Gene targeting is enhanced in human cells overexpressing hRAD51

Yáñez, R. J.; Porter, Andrew C.G.

doi:10.1038/sj.gt.3300945

Cited by 91 publications

(42 citation statements)

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“…Therefore to optimise gene correction, we created a reporter system that allows us to measure gene repair directly in living cells. Gene repair reporters have proved effective [11][12][13][15][16][17][18][19][20][21][22] and we hope that this system will allow application of this technology to living cells (see also Liu et al 14 ). A single base substitution was introduced into the EGFP gene (from Clontech's pHygEGFP vector (Clontech, Palo Alto, CA, USA)) to create a premature stop codon (GFP W399X) which inactivates the green fluorescence of EGFP.…”

Section: Resultsmentioning

confidence: 99%

Optimising gene repair strategies in cell culture

2002

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Section: Resultsmentioning

confidence: 99%

Optimising gene repair strategies in cell culture

2002

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“…Southern blots were washed with 2ϫ SSC (1ϫ SSC is 150 mM NaCl, 15 mM trisodium citrate), 0.5% (wt/vol) SDS for 10 min at room temperature followed by 0.2ϫ SSC, 0.5% (wt/vol) SDS for 10 min at 68°C. Western blots were as described previously (Yáñ ez and Porter, 1999). Antibodies are described in Table 1.…”

Section: Blots and Flow Cytometrymentioning

confidence: 99%

Construction, Characterization, and Complementation of a Conditional-Lethal DNA Topoisomerase IIα Mutant Human Cell Line

Carpenter

Porter

2004

MBoC

136

175

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DNA Topoisomerase II␣ (topoII␣) is a DNA decatenating enzyme, abundant constituent of mammalian mitotic chromosomes, and target of numerous antitumor drugs, but its exact role in chromosome structure and dynamics is unclear. In a powerful new approach to this important problem, with significant advantages over the use of topoII inhibitors or RNA interference, we have generated and characterized a human cell line (HTETOP) in which >99.5% topoII␣ expression can be silenced in all cells by the addition of tetracycline. TopoII␣-depleted HTETOP cells enter mitosis and undergo chromosome condensation, albeit with delayed kinetics, but normal anaphases and cytokineses are completely prevented, and all cells die, some becoming polyploid in the process. Cells can be rescued by expression of topoII␣ fused to green fluorescent protein (GFP), even when certain phosphorylation sites have been mutated, but not when the catalytic residue Y805 is mutated. Thus, in addition to validating GFP-tagged topoII␣ as an indicator for endogenous topoII␣ dynamics, our analyses provide new evidence that topoII␣ plays a largely redundant role in chromosome condensation, but an essential catalytic role in chromosome segregation that cannot be complemented by topoII␤ and does not require phosphorylation at serine residues 1106, 1247, 1354, or 1393. INTRODUCTIONType II topoisomerases are ubiquitous, highly conserved proteins that catalyze the passage of one DNA duplex through another, creating a transient double-strand break (DSB) in the latter (Watt and Hickson, 1994;Wang, 2002). Studies in a variety of systems indicate that topoII is essential for cellular viability and that it plays a key role in chromosome segregation. Thus, the abnormal cell divisions seen in yeast top2 mutants (Holm et al., 1985;Uemura and Yanagida, 1986), and in vertebrate cells treated with topoII inhibitors (Downes et al., 1991;Ishida et al., 1991Ishida et al., , 1994Haraguchi et al., 2000) are consistent with the unique ability of topoII to resolve the sister chromatids catenations generated when DNA replication forks meet (Wang, 2002). TopoII has been implicated in mammalian centromere function (Floridia et al., 2000;Spence et al., 2002), and it has been suggested that topoII in yeast may play a role in centromeric chromatin structure (Bachant et al., 2002). Further work is required to understand the relationship between any such structural role, topoII-mediated chromatid decatenation, and the role of the cohesin complex (Nasmyth, 2002) in maintaining sister chromatid cohesion until anaphase (Bernard and Allshire, 2002).TopoII also has been implicated in DNA recombination, replication, transcription, and chromosome condensation (Watt and Hickson, 1994;Wang, 2002). Of these, chromosome condensation is the only process in which topoII is widely reported to play an essential role (Koshland and Strunnikov, 1996;Losada and Hirano, 2001;Swedlow and Hirano, 2003), but the data are equivocal. Genetic analyses suggest that topoII is required for chromosome condensation in Sc...

