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    ABSTRACT: Electron microscopy image of ependymal cells of the telencephalic ChP. From Failure of centrosome migration causes a loss of motile cilia in talpid3 mutants, Louise A. Stephen, Gemma M. Davis, Katie E. McTeir, John James, Lynn McTeir, Martin Kierans, Andrew Bain, Megan G. Davey, Developmental Dynamics 242:923-931.
    Developmental Dynamics 08/2013; 242(8):C1. DOI:10.1002/dvdy.24012
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    ABSTRACT: Genetically engineered (GE) livestock have existed since the mid-1980s, and, since then, a range of methods for delivery of the transgene have been developed, each with advantages and limitations. In regards to the wealth of possible methods for the production of GE animals, two general approaches have emerged. Historically first, the direct manipulation of the zygote (including manipulation of germ cells) now comes in many flavours, from direct pronuclear injection of double-stranded DNA constructs to the use of vectors to deliver the transgene. The second approach utilises an in vitro stage where cells are engineered in culture before being introduced in some way to the developing embryo. The former suffers from lack of control of transgene integration, which can expose the transgene to position effects. The latter can be exploited through homologous recombination to engineer specific genetic loci; with somatic cell cloning being the most widely used method. Both approaches can now be combined with the exciting new editor technologies to enable precise genome editing which in some cases does not involve the incorporation of a transgene. For methods involving the zygote, the use of specific vectors can be of advantage, and the same can be true for the manipulation of cells; however, many delivery strategies are possible for this process. Overall, the drivers for delivery method development have revolved around efficiency and specificity. With regard to viral vectors, and possibly nonviral nanoparticle formulations in the future, spectacular increases in transgenesis rates can be achieved. With the most widely used vector, based on a lentivirus genome, in some cases all animals born from injected zygotes can be transgenic. In livestock, where gestation and breeding times are long, this dramatically reduces the time to proof-of-concept for a given project. In addition, these founder animals will carry different transgene copy-numbers, which is associated with different levels of transgene expression. This strategy can be exploited to quickly produce a large cohort of animals that enable modelling of the range of phenotype observed in a population for a given disease. In addition to the delivery of a transgene, such vectors can also be beneficial for the delivery of reagents that facilitate genome engineering, the most exciting of which are the genome editors. In this situation, either the editor and/or any DNA sequence to be incorporated can be efficiently delivered if not essential for broad uptake of this approach. It is likely that nonintegrating vectors will be desirable. In summary, viral vectors have a broad utility in facilitating the production of GE animals. In the future, nonviral nanoparticles may offer similar opportunities. Given the breadth of methodologies available and with the anticipated use of GE livestock in both agricultural and biomedical applications gaining momentum, we are entering an era of unparalleled opportunity in this area of animal biotechnology.
    Reproduction Fertility and Development 12/2012; 25(1):317. DOI:10.1071/RDv25n1Ab338
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    ABSTRACT: Linear bone growth is widely recognized to be adversely affected in children with chronic kidney disease (CKD) and other chronic inflammatory disorders. The growth hormone (GH)/insulin-like growth factor-1 (IGF-1) pathway is anabolic to the skeleton and inflammatory cytokines compromise bone growth through a number of different mechanisms, which include interference with the systemic as well as the tissue-level GH/IGF-1 axis. Despite attempts to promote growth and control disease, there are an increasing number of reports of the persistence of poor growth in a substantial proportion of patients receiving rhGH and/or drugs that block cytokine action. Thus, there is an urgent need to consider better and alternative forms of therapy that are directed specifically at the mechanism of the insult which leads to abnormal bone health. Suppressor of cytokine signaling 2 (SOCS2) expression is increased in inflammatory conditions including CKD, and is a recognized inhibitor of GH signaling. Therefore, in this review, we will focus on the premise that SOCS2 signaling represents a critical pathway in growth plate chondrocytes through which pro-inflammatory cytokines alter both GH/IGF-1 signaling and cellular function.
