression and object recognition deficiency. PMID:21255268

  9. Screening Methods to Identify TALEN-Mediated Knockout Mice

    PubMed Central

    Nakagawa, Yoshiko; Yamamoto, Takashi; Suzuki, Ken-Ichi; Araki, Kimi; Takeda, Naoki; Ohmuraya, Masaki; Sakuma, Tetsushi

    2014-01-01

    Genome editing with site-specific nucleases, such as zinc-finger nucleases or transcription activator-like effector nucleases (TALENs), and RNA-guided nucleases, such as the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system, is becoming the new standard for targeted genome modification in various organisms. Application of these techniques to the manufacture of knockout mice would be greatly aided by simple and easy methods for genotyping of mutant and wild-type pups among litters. However, there are no detailed or comparative reports concerning the identification of mutant mice generated using genome editing technologies. Here, we genotyped TALEN-derived enhanced green fluorescent protein (eGFP) knockout mice using a combination of approaches, including fluorescence observation, heteroduplex mobility assay, restriction fragment length polymorphism analysis and DNA sequencing. The detection sensitivities for TALEN-induced mutations differed among these methods, and we therefore concluded that combinatorial testing is necessary for the screening and determination of mutant genotypes. Since the analytical methods tested can be carried out without specialized equipment, costly reagents and/or sophisticated protocols, our report should be of interest to a broad range of researchers who are considering the application of genome editing technologies in various organisms. PMID:24521866

  10. 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

  11. 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. PMID:24973446

  12. 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. PMID:24102798

  13. Study of 19C by One-Neutron Knockout

    NASA Astrophysics Data System (ADS)

    Hwang, Jongwon; Kim, Sunji; Satou, Yoshiteru; Orr, Nigel A.; Nakamura, Takashi; Kondo, Yosuke; Gibelin, Julien; Achouri, N. Lynda; Aumann, Thomas; Baba, Hidetada; Delaunay, Franck; Doornenbal, Pieter; Fukuda, Naoki; Inabe, Naohito; Isobe, Tadaaki; Kameda, Daisuke; Kanno, Daiki; Kobayashi, Nobuyuki; Kobayashi, Toshio; Kubo, Toshiyuki; Leblond, Sylvain; Lee, Jenny; Marqués, F. Miguel; Minakata, Ryogo; Motobayashi, Tohru; Murai, Daichi; Murakami, Tetsuya; Muto, Kotomi; Nakashima, Tomohiro; Nakatsuka, Noritsugu; Navin, Alahari; Nishi, Seijiro; Ogoshi, Shun; Otsu, Hideaki; Sato, Hiromi; Shimizu, Yohei; Suzuki, Hiroshi; Takahashi, Kento; Takeda, Hiroyuki; Takeuchi, Satoshi; Tanaka, Ryuki; Togano, Yasuhiro; Tuff, Adam G.; Vandebrouck, Marine; Yoneda, Ken-ichiro

    2016-03-01

    The spectroscopic structure of 19C, a prominent one-neutron halo nucleus, has been studied with a 20C secondary beam at 290 MeV/nucleon and a carbon target. Neutron-unbound states populated by the one-neutron knockout reaction were investigated by means of the invariant mass method. The preliminary relative energy spectrum and parallel momentum distribution of the knockout residue, 19C*, were reconstructed from the measured four momenta of the 18C fragment, neutron, and beam. Three resonances were observed in the spectrum, which correspond to the states at Ex = 0.62(9), 1.42(10), and 2.89(10) MeV. The parallel momentum distributions for the 0.62-MeV and 2.89-MeV states suggest spin-parity assignments of 5/2+ and 1/2-, respectively. The 1.42-MeV state is in line with the reported 5/22+ state.

