Efficient homologous recombination of linear DNA substrates after injection into Xenopus laevis oocytes.

Carroll, Dana; Wright, Scott H.; Wolff, Roger K.; Grzesiuk, Elżbieta; Maryon, Edward B.

doi:10.1128/mcb.6.6.2053

Cited by 60 publications

(102 citation statements)

References 61 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The strong DNA-end joining activity is characteristic for frog eggs and early embryos; it was hardly, if at all, detectable in oocytes, and there is a dramatic boost of the activity when oocytes mature into unfertilized eggs (Goedecke et al, 1992). From studies by D. Carroll and colleagues (Carroll, 1983;Carroll et al, 1986) it is also known that Xenopus oocytes, like most cells studied so far, are able to join two overlapping identical sequences via homologous recombination. From these independent sets of data one would tentatively conclude that in oocytes and eggs, homologous and nonhomologous recombination would predominate, respectively.…”

Section: Discussion Homologous Recombination and Nonhomologous Dna-enmentioning

confidence: 99%

Dramatic Changes in the Ratio of Homologous Recombination to Nonhomologous DNA-End Joining in Oocytes and Early Embryos ofXenopus laevis

Hagmann¹,

Adlkofer²,

Pfeiffer³

et al. 1996

Biological Chemistry Hoppe-Seyler

View full text Add to dashboard Cite

We have developed a versatile plasmid vector (pReco80% of the homologously recombined product, whereas in eggs a highly efficient DMA-end joining activity predominates Both reactions, homologous recombination and DMA-end joining, are shown to occur quickly, with the majority of the respective products being formed within the first 20 minutes of incubation under optimal conditions. In fertilized eggs, up to 50% of all injected linear DMA molecules are recircularized by DMA-end joining. With high amounts of injected DMA per fertilized egg, DMA-end joining is reduced, presumably due to competition for essential factors, and homologous recombination becomes readily detectable.As there is a sequence of rapid cleavage divisions after fertilization of the egg, the fast and highly efficient DMA-end joining, even though it is error-prone at the junction site, seems to be best suited to cope 1 present address: Institut für Zellbiologie, Swiss Federal Institute of Technology, Zürich-Hönggerberg, CH-8093 Zürich, Switzerland with DMA double-strand breaks that might occur in the genome during early embryogenesis. On the other hand, the long-lived oocytes seem to repair DMA double-strand breaks via homologous recombination. This latter property may be exploited both in Xenopus and in other organisms to achieve homologous integration of exogenous DMA into germ cells for gene targeting.

show abstract

Section: Discussion Homologous Recombination and Nonhomologous Dna-enmentioning

confidence: 99%

Dramatic Changes in the Ratio of Homologous Recombination to Nonhomologous DNA-End Joining in Oocytes and Early Embryos ofXenopus laevis

Hagmann¹,

Adlkofer²,

Pfeiffer³

et al. 1996

Biological Chemistry Hoppe-Seyler

View full text Add to dashboard Cite

show abstract

“…These enormous cells have a large capacity for homologous recombination that is readily accessed by microinjection of appropriate substrates (5) and that proceeds by the same single-strand annealing mechanism that is the principal pathway available to exogenous DNAs in cultured mammalian cells (5,53). Injected linear DNAs undergo efficient recombination if they carry appropriately placed homologous sequences, and a single oocyte can process more than 10 9 molecules into completed recombination products in a few hours (6,35,36). Injected circular DNAs are assembled into apparently normal chromatin and are inert for recombination, but they can be induced to interact with a homologous partner, if they are cleaved (48).…”

mentioning

confidence: 99%

Stimulation of Homologous Recombination through Targeted Cleavage by Chimeric Nucleases

