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  • 1
    Keywords: Life sciences ; Cell Biology ; Life sciences ; Cell Biology ; Springer eBooks
    Description / Table of Contents: Isolation, Proteomic Analysis and Microscopy Confirmation of the Liver Nuclear Envelope Proteome -- Exploring the Protein Composition of the Plant Nuclear Envelope -- High Efficiency Isolation of Nuclear Envelope Protein Complexes from Trypanosomes -- Super Resolution Microscopy of the Nuclear Envelope and Associated Proteins -- Analyses of the Dynamic Properties of Nuclear Lamins by Fluorescence Recovery after Photobleaching (FRAP) and Fluorescence Correlation Spectroscopy (FCS) -- Probing Protein Distribution Along the Nuclear Envelope In Vivo by Using Single-Point FRAP -- The Use of 2-Photon FLIM/FRET to Study Protein Interactions during Nuclear Envelope Fusion In Vivo and In Vitro -- Identifying Protein-Protein Associations at the Nuclear Envelope with BioID -- In Situ Detection of Interactions between Nuclear Envelope Proteins and Partners -- Methods for Single-Cell Pulse-Chase Analysis of Nuclear Components -- Analysis of Nuclear Lamina Proteins in Myoblast Differentiation by Functional Complementation -- Analysis of Meiotic Telomere Behavior in the Mouse -- Identification and Validation of Putative Nesprin Variants -- Detection of Diverse and High Molecular Weight Nesprin-1 and Nesprin-2 Isoforms Using Western Blotting -- The Use of Polyacrylamide Hydrogels to Study the Effects of Matrix Stiffness on Nuclear Envelope Properties -- Cell Microharpooning to Study Nucleo-Cytoskeletal Coupling -- Wound Healing Assays to Study Mechanisms of Nuclear Movement in Fibroblasts and Myoblasts -- Methods for Assessing Nuclear Rotation and Nuclear Positioning in Developing Skeletal Muscle Cells -- Imaging Approaches to Investigate Myonuclear Positioning in Drosophila -- Mapping Nuclear Lamin-Genome Interactions by Chromatin Immunoprecipitation of Nuclear Lamins -- Lamin ChIP from Chromatin Prepared by Micrococcal Nuclease Digestion -- DamID Analysis of Nuclear Organization in Caenorhabditis elegans -- The Application of DamID to Identify Peripheral Gene Sequences in Differentiated and Primary Cells -- Visualizing the Spatial Relationship of the Genome with the Nuclear Envelope Using Fluorescence in situ Hybridization -- Visualization of Genomic Loci in Living Cells with a Fluorescent CRISPR/Cas9 System -- Methods to Monitor DNA Repair Defects and Genomic Instability in the Context of a Disrupted Nuclear Lamina -- High Resolution Scanning Electron Microscopy and Immuno-Gold Labeling of the Nuclear Lamina and Nuclear Pore Complex -- An In Vitro Assay to Study Targeting of Membrane Proteins to the Inner Nuclear Membrane -- Nuclear Protein Transport in Digitonin Permeabilized Cells -- Analysis of CRM1-Dependent Nuclear Export in Permeabilized Cells -- SPEED Microscopy and Its Application in Nucleocytoplasmic Transport
    Abstract: This volume provides a wide range of protocols used in studying the nuclear envelope, with special attention to the experimental adjustments that may be required to successfully investigate this complex organelle in cells from various organisms. The Nuclear Envelope: Methods and Protocols is divided into five sections: Part I – Nuclear Envelope Isolation; Part II – Nuclear Envelope Protein Interactions, Localization, and Dynamics; Part III – Nuclear Envelope Interactions with the Cytoskeleton; Part IV – Nuclear Envelope-Chromatin Interactions; and Part V – Nucleo-Cytoplasmic Transport. Many of the modifications discussed in this book have only been circulated within laboratories that have conducted research in this field for many years. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting edge and thorough, The Nuclear Envelope: Methods and Protocols is a timely resource for researchers who have joined this dynamic and rapidly growing field
    Pages: XIV, 523 p. 103 illus., 77 illus. in color. : online resource.
