Gene Targeting Protocols: (133 Methods in Molecular Biology)

The potential now exists in many experimental systems to transfer a cloned, modified gene back into the genome of the host organism. In the ideal situation, the cloned gene is returned to its homologous location in the genome and becomes inserted at the target locus. This process is a controlled means for the repair of DNA damage and ensures accurate chromosome disjunction during meiosis. The paradigm for thinking about the mechanism of this p- cess has emerged primarily from two sources: (1) The principles of reaction mechanics have come from detailed biochemical analyses of the RecA protein purified from Escherichia coli; and (2) the principles of information transfer have been derived from genetic studies carried out in bacteriophage and fungi. A compelling picture of the process of homologous pairing and DNA strand exchange has been influential in directing investigators interested in gene t- geting experiments. The ability to find and pair homologous DNA molecules enables ac- rate gene targeting and is the central phenomenon underlying genetic recombi- tion. Biochemically, the overall process can be thought of as a series of steps in a reaction pathway whereby DNA molecules are brought into homologous register, the four-stranded Holliday structure intermediate is formed, hete- duplex DNA is extended, and DNA strands are exchanged. Not much is known about the biochemical pathway leading to homologous recombination in euka- otes.

Table of Contents:
Nucleic Acid Transfer Using Cationic Lipids.- Optimizing Polyethylenimine-Based Gene Transfer into Mammalian Brain for Analysis of Promoter Regulation and Protein Function.- Gene Transfer and Drug Delivery by Electronic Pulse Delivery.- Strategies for Improving the Frequency and Assessment of Homologous Recombination.- Effective Gene Transfer Using Viral Vectors Based on SV40.- Rapid Generation of Isogenic Mammalian Cell Lines Expressing Recombinant Transgenes by Use of Cre Recombinase.- Site-Directed Alteration of Genomic DNA by Small-Fragment Homologous Replacement.- Mutation Correction by Homologous Recombination with an Adenovirus Vector.- Site-Specific Targeting of DNA Plasmids to Chromosome 19 Using AAV Cis and Trans Sequences.- Adeno-Associated Virus Based Gene Therapy in Skeletal Muscle.- Rapid Establishment of Myeloma Cell Lines Expressing Fab(Tac)-Protamine, a Targetable Protein Vector, Directed Against High-Affinity ?-Chain of Human Interleukin-2 Receptor.- EBV-Derived Episomes to Probe Chromatin Structure and Gene Expression in Human Cells.- Triplex-Directed Site-Specific Genome Modification.- Use of Quantitative Ligation-Mediated Polymerase Chain Reaction to Detect Gene Targeting by Alkylating Oligodeoxynucleotides.- Gene Targeting in Plants via Site-Directed Mutagenesis.- Antisense Oligonucleotides as Modulators of Pre-mRNA Splicing.