遗传学实验技术

Escherichia coli is a versatile and popular tool for heterologous protein production. Some of the reasons for its popularity include rapid growth, a variety of portable vectors, relatively simple genetics, and the potential for high-density cultivation. In addition, the extensive la ...

Heterologously expressed proteins in Escherichia coli may undergo unwanted N-terminal processing by methionine and proline aminopeptidases. To overcome this problem, we present a system where the gene of interest is cloned as a fusion to a self-splicing mini-intein. This fusion cons ...

The heterologous expression of functional mammalian integral membrane proteins still represents a significant hurdle towards evaluating the relationship between their structure and function. We have therefore utilised the OmpA signal sequence to deliberately target the ...

Biological samples may contain contaminants that interfere with analysis by twodimensional (2-D) electrophoresis. Lysates or biological fluids are complex mixtures that contain a wide variety of nonprotein substances in addition to the proteins to be analyzed. These substances ...

The concentration of protein in a sample prepared for two dimensional (2-D) electrophoretic analysis is usually determined by protein assay. Reasons for this include the following. (1) Protein quantitation ensures that the amount of protein to be separated is appropriate for the gel size a ...

Before two-dimensional electrophoresis (2-DE), proteins of the sample must be denatured, reduced, disaggregated, and solubilized. Sample solubilization is usually carried out in a buffer containing chaotropes (typically 9.5 M urea, or 5–8 M urea and 2 M thiourea), 2–4% nonionic and/or zwit ...

Enzymatic methods provide a convenient alternative for overcoming technical disadvantages of mechanical disruption. Protocols for protein extraction from bacteria and Saccharomyces cerevisiae using lytic enzymes are presented in this chapter. Adaptation of the yeast pr ...

This chapter covers the various methods of mechanical cell disruption and tissue homogenization that are currently commercially available for processing minute samples (

Somatic cell nuclear transfer (SCNT) is a powerful tool for the investigation of the mechanisms of nuclear remodeling. In addition, SCNT may offer the possibility of introducing targeted mutations by homologous recombination in species for which ES cell technology is not available. The r ...

Rats have important advantages over mice as an experimental system for physiological and pharmacological investigations. Their embryonic stem (ES) cells, after differentiation into each tissue or organ, are applied in regenerative medicine, which enables examination of the ef ...

The rat is one of the most preferred model organisms in biomedical research and has been extremely useful for linking physiology and pathology to the genome. However, approaches to genetically modify specific genes in the rat germ line remain relatively scarce. To date, the most efficient appr ...

Embryonic stem (ES) cells have been used extensively for site-specific gene targeting in the mouse. The resulting knock-out and knock-in mouse models generated so far have demonstrated their usefulness in biomedical research. However, for many diseases and fields of study, the rat still re ...

The genetic dissection of physiological and pathological traits in laboratory model organisms is accelerated by the ability to engineer loss-of-function mutations at investigator-specified loci. This chapter describes the use of zinc-finger nucleases (ZFNs) for the targeted ...

With the lack of tools available to manipulate the rat genome, alternative technologies have been investigated to generate loss-of-function rat models by gene invalidation. The recent demonstration that RNA interference (RNAi)-mediated gene silencing occurs in rodents has open ...

The field of toxicogenomics, which is becoming an important sub-discipline of toxicology, resulted from the natural convergence of the field of conventional toxicological research and the emergent field of functional genomics. One technology that has played a significant role in the ...

Congenic strains are routinely used for positional mapping of quantitative trait loci; while conplastic strains, derived by substitution of different mitochondrial genomes on the same nuclear genetic background of inbred rodent strains, provide a way to unambiguously isolate e ...

Congenic and consomic rat strains are inbred strains containing in their genome a given genomic region (congenic) or a whole chromosome (consomic) from another strain. They are nowadays invaluable tools for the identification of genes and mechanisms of multifactorial diseases, one of t ...

It is extremely useful to define a rapid and accurate method for identifying homozygous and heterozygous transgenic animals prior to setting up breeding programs for transgenic colonies and in experiments in which gene dosage effects could have a functional impact. Southern-blotti ...

Currently, most genetically engineered rat strains are created by methods that involve random integration of transgenes into the genome. The ability to identify the chromosomal location of the transgene insertion site enables the development of efficient genotyping assays, all ...

The rat evokes fear and disgust in a large percentage of people around the world. Yet, other people are fascinated by this amazing creature that is raised as a pet, has an important place in several religions, and is a prominent model for biomedical research. This book focuses on a variety of methodologies ...