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A. John Clark

Bio: A. John Clark is an academic researcher from The Roslin Institute. The author has contributed to research in topics: Transgene & Embryonic stem cell. The author has an hindex of 15, co-authored 31 publications receiving 1464 citations. Previous affiliations of A. John Clark include Western Infirmary & University of Edinburgh.

Papers
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Journal ArticleDOI
TL;DR: It is reported here that mice lacking heart‐type FABP (H‐FABP) exhibit a severe defect of peripheral (non‐hepatic, non‐fat) LCFA utilization, establishing a requirement for H‐FabP in cardiac intracellular lipid transport and fuel selection and a major role in metabolic homeostasis.
Abstract: Nonenzymatic cytosolic fatty acid binding proteins (FABPs) are abundantly expressed in many animal tissues with high rates of fatty acid metabolism. No physiological role has been demonstrated for any FABP, although these proteins have been implicated in transport of free long-chain fatty acids (LCFAs) and protection against LCFA toxicity. We report here that mice lacking heart-type FABP (H-FABP) exhibit a severe defect of peripheral (nonhepatic, non-fat) LCFA utilization. In these mice, the heart is unable to efficiently take up plasma LCFAs, which are normally its main fuel, and switches to glucose usage. Altered plasma levels of LCFAs, glucose, lactate and beta-hydroxybutyrate are consistent with depressed peripheral LCFA utilization, intensified carbohydrate usage, and increased hepatic LCFA oxidation; these changes are most pronounced under conditions favoring LCFA oxidation. H-FABP deficiency is only incompletely compensated, however, causing acute exercise intolerance and, at old age, a localized cardiac hypertrophy. These data establish a requirement for H-FABP in cardiac intracellular lipid transport and fuel selection and a major role in metabolic homeostasis. This new animal model should be particularly useful for investigating the significance of peripheral LCFA utilization for heart function, insulin sensitivity, and blood pressure.

292 citations

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TL;DR: It is concluded that hES cell nuclei have a distinct nuclear architecture, especially at loci involved in maintaining pluripotency, which provides a framework within which other large-scale chromatin changes that may accompany differentiation can be considered.
Abstract: Nuclear organisation is thought to be important in regulating gene expression. Here we investigate whether human embryonic stem cells (hES) have a particular nuclear organisation, which could be important for maintaining their pluripotent state. We found that whereas the nuclei of hES cells have a general gene-density-related radial organisation of chromosomes, as is seen in differentiated cells, there are also distinctive localisations for chromosome regions and gene loci with a role in pluripotency. Chromosome 12p, a region of the human genome that contains clustered pluripotency genes including NANOG, has a more central nuclear localisation in ES cells than in differentiated cells. On chromosome 6p we find no overall change in nuclear chromosome position, but instead we detect a relocalisation of the OCT4 locus, to a position outside its chromosome territory. There is also a smaller proportion of centromeres located close to the nuclear periphery in hES cells compared to differentiated cells. We conclude that hES cell nuclei have a distinct nuclear architecture, especially at loci involved in maintaining pluripotency. Understanding this level of hES cell biology provides a framework within which other large-scale chromatin changes that may accompany differentiation can be considered.

170 citations

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TL;DR: Five founder transgenic sheep described carry genes designed to direct the production of human clotting factor IX or human αl–antitrypsin in milk and may ultimately provide a new source of these and other therapeutic proteins.
Abstract: Gene transfer into animals has considerable potential for livestock improvement. If this potential is to be realized, the ease of generation of transgenic livestock will be of major importance. We report here the production of six transgenic sheep by microinjection of DNA into early embryos (1.2% of embryos transferred). Three different gene constructs were injected and transgenic sheep were obtained with each. The transgenic animals have all incorporated the DNA without detectable rearrangement, and where multiple copies were integrated, they are present in arrays of tandem repeats. Transmission of transferred genes to progeny of three of the sheep has been demonstrated. Five founder transgenic sheep described carry genes designed to direct the production of human clotting factor IX or human αl–antitrypsin in milk. Transgenic animals carrying such genes may ultimately provide a new source of these and other therapeutic proteins.

153 citations

Journal ArticleDOI
TL;DR: These OCT4‐EGFP clonal cell lines exhibit features similar to parental hESCs, are pluripotent, and are able to produce all three embryonic germ layer cells, and they will be invaluable for studying not only OCT4 function in hESC self‐renewal and differentiation but also the factors required for maintenance of undifferentiated h ESCs in culture.
Abstract: Human embryonic stem cells (hESCs) are derived from the inner cell mass of preimplantation embryos; they can be cultured indefinitely and differentiated into many cell types in vitro. These cells therefore have the ability to provide insights into human disease and provide a potential unlimited supply of cells for cell-based therapy. Little is known about the factors that are important for maintaining undifferentiated hESCs in vitro, however. As a tool to investigate these factors, transfected hES clonal cell lines were generated; these lines are able to express the enhanced green fluorescent protein (EGFP) reporter gene under control of the OCT4 promoter. OCT4 is an important marker of the undifferentiated state and a central regulator of pluripotency in ES cells. These OCT4-EGFP clonal cell lines exhibit features similar to parental hESCs, are pluripotent, and are able to produce all three embryonic germ layer cells. Expression of OCT4-EGFP is colocalized with endogenous OCT4, as well as the hESC surface antigens SSEA4 and Tra-1-60. In addition, the expression is retained in culture for an extensive period of time. Differentiation of these cells toward the neural lineage and targeted knockdown of endogenous OCT4 expression by RNA interference downregulated the EGFP expression in these cell lines, and this correlates closely with the reduction of endogenous OCT4 expression. Therefore, these cell lines provide an easy and noninvasive method to monitor expression of OCT4 in hESCs, and they will be invaluable for studying not only OCT4 function in hESC self-renewal and differentiation but also the factors required for maintenance of undifferentiated hESCs in culture.

