(NAS Colloquium) Links Between Recombination and Replication: Vital Roles of Recombination

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Colloquium on Links Between Recombination and Replication: Vital Roles of Recombination

Colloquium

Manipulating the mammalian genome by homologous recombination

Karen M.Vasquez*, Kathleen Marburger^†, Zsofia Intody^†‡, and John H.Wilson^†^§

*Science Park Research Division, M.D. Anderson Cancer Center, Smithville, TX 78957; ^‡Semmelwies University of Medicine, Department of Ophthalmology No. 1, Budapest, Hungary H-1083; and ^†Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030

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 gene-specific DNA damage can stimulate local homologous recombination, and we discuss recent results with two general methodologies—chimeric nucleases and triplex-forming oligonucleotides— for stimulating recombination in cells.

Homologous recombination (HR) provides a precise mechanism for targeting defined modifications to genomes in living cells. In the 15 years since gene targeting was demonstrated in vertebrate cells (1–4), it has been used extensively to investigate gene function and to create mouse models of human diseases. Thus, gene targeting is now a standard tool of somatic cell genetics, as it has been in yeast for many years. Calling it a standard tool, however, does not mean that gene targeting is easy or that success is assured. Indeed, its application requires a certain persistence of effort that is not necessary, for example, in Saccharomyces cerevisiae. Any approach that would simplify the process in mammalian cells would be welcomed. Does our current knowledge of recombination in somatic cells offer any promising new strategies for gene targeting? We address this question here.

Various aspects of HR and nonhomologous end joining (NHEJ) have been covered in recent reviews (5–10), as have strategies for gene targeting (11–16). Space limitations preclude discussion of other promising approaches to gene correction, including targeting with small DNA fragments (17, 18) and RNA/DNA chimeras (19, 20).

Current protocols for gene targeting rely on the cell’s enzymatic machinery to accomplish HR, which generally occurs at a frequency of roughly one event per 10⁵ to 10⁷ treated cells (14). This low frequency of targeting probably reflects an average low frequency of recombination in every cell, rather than the presence of rare, HR-competent cells in the population. Early experiments using microinjection obtained targeted recombinants at about 1 per 1,000 injected cells (2). Moreover, recent experiments designed to stimulate HR, as discussed below, generated recombinants in several percent of treated cells (21). An average capability per cell is, of course, an oversimplification because there are clear indications of cell cycle-dependent and damage-induced expression of proteins involved in recombinational processes (5, 22–25)

The principal barrier to facile gene targeting in vertebrate cells is not the low frequency of HR, but rather the high frequency of random (nonhomologous) integration, which occurs in about one cell per 10² to 10⁴ treated cells (26). For most cells, targeted recombinants are obscured by more than a 1,000-fold higher frequency of random integrants (14). Random integration is thought to occur by NHEJ, although analysis of multiple integration junctions indicates that more homology is used than is common for NHEJ (27). Several tricks have been devised to suppress the number of random integrants that survive selection and thereby improve the ratio of targeted recombinants to random integrants. These include positive-negative selection, promoter and polyadenylation trap strategies, and marker-target gene fusions (28–30). Positive-negative selection—the most commonly used approach—works well in mouse embryonic stem (ES) cells and has made gene targeting fairly routine in those cells.

For many purposes, it would be useful to target genes in established cell lines, which are widely used as model systems. With rare exceptions (31) positive-negative selection in cell lines enriches targeted recombinants less than 5-fold relative to random integrants (32). This low degree of enrichment, coupled with the lower starting ratio of targeted recombinants to random integrants that is typical for cell lines, means that many colonies must be screened to find targeted recombinants—a substantial barrier to routine targeting. Promoter trap strategies can give a significantly better enrichment in cell lines when careful attention is paid to matching the expression level of the selectable marker to that of the target gene and to applying the correct stringency of selection (32, 33). Additionally, there is often uncertainty as to the number of genes to be targeted because most cell lines are not perfect diploids. Thus, obtaining targeted recombinants in cell lines currently requires significant up-front characterization or extensive downstream screening.

The avian leukosis virus-induced chicken B cell line DT40 deserves special mention. DT40 cells have slightly elevated levels of HR and much reduced levels of random integration, which together yield a targeting ratio of 10–100% without the need for selection tricks (34). The ease of targeting in DT40 cells has made them an increasingly important model system for studying vertebrate cell biology and has contributed enormously to our knowledge of HR (5). Although DT40 cells have specialized

This paper results from the National Academy of Sciences colloquium, “Links Between Recombination and Replication: Vital Roles of Recombination,” held November 10–12, 2000, in Irvine, CA.

Abbreviations: TFO, triplex-forming oligonucleotide; NHEJ, nonhomologous end joining; HR, homologous recombination; ES, embryonic stem; ATM, ataxia telangiectasia mutated; PARP, poly (ADP-ribose) polymerase.

^§	To whom reprint requests should be addressed. E-mail: jwilson@bcm.tmc.edu.

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