  14. In vivo identification of essential nucleotides in tRNALeu to its functions by using a constructed yeast tRNALeu knockout strain

    PubMed Central

    Huang, Qian; Yao, Peng; Eriani, Gilbert; Wang, En-Duo

    2012-01-01

    The fidelity of protein biosynthesis requires the aminoacylation of tRNA with its cognate amino acid catalyzed by aminoacyl-tRNA synthetase with high levels of accuracy and efficiency. Crucial bases in tRNALeu to aminoacylation or editing functions of leucyl-tRNA synthetase have been extensively studied mainly by in vitro methods. In the present study, we constructed two Saccharomyces cerevisiae tRNALeu knockout strains carrying deletions of the genes for tRNALeu(GAG) and tRNALeu(UAG). Disrupting the single gene encoding tRNALeu(GAG) had no phenotypic consequence when compared to the wild-type strain. While disrupting the three genes for tRNALeu(UAG) had a lethal effect on the yeast strain, indicating that tRNALeu(UAG) decoding capacity could not be compensated by another tRNALeu isoacceptor. Using the triple tRNA knockout strain and a randomly mutated library of tRNALeu(UAG), a selection to identify critical tRNALeu elements was performed. In this way, mutations inducing in vivo decreases of tRNA levels or aminoacylation or editing ability by leucyl-tRNA synthetase were identified. Overall, the data showed that the triple tRNA knockout strain is a suitable tool for in vivo studies and identification of essential nucleotides of the tRNA. PMID:22917587

  15. trkA modulation of developing somatosensory neurons in oro-facial tissues: tooth pulp fibers are absent in trkA knockout mice.

    PubMed

    Matsuo, S; Ichikawa, H; Henderson, T A; Silos-Santiago, I; Barbacid, M; Arends, J J; Jacquin, M F

    2001-01-01

    To investigate the nerve growth factor requirement of developing oro-facial somatosensory afferents, we have studied the survival of sensory fibers subserving nociception, mechanoreception or proprioception in receptor tyrosine kinase (trkA) knockout mice using immunohistochemistry. trkA receptor null mutant mice lack nerve fibers in tooth pulp, including sympathetic fibers, and showed only sparse innervation of the periodontal ligament. Ruffini endings were formed definitively in the periodontal ligament of the trkA knockout mice, although calcitonin gene-related peptide- and substance P-immunoreactive fibers were reduced in number or had disappeared completely. trkA gene deletion had also no obvious effect on the formation of Meissner corpuscles in the palate. In the vibrissal follicle, however, some mechanoreceptive afferents were sensitive for trkA gene deletion, confirming a previous report [Fundin et al. (1997) Dev. Biol. 190, 94-116]. Moreover, calretinin-positive fibers innervating longitudinal lanceolate endings were completely lost in trkA knockout mice, as were the calretinin-containing parent cells in the trigeminal ganglion.These results indicate that trkA is indispensable for developing nociceptive neurons innervating oral tissues, but not for developing mechanoreceptive neurons innervating oral tissues (Ruffini endings and Meissner corpuscles), and that calretinin-containing, trkA dependent neurons in the trigeminal ganglion normally participate in mechanoreception through longitudinal lanceolate endings of the vibrissal follicle. PMID:11516838

