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A Modified Yeast One-Hybrid System for Genome-Wide Identification of Transcription Factor Binding Sites

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 ...

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Rat Spermatogonial Stem Cell-Mediated Gene Transfer

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 ...

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Vertebrate Transgenesis by Transposition

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 ...

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Generation of Transgenic Animals with Lentiviral Vectors

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 ...

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Patent and Licensing Issues in Transgenic Technology

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 ...

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BAC Transgenes, DNA Purification, and Transgenic Mouse Production

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 ...

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Generation of Transgenic Animals by Use of YACs

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 ...

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Generation of Transgenic Rats Using Microinjection of Plasmid DNA or Lentiviral Vectors

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 ...

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Generation of Transgenic Mice by Pronuclear Microinjection

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 ...

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Transgenic Production Benchmarks

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 ...

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Gene Targeting Vector Design for Embryonic Stem Cell Modifications

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 ...

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Designing Transgenes for Optimal Expression

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 ...

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Global Resources: Including Gene Trapped ES Cell Clones - Is Your Gene Already Knocked Out

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 ...

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Refinement, Reduction, and Replacement

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 ...

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Pathogen-Free Mouse Rederivation by IVF, Natural Mating and Hysterectomy

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 ...

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Shipment of Mice and Embryos

Following the advent of transgenic technology and the subsequent establishment of international consortia for broad-based genomic analysis of the mouse, the need for sharing of resources in the exchange of mouse models and material has reached an all time high. Live animals, embryos, and g ...

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Cryopreservation

Cryopreservation is the method of choice not only for archiving mouse mutant models at the end of an experiment, but also to create a security back up during their development. Three key factors need to be considered in cryopreservation: why, when, and how. In this chapter, we intend to discuss these fac ...

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Colony Management

Technical advances in the generation of genetically modified (GM) mice and the efforts of large-scale consortia have provided a wealth of resources to the biomedical research community. It has never been easier to obtain a specifically modified allele. However, the establishment and pr ...

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The Preparation and Analysis of DNA for Use in Transgenic Technology

The techniques of pronuclear and blastocyst injection are now routine procedures for making transgenic and gene-targeted mice, respectively. For many institutions, Core Facilities are the standard venue for producing such genetically modified animals for use in both basic and bio ...

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Induced Pluripotency: Generation of iPS Cells from Mouse Embryonic Fibroblasts

The ability to directly reprogram mammalian adult somatic cells to an undifferentiated pluripotent stage similar to that of embryonic stem cells by introduction of a reduced number of transcription factors has opened new venues in many fields of Biology and Medicine. These reprogramm ...

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