细胞技术

The stromal compartment of mesenchymal tissues is thought to harbor stem cells that display extensive proliferative capacity and multilineage potential. Stromal stem cells offer a potentially large therapeutic potential in the field of regenerative medicine. Adipose tissue c ...

Cloning by nuclear transfer in mammals has revealed the remarkable ability of an oocyte to reprogram transferred cell nuclei and induce them to recapitulate the developmental program. This chapter summarizes the method used since 1998 for mouse cloning, which differs from that for large a ...

RNA interference (RNAi) is an evolutionary conserved gene-silencing pathway that can be efficiently utilized as a tool to study gene function. RNAi is initiated by long double-stranded RNAs (dsRNAs), which are processed into small duplexes called small-interfering RNAs (siRNAs). In t ...

Fluorescently tagged proteins have become a crucial weapon in the armory of a successful cell biology laboratory. This chapter describes how to produce cRNA coding for a fluorescently tagged protein of choice, such that it is suitable for microinjection and subsequent expression studi ...

Oocytes are extremely large cells that have to coordinate accurate chromosome segregation, asymmetric cytoplasm partitioning together with their own development as fertilizable gametes. For this, they undergo both global (cell cycle progression related) and local changes. It ...

The first meiotic division of mammalian oocytes physiologically occurs in the ovary in the hours preceding ovulation. Fortunately, oocytes removed from their follicular environment will readily undergo this process in culture. Their large size, optical transparency, and effic ...

Protein phosphorylation plays a pivotal role in cell cycle regulation. MPF (M-phase Promoting Factor) and MAPK (Mitogen-activated protein kinase) are two major kinases driving oocyte maturation and early embryonic divisions. Their activities can be measured experimentally wi ...

The development of primordial germ cells into oocytes within primordial follicles involves a complex sequence of proliferation, developmental commitment, entry and arrest in meiosis, and association with surrounding somatic cells. These processes occur over the first few mont ...

Distinct meiotic cohesin complexes play fundamental roles in various meiosis-specific chromosomal events in spatiotemporally different manners during mammalian meiotic prophase. Immunostaining is one of the essential methods to study meiotic cohesin dynamics. For the s ...

The nuclear transfer (NT) technique in the mouse has enabled us to generate cloned mice and to establish NT embryonic stem (ntES) cells. Direct nuclear injection into mouse oocytes with a piezo impact drive unit can aid in the bypass of several steps of the original cell fusion procedure. It is important ...

Meiosis is the specialized cell division in sexually reproducing organisms in which haploid gametes are produced. Meiotic prophase I is the defining stage of meiosis, when pairing and synapsis occur between homologous chromosomes, concurrent with reciprocal recombination (or c ...

The cytogenetic analysis of single cells, such as oocytes and polar bodies, is extremely challenging. The main problem is low probability of obtaining a metaphase preparation in which all of the chromosomes are sufficiently well spread to permit accurate analysis (no overlapping chrom ...

Mouse oocytes and zygotes are semitransparent and large cells approximately 80 μm in diameter. Bisection is one of the easiest ways for performing micromanipulations on such cells. It allows living sister halves or smaller fragments to be obtained, which can be cultured and observed for long ...

Chromosomal spreads are an established method to assess ploidy in different cell types. However, many traditional chromosome-spreading techniques require dissolution of the cell and can only be used to assess hyperploidy because of potential chromosome loss inherent in the proce ...

At fertilization mammalian eggs are activated by a prolonged series of oscillations in the intracellular free Ca2+ concentration. These oscillations can be monitored with any number of Ca2+-sensitive fluorescent dyes. The oscillations last for several hours at fertilization and so ...

The visualization of subcellular organelles and structures is a valuable tool for understanding cellular changes that occur in oocytes and early embryos as a result of genetic alterations, incubation conditions, drug treatments, and many other manipulations. Preparing oocytes f ...

This chapter describes a technique for performing chromosome spreads from mouse oocytes. It is based on a previously described protocol (Hodges and Hunt, Chromosoma 111: 165–169, 2002), which we have modified. Chromosomes are stained with either Propidium Iodide or Hoechst. This spread ...

We previously isolated Aurora-C (Aurkc/Aie1) in a screen for kinases expressed in mouse sperm and eggs. Aurora-C kinase was reported to be a chromosomal passenger protein that plays critical roles in chromosome alignment, segregation, kinetochore-microtubule attachment, and cyt ...

Understanding how human oocytes execute chromosome segregation is of paramount importance as errors in this process account for the overwhelming majority of human aneuploidies and increase exponentially with advancing female age. The spindle is the cellular apparatus respon ...

Radiolabeled compounds that are substrates for transmembrane transporters can be used to study transport and metabolism in mammalian oocytes and preimplantation embryos. Because even very small amounts of radioisotopes can be detected, these techniques are feasible to use with o ...