  16. The transforming growth factor-beta 3 knock-out mouse: an animal model for cleft palate.

    PubMed

    Koo, S H; Cunningham, M C; Arabshahi, B; Gruss, J S; Grant, J H

    2001-09-15

    The recent report of a transforming growth factor-beta 3 (TGF-beta 3) knock-out mouse in which 100 percent of the homozygous pups have cleft palate raised the question as to the potential usefulness of these animals as a model for cleft palate research. The specific aim in this study was to carefully document the anatomy of the cleft palate in the TGF-beta 3 knock-out mice as compared with wild type controls. Special attention was paid to the levator veli palatini muscle, the tensor veli palatini muscle, and their respective innervation. Because the TGF-beta 3 knock-out is lethal in the early perinatal period and because the heterozygotes are phenotypically normal, polymerase chain reaction was required to genotype the animals before mating. Time-mated pregnancies between proven heterozygotes were then delivered by cesarean section at gestational day 18.5 to prevent maternal cannibalism of homozygote pups. All delivered pups were killed and their tails processed by polymerase chain reaction to verify genotype. The heads were then fixed and sectioned in axial, coronal, or sagittal planes. Sections were stained with hematoxylin and eosin or processed for immunohistochemistry with nerve specific protein gene product 9.5 and calcitonin gene-related peptide antibodies. Sections were analyzed in a serial fashion. Nine wild type control animals were analyzed along with nine TGF-beta 3 knock-out homozygotes. Time matings between proven heterozygotes yielded wild type pups, heterozygote pups, and homozygote knock-out pups in the expected mendelian ratios (28 percent to 46 percent to 26 percent; n = 43). The results demonstrated 100 percent clefting in the homozygous TGF-beta 3 knock-out pups. Complete clefting of the secondary palate was seen in four of nine and incomplete clefting was seen in five of nine. The levator veli palatini and tensor veli palatini muscles were demonstrated coursing parallel to the cleft margin in all cleft mice. The orientation of these muscles

  17. Completely Humanizing Prolactin Rescues Infertility in Prolactin Knockout Mice and Leads to Human Prolactin Expression in Extrapituitary Mouse Tissues

    PubMed Central

    Christensen, Heather R.; Murawsky, Michael K.; Horseman, Nelson D.; Willson, Tara A.

    2013-01-01

    A variety of fundamental differences have evolved in the physiology of the human and rodent prolactin (PRL) systems. The PRL gene in humans and other primates contains an alternative promoter, 5.8 kbp upstream of the pituitary transcription start site, which drives expression of PRL in “extrapituitary” tissues, where PRL is believed to exert local, or paracrine, actions. Several of these extrapituitary PRL tissues serve a reproductive function (eg, mammary gland, decidua, prostate, etc), consistent with the hypothesis that local PRL production may be involved in, and required for, normal reproductive physiology in primates. Rodent research models have generated significant findings regarding the role of PRL in reproduction. Specifically, disruption (knockout) of either the PRL gene or its receptor causes profound female reproductive defects at several levels (ovaries, preimplantation endometrium, mammary glands). However, the rodent PRL gene differs significantly from the human, most notably lacking the alternative promoter. Understanding of the physiological regulation and function of extrapituitary PRL has been limited by the absence of a readily accessible experimental model, because the rodent PRL gene does not contain the alternative promoter. To overcome these limitations, we have generated mice that have been “humanized” with regard to the structural gene and tissue expression of PRL. Here, we present the characterization of these animals, demonstrating that the human PRL transgene is responsive to known physiological regulators both in vitro and in vivo. More importantly, the expression of the human PRL transgene is able to rescue the reproductive defects observed in mouse PRL knockout (mPRL−) females, validating their usefulness in studying the function or regulation of this hormone in a manner that is relevant to human physiology. PMID:24029242

  18. The transcobalamin receptor knockout mouse: a model for vitamin B12 deficiency in the central nervous system

    PubMed Central

    Lai, Shao-Chiang; Nakayama, Yasumi; Sequeira, Jeffrey M.; Wlodarczyk, Bogdan J.; Cabrera, Robert M.; Finnell, Richard H.; Bottiglieri, Teodoro; Quadros, Edward V.

