细胞技术

In vitro techniques for the study of chondrogenic differentiation of embryonic limb mesenchymal cells have been available for some time. Early methods require highdensity confluent monolayer cell cultures (1,2). The micromass culture method developed by Ahrens et al. (3) represent ...

Electroporation has been frequently adopted as a means to transfect primary cells or cell lines that demonstrate resistance to other commonly used methods of transfection, e.g., DEAE-dextran or calcium phosphate precipitation or liposome-mediated transfection. Electropor ...

A common goal of cell biologists is the establishment of in vitro model systems that faithfully recapitulate a particular biological process that occurs in vivo. Currently, numerous methodologies exist for in vitro analysis of osteoblastic cells, however, many of these methods depend on ...

Bone, muscle, fat, and cartilage are differentiated adult tissues derived from embryonic mesoderm. Under the control of specific molecular and cellular signals, mesenchymal stem cells differentiate into one of these mature tissues. The involvement of the master genes myoD, myogenin, ...

The process of hematopoiesis occurs very early during embryogenesis, with the appearance of red blood cells within the yolk sac (1,2). It is at the gastrula stage when hematopoietic stem cells arise from newly formed mesoderm (3,4). Only mesoderm cells found within the extraembryonic area opa ...

The isolation and culture of germ cells has allowed both the analysis of gene expression in these cells as well as studies of their behavior and growth requirements. Some of the factors found to be important for the in vitro culture of germ cells have subsequently been found to be physiologically rele ...

In this chapter, we describe the preparation and use of b-PG, a biotinylated complex that specifically binds hyaluronan (1,2). The b-PG is derived from cartilage and consists of a trypsin fragment of the proteoglycan core protein and one of the link proteins. Because of its ability to bind to hyaluron ...

Cadherins constitute a family of transmembrane glycoproteins (Table 1) that mediate cell-cell adhesion by their ability to self-associate (1–4). For example, E-cadherin binds only to E-cadherin and not to N-cadherin. The homotypic interaction of cadherins is important for the sorting ...

The introduction of microinjection of trace amounts of a fluorescently labeled protein (1,2) into cultured cells leads to its incorporation into the cell’s pool of endogenous protein. Provided the microinjected protein has retained the properties of the native protein, it will become i ...

The manipulation of mice genome is a powerful technique to analyze biological processes that cannot be studied in tissue culture (1). The understanding of many developmental processes is today improving because of such techniques. This is also changing the way human genetic diseases are s ...

Formation of the vertebrate skeleton is an intricately orchestrated, multistep process. Mechanisms underlying skeletogenesis need to account for the global specification of skeletal pattern, commitment of cells to skeletal and hematopoietic lineages, local control of skel ...

Pax3, a transcription factor expressed in the developing embryo, is a critical factor for the proper formation of the mammalian nervous, cardiovascular, and muscular systems. In the mouse, spontaneous mutations in Pax3 resulting in complete loss of function have provided important mod ...

Disorders affecting craniofacial development represent a large fraction of birth defects (1). The advent of transgenic and gene knockout technology has led to the identification of a variety of genes that have roles in craniofacial development. These technologies, together with the t ...

The low solubility of a protein is one of the most frequent impediments for its structural and functional analysis and, on a more practical aspect, for its application as an industrial enzyme. The reason for low solubility can lie in low conformational stability (1), in a high number of surface-expos ...

For many applications proteins must retain their function for a long time and under a wide range of conditions, sometimes at elevated temperatures. However, the stability of natural proteins is often very low, just sufficient to ensure proper functioning under cellular conditions (1). It is s ...

Protein-protein interactions are involved in most biological processes and are an important target for drug design. Over the past decade, there has been an increased interest in the design of small molecules that mimic functional epitopes in protein-protein interactions. However, the ...

It is often possible to screen libraries of variant enzymes directly on agar plates. Colony-based solid-phase screening is an attractive option because of its relative ease and high throughput compared to liquid-phase screening in multi-well plates. As with any high throughput screeni ...

Enzyme thermostability is a property of great importance in the era of designed biocatalysts. While enzymes are capable of catalyzing reactions with exquisite specificity and selectivity, they are often limited by insufficient stability. Improvements in enzyme activity throu ...

We describe a high throughput screening setup for measuring α-amylase activity at temperatures far above the boiling point of water. The system consists of a sealed aluminum 384-well assay plate incubated between two preheated aluminum blocks. Samples consisting of starch solution and ...

Cytochromes P450 are a superfamily of enzymes that catalyze the monoxygenation of a variety of substrates, including aliphatic and aromatic compounds (1). They contain a noncovalently bound protoporphyrin IX, giving these enzymes characteristic spectral properties. This heme ...