细胞技术

Here we describe the methods for the expression of human and murine soluble tumor necrosis factor (hTNF and mTNF) in Escherichia coli cells and for their extraction, purification, and characterization. The expression and purification procedure takes about 2 wk. Human and murine TNF can be pu ...

Tumor necrosis factor (TNF) was originally described as a molecule with antitumor properties released by macrophages stimulated with bacterial products. Almost at the same time that TNF was cloned, it was found to be identical to cachectin, a mediator of cachexia. After the finding of this sec ...

A double-labeling assay for detecting apoptotic cells, using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) assay, and antigens of interest, using immunofluorescence, is described. The assay has been used successfully on fixed, cult ...

Widespread inherent or acquired resistance to cytotoxic drugs is a major limitation to chemotherapy. There are many mechanisms that contribute to such resistance. In neuroblastomas there is evidence that acquired drug resistance may be associated with altered response to growth fa ...

The ability to examine gene expression differences during cancer progression is crucial to our understanding of the biological events that lead to uncontrolled growth of malignant cells. Differential expression screening of complementary DNA libraries is described in detail h ...

Oxidative stress has been shown to have a myriad of effects on cells. Treatment of cells with oxidants, such as hydrogen peroxide or agents that induce reactive oxygen intermediates, has been shown to induce many cellular signaling pathways and, in some cases, cell apoptosis. Many chemotherap ...

Gene targeting is one of the most powerful tools to define the role of signaling molecules in animal development and disease etiology. By using this technique, nearly 1000 knockout mice have been produced over the last two decades. Generating knockout mice, however, is a time-consuming proced ...

Molecular analysis of prostate cancer specimens is complicated by tumor heterogeneity and admixture of malignant cells with benign epithelium, stroma and inflammatory cells. Laser Capture Microdissection allows procurement of selected groups of cells from specific areas of t ...

Coimmunoprecipitation is a powerful tool to study protein-protein interactions and can be used to test for the physical association between a known protein kinase and its substrate. In this chapter, the author describes a protocol for the preparation of a cell lysate, the immunoprecipita ...

Acquiring functional knowledge from protein-protein interaction studies often necessitates the production of binding-impaired mutants and the study of their effects in biological systems. In many cases, multiple ligands compete for binding to the same protein domain and it beco ...

Development of sensitive methods to monitor and quantitatively assess the expression levels of endogenous genes and the association-interaction of proteins in living cells and whole organisms is a complex and challenging problem. In this chapter, we have described basic methods for ...

This chapter will focus on understanding how various wild type (WT), dominant negative (DN), constitutively active (CA), and conditionally active (COND) oncogenes, as well as antisense (AS) genes contained in retroviral vectors may be used to elucidate signal transduction pathways. We w ...

This chapter will focus on introduction of various wild type (WT) and mutant genes into cells by DNA transfection. Techniques for analysis of the inheritance, expression, and biological effects of the introduced genes will be described. Various strong and weak points about three different ...

Investigation of transcription factor activity in animal tissue during the early stage of cancer development can be difficult because of a low number of affected cells in a background of a large number of normal cells. We have used a well-established multistage skin carcinogenesis model to st ...

The ability to rescue viable prostate precursor tissue from Rb−/− fetal mice has allowed for the generation of Rb−/− prostate tissue and Rb−/− prostate epithelial cell lines. Herein, we provide a protocol for the rescue of urogenital precursor tissue from mouse embryos harboring the lethal Rb−/− mu ...

In order to be properly divisible, the cell membrane has to be remodeled and intracellular membranes must be converted into a vesiculated state prior to mitosis. Phospholipases A2, C, and D (PLA2, PLC, and PLD) are involved in regulatory events of intracellular mitogen signaling pathways. We des ...

Immunoprecipitation is one of the most commonly used techniques to study protein-protein interaction in vivo. There are three major steps involved in an immunoprecipitation procedure: 1) lysis of the cells to make the antigen of interest available; 2) formation of the antibody-antigen c ...

Alterations of protein tyrosine kinase and tyrosine phosphatase are often associated with uncontrolled cell growth and cellular transformation. Because of the large number of tyrosine kinase/phosphatase genes in such gene family, it is essential to use an efficient and simple appr ...

Loss of tumor suppressors contributes to numerous cancer types. Many, but not all, proteins encoded by tumor suppressor genes have antiproliferative activity and halt cell-cycle progression. In this chapter, we present three methods that have been utilized to monitor the antimitogen ...

In 1999 it was estimated that renal cell carcinoma (RCC) would account for 29,990 new cancer cases diagnosed in the United States (61% in men and 39% in women), and lead to 11,600 deaths. RCC accounts for 2–3% of all malignancies in adults and causes 2.3% of all cancer deaths in the United States annually (1). Approx 4% of ...