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Repair at single targeted DNA double-strand breaks in pluripotent and differentiated human cells.

Fung H, Weinstock DM - PLoS ONE (2011)

Bottom Line: Differences in ex vivo cell culture conditions can drastically affect stem cell physiology.DSBs in mammalian cells are primarily repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ).We show that the frequency of HR at a single DSB differs up to 20-fold between otherwise isogenic human embryonic stem cells (hESCs) based on the site of the DSB within the genome.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
Differences in ex vivo cell culture conditions can drastically affect stem cell physiology. We sought to establish an assay for measuring the effects of chemical, environmental, and genetic manipulations on the precision of repair at a single DNA double-strand break (DSB) in pluripotent and somatic human cells. DSBs in mammalian cells are primarily repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). For the most part, previous studies of DSB repair in human cells have utilized nonspecific clastogens like ionizing radiation, which are highly nonphysiologic, or assayed repair at randomly integrated reporters. Measuring repair after random integration is potentially confounded by locus-specific effects on the efficiency and precision of repair. We show that the frequency of HR at a single DSB differs up to 20-fold between otherwise isogenic human embryonic stem cells (hESCs) based on the site of the DSB within the genome. To overcome locus-specific effects on DSB repair, we used zinc finger nucleases to efficiently target a DSB repair reporter to a safe-harbor locus in hESCs and a panel of somatic human cell lines. We demonstrate that repair at a targeted DSB is highly precise in hESCs, compared to either the somatic human cells or murine embryonic stem cells. Differentiation of hESCs harboring the targeted reporter into astrocytes reduces both the efficiency and precision of repair. Thus, the phenotype of repair at a single DSB can differ based on either the site of damage within the genome or the stage of cellular differentiation. Our approach to single DSB analysis has broad utility for defining the effects of genetic and environmental modifications on repair precision in pluripotent cells and their differentiated progeny.

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Related in: MedlinePlus

Site-specific targeting without sequence homology.A. DR-GFP can be integrated at the p84 locus in either orientation. B. PCR with p84-5′ and GFP-3′ primers demonstrates three 293T clones with site-specific integration. λ indicated DNA ladder. C. FISH demonstrates colocalization of probes against DR-GFP (green) and spanning the p84 locus (orange) in 293T cells. D. Sequences at 5′ and 3′ borders of integrated DR-GFP. Red sequence is from the p84 locus and blue sequence is from the DR-GFP construct. Integration with no sequence modification is indicated as ‘unmod’.
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pone-0020514-g002: Site-specific targeting without sequence homology.A. DR-GFP can be integrated at the p84 locus in either orientation. B. PCR with p84-5′ and GFP-3′ primers demonstrates three 293T clones with site-specific integration. λ indicated DNA ladder. C. FISH demonstrates colocalization of probes against DR-GFP (green) and spanning the p84 locus (orange) in 293T cells. D. Sequences at 5′ and 3′ borders of integrated DR-GFP. Red sequence is from the p84 locus and blue sequence is from the DR-GFP construct. Integration with no sequence modification is indicated as ‘unmod’.

Mentions: To target DR-GFP, we first attempted an NHEJ-based gene capture strategy. The HPRT-DRGFP reporter [7], which contains DR-GFP flanked by sequence from the murine Hprt gene was linearized and co-transfected with ZFNs targeting the p84 locus [27] in 293T cells. No sequence homology is present between HPRT-DRGFP and p84, so integration results solely through NHEJ. Integrants were analyzed by PCR and fluorescence in situ hybridization (FISH) for targeting (Figure 2). Overall, 7 (3.6%) of 194 integrants had undergone site-specific targeting, including 4 in the same orientation as p84 coding sequence and 3 in the opposite orientation (Figure 2). Thus, even in the absence of any sequence homology, targeting of the 9.6 kb DR-GFP sequence can be performed through ZFN-directed NHEJ.