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“…[13][14][15] Shesely et al 13 first described the production of a targeted mutation of the human ␤-globin locus with homologous recombination in mouse erythroleukemia (MEL) cells containing a single copy of human chromosome 11. In that experiment, the authors used a targeting vector that contained 4.7 kb nonisogenic human DNA in the targeting arms.…”

Section: Introductionmentioning

confidence: 99%

Lack of neighborhood effects from a transcriptionally active phosphoglycerate kinase–neo cassette located between the murine β-major and β-minor globin genes

Kaufman

Behl

et al. 2001

Blood

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For the treatment of ␤-globin gene defects, a homologous recombination-mediated gene correction approach would provide advantages over random integrationbased gene therapy strategies. However, "neighborhood effects" from retained selectable marker genes in the targeted locus are among the key issues that must be taken into consideration for any attempt to use this strategy for gene correction. An Ala-to-Ile mutation was created in the ␤6 position of the mouse ␤-major globin gene (␤ 6I ) as a step toward the development of a murine model system that could serve as a platform for therapeutic gene correction studies. The marked ␤-major gene can be tracked at the level of DNA, RNA, and protein, allowing investigation of the impact of a retained phosphoglycerate kinase (PGK)-neo cassette located between the mutant ␤-major and ␤-minor globin genes on expression of these 2 neighboring genes. Although the PGK-neo cassette was expressed at high levels in adult erythroid cells, the abundance of the ␤ 6I mRNA was indistinguishable from that of the wildtype counterpart in bone marrow cells. Similarly, the output from the ␤-minor globin gene was also normal. Therefore, in this specific location, the retained, transcriptionally active PGK-neo cassette does not disrupt the regulated expression of the adult ␤-globin genes. IntroductionSince the molecular cloning and sequencing of the globin genes more than 20 years ago, many investigators have explored gene therapy as a potential mechanism for treating ␤-globin gene defects. 1 The gene therapy approach that has been most carefully studied makes use of retroviral vectors to introduce ␤-globin genes into hematopoietic stem or progenitor cells, where the ␤-globin gene randomly integrates into the host cell genome (reviewed in 2 ). This approach has met with limited success for a variety of reasons, including low efficiency of progenitor cell transduction, 3,4 variegation of expression of the integrated ␤-globin genes, and silencing of the integrated ␤-globin genes that occurs as a function of time. 5,6 When the locus control region was identified more than a decade ago, the core regions of the hypersensitive sites were added to retroviral vectors to reduce or eliminate the integration site-specific variegation of ␤-globin gene expression. [7][8][9] Unfortunately, this change in the design of these vectors led to instability in the viral genomes on integration, which limited the usefulness of the locus control region (LCR) in retroviral systems. 7,8 Although improvements in the system have recently been described using lentivirusbased vectors, fundamental problems with regulated ␤-globin gene expression still exist. 10 In addition, retroviral vectors necessarily cause mutations at sites of integration, and the results of these mutations in any given recipient cell cannot be predicted. The mutagenesis caused by random integration remains a problem for most gene therapy strategies.Alternative approaches for the gene therapy of ␤-globin disorders include "gene correction" 11,12 and h...

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Gene targeting is enhanced in human cells overexpressing hRAD51

Cited by 91 publications

References 50 publications

Optimising gene repair strategies in cell culture

Optimising gene repair strategies in cell culture

Construction, Characterization, and Complementation of a Conditional-Lethal DNA Topoisomerase IIα Mutant Human Cell Line

Lack of neighborhood effects from a transcriptionally active phosphoglycerate kinase–neo cassette located between the murine β-major and β-minor globin genes

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