细胞技术

Xenopus laevis has been used for many decades to study oocyte development and maturation. The Xenopus oocytes’ large size, relative abundance, and clearly defined progression of physical characteristics from oogonia to eggs make them ideal for studying oogenesis. In addition, the abil ...

Protein kinase A (PKA) activity is regulated by intracellular cyclic adenosine mono-phosphate. Conventional protein kinase assays after cell lysis are hence not suitable for analyzing PKA activities. In this chapter, we describe a new method for monitoring PKA activity in live cells. A tr ...

Xenopus oocytes are naturally arrested at G2/M in prophase I of meiosis. Stimulation with progesterone initiates a nontranscriptional signaling pathway that culminates in the activation of Cdc2/cyclin B and reentry into meiosis. This pathway presents a paradigm for nongenomic sig ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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.

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 ...

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 ...

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 ...

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 ...

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 ...