Repair at single targeted DNA double-strand breaks in pluripotent and differentiated human cells.

Fung H, Weinstock DM - PLoS ONE (2011)

Site-specific targeting without sequence homology.A. DR-GFP can be integrated at the p84 locus in either orientation. B. PCR with p84-5′ and GFP-3′ primers demonstrates three 293T clones with site-specific integration. λ indicated DNA ladder. C. FISH demonstrates colocalization of probes against DR-GFP (green) and spanning the p84 locus (orange) in 293T cells. D. Sequences at 5′ and 3′ borders of integrated DR-GFP. Red sequence is from the p84 locus and blue sequence is from the DR-GFP construct. Integration with no sequence modification is indicated as ‘unmod’.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102116&req=5

pone-0020514-g002: Site-specific targeting without sequence homology.A. DR-GFP can be integrated at the p84 locus in either orientation. B. PCR with p84-5′ and GFP-3′ primers demonstrates three 293T clones with site-specific integration. λ indicated DNA ladder. C. FISH demonstrates colocalization of probes against DR-GFP (green) and spanning the p84 locus (orange) in 293T cells. D. Sequences at 5′ and 3′ borders of integrated DR-GFP. Red sequence is from the p84 locus and blue sequence is from the DR-GFP construct. Integration with no sequence modification is indicated as ‘unmod’.
Mentions: To target DR-GFP, we first attempted an NHEJ-based gene capture strategy. The HPRT-DRGFP reporter [7], which contains DR-GFP flanked by sequence from the murine Hprt gene was linearized and co-transfected with ZFNs targeting the p84 locus [27] in 293T cells. No sequence homology is present between HPRT-DRGFP and p84, so integration results solely through NHEJ. Integrants were analyzed by PCR and fluorescence in situ hybridization (FISH) for targeting (Figure 2). Overall, 7 (3.6%) of 194 integrants had undergone site-specific targeting, including 4 in the same orientation as p84 coding sequence and 3 in the opposite orientation (Figure 2). Thus, even in the absence of any sequence homology, targeting of the 9.6 kb DR-GFP sequence can be performed through ZFN-directed NHEJ.

Bottom Line: Differences in ex vivo cell culture conditions can drastically affect stem cell physiology.DSBs in mammalian cells are primarily repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ).We show that the frequency of HR at a single DSB differs up to 20-fold between otherwise isogenic human embryonic stem cells (hESCs) based on the site of the DSB within the genome.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
Differences in ex vivo cell culture conditions can drastically affect stem cell physiology. We sought to establish an assay for measuring the effects of chemical, environmental, and genetic manipulations on the precision of repair at a single DNA double-strand break (DSB) in pluripotent and somatic human cells. DSBs in mammalian cells are primarily repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). For the most part, previous studies of DSB repair in human cells have utilized nonspecific clastogens like ionizing radiation, which are highly nonphysiologic, or assayed repair at randomly integrated reporters. Measuring repair after random integration is potentially confounded by locus-specific effects on the efficiency and precision of repair. We show that the frequency of HR at a single DSB differs up to 20-fold between otherwise isogenic human embryonic stem cells (hESCs) based on the site of the DSB within the genome. To overcome locus-specific effects on DSB repair, we used zinc finger nucleases to efficiently target a DSB repair reporter to a safe-harbor locus in hESCs and a panel of somatic human cell lines. We demonstrate that repair at a targeted DSB is highly precise in hESCs, compared to either the somatic human cells or murine embryonic stem cells. Differentiation of hESCs harboring the targeted reporter into astrocytes reduces both the efficiency and precision of repair. Thus, the phenotype of repair at a single DSB can differ based on either the site of damage within the genome or the stage of cellular differentiation. Our approach to single DSB analysis has broad utility for defining the effects of genetic and environmental modifications on repair precision in pluripotent cells and their differentiated progeny.

Show MeSH
Related in: MedlinePlus