    ISBN: 9781493935307
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  • 2
    ISSN: 1573-9368
    Keywords: transgenesis ; nuclear import ; fluorescence in situ hybridization ; Danio rerio
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We report that cytoplasmic injection into zebrafish eggs of 104 copies of plasmid DNA complexed to nuclear localization signal (NLS) peptides, as compared to 106 copies of naked DNA, increased nuclear uptake of transgene DNA early during embryo development and enhanced transgene integration frequency into the germline of founders. Monitoring the dynamics of nuclear uptake of DNA-NLS complexes by fluorescence in situ hybridization (FISH) of interphase nuclei indicates that NLS enhances both the proportion of nuclei importing DNA during early embryo development, and the amount of DNA imported by individual nuclei. The use of NLS increases the proportion of germline transgenic founders from 14 to 43% (P 〈 0.01) as assessed by polymerase chain reaction analysis of F1s. From germline transgenic DNA-NLS-injected founders, 47% transgenic F1s are obtained in wild-type crosses, as opposed to 6% from naked DNA-injec...
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1436-2236
    Keywords: Key words: gene gun, DNA transfer, expression, zebrafish, luciferase, GFP.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract: We describe the use of gene-gun-mediated transfer of luciferase and green fluorescent protein (GFP) reporter genes in zebrafish (Danio rerio). Optimization of DNA transfer parameters indicated highest overall luciferase expression in epidermis and dermis using 1-μm microcarriers and 1 μg of pCMVL plasmid DNA at a delivery pressure of 200 psi. Time course studies revealed luciferase activity peaking at 18 hours and decreasing to 30% of the maximum at day 8 after DNA transfer. Onset of reporter gene (GFP) expression was detected at 13 minutes after DNA delivery, and by 65 minutes approximately 100% of the cells in the target area exhibited GFP expression. No germline association or integration events were detected in a screen of approximately 250,000 zebrafish sperm cells by fluorescence in situ hybridization at 15 or 30 days after delivery of 1 μg of pCMVL DNA, suggesting incidental male germline integration should not be considered as a risk factor when using the biolistic DNA delivery parameters and target tissues described.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1040-452X
    Keywords: Oocyte activation ; Calcium ; Maturation promoting factor ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The present study investigated the role of intracellular Ca2+ (Ca2+i) elevation on the inactivation of maturation promoting factor (MPF) in rabbit oocytes. The effects of the number of Ca2+ stimulations and of the amplitude of Ca2+i elevation on the profile of histone H1 kinase activity were determined. A Ca2+ stimulation consisted of transferring mature oocytes from culture medium to 0.3 M mannitol containing 0.1-1.0 mM CaCl2, and pulsing them at 1.25 kV/cm for 10 μsec, or microinjecting 2-8 mM CaCl2 into the oocyte cytoplasm. The number of electrically-induced Ca2+ stimulations was varied, and amplitude of the Ca2+i rise was controlled by altering Ca2+ concentration in the pulsing medium or the injection pipette. Ca2+i concentration was determined with fura-2 dextran; oocytes were snap-frozen at indicated time points and assayed for H1 kinase activity. The activity was quantified by densitometry and expressed as a fraction of activity in nonstimulated oocytes. Electrically-mediated Ca2+i rises inactivated H1 kinase in a manner dependent on the number of Ca2+ stimulations. A single Ca2+ stimulation inactivated H1 kinase to 30-40% of its initial activity. However, H1 kinase inactivation was only transient, regardless of the amplitude of the electrically- or injection-mediated Ca2+i elevation. Increasing the number of Ca2+ stimulations helped to maintain H1 kinase activity at basal (pronuclear) levels. The results show the necessity of a threshold of Ca2+i concentration to trigger MPF inactivation, and suggest a role for the extended period of time over which Ca2+i oscillates at fertilization. © 1995 Wiley-Liss, Inc.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1573-9368
    Keywords: luciferase activity ; transient expression ; microinjection ; nuclear localization signal ; Danio rerio
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We report luciferase expression in zebrafish embryos after cytoplasmic injection of low copy numbers of plasmid DNA coupled to the SV40 T antigen nuclear localization sequence (NLS). Binding of NLS to plasmid DNA (pCMVL) occurs at room temperature in 0.25m KCl, as assayed by gel retardation at molar ratios of NLS:pCMVL of at least 100:1. Luciferase expression is induced in 35% of embryos with as low as 103 NLS-bound pCMVL copies. With 104 copies, the proportion of expression increases from 6% at 0:1 to 70% 100:1 NLS:pCMVL (p〈0.01). The beneficial effect of NLS is abolished at DNA concentrations promoting high frequencies of transgene expression without NLS. Regardless of the DNA concentration, the use of NLS does not affect embryo viability for at least up to 10 days: The specificity of NLS on luciferase expression was tested by using a nuclear import deficient reverse NLS peptide (revNLS). revNLS binds to pCMVL, causing gel retardation similarly to NLS, but does not promote transgene expression. Binding of equimolar amounts of revNLS and NLS to DNA reduces by 50% the beneficial effect of NLS on transgene expression. The results suggest efficient targeting of NLS-bound plasmid DNA to the nucleus, and subsequent enhanced uptake of DNA by the nucleus. The data suggest that the use of NLS may reduce the need for using elevated DNA copy numbers in some gene transfer applications.