    2013-01-01

    The membrane receptor (TCblR/CD320) for transcobalamin (TC)-bound cobalamin (Cbl) facilitates the cellular uptake of Cbl. A genetically modified mouse model involving ablation of the CD320 gene was generated to study the effects on cobalamin homeostasis. The nonlethal nature of this knockout and the lack of systemic cobalamin deficiency point to other mechanisms for cellular Cbl uptake in the mouse. However, severe cobalamin depletion in the central nervous system (CNS) after birth (P<0.01) indicates that TCblR is the only receptor responsible for Cbl uptake in the CNS. Metabolic Cbl deficiency in the brain was evident from the increased methylmalonic acid (P<0.01–0.04), homocysteine (P<0.01), cystathionine (P<0.01), and the decreased S-adenosylmethionine/S-adenosyl homocysteine ratio (P<0.01). The CNS pathology of Cbl deficiency seen in humans may not manifest in this mouse model; however, it does provide a model with which to evaluate metabolic pathways and genes affected.—Lai, S.-C., Nakayama, Y., Sequeira, J. M., Wlodarczyk, B. J., Cabrera, R. M., Finnell, R. H., Bottiglieri, T., Quadros, E. V. The transcobalamin receptor knockout mouse: a model for vitamin B12 deficiency in the central nervous system. PMID:23430977

  19. One-step generation of triple knockout CHO cell lines using CRISPR/Cas9 and fluorescent enrichment.

    PubMed

    Grav, Lise Marie; Lee, Jae Seong; Gerling, Signe; Kallehauge, Thomas Beuchert; Hansen, Anders Holmgaard; Kol, Stefan; Lee, Gyun Min; Pedersen, Lasse Ebdrup; Kildegaard, Helene Faustrup

    2015-09-01

    The CRISPR/Cas9 genome editing technology has previously been shown to be a highly efficient tool for generating gene disruptions in CHO cells. In this study we further demonstrate the applicability and efficiency of CRISPR/Cas9 genome editing by disrupting FUT8, BAK and BAX simultaneously in a multiplexing setup in CHO cells. To isolate Cas9-expressing cells from transfected cell pools, GFP was linked to the Cas9 nuclease via a 2A peptide. With this method, the average indel frequencies generated at the three genomic loci were increased from 11% before enrichment to 68% after enrichment. Despite the high number of genome editing events in the enriched cell pools, no significant off-target effects were observed from off-target prediction followed by deep sequencing. Single cell sorting of enriched multiplexed cells and deep sequencing of 97 clones revealed the presence of four single, 23 double and 34 triple gene-disrupted cell lines. Further characterization of selected potential triple knockout clones confirmed the removal of Bak and Bax protein and disrupted fucosylation activity as expected. The knockout cell lines showed improved resistance to apoptosis compared to wild-type CHO-S cells. Taken together, multiplexing with CRISPR/Cas9 can accelerate genome engineering efforts in CHO cells even further. PMID:25864574

  20. Increased fronto-hippocampal connectivity in the Prrxl1 knockout mouse model of congenital hypoalgesia.

    PubMed

    Monteiro, Clara; Cardoso-Cruz, Helder; Matos, Mariana; Dourado, Margarida; Lima, Deolinda; Galhardo, Vasco

    2016-09-01

    Despite the large number of studies addressing how prolonged painful stimulation affects brain functioning, there are only a handful of studies aimed at uncovering if persistent conditions of reduced pain perception would also result in brain plasticity. Permanent hypoalgesia induced by neonatal injection of capsaicin or carrageenan has already been shown to affect learning and memory and to induce alterations in brain gene expression. In this study, we used the Prrxl1 model of congenital mild hypoalgesia to conduct a detailed study of the neurophysiological and behavioral consequences of reduced pain experience. Prrxl1 knockout animals are characterized by selective depletion of small diameter primary afferents and abnormal development of the superficial dorsal laminae of the spinal cord, resulting in diminished pain perception but normal tactile and motor behaviour. Behavioral testing of Prrxl1 mice revealed that these animals have reduced anxiety levels, enhanced memory performance, and improved fear extinction. Neurophysiological recordings from awake behaving Prrxl1 mice show enhanced altered fronto-hippocampal connectivity in the theta- and gamma-bands. Importantly, although inflammatory pain by Complete Freund Adjuvant injection caused a decrease in fronto-hippocampal connectivity in the wild-type animals, Prrxl1 mice maintained the baseline levels. The onset of inflammatory pain also reverted the differences in forebrain expression of stress- and monoamine-related genes in Prrxl1 mice. Altogether our results suggest that congenital hypoalgesia may have an effect on brain plasticity that is the inverse of what is usually observed in animal models of chronic pain. PMID:27168359