152 citations

Journal ArticleDOI
TL;DR: Efficient generation of transgenic pigs using an equine infectious anaemia virus derived vector is demonstrated, indicating that lentiviral transgene delivery may be a general tool with which to efficiently generate transgenic mammals.

143 citations


Cited by
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Journal ArticleDOI
12 Jun 2008-Nature
TL;DR: A high-resolution map of the interaction sites of the entire genome with NL components in human fibroblasts is constructed and demonstrates that the human genome is divided into large, discrete domains that are units of chromosome organization within the nucleus.
Abstract: The architecture of human chromosomes in interphase nuclei is still largely unknown. Microscopy studies have indicated that specific regions of chromosomes are located in close proximity to the nuclear lamina (NL). This has led to the idea that certain genomic elements may be attached to the NL, which may contribute to the spatial organization of chromosomes inside the nucleus. However, sequences in the human genome that interact with the NL in vivo have not been identified. Here we construct a high-resolution map of the interaction sites of the entire genome with NL components in human fibroblasts. This map shows that genome-lamina interactions occur through more than 1,300 sharply defined large domains 0.1-10 megabases in size. These lamina-associated domains (LADs) are typified by low gene-expression levels, indicating that LADs represent a repressive chromatin environment. The borders of LADs are demarcated by the insulator protein CTCF, by promoters that are oriented away from LADs, or by CpG islands, suggesting possible mechanisms of LAD confinement. Taken together, these results demonstrate that the human genome is divided into large, discrete domains that are units of chromosome organization within the nucleus.

1,762 citations

Journal ArticleDOI
TL;DR: The central role of lipid chaperones — the fatty acid-binding proteins (FABPs) — in lipid-mediated biological processes and systemic metabolic homeostasis through the regulation of diverse lipid signals is discussed, and their therapeutic significance is highlighted.
Abstract: Lipids are vital components of many biological processes and crucial in the pathogenesis of numerous common diseases, but the specific mechanisms coupling intracellular lipids to biological targets and signalling pathways are not well understood. This is particularly the case for cells burdened with high lipid storage, trafficking and signalling capacity such as adipocytes and macrophages. Here, we discuss the central role of lipid chaperones — the fatty acid-binding proteins (FABPs) — in lipid-mediated biological processes and systemic metabolic homeostasis through the regulation of diverse lipid signals, and highlight their therapeutic significance. Pharmacological agents that modify FABP function may provide tissue-specific or cell-type-specific control of lipid signalling pathways, inflammatory responses and metabolic regulation, potentially providing a new class of drugs for diseases such as obesity, diabetes and atherosclerosis.

1,320 citations

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TL;DR: It is suggested that hyperdynamic binding of structural chromatin proteins is a functionally important hallmark of pluripotent ES cells that contributes to the maintenance of plasticity in undifferentiated ES cells and to establishing higher-order chromatin structure.

1,003 citations

Journal ArticleDOI
TL;DR: This work shows that allelic loss of beclin1 and defective autophagy sensitized mammary epithelial cells to metabolic stress and accelerated lumen formation in mammary acini and proposed that autophagic limits metabolic stress to protect the genome.
Abstract: Autophagy is a catabolic process involving self-digestion of cellular organelles during starvation as a means of cell survival; however, if it proceeds to completion, autophagy can lead to cell death. Autophagy is also a haploinsufficient tumor suppressor mechanism for mammary tumorigenesis, as the essential autophagy regulator beclin1 is monoallelically deleted in breast carcinomas. However, the mechanism by which autophagy suppresses breast cancer remains elusive. Here we show that allelic loss of beclin1 and defective autophagy sensitized mammary epithelial cells to metabolic stress and accelerated lumen formation in mammary acini. Autophagy defects also activated the DNA damage response in vitro and in mammary tumors in vivo, promoted gene amplification, and synergized with defective apoptosis to promote mammary tumorigenesis. Therefore, we propose that autophagy limits metabolic stress to protect the genome, and that defective autophagy increases DNA damage and genomic instability that ultimately facilitate breast cancer progression.

800 citations

Journal ArticleDOI
TL;DR: This work discusses how unique properties of chromatin in ES cells contribute to the maintenance of pluripotency and the determination of differentiation properties.
Abstract: What makes a stem cell is still poorly understood. Recent studies have uncovered that chromatin might hold some of the keys to how embryonic stem cells maintain their pluripotency, their ability to self-renew and induce lineage specification. Embryonic stem (ES) cells are unique in that they are pluripotent and have the ability to self-renew. The molecular mechanisms that underlie these two fundamental properties are largely unknown. We discuss how unique properties of chromatin in ES cells contribute to the maintenance of pluripotency and the determination of differentiation properties.

707 citations