I-SceI-mediated double-strand break does not increase the frequency of homologous recombination at the Dct locus in mouse embryonic stem cells.

Targeted induction of double-strand breaks (DSBs) at natural endogenous loci was shown to increase the rate of gene replacement by homologous recombination in mouse embryonic stem cells. The gene encoding dopachrome tautomerase (Dct) is specifically expressed in melanocytes and their precursors. To...

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Autores principales:	Myriam Fenina, Dominique Simon-Chazottes, Sandrine Vandormael-Pournin, Jihane Soueid, Francina Langa, Michel Cohen-Tannoudji, Bruno A Bernard, Jean-Jacques Panthier
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/a25886e9225b4dca9b0f56c4bd817d46
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spelling	oai:doaj.org-article:a25886e9225b4dca9b0f56c4bd817d462021-11-18T07:14:19ZI-SceI-mediated double-strand break does not increase the frequency of homologous recombination at the Dct locus in mouse embryonic stem cells.1932-620310.1371/journal.pone.0039895https://doaj.org/article/a25886e9225b4dca9b0f56c4bd817d462012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22761925/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Targeted induction of double-strand breaks (DSBs) at natural endogenous loci was shown to increase the rate of gene replacement by homologous recombination in mouse embryonic stem cells. The gene encoding dopachrome tautomerase (Dct) is specifically expressed in melanocytes and their precursors. To construct a genetic tool allowing the replacement of Dct gene by any gene of interest, we generated an embryonic stem cell line carrying the recognition site for the yeast I-SceI meganuclease embedded in the Dct genomic segment. The embryonic stem cell line was electroporated with an I-SceI expression plasmid, and a template for the DSB-repair process that carried sequence homologies to the Dct target. The I-SceI meganuclease was indeed able to introduce a DSB at the Dct locus in live embryonic stem cells. However, the level of gene targeting was not improved by the DSB induction, indicating a limited capacity of I-SceI to mediate homologous recombination at the Dct locus. These data suggest that homologous recombination by meganuclease-induced DSB may be locus dependent in mammalian cells.Myriam FeninaDominique Simon-ChazottesSandrine Vandormael-PourninJihane SoueidFrancina LangaMichel Cohen-TannoudjiBruno A BernardJean-Jacques PanthierPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 6, p e39895 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Myriam Fenina Dominique Simon-Chazottes Sandrine Vandormael-Pournin Jihane Soueid Francina Langa Michel Cohen-Tannoudji Bruno A Bernard Jean-Jacques Panthier I-SceI-mediated double-strand break does not increase the frequency of homologous recombination at the Dct locus in mouse embryonic stem cells.
description	Targeted induction of double-strand breaks (DSBs) at natural endogenous loci was shown to increase the rate of gene replacement by homologous recombination in mouse embryonic stem cells. The gene encoding dopachrome tautomerase (Dct) is specifically expressed in melanocytes and their precursors. To construct a genetic tool allowing the replacement of Dct gene by any gene of interest, we generated an embryonic stem cell line carrying the recognition site for the yeast I-SceI meganuclease embedded in the Dct genomic segment. The embryonic stem cell line was electroporated with an I-SceI expression plasmid, and a template for the DSB-repair process that carried sequence homologies to the Dct target. The I-SceI meganuclease was indeed able to introduce a DSB at the Dct locus in live embryonic stem cells. However, the level of gene targeting was not improved by the DSB induction, indicating a limited capacity of I-SceI to mediate homologous recombination at the Dct locus. These data suggest that homologous recombination by meganuclease-induced DSB may be locus dependent in mammalian cells.
format	article
author	Myriam Fenina Dominique Simon-Chazottes Sandrine Vandormael-Pournin Jihane Soueid Francina Langa Michel Cohen-Tannoudji Bruno A Bernard Jean-Jacques Panthier
author_facet	Myriam Fenina Dominique Simon-Chazottes Sandrine Vandormael-Pournin Jihane Soueid Francina Langa Michel Cohen-Tannoudji Bruno A Bernard Jean-Jacques Panthier
author_sort	Myriam Fenina
title	I-SceI-mediated double-strand break does not increase the frequency of homologous recombination at the Dct locus in mouse embryonic stem cells.
title_short	I-SceI-mediated double-strand break does not increase the frequency of homologous recombination at the Dct locus in mouse embryonic stem cells.
title_full	I-SceI-mediated double-strand break does not increase the frequency of homologous recombination at the Dct locus in mouse embryonic stem cells.
title_fullStr	I-SceI-mediated double-strand break does not increase the frequency of homologous recombination at the Dct locus in mouse embryonic stem cells.
title_full_unstemmed	I-SceI-mediated double-strand break does not increase the frequency of homologous recombination at the Dct locus in mouse embryonic stem cells.
title_sort	i-scei-mediated double-strand break does not increase the frequency of homologous recombination at the dct locus in mouse embryonic stem cells.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/a25886e9225b4dca9b0f56c4bd817d46
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I-SceI-mediated double-strand break does not increase the frequency of homologous recombination at the Dct locus in mouse embryonic stem cells.

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