    Pediatric Nephrology 08/2012; 28(4). DOI:10.1007/s00467-012-2271-0
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    ABSTRACT: Little is known about the involvement of microRNAs (miRNAs) in the follicular-luteal transition. The aim of this study was to identify genome-wide changes in miRNAs associated with follicular differentiation in sheep. miRNA libraries were produced from samples collected at defined stages of the ovine oestrous cycle and representing healthy growing follicles, (diameter, 4.0-5.5  mm), pre-ovulatory follicles (6.0-7.0  mm), early corpora lutea (day 3 post-oestrus) and late corpora lutea (day 9). A total of 189 miRNAs reported in sheep or other species and an additional 23 novel miRNAs were identified by sequencing these libraries. miR-21, miR-125b, let-7a and let-7b were the most abundant miRNAs overall, accounting for 40% of all miRNAs sequenced. Examination of changes in cloning frequencies across development identified nine different miRNAs whose expression decreased in association with the follicular-luteal transition and eight miRNAs whose expression increased during this transition. Expression profiles were confirmed by northern analyses, and experimentally validated targets were identified using miRTarBase. A majority of the 29 targets identified represented genes known to be actively involved in regulating follicular differentiation in vivo. Finally, luteinisation of follicular cells in vitro resulted in changes in miRNA levels that were consistent with those identified in vivo, and these changes were temporally associated with changes in the levels of putative miRNA targets in granulosa cells. In conclusion, this is the first study to characterise genome-wide miRNA profiles during different stages of follicle and luteal development. Our data identify a subset of miRNAs that are potentially important regulators of the follicular-luteal transition.
    Reproduction 05/2012; 144(2):221-33. DOI:10.1530/REP-12-0025
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    ABSTRACT: The derivation of germ-line competent avian primordial germ cells establishes a cell-based model system for the investigation of germ cell differentiation and the production of genetically modified animals. Current methods to modify primordial germ cells using DNA or retroviral vectors are inefficient and prone to epigenetic silencing. Here, we validate the use of transposable elements for the genetic manipulation of primordial germ cells. We demonstrate that chicken primordial germ cells can be modified in vitro using transposable elements. Both piggyBac and Tol2 transposons efficiently transpose primordial germ cells. Tol2 transposon integration sites were spread throughout both the macro- and microchromosomes of the chicken genome and were more prevalent in gene transcriptional units and intronic regions, consistent with transposon integrations observed in other species. We determined that the presence of insulator elements was not required for reporter gene expression from the integrated transposon. We further demonstrate that a gene-trap cassette carried in the Tol2 transposon can trap and mutate endogenous transcripts in primordial germ cells. Finally, we observed that modified primordial germ cells form functional gametes as demonstrated by the generation of transgenic offspring that correctly expressed a reporter gene carried in the transposon. Transposable elements are therefore efficient vectors for the genetic manipulation of primordial germ cells and the chicken genome.
    Proceedings of the National Academy of Sciences 05/2012; 109(23):E1466-72. DOI:10.1073/pnas.1118715109
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    ABSTRACT: Suppressor of Cytokine Signaling-2 (SOCS2) is a negative regulator of growth hormone (GH) signaling and bone growth via inhibition of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway. This has been classically demonstrated by the overgrowth phenotype of SOCS2(-/-) mice, which has normal systemic insulin-like growth factor 1 (IGF-1) levels. The local effects of GH on bone growth are equivocal, and therefore this study aimed to understand better the SOCS2 signaling mechanisms mediating the local actions of GH on epiphyseal chondrocytes and bone growth. SOCS2, in contrast to SOCS1 and SOCS3 expression, was increased in cultured chondrocytes after GH challenge. Gain- and loss-of-function studies indicated that GH-stimulated chondrocyte STATs-1, -3, and -5 phosphorylation was increased in SOCS2(-/-) chondrocytes but not in cells overexpressing SOCS2. This increased chondrocyte STAT signaling in the absence of SOCS2 is likely to explain the observed GH stimulation of longitudinal growth of cultured SOCS2(-/-) embryonic metatarsals and the proliferation of chondrocytes within. Consistent with this metatarsal data, bone growth rates, growth plate widths, and chondrocyte proliferation were all increased in SOCS2(-/-) 6-week-old mice as was the number of phosphorylated STAT-5-positive hypertrophic chondrocytes. The SOCS2(-/-) mouse represents a valid model for studying the local effects of GH on bone growth.
    Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 05/2012; 27(5):1055-66. DOI:10.1002/jbmr.1544
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    ABSTRACT: For the majority of animals, males and females are obviously different in terms of appearance, behaviour and physiology, and until recently, these differences were considered to be the result of hormone actions. However, there is now considerable evidence that the development of some sexually dimorphic structures/behaviours is a function of properties inherent to male and female cells (hormone independent). The relative contribution of hormones and cellular identity to the development of the phenotype is not clear and is likely to vary from species to species. The study of gynandromorph birds and chimeric embryos has greatly assisted efforts to distinguish between the effects of hormones and inherent cellular factors on phenotype. It is now clear that in birds, male/female differences are not primarily the result of hormone action and that male and female somatic cells possess a cell autonomous sex identity (CASI). Here, we review evidence for CASI in birds and discuss the implications for the process of sex determination.
    Chromosome Research 11/2011; 20(1):177-90. DOI:10.1007/s10577-011-9257-9
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    ABSTRACT: Vascular calcification has severe clinical consequences and is considered an accurate predictor of future adverse cardiovascular events. Vascular calcification refers to the deposition of calcium phosphate mineral, most often hydroxyapatite, in arteries. Extensive calcification of the vascular system is a key characteristic of aging. In this article, we outline the mechanisms governing vascular calcification and highlight its association with cellular senescence. This review discusses the molecular mechanisms of cellular senescence and its affect on calcification of vascular cells, the relevance of phosphate regulation and the function of FGF23 and Klotho proteins. The association of vascular calcification and cellular senescence with the rare human aging disorder Hutchison-Gilford Progeria Syndrome (HGPS) is highlighted and the mouse models used to try to determine the underlying pathways are discussed. By understanding the pathways involved in these processes novel drug targets may be elucidated in an effort to reduce the effects of cellular aging as a risk factor in cardiovascular disease.
    Current Aging Science 07/2011; 4(2):128-36. DOI:10.2174/1874609811104020128
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    ABSTRACT: We use both large and small animal models in our pre-clinical evaluation of gene transfer agents (GTAs) for cystic fibrosis (CF) gene therapy. Here, we report the use of a large animal model to assess three non-viral GTAs: 25 kDa-branched polyethyleneimine (PEI), the cationic liposome (GL67A) and compacted DNA nanoparticle formulated with polyethylene glycol-substituted lysine 30-mer. GTAs complexed with plasmids expressing human cystic fibrosis transmembrane conductance regulator (CFTR) complementary DNA were administered to the sheep lung (n = 8 per group) by aerosol. All GTAs gave evidence of gene transfer and expression 1 day after treatment. Vector-derived mRNA was expressed in lung tissues, including epithelial cell-enriched bronchial brushing samples, with median group values reaching 1-10% of endogenous CFTR mRNA levels. GL67A gave the highest levels of expression. Human CFTR protein was detected in small airway epithelial cells in some animals treated with GL67A (two out of eight) and PEI (one out of eight). Bronchoalveolar lavage neutrophilia, lung histology and elevated serum haptoglobin levels indicated that gene delivery was associated with mild local and systemic inflammation. Our conclusion was that GL67A was the best non-viral GTA currently available for aerosol delivery to the sheep lung, led to the selection of GL67A as our lead GTA for clinical trials in CF patients. Gene Therapy (2011) 18, 996-1005; doi:10.1038/gt.2011.55; published online 21 April 2011
    Gene therapy 04/2011; 18(10):996-1005. DOI:10.1038/gt.2011.55
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    ABSTRACT: Transgenic technologies can provide important animal models for studying drug-metabolizing enzymes. Our overall aim was to generate versatile cell and animal systems that exhibited varying levels of cytochrome P450 oxidoreductase (POR) activity, more accurately modelling the human population for pharmacological and toxicology studies. Towards this goal we evaluated RNA-interference constructs designed for use in vitro and in vivo for reducing POR activity in hepatocytes. This study clearly demonstrates that both POR protein level and reductase activity can be significantly knocked down in Hepa-1 cells in vitro, while highlighting the difficulty in predicting knockdown efficiency in transgenic animals. The high levels of embryonic lethality observed, and inability to produce multi-copy transgenic animals indicates that high levels of shRNA expression may be detrimental to embryonic development.
    11/2010; 6(2):416-21.
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