实验基础

We describe a human cell display strategy to isolate high-affinity single-chain antibody fragments (scFvs) specific for CD22 for the treatment of B-cell malignancies. Our strategy uses flow cytometry and human embryonic kidney 293T (HEK-293T) cells that are widely used for transient p ...

Phage display has the capacity to rapidly isolate recombinant antibodies against protein targets and other molecules of significant size. However, there is no obvious lower limit to the power of the selection methods: this chapter describes how the techniques of phage display can be adapt ...

The cartilaginous fish (sharks, skates, and rays) are the oldest phylogenetic group in which a human-type adaptive immune system and immunoglobulins (Igs) have been found. In addition to their conventional (heavy-light chain heterodimeric) isotypes, IgM and IgW, sharks produce the nov ...

This chapter describes the construction and screening of a library of single-chain variable fragments (svFv) derived from patients with autoimmune disease. The methods cover the isolation of mononuclear cells from peripheral blood, preparation of RNA, and recovery of immunoglob ...

A method for the construction of West Nile virus immune donor antibody repertoires is described. B cells are harvested from a suitable donor and the antibody variable genes are amplified using polymerase chain reaction (PCR). The PCR fragments are cloned in a phage display vector to construct a r ...

A method is described for affinity isolation of antigen-specific circulating B cells of interest for subsequent generation of immune antibody phage display libraries. This approach should overcome the problem of low yields of monoclonal antibodies of interest in the libraries gene ...

Organisms have the ability to counteract environmental perturbations and keep certain components within a cell homeostatically regulated. Closely related to homeostasis is the behavior of perfect adaptation where an organism responds to a step-wise perturbation by regulati ...

In this chapter, stochasticity in gene expression is investigated using -expansion technique. Two theoretical models are considered here, one concern the stochastic fluctuations in a single-gene network with negative feedback regulation, and the other the additivity of noise pr ...

To understand the behavior of genes, it is important to explore how the patterns of gene expression change over a period of time because biologically related gene groups can share the same change patterns. In this study, the problem of finding similar change patterns is induced to clustering with t ...

Determining the in vivo kinetics of a signaling pathway is a challenging task. We can measure a property we termed pathway bandwidth to put in vivo bounds on the kinetics of the mitogen-activated protein kinase (MAPk) signaling cascade in Saccharomyces cerevisiae that responds to hyperosm ...

Gene functional studies consist of phenotyping cells with altered gene expression. Improving the precision of current gene expression control techniques would enable more detailed studies of gene function. Here, we provide protocols for building synthetic gene constructs for t ...

This chapter describes a method for generating yeast respiratory oscillations in continuous culture and monitoring rhythmic promoter activity of the culture by automated real-time recording of luminescence. These techniques chiefly require the use of a strain of Saccharomyces ...

Promoters contain a large number of binding sites for transcriptional factors transmitting signals from a variety of cellular pathways. The promoter processes these input signals and sets the level of gene expression, the output of the gene. Here, we describe how to design genetic construc ...

Gene transcription is a dynamic process in which the desired amount of an mRNA is obtained by the equilibrium between its transcription (TR) and degradation (DR) rates. The control mechanism at the RNA polymerase level primarily causes changes in TR. Despite their importance, TRs have been rar ...

Turnover of mRNA is an important level of gene regulation. Individual mRNAs have different intrinsic stabilities. Moreover, mRNA stability changes dynamically with conditions such as hormonal stimulation or cellular stress. While accurate methods exist to measure the half-life ...

Over the past decade, researchers have recognized the need to study biological systems as integrated systems. While the reductionist approaches of the past century have made remarkable advances of our understanding of life, the next phase of understanding comes from systems-level inv ...

Genetic networks underlying many biological processes, such as vertebrate somitogenesis, cell cycle, hormonal signaling, and circadian rhythms, are characterized by oscillations in gene expression. It has been recognized that the frequency and amplitude of gene expression os ...

Although bakers and wine makers constantly select, compare, and hunt for new wild strains of Saccharomyces cerevisiae, yeast geneticists have long focused on a few “standard” strains to ensure reproducibility and easiness of experimentation. And so far, the wonderful natural resource ...

The budding yeast Kluyveromyces lactis has diverged from the Saccharomyces lineage before the whole-genome duplication and its genome sequence reveals lower redundancy of many genes. Moreover, it shows lower preference for fermentative carbon metabolism and a broader substra ...

Analysis of gene function often involves detailed studies of when a given gene is expressed or silenced. Transposon mutagenesis is a powerful tool to generate insertional mutations that provide with a selectable marker and a reporter gene that can be used to analyze the transcriptional act ...