23 Targeted Gene Repair in Mammalian Cells Using Chimeric RNA/DNA Oligonucleotides

Abstract

BACKGROUND AND EARLY EXPERIMENTAL STUDIES
The potential now exists in many experimental systems to transfer a cloned, modified gene into the genome of the host organism. In the ideal situation, the cloned gene is returned to its homologous location and is inserted at the target locus. The process is a controlled means for repairing DNA damage and ensuring accurate chromosome disjunction during meiosis. The paradigm for understanding this process is derived from detailed biochemical analyses of the RecA protein purified from Escherichia coli and 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-targeting experiments.

A body of information has emerged from these studies on transformation in lower eukaryotes that has been crucial in developing strategies for gene targeting in higher organisms. Unfortunately, the limited biochemical data, as well as the often confusing and sometimes contradictory results from the genetic studies, have not provided a thorough mechanistic foundation for experimentation. For example, it is unclear from the transformation studies that information on the genetic control of integration will be generally applicable to higher eukaryotic systems. The significance of the functionally independent, yet structurally redundant, RecA-like Rad51, Rad55, Rad57, and Dmc1 genes in yeast is not totally resolved (Story et al. 1993). In addition, the virtual absence of the illegitimate integration events during plasmid transformation commonly observed in many other eukaryotic systems raises certain caveats as to the generality of the...