遗传学实验技术

The structural and functional analyses of proteins have benefited enormously from the use of technologies of recombinant gene expression. The recombinant vaccinia virus system has been widely employed to express genes from eukaryotic, prokaryotic, and viral origins (for reviews, ...

The utility of a recombinant vaccinia virus expression system for transient expression of genes was demonstrated in 1982 (1,2). Among a number of useful characteristics of this expression system are the capacity of the vaccinia virus genome to accommodate large genes (20 kb pairs), availab ...

Several characteristics of amphotropic murine retroviruses have made them useful as vectors for gene transfer with some distinct advantages over other methods of transduction. First, the normal replication cycle of retroviruses includes integration of the viral genome into the h ...

Genetic intervention is becoming increasingly useful in elucidating the molecular basis of various biological processes, including those of the brain. Many genes have now been isolated which encode key regulatory molecules such as signal transduction components or transcript ...

The baculovirus expression vector system is a helper-independent system that has found extensive use in the past decade for the expression of heterologous genes. Its popularity stems from a combination of high levels of expression with the ability to carry out most eukaryotic posttransl ...

Molecular biology of gene expression in early amphibian embryogenesis began in 1964, when undegraded, as opposed to alkaline-hydrolyzed, PCA (perchloric acid), or TCA (trichloroacetic acid)-degraded, RNAs were extracted by Brown and Littna (1–3), Shiokawa and Yamana (4,5), and Woodl ...

Like Xenopus oocytes, Xenopus cleavage blastomeres are ideal synthetic factories for the expression of heterologous gene products. For 6 h after fertilization, the embryos are transcriptionally quiescent (1), but mRNA is recruited for translation at a rate greater than in the oocyte, and ...

The technology of expressing foreign genes in mammalian cells has become increasingly important to study a number of biological questions and as a primary method for production of proteins for pharmaceutical use. Mammalian cells are frequently used as a host for expression of foreign gen ...

Transfection of foreign DNA into mammalian cells is one of the most common procedures performed by investigators next to cell culture itself. Whether the goal is to express mammalian or viral genes transiently or by the generation of stable cells lines, researchers have a variety of transfect ...

Electroporation is a method which utilizes an electric field to introduce DNA or other macromolecules into cells (For review, see ref. 1). When a cell is exposed to a pulse of high electric field, its cell membrane quickly becomes permeabilized. During this permeabilized state, macromolecul ...

The ability to transfect cells with native genometic DNA and engineered gene/vector constructs has played a leading role in accelerating our understanding of gene regulation and function. Yet, despite significant diversification of gene delivery strategies, efforts to expand the ...

DNA-mediated gene transfer techniques have revolutionalized molecular biology and are used extensively to study the function and regulation of eukaryotic genes in a variety of cell types. In general, expression of genes in mammalian cells can be studied by either stable transformati ...

Although optimization of recombinant protein production is an important part of expression, it is difficult to provide “cookbook” techniques. We will instead outline general approaches to optimization with specific methods described where appropriate. Optimal titers are rea ...

There are many applications for gene transfer and gene expression technology in mammalian somatic cell biology. Whether for functional analysis of a newly cloned gene or for purifying large amounts of a protein for its subsequent biochemical characterization, it is often important to ov ...

Microprojectile bombardment or biolistic techniques have been widely used for cereal transformation. These methods rely on the acceleration of gold particles, coated with plasmid DNA, into plant cells as a method of directly introducing the DNA. The first report of the generation of fert ...

This chapter details a reproducible method for the transformation of spring wheat using Agrobacterium tumefaciens via the direct inoculation of bacteria into immature seeds in planta as described in patent WO 00/63398(1. Transformation efficiencies from 1 to 30% have been obtained and ...

The design of reverse genetic experiments that utilize transgenic approaches often requires transgenes to be expressed in a predefined pattern and there is limited information regarding the gene expression profile for specific promoters. It is important that expression patter ...

Methods for the transformation of barley using Agrobacterium-mediated techniques have been available for the past 10 years. Agrobacterium offers a number of advantages over biolistic-mediated techniques in terms of efficiency and the quality of the transformed plants produced. ...

Down-regulation of endogenous genes via post-transcriptional gene silencing (PTGS) is a key to the characterization of gene function in plants. Many RNA-based silencing mechanisms such as post-transcriptional gene silencing, co-suppression, quelling, and RNA interference ( ...

During the past 25 years, the molecular analysis of transgene insertion patterns and sites in plants has greatly contributed to our understanding of the mechanisms underlying transgene integration, expression, and stability in the nuclear genome. Molecular characterization is a ...