Eukaryotic DNA and core histones form the fundamental repeating units of chromatin. Condensed c�hromatin, which has higher-order structures, prevents transcriptional complexes from accessing their target genes. Epigenetic regulation, including structural changes of c ...

Affinity purification by pulldown methods using target-bound gel beads provides a powerful approach for purifying endogenous protein complexes. Such methods can be improved by using nanoparticle-based probe, coupled with immunoblot analysis or quantitative proteomics me ...

In traditional electrophoresis mobility shift assay (EMSA) a single 32P-labeled double-stranded DNA oligonucleotide or a restriction fragment bound to a protein is separated from the unbound DNA by polyacrylamide gel electrophoresis (PAGE) under nondenaturing conditions. ...

Gene expression is in part regulated by transcription factors that bind specific sequence motifs in genomic DNA. Transcription factors cooperate with the basal machinery to upregulate or downregulate transcription. Experimental data have revealed the importance of interact ...

A comprehensive identification of protein–DNA interactions that drive processes such as transcription and replication, both in prokaryotic and eukaryotic organisms, remains a major technical challenge. In this chapter, we present a SILAC-based DNA affinity purification met ...

The yeast one-hybrid system is a powerful genetic method to identify DNA–protein interactions, but there is a major limitation inherent to the system. Namely, frequency of false positives generated by yeast endogenous transcription factors has been thought to be higher than that of true po ...

More than 20 years have passed since the advent of genetic manipulation of the mouse germline using cultures of pluripotent embryonic stem cells. Still, despite remarkable successes in the mouse, the application of stem cell cultures for transgenesis in other mammalian species has been co ...

Transposable elements represent a class of DNA molecules that have the ability to move genetic material from one location to another. Since the first recognition and description of their activities by Barbara McClintock beginning in the 1950s, researchers have been harnessing their mo ...

In this chapter, we will discuss the use of lentiviral vectors to generate transgenic animals. Specifically, we will review the technology as applied to the generation of rodents and birds. But many of the procedures can be applied in other species. We review the production of vectors, their design a ...

The use and study of transgenic organisms raises legal issues, particularly the potential for patent infringement if a necessary license is not obtained. Scientists, regardless of whether they practice in an academic or corporate setting, should be aware of patents relating to the resear ...

Transgenic mouse models open new avenues of research to understand gene function, to mark cells, and to model human genetic diseases. The use of large DNA transgenes provides more information to cells and tissues in the mouse so that gene expression occurs at physiological levels in the appropr ...

The use of genomic-type DNA constructs ensures optimal transgene expression, once inserted into the host genome, because their large size includes most if not all the regulatory elements that are needed for correct gene expression. Large heterologous DNA molecules can be easily manipul ...

The rat is an important system for modeling of human disease. The use of transgenesis is relatively uncommon in rats. In this chapter, we focus on describing efficient techniques for the generation of transgenic rats by microinjection of plasmid DNA into pronuclei and the injection of human imm ...

The introduction of a transgene into a fertilized oocyte by pronuclear microinjection of a solution containing the construct of choice is probably the most straightforward method to generate a genetically modified organism. This technique has been adapted to a number of vertebrate and ...

The efficiency with which transgenic mice can be produced via pronuclear injection of DNA constructs is subject to a large number of variables ranging from human to mechanical to biological. Transgenic core facilities, which are often run like small businesses that must attract and satisfy ...

The use of genetically engineered mice to understand gene function is widespread. Changes to the mouse genome can be introduced with gene targeting vectors or with transgenes. Targeting vectors are usually used to ablate gene expression while transgenes are designed to express proteins ...

In theory, designing a DNA construct to be used for transgene purposes, for standard pronuclear microinjection, would seem a rather easy task. The combination of a given promoter and some regulatory elements of choice, driving the expression of the construct to the desired tissue, with a suitab ...

The design of any new mouse genetic modification today should start with careful scrutiny of the resources that are already available, through the internet, for information relating to your gene of interest. International mouse consortia are constantly providing new genetically mo ...

In their 1959 publication The Principles of Humane Experimental Technique, Russell and Burch defined three criteria to be used to alleviate the sources and incidences of inhumanity when performing animal experimentation. These include reduction, replacement, and refinement. To ...

The increased popularity of genetically modified animals in collaborative studies has stimulated the widespread interchange of mice among institutions with inconsistent health standards. While the presence of certain organisms may be tolerated at one institution, the impact ...