  1. Dynamin triple knockout cells reveal off target effects of commonly used dynamin inhibitors

    PubMed Central

    Park, Ryan J.; Shen, Hongying; Liu, Lijuan; Liu, Xinran; Ferguson, Shawn M.; De Camilli, Pietro

    2013-01-01

    Summary Dynamin, which is encoded by three genes in mammals, is a GTPase implicated in endocytic membrane fission. Dynamin 1 and 3 are predominantly expressed in brain, whereas dynamin 2 is ubiquitously expressed. With the goal of assessing the impact of the lack of dynamin on cell physiology, we previously generated and characterized dynamin 1 and 2 double knockout (DKO) fibroblasts. These DKO cells were unexpectedly viable in spite of a severe impairment of clathrin-mediated endocytosis. As low-level expression of the dynamin 3 gene in these cells could not be excluded, we have now engineered dynamin 1, 2 and 3 triple KO (TKO) fibroblasts. These cells did not reveal any additional defects beyond what was previously observed in DKO fibroblasts. Surprisingly, although fluid-phase endocytosis and peripheral membrane ruffling were not impaired by the lack of all three dynamins, two structurally similar, widely used dynamin inhibitors, dynasore and Dyngo-4a, robustly inhibited these two processes both in wild-type and TKO cells. Dynamin TKO cells will be useful tools for the further exploration of dynamin-dependent processes and the development of more specific dynamin inhibitors. PMID:24046449

  2. Resistance of R-Ras knockout mice to skin tumour induction

    PubMed Central

    May, Ulrike; Prince, Stuart; Vähätupa, Maria; Laitinen, Anni M.; Nieminen, Katriina; Uusitalo-Järvinen, Hannele; Järvinen, Tero A. H.

    2015-01-01

    The R-ras gene encodes a small GTPase that is a member of the Ras family. Despite close sequence similarities, R-Ras is functionally distinct from the prototypic Ras proteins; no transformative activity and no activating mutations of R-Ras in human malignancies have been reported for it. R-Ras activity appears inhibitory towards tumour proliferation and invasion, and to promote cellular quiescence. Contrary to this, using mice with a deletion of the R-ras gene, we found that R-Ras facilitates DMBA/TPA-induced skin tumour induction. The tumours appeared in wild-type (WT) mice on average 6 weeks earlier than in R-Ras knockout (R-Ras KO) mice. WT mice developed almost 6 times more tumours than R-Ras KO mice. Despite strong R-Ras protein expression in the dermal blood vessels, no R-Ras could be detected in the epidermis from where the tumours arose. The DMBA/TPA skin tumourigenesis-model is highly dependent upon inflammation, and we found a greatly attenuated skin inflammatory response to DMBA/TPA-treatment in the R-Ras KO mice in the context of leukocyte infiltration and proinflammatory cytokine expression. Thus, these data suggest that despite its characterised role in promoting cellular quiescence, R-Ras is pro-tumourigenic in the DMBA/TPA tumour model and important for the inflammatory response to DMBA/TPA treatment. PMID:26133397

