Genomic sequence correction by single‐stranded DNA oligonucleotides: role of DNA synthesis and chemical modifications of the oligonucleotide ends

Olsen, Petter Angell; Randøl, Markus; Luna, Luisa; Brown, Tom; Krauß, Stefan

doi:10.1002/jgm.804

Cited by 57 publications

(79 citation statements)

References 40 publications

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“…30,[41][42][43][44][45][46] Largely, mechanistic studies have utilized a mutated eGFP gene as the target and a functional eGFP is produced if the gene is repaired; then, the corrected cells express eGFP and display green fluorescence. In the standard model system, a single copy of the eGFP gene with a single base mutation -TAC → TAG -generating a stop codon has been stably integrated in the sequence of HCT116 cells.…”

Section: Recovery and Proliferation Of Genetically Modified Cancer Cellsmentioning

confidence: 99%

Electrospun fiber membranes enable proliferation of genetically modified cells

Borjigin¹,

Eskridge²,

Niamat³

et al. 2013

IJN

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Polycaprolactone (PCL) and its blended composites (chitosan, gelatin, and lecithin) are well-established biomaterials that can enrich cell growth and enable tissue engineering. However, their application in the recovery and proliferation of genetically modified cells has not been studied. In the study reported here, we fabricated PCL-biomaterial blended fiber membranes, characterized them using physicochemical techniques, and used them as templates for the growth of genetically modified HCT116-19 colon cancer cells. Our data show that the blended polymers are highly miscible and form homogenous electrospun fiber membranes of uniform texture. The aligned PCL nanofibers support robust cell growth, yielding a 2.5-fold higher proliferation rate than cells plated on standard plastic plate surfaces. PCL-lecithin fiber membranes yielded a 2.7-fold higher rate of proliferation, while PCL-chitosan supported a more modest growth rate (1.5-fold higher). Surprisingly, PCL-gelatin did not enhance cell proliferation when compared to the rate of cell growth on plastic surfaces.

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Section: Recovery and Proliferation Of Genetically Modified Cancer Cellsmentioning

confidence: 99%

Electrospun fiber membranes enable proliferation of genetically modified cells

Borjigin¹,

Eskridge²,

Niamat³

et al. 2013

IJN

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“…Indeed, studies in E. coli demonstrated that ssODNs corresponding in sequence to the lagging strand consistently performed better, irrespective of the direction of transcription, thereby establishing a direct link between ssODN polarity and the direction of replication through the target locus. 14,20,21,28,29 In mammalian cells, a direct link between the polarity of the ssODN and the direction of replication could not be established, 16,30 as the direction of the replication fork through the target locus is difficult to determine and may vary because of the firing of different origins in subsequent cell cycles.…”

Section: Mechanistic Models For Ssodn-mediated Gene Targetingmentioning

confidence: 99%

“…Igoucheva et al 31 suggested a model based on the differential accessibility of the DNA strands during transcription to explain the often-observed strand bias in favor of anti-sense ssODNs. 10,11,13,27,30 During transcription, ssDNA regions are provided that facilitates annealing of complementary ssODN. The increased accessibility of the non-transcribed strand could promote annealing of anti-sense ssODNs, whereas the RNA polymerase activity on the transcribed strand could lead to displacement of sense ssODNs.…”

Section: Mechanistic Models For Ssodn-mediated Gene Targetingmentioning

confidence: 99%

“…19 As a result, unmodified ssODNs yielded higher targeting frequencies than PTO-modified ssODNs in long-term assays, 9,16 although PTO-modified ssODNs initially seemed to be superior in short-term assays. 30,57 PROSPECTS Challenges for therapeutic application of ssODN-mediated gene targeting The possibility to site-specifically alter endogenous genes may make ssODN-mediated gene targeting an attractive tool for therapeutic applications. Thus far, several reports have described successful in vivo ssODN-directed gene modification in mice.…”

Section: Consequences Of Ssodn-mediated Gene Targeting On Cell Viabilitymentioning

confidence: 99%

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Progress and prospects: oligonucleotide-directed gene modification in mouse embryonic stem cells: a route to therapeutic application

Aarts

Riele

2010

Gene Ther

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Gene targeting by single-stranded oligodeoxyribonucleotides (ssODNs) is a promising technique for introducing site-specific sequence alterations without affecting the genomic organization of the target locus. Here, we discuss the significant progress that has been made over the last 5 years in unraveling the mechanisms and reaction parameters underlying ssODN-mediated gene targeting. We will specifically focus on ssODN-mediated gene targeting in murine embryonic stem cells (ESCs) and the impact of the DNA mismatch repair (MMR) system on the targeting process. Implications of novel findings for routine application of ssODN-mediated gene targeting and challenges that need to be overcome for future therapeutic applications are highlighted.

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“…Some labs have reported different clonal efficiencies after illegitimate integration 22,23 or different efficiencies for point mutations of different mismatches. 12 We sought to systematically probe these two sources of variability.…”

Section: Correction Efficiency Varies With Esc Clonal Line and Ssodn mentioning

confidence: 99%

Delivery and mechanistic considerations for the production of knock-in mice by single-stranded oligonucleotide gene targeting

et al. 2006

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Single-stranded oligodeoxynucleotide (ssODN) gene targeting may facilitate animal model creation and gene repair therapy. Lipofection of ssODN can introduce point mutations into target genes. However, typical efficiencies in mouse embryonic stem cells (ESC) are o10 À4 , leaving corrections too rare to effectively identify. We developed ESC lines with an integrated mutant neomycin resistance gene (Tyr22Ter). After targeting with ssODN, repaired cells survive selection in G418. Correction efficiencies varied with different lipofection procedures, clonal lines, and ssODN designs, ranging from 1 to 100 corrections per million cells plated. Uptake studies using cell sorting of Cy5-labelled ssODN showed 40% of the corrections concentrated in the best transfected 22% of cells. Four different basepair mismatches were tested and results show that the basespecificity of the mismatch is critical. Dual mismatch ssODN also showed mismatch preferences. These ESC lines may facilitate development of improved ssODN targeting technologies for either animal production or ex vivo gene therapy.

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Genomic sequence correction by single‐stranded DNA oligonucleotides: role of DNA synthesis and chemical modifications of the oligonucleotide ends

Cited by 57 publications

References 40 publications

Electrospun fiber membranes enable proliferation of genetically modified cells

Electrospun fiber membranes enable proliferation of genetically modified cells

Progress and prospects: oligonucleotide-directed gene modification in mouse embryonic stem cells: a route to therapeutic application

Delivery and mechanistic considerations for the production of knock-in mice by single-stranded oligonucleotide gene targeting

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