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

Genomic sequence correction by single-stranded DNA oligonucleotides: role of DNA synthesis and chemical modifications of the oligonucleotide ends
Genomic sequence correction by single-stranded DNA oligonucleotides: role of DNA synthesis and chemical modifications of the oligonucleotide ends
Background Single-stranded oligonucleotides (ssODN) can induce site-specific genetic alterations in selected mammalian cells, but the involved mechanisms are not known.
Methods We corroborate the potential of genomic sequence correction by ssODN using chromosomally integrated mutated enhanced green fluorescent protein (mEGFP) reporter genes in CHO cell lines. The role of integration site was studied in a panel of cell clones with randomly integrated reporters and in cell lines with site-specific single copy integration of the mEGFP reporter in opposite orientations. Involvement of end modification was examined on ssODN with unprotected or phosphorothioate (PS) protected ends. Also ssODN containing octyl or hexaethylene glycol (HEG) end blocking groups were tested. The significance of DNA synthesis was investigated by cell cycle analysis and by the DNA polymerases alpha, delta and epsilon inhibitor aphidicolin.
Results Correction rates of up to 5% were observed upon a single transfection of ssODN. Independent of the mEGFP chromosomal integration site and of its orientation towards the replication fork, antisense ssODN were more effective than sense ssODN. When ssODN ends were blocked by either octyl or HEG groups, correction rates were reduced. Finally, we demonstrate a dependence of the process on DNA synthesis.
Conclusions We show that, on a chromosomal level, the orientation of the replication fork towards the targeted locus is not central in the strand bias of ssODN-based targeted sequence correction. We demonstrate the importance of accessible ssODN ends for sequence alteration. Finally, we provide evidence for the involvement of DNA synthesis in the process.
targeted sequence alteration, single-stranded oligonucleotides, dna replication, dna synthesis, chinese hamster ovary cells targeted gene repair, cell-cycle progression, embryonic stem-cells, mammalian-cells, in-vivo, homologous recombination, directed mutagenesis, escherichia-coli, polymerase-alpha, transcription
1099-498X
1534-1544
Olsen, Petter Angell
31d63c95-2f34-45d0-a5ad-4d701760d3d0
Randol, Markus
927d157d-c9f6-46d8-9371-927139a680f6
Luna, Luisa
a71dac07-c621-4b3a-8986-df91d1631153
Brown, Tom
1cd7df32-b945-4ca1-8b59-a51a30191472
Krauss, Stefan
d382e4cd-1a12-4d39-9ce5-4fc2e37982af
Olsen, Petter Angell
31d63c95-2f34-45d0-a5ad-4d701760d3d0
Randol, Markus
927d157d-c9f6-46d8-9371-927139a680f6
Luna, Luisa
a71dac07-c621-4b3a-8986-df91d1631153
Brown, Tom
1cd7df32-b945-4ca1-8b59-a51a30191472
Krauss, Stefan
d382e4cd-1a12-4d39-9ce5-4fc2e37982af

Olsen, Petter Angell, Randol, Markus, Luna, Luisa, Brown, Tom and Krauss, Stefan (2005) Genomic sequence correction by single-stranded DNA oligonucleotides: role of DNA synthesis and chemical modifications of the oligonucleotide ends. The Journal of Gene Medicine, 7 (12), 1534-1544. (doi:10.1002/jgm.804).

Record type: Article

Abstract

Background Single-stranded oligonucleotides (ssODN) can induce site-specific genetic alterations in selected mammalian cells, but the involved mechanisms are not known.
Methods We corroborate the potential of genomic sequence correction by ssODN using chromosomally integrated mutated enhanced green fluorescent protein (mEGFP) reporter genes in CHO cell lines. The role of integration site was studied in a panel of cell clones with randomly integrated reporters and in cell lines with site-specific single copy integration of the mEGFP reporter in opposite orientations. Involvement of end modification was examined on ssODN with unprotected or phosphorothioate (PS) protected ends. Also ssODN containing octyl or hexaethylene glycol (HEG) end blocking groups were tested. The significance of DNA synthesis was investigated by cell cycle analysis and by the DNA polymerases alpha, delta and epsilon inhibitor aphidicolin.
Results Correction rates of up to 5% were observed upon a single transfection of ssODN. Independent of the mEGFP chromosomal integration site and of its orientation towards the replication fork, antisense ssODN were more effective than sense ssODN. When ssODN ends were blocked by either octyl or HEG groups, correction rates were reduced. Finally, we demonstrate a dependence of the process on DNA synthesis.
Conclusions We show that, on a chromosomal level, the orientation of the replication fork towards the targeted locus is not central in the strand bias of ssODN-based targeted sequence correction. We demonstrate the importance of accessible ssODN ends for sequence alteration. Finally, we provide evidence for the involvement of DNA synthesis in the process.

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More information

Published date: 2005
Keywords: targeted sequence alteration, single-stranded oligonucleotides, dna replication, dna synthesis, chinese hamster ovary cells targeted gene repair, cell-cycle progression, embryonic stem-cells, mammalian-cells, in-vivo, homologous recombination, directed mutagenesis, escherichia-coli, polymerase-alpha, transcription

Identifiers

Local EPrints ID: 44515
URI: http://eprints.soton.ac.uk/id/eprint/44515
ISSN: 1099-498X
PURE UUID: 507674c1-40ac-46f4-a267-82e797473fb1

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Date deposited: 06 Mar 2007
Last modified: 13 Mar 2019 21:07

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Contributors

Author: Petter Angell Olsen
Author: Markus Randol
Author: Luisa Luna
Author: Tom Brown
Author: Stefan Krauss

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