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丁香实验推荐阅读
Oocyte Extracts for the Study of Meiotic M-M Transition

In meiotic cell cycles, meiosis I (MI) is followed by meiosis II (Mil) without an intervening S phase, whereas in mitotic cell cycles, an S phase necessarily alternates with an M phase. For the study of mitotic cell cycles, extracts prepared from unfertilized and parthenogenetically activated X ...

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Methods for Studying Spindle Assembly and Chromosome Condensation in Xenopus Egg Extracts

Methods are presented for preparing cytoplasmic extracts from Xenopus laevis eggs and their utilization to reconstitute and monitor events of the cell cycle in vitro. Addition of sperm nuclei to crude extracts and “cycling” of the reaction through interphase and back into metaphase prom ...

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Oocyte Isolation and Enucleation

Xenopus laevis oocytes are popular cells in experimental biology. Fully grown oocytes are large (∼1.3-mm diameter) with an enormous nucleus (∼300-�m diameter). Oocytes are generally isolated by either manual dissection (manual defolliculation) or enzymatic (mainly with collage ...

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Xenopus tropicalis Oocytes: More Than Just a Beautiful Genome

For more than 30 yr, Xenopus laevis has been the animal of choice for studying the biochemical regulation of the meiotic and early mitotic vertebrate cell cycles. Attracted by its diploid genome, several laboratories have begun using the similar, although evolutionarily distinct, frog Xe ...

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Oocyte Expression With Injection of Purified T7 RNA Polymerase

The Xenopus oocyte is a widely used system for protein expression. Investigators have had the choice between two different techniques: injection into the cytoplasm of in vitro transcribed complementary RNA (cRNA) or injection into the nucleus of complementary DNA (cDNA). We report on a thi ...

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Multiphoton Laser Scanning Microscopy as a Tool for Xenopus Oocyte Research

Multiphoton laser scanning microscopy (MPLSM) has become an increasingly invaluable tool in fluorescent optical imaging. There are several distinct advantages to implementing MPLSM as a Xenopus oocyte research tool. MPLSM increases signal-to-noise ratio and therefore incr ...

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Visualization of the Cytoskeleton in Xenopus Oocytes and Eggs by Confocal Immunofluorescence Microscopy

Xenopus oocytes and eggs are popular models for studying the developmental and cellular mechanisms of RNA localization, axis specification and establishment, and nuclear envelope assembly/disassembly. However, their large size and opacity hamper application of many techni ...

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Imaging Ca2+ Signals in Xenopus Oocytes

Xenopus oocytes have become a favored preparation in which to study the spatiotemporal dynamics of intracellular Ca2+ signaling. Advantages of the oocyte as a model cell system include its large size, lack of intracellular Ca2+ release channels other than the type 1 inositol trisphosphate ...

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Chromosomal DNA Replication in a Soluble Cell-Free System Derived From Xenopus Eggs

Cytoplasmic egg extracts from the frog Xenopus laevis represent a powerful cell-free system to study eukaryotic chromosomal DNA replication. In the classical approach, sperm chromatin is added to unfractionated egg cytoplasm, leading to the assembly of transport-competent nuc ...

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Transgenic Systems in Nuclear Reprogramming

Transgenic reporters have proved to be invaluable in the study of nuclear reprogramming, from demonstrating revival or silencing of gene expression in fusion hybrids to providing a means to display levels and distribution of specific gene products after nuclear transfer. Here, the met ...

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Using Immunofluorescence to Observe Methylation Changes in Mammalian Preimplantation Embryos

DNA methylation is an important epigenetic mark involved in gene silencing, X chromosome and transposon inactivation, genomic imprinting, and chromosome stability. Recently, it has been increasingly recognized that DNA methylation plays an essential regulatory function in m ...

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Observing S-Phase Dynamics of Histone Modifications With Fluorescently Labeled Antibodies

Histone modifications are central to epigenetic regulation and must be reestablished with each round of DNA replication. Here we describe methods to localize these modifications within mammalian nuclei and to relate them to specific spatiotemporal patterns of DNA replication.

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Quantitative Analysis of Telomerase Activity and Telomere Length in Domestic Animal Clones

It has been speculated that incomplete epigenetic reprogramming of the somatic cell genome is the primary reason behind the developmental inefficiencies and postnatal abnormalities observed after nuclear transplantation in domestic animal clones. One chromosome struct ...

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Pluripotency: Capacity for In Vitro Differentiation of Undifferentiated Embryonic Stem Cells

Embryonic stem (ES) cells, the pluripotent cells of early embryos have been successfully cultured as undifferentiated cells. The cells are characterized by two unique properties, unlimited self-renewal capacity and the ability to differentiate into all cells of the body. Because of the ...

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Staining Embryonic Stem Cells Using Monoclonal Antibodies to Stage-Specific Embryonic Antigens

Stage-specific embryonic antigens (SSEAs) are cell-surface molecules that exhibit lineage-restricted patterns of expression during development. These antigens provide useful markers for identifying embryonic stem cells and their differentiated derivatives. SSEA ...

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Analysis of the Nucleolar Compartment of the Nucleus as an Indicator of Nuclear Reprogramming After Nuclear Transfer

When analyzing reprogramming after nuclear transfer, it is interesting to focus on the nucleolar compartment, which is the most morphologically well-defined compartment in the nucleus. As with many messenger RNA-encoding genes, the ribosomal RNA genes are expressed in the nuclei of ce ...

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Methylation-Sensitive Polymerase Chain Reaction

Here, we describe a robust and reproducible methylation-sensitive polymerase chain reaction (MS-PCR) method to detect the percentage methylation in repeat sequences of individual pre-implantation ovine embryos produced by different embryo technologies. This method allo ...

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Chromatin Immunoprecipitation Assay for Mammalian Tissues

In this postgenome era, understanding how a cell regulates access to information encoded in the deoxyribonucleic acid (DNA) is essential. In eukaryotic cells, DNA is bound to histone proteins to form chromatin fibers. Numerous studies have now shown that post-translational histone mod ...

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Analysis of DNA Methylation Profiles in Preimplantation Embryos Using Bisulfite Mutagenesis

For developmental competence of mammalian embryos, dynamic epigenetic changes to both the maternally and paternally derived contributions to the genome of the zygote should be brought about in the early cleavage stages. DNA methylation is a typical epigenetic mark modified during pr ...

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Nuclear Transplantation in Xenopus

Nuclear transplantation in amphibia started in 1952. By this is meant, sexually mature cloned frogs can be obtained from the nuclei of embryo cells, differentiating cells, and larval-differentiated cells. Transplanted nuclei are reprogrammed to entirely new patterns of gene expres ...

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