Isolation and Maintenance.- Isolation and Differentiation of Medaka Embryonic Stem Cells.- Maintenance of Chicken Embryonic Stem Cells In Vitro.- Derivation and Culture of Mouse Trophoblast Stem Cells In Vitro.- Derivation, Maintenance, and Characterization of Rat Embryonic Stem Cells In Vitro.- Derivation, Maintenance, and Induction of the Differentiation In Vitro of Equine Embryonic Stem Cells.- Generation and Characterization of Monkey Embryonic Stem Cells.- Derivation and Propagation of Embryonic Stem Cells in Serum- and Feeder-Free Culture.- Signaling in Embryonic Stem Cell Differentiation.- Internal Standards in Differentiating Embryonic Stem Cells In Vitro.- Matrix Assembly, Cell Polarization, and Cell Survival.- Phosphoinositides, Inositol Phosphates, and Phospholipase C in Embryonic Stem Cells.- Cripto Signaling in Differentiating Embryonic Stem Cells.- The Use of Embryonic Stem Cells to Study Hedgehog Signaling.- Transfection and Promoter Analysis in Embryonic Stem Cells.- SAGE Analysis to Identify Embryonic Stem Cell-Predominant Transcripts.- Utilization of Digital Differential Display to Identify Novel Targets of Oct3/4.- Gene Silencing Using RNA Interference in Embryonic Stem Cells.- Genetic Manipulation of Embryonic Stem Cells.- Efficient Transfer of HSV-1 Amplicon Vectors Into Embryonic Stem Cells and Their Derivatives.- Lentiviral Vector-Mediated Gene Transfer in Embryonic Stem Cells.- Use of the Cytomegalovirus Promoter for Transient and Stable Transgene Expression in Mouse Embryonic Stem Cells.- Use of Simian Immunodeficiency Virus Vectors for Simian Embryonic Stem Cells.- Generation of Green Fluorescent Protein-Expressing Monkey Embryonic Stem Cells.- DNA Damage Response and Mutagenesis in Mouse Embryonic Stem Cells.- Ultraviolet-Induced Apoptosis in Embryonic Stem Cells In Vitro.- Use of Embryonic Stem Cells in Pharmacological and Toxicological Screens.- Use of Differentiating Embryonic Stem Cells in Pharmacological Studies.- Embryonic Stem Cells as a Source of Differentiated Neural Cells for Pharmacological Screens.- Use of Murine Embryonic Stem Cells in Embryotoxicity Assays.- Use of Chemical Mutagenesis in Mouse Embryonic Stem Cells.- Epigenetic Analysis of Embryonic Stem Cells.- Nuclear Reprogramming of Somatic Nucleus Hybridized With Embryonic Stem Cells by Electrofusion.- Methylation in Embryonic Stem Cells In Vitro.- Tumor-Like Properties.- Identification of Genes Involved in Tumor-Like Properties of Embryonic Stem Cells.- In Vivo Tumor Formation From Primate Embryonic Stem Cells.- Animal Models and Therapy.- Directed Differentiation and Characterization of Genetically Modified Embryonic Stem Cells for Therapy.- Use of Differentiating Embryonic Stem Cells in the Parkinsonian Mouse Model.

Isolation and characterization

Escherichia coli-based artificial chromosomes have become important tools for physical mapping and sequencing in various genome projects. The lack of a general method to modify these large bacterial clones, however, has limited their utility in functional studies. We developed a simple method to modify bacterial artificial chromosomes directly in the recombination-deficient E. coli host strain by homologous recombination for in vivo studies. The IRES-LacZ marker gene was introduced into a 131 kb BAC containing the murine zinc finger gene, RU49. No rearrangements or deletions were detected in the modified BACs. Furthermore, transgenic mice were generated by pronuclear injection of the modified BAC, and germline transmission of the intact BAC has been obtained. Proper expression of the lacZ transgene in the brain has been observed, which could not be obtained with conventional transgenic constructs.

Homologous recombination based modification in Escherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome.

Construction and characterization of a human bacterial artificial chromosome library

Human BAC library: construction and rapid screening

Working with bacteriophages: common techniques and methodological approaches

Construction and characterization of a human chromosome 2-specific BAC library.

Publisher Summary Pulsed-field gel electrophoresis (PFGE) of agarose gels enables the reproducible separation of large DNA fragments. In concept, PFGE is an extension of conventional electrophoresis, in which two alternating (or pulsed) electric fields are used instead of the traditional single static field. Separation occurs when these fields are oriented at an obtuse angle to one another. In a pulsed-field gel, the end of each molecule migrates in a new direction, with each change of the electric fields. The DNA molecules, thus, migrate through the agarose matrix in a zigzag motion. The tardiness of the larger molecules, in turning corners (e.g., in PFGE) or in running forward and backward [e.g., in field-inversin gel electrophoresis (FIGE)], separates them from the smaller size fragments. The effectiveness of PFGE, however, is not limited to the separation of very large DNA molecules. PFGE can improve the resolution of DNA molecules of only a few hundred bases and permits separation up to 12,000 kilobase pairs (kb). A number of models and theories have been proposed to explain some of the more complex behavior of DNA molecules in PFGE. However, biologists rarely need to consult these physical models or equations for practical PFGE applications. This chapter discusses the optimum PFG electrophoretic conditions for the separation of DNA fragments from 1 to 6000 kb.

Janet C. Wrestler

Papers

Construction and characterization of a human chromosome 2-specific BAC library.

Pulsed-field gel electrophoresis.