遗传学实验技术

Understanding the genetic basis for tumor formation is crucial for treating cancer. Forward genetic screens using insertional mutagenesis technologies have identified many important tumor suppressor genes and oncogenes in mouse models of human cancer. Traditionally, retr ...

In recent years, methods to address the simplification of targeting vector (TV) construction have been developed and validated. Based on in vivo recombination in Escherichia coli, these protocols have reduced dependence on restriction endonucleases, allowing the fabrication of ...

Zinc-finger nucleases (ZFNs) are promising new tools for enhancing the efficiency of gene targeting in many organisms. Because of the flexibility of zinc finger DNA recognition, ZFNs can be designed to bind many different genomic sequences. The double-strand breaks they create are repai ...

Homologous recombination is the most precise way to manipulate the genome. As a tool it has been used extensively in bacteria, yeast, murine embryonic stem cells, and a few other specialized cell lines but has not been available to researchers in other systems, such as for mammalian somatic cell gen ...

Cell line development for protein production or for the screening of drug targets requires the reproducible and stable expression of transgenes. Such cell lines can be engineered with meganucleases, sequence-specific endonucleases that recognize large DNA target sites. These pr ...

Gene targeting provides a powerful means for studying gene function by a reverse genetic approach. Despite recent rapid progress in gene knockdown technologies, gene knockout studies using human somatic cells will be of greater importance for analyzing the functions of human genes in gr ...

Chinese hamster ovary (CHO) cells are the most common host cells and are widely used in the manufacture of approved recombinant therapeutics. They represent a major new class of universal hosts in biopharmaceutical production. However, there remains room for improvement to create more id ...

Clostridium perfringens is a major natural pathogen of human and domestic animals owing to the production of multiple toxins. Defined clostridial mutants are essential for studying the role of toxins in disease pathogenesis. However, it has been very difficult to introduce mutations i ...

Although substantial advances have been made in mycobacterial genetics over the past 15 yr, manipulation of mycobacterial genomes and Mycobacterium tuberculosis in particular, continues to be hindered by problems of relatively poor DNA uptake, slow growth rate, and high levels of ill ...

Mycobacterium tuberculosis has been studied since the 19th century, but genetic manipulation of this organism has only become possible within the last decade of the 20th century. One key methodology is the allelic exchange of unmarked, in-frame deletion mutations and point mutations us ...

Gene targeting with DNA-binding molecules such as triplex-forming oligonucleotides or peptide nucleic acids can be utilized to direct mutagenesis or induce recombination site-specifically. In this chapter, several detailed protocols are described for the design and use of tri ...

Efficient linking of primary DNA sequence information to gene functions in vertebrate models requires that genetic modifications and their effects are analyzed in an efficacious, controlled, and scalable manner. Thus, to facilitate analysis of gene function, new genetic tools and s ...

The knowledge about the complete genome sequences of mouse, human, and other organisms is only the first step toward the functional annotation of all genes. It facilitates the recognition of sequence conservation, which helps to distinguish between important and not important and also co ...

Immunoprecipitation (IP) uses the specificity of antibodies to isolate target proteins (antigens) out of complex sample mixtures. Three different approaches for performing IP will be discussed; traditional (classical) method, oriented affinity method and direct affinity me ...

This protocol describes the purification of mitochondria from rat liver with the aid of zone electrophoresis in a free flow device (ZE-FFE). Starting from liver homogenate, cell debris and nuclei are removed by low speed centrifugation. A crude mitochondrial fraction is obtained by medium ...

Advanced prefractionation strategies, in combination with highly sensitive and accurate mass spectrometers provide powerful means to detect and analyze low abundant proteins on the subcellular and organelle-specific level. Among enrichment techniques, subcellular fr ...

Preparative electrophoresis is a protein fractionation approach useful for the enrichment of low-abundance gene products. Preparative electrophoresis is usually performed in the PrepCell apparatus. Proteins are separated according to their size in a cylindrical gel in the pr ...

This chapter describes the technology of free flow electrophoresis (FFE) and protocols to separate human plasma for proteome analysis. FFE is a highly versatile technology applied in the field of proteomics because of its continuous processing of sample and high resolution in separati ...

Alteration of protein structure and function by introducing unusual amino acids has great potential to develop new biological tool and to produce novel therapeutic agents. Lantibiotics produced by Gram-positive bacteria are ribosomally synthesized and post-translationa ...

While Escherichia coli is in wide use as a host organism for preparative protein production, problems with the folding of the recombinant gene product as well as protein aggregation, i.e., formation of inclusion bodies, are frequently encountered. This is particularly true for proteins th ...