DNA replication and transcription direct a DNA strand bias in the process of targeted gene repair in mammalian cells

Brachman, Erin E.; Kmiec, Eric B.

doi:10.1242/jcs.01250

Cited by 44 publications

(60 citation statements)

References 39 publications

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“…Clearly, the level of transcriptional activity 34 and/or the movement of DNA replication forks 35,36 could influence the route taken by the oligonucleotide. One pathway (Figure 1a) involves a direct nucleotide exchange reaction that could be catalyzed by mismatch repair or nucleotide excision repair activities.…”

Section: Prospects: Regulatory Pathways Will Guide Experimental Desigmentioning

confidence: 99%

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Gene therapy progress and prospects: targeted gene repair

et al. 2005

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The capacity to correct a mutant gene within the context of the chromosome holds great promise as a therapy for inherited disorders but fulfilling this promise has proven to be challenging. However, steady progress is being made and the development of gene repair as a viable and robust approach is underway. Here, we present some of the recent advances that are helping to shape our thinking about the feasibility and the limitations of this technique. For the most part, these advances center on understanding the regulation of the reaction and validating its application in animal models. Gene Therapy (2005) 12, 639-646.

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Section: Prospects: Regulatory Pathways Will Guide Experimental Desigmentioning

confidence: 99%

“…This model would also predict that cells in S phase are more amenable to gene repair activity and recent data strongly suggest that this is the case. 24,36,42,48 Correction-yes, but at what price?…”

Section: The Dna Replication Process Regulates Gene Repair Activitymentioning

confidence: 99%

Gene therapy progress and prospects: targeted gene repair

et al. 2005

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“…3,9,10 Also in mammalian cells, a modestly increased efficiency of lagging strand over leading strand oligonucleotides was observed and the efficacy of oligonucleotide-mediated gene modification was dependent on the phase of the cell cycle, the highest frequencies being found when oligonucleotides were delivered to cells immediately after release of a G 1 /S block. 11,12 Furthermore, the efficacy could be improved by slowing down progression of replication forks again suggesting that also in mammalian cells at least one mechanism of oligonucleotide-mediated gene modification involves replication of DNA. 13,14 We have previously identified another parameter affecting the frequency of oligonucleotide-mediated gene modification: DNA mismatch repair (MMR).…”

Section: Introductionmentioning

confidence: 99%

Effective oligonucleotide-mediated gene disruption in ES cells lacking the mismatch repair protein MSH3

et al. 2006

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We have previously demonstrated that site-specific insertion, deletion or substitution of one or two nucleotides in mouse embryonic stem cells (ES cells) by single-stranded deoxyribo-oligonucleotides is several hundred-fold suppressed by DNA mismatch repair (MMR) activity. Here, we have investigated whether compound mismatches and larger insertions escape detection by the MMR machinery and can be effectively introduced in MMR-proficient cells. We identified several compound mismatches that escaped detection by the MMR machinery to some extent, but could not define general rules predicting the efficacy of complex base-pair substitutions. In contrast, we found that fournucleotide insertions were largely subject to suppression by the MSH2/MSH3 branch of MMR and could be effectively introduced in Msh3-deficient cells. As these cells have no overt mutator phenotype and Msh3-deficient mice do not develop cancer, Msh3-deficient ES cells can be used for oligonucleotide-mediated gene disruption. As an example, we present disruption of the Fanconi anemia gene Fancf. Gene Therapy (2006) 13, 686-694.

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“…Finally, in contrast to procaryotic systems, 19 a model based on the integration of a ssODN into the lagging strand during replication could not be established in mammalian cells using episomal assays. 20 To achieve high rates of ssODN-based sequence correction that would allow an improved tool for in vivo analysis; two CHO cell lines were created that contained multiple copies of a chromosomally integrated mutated EGFP (mEGFP-a and -b) reporter gene. Such a strategy was essential since the literature shows conflicting results between in vivo assays on chromosomal DNA, in vivo assays on episomal DNA and in vitro experiments carried out on cell extracts.…”

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Implications of cell cycle progression on functional sequence correction by short single-stranded DNA oligonucleotides

Olsen¹,

Randøl²,

Krauß³

2005

Gene Ther

106

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Oligonucleotide-based sequence alteration in living cells is a substantial methodological challenge in gene therapy. Here, we demonstrate that using corrective single-stranded oligonucleotides (ssODN), high and reproducible sequence correction rates can be obtained. CHO cell lines with chromosomally integrated multiple copy EGFP reporter genes routinely show rates of 4.5% targeted sequence correction after transfection with ssODN. We demonstrate that the cell cycle influences the rates of targeted sequence correction in vivo, with a peak in the early S phase during ssODN exposure. After cell division, the altered genomic sequence is predominantly passed to one daughter cell, indicating that targeted sequence alteration occurs after the replication fork has passed over the targeted site. Although high initial correction rates can be obtained by this method, we show that a majority of the corrected cells arrest in the G2/M cell cycle phase, although 1-2% of the corrected cells form viable colonies. The G2/M arrest observed after targeted sequence correction can be partially released by caffeine, pentoxifylline or Gö 6976 exposure. Despite substantial remaining challenges, targeted sequence alteration based on ssODN increasingly promises to become a powerful tool for functional gene alterations. Gene Therapy (2005) Various strategies have been developed to achieve therapeutic targeted gene repair, including small fragment homologous sequence 1,2 ssODN, 3-6 triple helixforming oligonucleotides containing a reactive group, 7-9 bifunctional oligonucleotides based on a triple helixforming DNA recognition domain 10,11 and branched oligonucleotides. 12 Although a general proof of principle for oligonucleotide-based sequence alteration appears to be established, the paradigm for oligonucleotide-based genome alteration remain largely unknown. 6 The central problem in studying this phenomenon are low rates of in vivo sequence correction rates in cell culture. Generally, observed sequence alteration rates on in vivo chromosomal templates in selected cell lines are in the range of 0.1%, 6 while correction rates in untransformed primary cells are substantially lower. [13][14][15] In the absence of a suitable selection system, such low rates allow only limited functional analysis, although Yoon and co-workers have recently suggested a selection system based on simultaneous targeting of two loci. 16 A further problem is the variety of model systems and assays that were used to measure targeted sequence correction which lead to confusing and often conflicting results. 6 Despite these obstacles, several consistent observations have been made: targeted sequence correction depends centrally on transcription, and ssODN that are complementary to the nontranscribed strand show in most cases substantially higher correction rates than ssODN complementary to the transcribed strand in both episomal and chromosomal assays. 17,18 The involvement of transcription-coupled repair has therefore been suggested. Furthermore, the absence of MSH2, ...

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DNA replication and transcription direct a DNA strand bias in the process of targeted gene repair in mammalian cells

Cited by 44 publications

References 39 publications

Gene therapy progress and prospects: targeted gene repair

Gene therapy progress and prospects: targeted gene repair

Effective oligonucleotide-mediated gene disruption in ES cells lacking the mismatch repair protein MSH3

Implications of cell cycle progression on functional sequence correction by short single-stranded DNA oligonucleotides

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