    Type of Medium: Electronic Resource
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  • 6
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 38 (1994), S. 264-267 
    ISSN: 1040-452X
    Keywords: Inner cell mass ; Granulosa cell ; Nuclear transfer ; Embryo ; Bovine ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The developmental potential of bovine inner cell mass (ICM) and somatic differentiated (granulosa cell) nuclei was investigated using nuclear transplantation. ICM blastomeres were isolated after immunosurgery of day 7 in vitro produced blastocysts and cumulus granulosa cells recovered from in vitro matured oocytes. Nuclear transplantation was carried out by microinjection of the lysed donor cells into enucleated mature oocytes. Oocytes were activated by three 0.2 kVcm-1/20 μs pulses in mannitol containing 100 μM Ca2+, with each pulse 22 min apart. Embryos were cultured in vitro for 7 days and blastocysts were transferred into recipients. ICM and granulosa cell donor nuclei directed 7% (20/304) and 9% (19/213) development to blastocysts, respectively. Fifteen blastocysts from ICM donors resulted in four pregnancies (27%) and two births. No pregnancy was detected with granulosa cell donors. The results illustrate the totipotency of ICM nuclei and indicate that granulosa cell nuclei promote preimplantation development of nuclear transplant embryos. © 1994 Wiley-Liss, Inc.
    Additional Material: 1 Tab.
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  • 7
    ISSN: 1040-452X
    Keywords: Spindle ; Oocyte ; Mammalian ; Centrosome ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Artificial activation and nuclear transfer in rabbit oocytes have been used in past years in an attempt to develop viable techniques for cloning in cattle. The procedures established in our laboratory, using the rabbit as a model, consistently lead to high rates of development to the blastocyst stage. However, the rate of embryos developing to term is considerably lower. In the present study, we undertook a detailed immunocytochemical study of the patterns of both microtubules and chromatin during the first cell cycle of electrical pulse-activated oocytes and of nuclear transfer embryos. Our goal was to investigate the responses of the cell to the different stimuli applied and to establish the sequence of events leading to first cleavage in the absence of normal fertilization. Our results show that, in both electrically activated oocytes and nuclear transfer embryos, although the initial development patterns are rather unusual, embryos become synchronized at the time of the formation of a pronuclear-like structure, and then organize metaphase spindles and cleave. These spindles consistently present small defects, suggesting that problems in the formation of the mitotic apparatus during the first cell cycle may have a long-term effect leading to embryo mortality. © 1993 Wiley-Liss, Inc.
    Additional Material: 4 Ill.
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  • 8
    ISSN: 1040-452X
    Keywords: Electrical pulse ; Ca2+ elevation ; Bovine parthenote ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The influence of electrical stimulation on the level of intracellular Ca2+ in bovine oocytes, as well as activation and extent of parthenogenetic development, was investigated. Mature oocytes were electrically stimulated at 29 hr of maturation, and intracellular Ca2+ concentration was determined with the Ca2+ indicator fura-2 dextran (fura-2 D). The Ca2+ response of oocytes to a given electrical pulse was variable. Oocytes responded with either no Ca2+ rise from baseline (≍ 12 nM), a short-duration Ca2+ rise (from 12 nM to 300 nM) that returned to baseline within 2 min of the pulse, or a long-duration Ca2+ rise (from 12 nM to 1,000-2,000 nM) that never returned to baseline during the 8 min period over which the oocytes were monitored. In these oocytes, Ca2+ level returned to baseline when oocytes were removed from 0.30 M mannitol and placed in an ionic medium. Increasing field strength or pulse duration tended to increase the proportion of oocytes displaying a Ca2+ rise, and at 1.0 kVcm-1 for 40 μsec, all oocytes displayed a long-duration Ca2+ elevation. Direct transfer of oocytes from culture medium to mannitol also triggered a Ca2+ rise. Multiple stimulations, either electrical or by transferring to mannitol, produced multiple Ca2+ rises. This mannitol-induced Ca2+ rise could be inhibited by first washing the oocytes in medium containing equal parts of 0.30 M mannitol and phosphate buffered saline (PBS). The level of Ca2+ stimulation affected activation and development of oocytes. Insufficient, or, conversely, excessive Ca2+ stimulation impaired development. Optimum development was obtained with (1) three pulses of 0.2 kVcm-1 for 20 μsec, each pulse 22 min apart, after direct transfer of oocytes from culture medium to mannitol (22% blastocysts) or (2) three pulses of 1.0 kVcm-1 for 20 μsec after transfer of oocytes from culture medium to medium containing equal parts mannitol and PBS, then to mannitol (24% blastocysts). This procedure avoided induction of a Ca2+ rise prior to the pulse. The results indicate that the level of Ca2+ stimulation can be regulated by incubation conditions prior to the pulse and, to some extent, by field strength and pulse duration. The level of electrical stimulation influenced oocyte Ca2+ response, activation, and parthenogenetic development. © 1993 Wiley-Liss, Inc.
