细胞技术

Telomerase is central to cellular immortality and is a key component of most cancer cells although this enzyme is rarely expressed to significant levels in normal cells. Therefore, the inhibition of telomerase has garnered considerable attention as a possible anticancer approach. Ma ...

The knockdown of genes that are over-expressed in cancer, and function in tumor onset and/or progression, is an attractive tool to impair the growth of tumor cells. Synthetic nucleic acids such as antisense oligodeoxynucleotides (AS-ODNs) or small-interfering RNAs (siRNAs) were appli ...

The method of RNA interference (RNAi) is an easy means of knocking down a gene without having to generate knockout mutants, which may prove to be difficult and time consuming. RNAi is a naturally occurring process that involves targeting the mRNA of a gene by introducing RNAs that are complementary to ...

RNA interference (RNAi) is one of the most commonly used procedures for gene targeting in today’s cutting edge technology and has great potential for use in clinical therapy. Using a plasmid construct that exogenously expresses short-hairpin RNAs (shRNAs) targeting a desired gene trans ...

Human telomerase reverse transcriptase (hTERT) represents a universal tumor-associated antigen to activate specific immune response in cancer immune therapy. Peptides derived from hTERT are presented by major histocompatibility complex (MHC) class I alleles to T lymphocyt ...

RNA interference (RNAi) has recently emerged as a reliable tool for studying the effects of knocking down or ablating the expression of specific genes. It is hoped that progress made in the laboratory toward in vitro down regulation of gene expression may be carried over into the clinic for treatme ...

Telomeres are the protective structures at the end of eukaryotic chromosomes. Telomerase is a ribonucleoprotein that contains both an RNA and a protein component for the maintenance of telomere length. Telomerase activity is detected in the majority of malignant tumors, but not in normal ...

Telomerase, a ribonucleoprotein enzyme, is detected in the vast majority of cancers, including malignant gliomas, but not in most normal somatic cells. To inhibit telomerase function effectively, we have adopted the 2′,5′-oligoadenylate (2-5A) antisense system. 2-5A is a mediator of one p ...

The methylotrophic yeast Pichia pastoris has been widely exploited for its high-level expression of heterologous proteins by recombination of gene sequences of interest with the methanol-inducible alcohol oxidase gene (AOX1) promoter (1–3). Secreted and cytoplasmic express ...

In optimizing expression of heterologous protein, the issue of proteolysis is often an important factor, since many peptides and proteins are susceptible to degradation by proteases produced in the host organism. In such cases even if the protein product is expressed at high levels, overall ...

Thorough characterization of recombinant strains of Pichia pastoris requires analysis of genomic DNA and RNA. The protocols in this chapter describe methods for isolation of high-quality genomic DNA suitable for hybridization experiments, crude genomic DNA suitable for use as a PCR ...

The extremely high levels of alcohol oxidase produced from the native AOX1 gene in Pichia pastoris (5–30% of cell protein on induction) suggested that single-copy AOX1 -promoter expression vectors would be sufficient for efficient foreign gene expression. Therefore, the first strate ...

The most commonly used vectors for heterologous protein expression in Pichia pastoris carry its wild-type HIS4 gene and the bacterial ampicillin resistance gene as selectable markers (1–6). The HIS4 gene is relatively large (3 kb), with ill-defined functional boundaries, and its use limi ...

The key to the molecular genetic manipulation of any organism is the ability to introduce and maintain DNA sequences of interest. For Pichia pastoris, the fate of introduced DNAs is generally similar to those described for Saccharomyces cerevisiae. Vectors can be maintained as autonomou ...

Immunohistochemistry is a powerful technique to localize proteins in tissues and cultured cells as well as in fractions of subcellular compartments, like mitochondria, endoplasmic reticulum, vesicles, and membranes. In most cases, the detection of a specific protein occurs by a sand ...

Research in the field of membrane proteins has undergone explosive growth during the last decade, primarily owing to the influence of the powerful techniques of modern molecular biology. Membrane proteins fulfill essential functions, such as communication, selective transport of ...

This chapter reviews the expression of two proteins that are naturally secreted: murine epidermal growth factor (mEGF) and the 120-kDa envelope protein from human immunodeficiency virus (HIV-1 ENV). Although the initial aims of these projects were to examine the secretion in Pichia past ...

The Pichia pastoris system has now been used successfully to express a large number of different intracellular and secreted proteins. In some cases, extremely high levels have been obtained (e.g., tumor necrosis factor, TNF ; human serum albumin, HSA ). The aim of this chapter is to illustrate some of t ...

Antibodies consist of two functional parts, the antigen binding site, a heterodimer composed of the VH and VL domains, and the Fc part, which facilitates in vivo the effector functions and stability of the antibody. Recombinant antibodies were first successfully expressed by secretion fr ...

A significant advantage of Pichia pastoris as an experimental system is the ability to bring to bear readily both classical and molecular genetic approaches to a research problem. Although the recent advent of yeast molecular genetics has introduced new and exciting capabilities, clas ...