Bibikova

Carroll

Segal

et al. 2001

Molecular and Cellular Biology

552

420

View full text Add to dashboard Cite

Chimeric nucleases that are hybrids between a nonspecific DNA cleavage domain and a zinc finger DNA recognition domain were tested for their ability to find and cleave their target sites in living cells. Both engineered DNA substrates and the nucleases were injected into Xenopus laevis oocyte nuclei, in which DNA cleavage and subsequent homologous recombination were observed. Specific cleavage required two inverted copies of the zinc finger recognition site in close proximity, reflecting the need for dimerization of the cleavage domain. Cleaved DNA molecules were activated for homologous recombination; in optimum conditions, essentially 100% of the substrate recombined, even though the DNA was assembled into chromatin. The original nuclease has an 18-amino-acid linker between the zinc finger and cleavage domains, and this enzyme cleaved in oocytes at paired sites separated by spacers in the range of 6 to 18 bp, with a rather sharp optimum at 8 bp. By shortening the linker, we found that the range of effective site separations could be narrowed significantly. With no intentional linker between the binding and cleavage domains, only binding sites exactly 6 bp apart supported efficient cleavage in oocytes. We also showed that two chimeric enzymes with different binding specificities could collaborate to stimulate recombination when their individual sites were appropriately placed. Because the recognition specificity of zinc fingers can be altered experimentally, this approach holds great promise for inducing targeted recombination in a variety of organisms.Procedures and reagents that allow the directed alteration of genes in situ constitute a powerful toolbox for experimental genetics and potentially for agricultural and therapeutic applications. In many organisms, however, and particularly in higher eukaryotes, the efficiency of recombination between an introduced DNA and the homologous chromosomal target is discouragingly low. For example, such events typically occur in mammalian cells at a frequency of only about 1 for each 10 6 cells treated (3, 31). We are interested in developing procedures that would substantially improve the frequency of gene targeting.A major impediment to efficient gene replacement is the status of the chromosomal target. Increasing the number of target sequences has little or no effect on targeting efficiency (54, 60). In contrast, making an intentional double-strand break (DSB) in the target DNA increases the yield of specific homologous recombination events up to 1,000-fold or more (10,11,14,44,46). It is believed that exonucleases act at broken ends to generate single-stranded tails that are recombinagenic in any of several pathways. In particular, the singlestrand annealing mechanism (33), by which homologous recombination involving exogenous DNA usually occurs in higher eukaryotes (53), cannot proceed unless both the donor and target have ends (5, 48).Whatever the mechanism of recombination, it is clear that the frequency of targeted recombination can be substantially improved by i...

show abstract

“…In many cases, the recombination substrates were introduced into cells together; in these studies, homologous recombination occurs efficiently and prior to chromosomal integration (1,6,20). In contrast with gene targeting, such extrachromosomal homologous recombination is efficient.…”

mentioning

confidence: 99%

Processing of DNA Prior to Illegitimate Recombination in Mouse Cells

Henderson

Simons

1997

Molecular and Cellular Biology

View full text Add to dashboard Cite

In mammalian cells, the predominant pathway of chromosomal integration of exogenous DNA is random or illegitimate recombination; integration by homologous recombination is infrequent. Homologous recombination is initiated at double-strand DNA breaks which have been acted on by single-strand exonuclease. To further characterize the relationship between illegitimate and homologous recombination, we have investigated whether illegitimate recombination is also preceded by exonuclease digestion. Heteroduplex DNAs which included strand-specific restriction markers at each of four positions were generated. These DNAs were introduced into mouse embryonic stem cells, and stably transformed clones were isolated and analyzed to determine whether there was any strand bias in the retention of restriction markers with respect to their positions. Some of the mismatches appear to have been resolved by mismatch repair. Very significant strand bias was observed in the retention of restriction markers, and there was polarity of marker retention between adjacent positions. We conclude that DNA is frequently subjected to 533 exonuclease digestion prior to integration by illegitimate recombination and that the length of DNA removed by exonuclease digestion can be extensive. We also provide evidence which suggests that frequent but less extensive 335 exonuclease processing also occurs.Most DNA introduced into mammalian cells is lost; that which is stably maintained is usually retained by virtue of its integration into nonhomologous chromosomal DNA sites by so-called illegitimate recombination. If the exogenous DNA includes sequences which are homologous with chromosomal sequences, a small minority of cells which integrate DNA will do so by homologous recombination. The practical application of homologous recombination between exogenous DNA and a chromosomal target (gene targeting) is making an enormous impact on mouse molecular genetics (4, 5). Highly efficient gene targeting would provide the ideal form of gene therapy, allowing deleterious mutations to be corrected rather than merely compensating for them (42), but at present the efficiency of gene targeting is several orders of magnitude too low for this to be feasible. Improvements in the frequency of homologous recombination and reductions in the proportion of integration which occurs by illegitimate recombination would be required before homologous recombination could be used for gene therapy. For such improvements to be made, it will be important to understand the mechanisms of gene targeting and illegitimate recombination and the relationships between these processes.There have been numerous studies of the mechanism of homologous recombination in mammalian cells. In many cases, the recombination substrates were introduced into cells together; in these studies, homologous recombination occurs efficiently and prior to chromosomal integration (1,6,20). In contrast with gene targeting, such extrachromosomal homologous recombination is efficient. The mechanism of extrachromosomal h...

show abstract

Efficient homologous recombination of linear DNA substrates after injection into Xenopus laevis oocytes.

Cited by 60 publications

References 61 publications

Dramatic Changes in the Ratio of Homologous Recombination to Nonhomologous DNA-End Joining in Oocytes and Early Embryos ofXenopus laevis

Dramatic Changes in the Ratio of Homologous Recombination to Nonhomologous DNA-End Joining in Oocytes and Early Embryos ofXenopus laevis

Stimulation of Homologous Recombination through Targeted Cleavage by Chimeric Nucleases

Processing of DNA Prior to Illegitimate Recombination in Mouse Cells

Contact Info

Product

Resources

About