    Additional Material: 5 Ill.
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  • 9
    ISSN: 1040-452X
    Keywords: MPF ; Ca2+ ; Electrical activation ; Cattle oocyte ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The influence of number of Ca2+ stimulations on the profile of histone H1 kinase activity in bovine oocytes was investigated. A Ca2+ stimulation consisted of transferring oocytes directly from culture medium to mannitol containing 100 μM Ca2+ and pulsing oocytes with a 0.2 kVcm-1, 20 μsec discharge. One, three, or six Ca2+ stimulations were given, each 22 min apart. Oocytes were frozen from 0 to 8 hr after the first stimulation at indicated time points and assayed for histone H1 kinase activity. H1 kinase activity was quantified using a densitometer and expressed as a percent of activity in nonpulsed metaphase II oocytes. Stimulating oocytes in the absence of Ca2+ in the pulsing medium did not inactivate H1 kinase. In the presence of Ca2+, however, H1 kinase was rapidly inactivated after stimulation. A single stimulation decreased H1 kinase activity to 44% ± 11% of its initial level in 1 hr. However, H1 kinase was dramatically reactivated at 2 hr after the stimulation and reached 122% ± 22% of the initial activity at 6 hr. With three stimulations, basal H1 kinase activity was 21% ± 3% and was obtained in 30 min. H1 kinase reactivation started at 4 hr after the first stimulation and level of activity reached 38% ± 15% at 8 hr. Six stimulations also led to rapid H1 kinase inactivation and to a basal activity of 14% ± 0.4%. With six stimulations, however, basal H1 kinase activity was maintained over at least 8 hr, and no reactivation occurred during this period. Basal H1 kinase activity obtained after six stimulations was similar to that of fertilized oocytes. Immunoprecipitation of p34cdc2 with an anti-cdc2 antibody strongly suggested an identity between histone H1 kinase and maturation-promoting factor. The data indicate that histone H1 kinase activity in oocytes could be regulated by the number of Ca2+ stimulations. A single Ca2+ stimulation led to H1 kinase inactivation, followed by reactivation of the kinase. Increasing the number of Ca2+ stimulations delayed the onset and reduced the extent of H1 kinase reactivation in the first parthenogenetic cell cycle. © 1993 Wiley-Liss, Inc.
    Additional Material: 7 Ill.
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  • 10
    ISSN: 1040-452X
    Keywords: Pronucleus ; Nuclear envelope ; Sea urchin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We demonstrate that complete sea urchin male pronuclear development in vitro is a two-step process involving membrane-independent chromatin decondensation and nuclear envelope-dependent pronuclear swelling. In the absence of cytoplasmic membrane vesicles (MVs), permeabilized sperm chromatin decondenses into a spherical nucleus of ≈4 μm in diameter. Pronuclear swelling to ≈7 μm requires an intact nuclear envelope, and the degree of swelling is limited by the amount of MVs assembled on the chromatin. Furthermore, after a nuclear envelope is formed, swelling can occur in the absence of additional cytoplasmic MVs. Nuclear swelling also requires ATP hydrolysis, Ca2+ and cytosolic factors, some of which are sensitive to heat and to the sulfhy-dryl alkylating agent, N-ethylmaleimide. The requirement for a nuclear envelope and the rate of pronuclear swelling are consistent with previous in vivo observations. © 1995 wiley-Liss, Inc.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
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