  3. Ethanol self-administration in serotonin transporter knockout mice: unconstrained demand and elasticity.

    PubMed

    Lamb, R J; Daws, L C

    2013-10-01

    Low serotonin function is associated with alcoholism, leading to speculation that increasing serotonin function could decrease ethanol consumption. Mice with one or two deletions of the serotonin transporter (SERT) gene have increased extracellular serotonin. To examine the relationship between SERT genotype and motivation for alcohol, we compared ethanol self-administration in mice with zero (knockout, KO), one (HET) or two copies (WT) of the SERT gene. All three genotypes learned to self-administer ethanol. The SSRI, fluvoxamine, decreased responding for ethanol in the HET and WT, but not the KO mice. When tested under a progressive ratio schedule, KO mice had lower breakpoints than HET or WT. As work requirements were increased across sessions, behavioral economic analysis of ethanol self-administration indicated that the decreased breakpoint in KO as compared to HET or WT mice was a result of lower levels of unconstrained demand, rather than differences in elasticity, i.e. the proportional decreases in ethanol earned with increasing work requirements were similar across genotypes. The difference in unconstrained demand was unlikely to result from motor or general motivational factors, as both WT and KO mice responded at high levels for a 50% condensed milk solution. As elasticity is hypothesized to measure essential value, these results indicate that KO value ethanol similarly to WT or HET mice despite having lower break points for ethanol. PMID:23927813

  4. 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

  5. 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. PMID

  6. 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 Central

    Spencer, Jacqueline F.; Tollefson, Ann E.; Sagartz, John E.; Kong, Il-Keun; Wang, Zhongde; Wold, William S. M.

    2015-01-01

    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. PMID

  7. Drop tests of the Three Mile Island knockout canister

    SciTech Connect

    Box, W D; Aaron, W S; Shappert, L B; Childress, P C; Quinn, G J; Smith, J V

    1987-01-01

    A type of Three Mile Island Unit 2 (TMI-2) defueling canister, called a ''knockout'' canister, was subjected to a series of drop tests at the Oak Ridge National Laboratory's Drop Test Facility. These tests confirmed the structural integrity of internal fixed neutron poisons in support of a request for NRC licensing of this type of canister for the shipment of TMI-2 reactor fuel debris to the Idaho National Engineering Laboratory (INEL) for the Core Examination R and D Program. This report presents the data generated and the results obtained from a series of four drop tests that included two drops with the test assembly in the vertical position and two drops with the assembly in the horizontal position.

  8. 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

  9. Nonsudden limits of heavy-ion induced knockout reactions.

    PubMed

    Flavigny, F; Obertelli, A; Bonaccorso, A; Grinyer, G F; Louchart, C; Nalpas, L; Signoracci, A

    2012-06-22

    We report on the single neutron and proton removal reactions from unstable nuclei with large asymmetry ΔS = S(n)-S(p) at incident energies below 80 MeV/nucleon. Strong nonsudden effects are observed in the case of deeply-bound-nucleon removal. The corresponding parallel momentum distributions exhibit an abrupt cutoff at high momentum that corresponds to an energy threshold occurring when the incident energy per particle is of comparable magnitude to the nucleon separation energy. A large low-momentum tail is related to both dissipative processes and the dynamics of the nucleon removal process. New limits for the applicability of the sudden and eikonal approximations in nucleon knockout are given. PMID:23004591

  10. Spectroscopy of 17C via one-neutron knockout reaction

    NASA Astrophysics Data System (ADS)

    Kim, Sunji; Hwang, Jongwon; Satou, Yoshiteru; Orr, Nigel A.; Nakamura, Takashi; Kondo, Yosuke; Gibelin, Julien; Achouri, N. Lynda; Aumann, Thomas; Baba, Hidetada; Delaunay, Franck; Doornenbal, Pieter; Fukuda, Naoki; Inabe, Naohito; Isobe, Tadaaki; Kameda, Daisuke; Kanno, Daiki; Kobayashi, Nobuyuki; Kobayashi, Toshio; Kubo, Toshiyuki; Leblond, Sylvain; Lee, Jenny; Marqués, F. Miguel; Minakata, Ryogo; Motobayashi, Tohru; Murai, Daichi; Murakami, Tetsuya; Muto, Kotomi; Nakashima, Tomohiro; Nakatsuka, Noritsugu; Navin, Alahari; Nishi, Seijiro; Ogoshi, Shun; Otsu, Hideaki; Sato, Hiromi; Shimizu, Yohei; Suzuki, Hiroshi; Takahashi, Kento; Takeda, Hiroyuki; Takeuchi, Satoshi; Tanaka, Ryuki; Togano, Yasuhiro; Tuff, Adam G.; Vandebrouck, Marine; Yoneda, Ken-ichiro

