Gene targeting in mammalian cells has proven invaluable in biotechnology, in studies of gene structure and function, and in understanding chromosome dynamics. It also offers a potential tool for gene-therapeutic applications. Two limitations constrain the current technology: the low rate of homologous recombination in mammalian cells and the high rate of random (nontargeted) integration of the vector DNA. Here we consider possible ways to overcome these limitations within the framework of our present understanding of recombination mechanisms and machinery. Several studies suggest that transient alteration of the levels of recombination proteins, by overexpression or interference with expression, may be able to increase homologous recombination or decrease random integration, and we present a list of candidate genes. We consider potentially beneficial modifications to the vector DNA and discuss the effects of methods of DNA delivery on targeting efficiency. Finally, we present work showing that genespecific DNA damage can stimulate local homologous recombination, and we discuss recent results with two general methodologies-chimeric nucleases and triplex-forming oligonucleotidesfor stimulating recombination in cells.

To examine the mechanisms of recombination governing the illegitimate integration of transfected DNA into a mammalian genome, we developed a cell system that selects for integration events in defined genomic regions. Cell lines with chromosomal copies of the 3 portion of the adenine phosphoribosyltransferase (APRT) gene (targets) were established. The 5 portion of the APRT gene, which has no homology to the integrated 3 portion, was then electroporated into the target cell lines, and selection for APRT gene function was applied. The reconstruction of the APRT gene was detected at frequencies ranging from less than 10 ؊7 to 10 ؊6 per electroporated cell. Twenty-seven junction sequences between the integrated 5 APRT and its chromosomal target were analyzed. They were found to be randomly distributed in a 2-kb region immediately in front of the 3 portion of the APRT gene. The junctions fell into two main classes: those with short homologies (microhomologies) and those with inserted DNA of uncertain origin. Three long inserts were shown to preexist elsewhere in the genome. Reconstructed cell lines were analyzed for rearrangements at the target site by Southern blotting; a variety of simple and complex rearrangements were detected. Similar analysis of individual clones of the parental cell lines revealed analogous types of rearrangement, indicating that the target sites are unstable. Given the high frequency of integration events at these sites, we speculate that transfected DNA may preferentially integrate at unstable mammalian loci. The results are discussed in relation to possible mechanisms of DNA integration.Mammalian genomes undergo many dynamic events and modifications. Chromosomal alterations under genetic control in normal cells include immune system rearrangements (31) and transposition events (75). In addition, genomic instability is a hallmark of tumor cell progression (24). Chromosomal deletions, translocations, and gene amplification are rearrangements commonly resulting from illegitimate (or nonhomologous) recombination in cancer cells. Although the processes that govern these rearrangements remain largely undefined, analysis of the DNA sequences at illegitimate recombination junctions has provided important information concerning the mechanisms involved (35).The dynamic properties of mammalian chromosomes are evident in the illegitimate (nonhomologous) integration of exogenously introduced DNA. Mammalian cells integrate foreign DNA widely throughout the genome in a process often referred to as random integration (27,58). The integration of DNA into the mammalian genome is efficient, with up to 20% of cells integrating microinjected DNA (10). Furthermore, integration events are usually associated with major chromosomal rearrangements that remain largely undefined (62). Fully characterized rearrangements include a 22-kb deletion (33) and a 5-kb duplication of the chromosomal sequence flanking the integrated DNA (76). Additional analysis of random integrants may provide insights into the processes involv...

The proportions of single-strand breaks and alkali-labile bonds produced by UV-light were investigated in covalently-closed circular 5-bromouracil (BrUra)-containing I-phage DNA. When BrUra--DNA was irradiated in 0.01 M Tris-0.001 M EDTA (pH 8.1) buffer, the Do was 11.7 J/m2 for single-strand breaks, 2.25 J/mz for total breaks, and 2.8 J/m2 for alkali-labile bonds. Thus. alkalilabile bonds were the predominant photochemical products. No double-strand breaks were observed aftcr exposure to 7.7 times the Do for neutral breakage. The photolability measured under both neutral and alkaline conditions was affected by the NaCl concentration in the irradiation solvent, with the greatest resistance to breakage exhibited at the lowest concentrations. The composition of the irradiation buffer also affected sensitivity. Exposure in 1/10 SSC yielded 4.4 (neutral) and 5.7 (alkaline) times the breakage produced in Tris-EDTA.

No abstract