    2016-03-01

    A spectroscopic study of 17C was performed via the one-neutron knockout reaction of 18C on a carbon target at RIKEN-RIBF. Three unbound states at excitation energies of 2.66(2), 3.16(5), and 3.97(3) MeV (preliminary) were observed. The energies are compared with shell-model calculations and existing measurements to deduce their spin-parities. From the comparison, the states at 2.66(2) and 3.97(3) MeV are suggested to be 1/2- and 3/2-, respectively. From its decay property, the state at 3.16(5) MeV is indicated to be 9/2+.

  11. 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.

  12. Fission product release from nuclear fuel by recoil and knockout

    NASA Astrophysics Data System (ADS)

    Lewis, B. J.

    1987-03-01

    An analytical model has been developed to describe the fission product release from nuclear fuel arising from the surface-fission release mechanisms of recoil and knockout. Release expressions are evaluated and compared to the short-lived activity measurements from in-reactor experiments with intact operating fuel. Recoil is shown to be an important process for releasing fission products from free UO 2 surfaces into the fuel-to-sheath gap. The model is also applied to tramp uranium in a power reactor primary heat transport circuit where it is demonstrated that recoil is the dominant release mechanism for small particles of fuel which are deposited on in-core surfaces. A methodology is established whereby release from surface contamination can be distinguished from that of fuel pin failure.

  13. Bioelectric characterization of epithelia from neonatal CFTR knockout ferrets.

    PubMed

    Fisher, John T; Tyler, Scott R; Zhang, Yulong; Lee, Ben J; Liu, Xiaoming; Sun, Xingshen; Sui, Hongshu; Liang, Bo; Luo, Meihui; Xie, Weiliang; Yi, Yaling; Zhou, Weihong; Song, Yi; Keiser, Nicholas; Wang, Kai; de Jonge, Hugo R; Engelhardt, John F

    2013-11-01

    Cystic fibrosis (CF) is a life-shortening, recessive, multiorgan genetic disorder caused by the loss of CF transmembrane conductance regulator (CFTR) chloride channel function found in many types of epithelia. Animal models that recapitulate the human disease phenotype are critical to understanding pathophysiology in CF and developing therapies. CFTR knockout ferrets manifest many of the phenotypes observed in the human disease, including lung infections, pancreatic disease and diabetes, liver disease, malnutrition, and meconium ileus. In the present study, we have characterized abnormalities in the bioelectric properties of the trachea, stomach, intestine, and gallbladder of newborn CF ferrets. Short-circuit current (ISC) analysis of CF and wild-type (WT) tracheas revealed the following similarities and differences: (1) amiloride-sensitive sodium currents were similar between genotypes; (2) responses to 4,4'-diisothiocyano-2,2'-stilbene disulphonic acid were 3.3-fold greater in CF animals, suggesting elevated baseline chloride transport through non-CFTR channels in a subset of CF animals; and (3) a lack of 3-isobutyl-1-methylxanthine (IBMX)/forskolin-stimulated and N-(2-Naphthalenyl)-((3,5-dibromo-2,4-dihydroxyphenyl)methylene)glycine hydrazide (GlyH-101)-inhibited currents in CF animals due to the lack of CFTR. CFTR mRNA was present throughout all levels of the WT ferret and IBMX/forskolin-inducible ISC was only observed in WT animals. However, despite the lack of CFTR function in the knockout ferret, the luminal pH of the CF ferret gallbladder, stomach, and intestines was not significantly changed relative to WT. The WT stomach and gallbladder exhibited significantly enhanced IBMX/forskolin ISC responses and inhibition by GlyH-101 relative to CF samples. These findings demonstrate that multiple organs affected by disease in the CF ferret have bioelectric abnormalities consistent with the lack of cAMP-mediated chloride transport. PMID:23782101

  14. 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 manif