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Direct Cloning of Isogenic Murine DNA in Yeast and Relevance of Isogenicity for Targeting in Embryonic Stem Cells

Efficient gene targeting in embryonic stem cells requires that modifying DNA sequences are identical to those in the targeted chromosomal locus. Yet, there is a paucity of isogenic genomic clones for human cell lines and PCR amplification cannot be used in many mutation-sensitive applications. Here,...

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Autores principales: Andréasson, Claes, Schick, Anna J., Pfeiffer, Susanne M., Sarov, Mihail, Stewart, Francis, Wurst, Wolfgang, Schick, Joel A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3772885/
https://www.ncbi.nlm.nih.gov/pubmed/24058528
http://dx.doi.org/10.1371/journal.pone.0074207
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author Andréasson, Claes
Schick, Anna J.
Pfeiffer, Susanne M.
Sarov, Mihail
Stewart, Francis
Wurst, Wolfgang
Schick, Joel A.
author_facet Andréasson, Claes
Schick, Anna J.
Pfeiffer, Susanne M.
Sarov, Mihail
Stewart, Francis
Wurst, Wolfgang
Schick, Joel A.
author_sort Andréasson, Claes
collection PubMed
description Efficient gene targeting in embryonic stem cells requires that modifying DNA sequences are identical to those in the targeted chromosomal locus. Yet, there is a paucity of isogenic genomic clones for human cell lines and PCR amplification cannot be used in many mutation-sensitive applications. Here, we describe a novel method for the direct cloning of genomic DNA into a targeting vector, pRTVIR, using oligonucleotide-directed homologous recombination in yeast. We demonstrate the applicability of the method by constructing functional targeting vectors for mammalian genes Uhrf1 and Gfap. Whereas the isogenic targeting of the gene Uhrf1 showed a substantial increase in targeting efficiency compared to non-isogenic DNA in mouse E14 cells, E14-derived DNA performed better than the isogenic DNA in JM8 cells for both Uhrf1 and Gfap. Analysis of 70 C57BL/6-derived targeting vectors electroporated in JM8 and E14 cell lines in parallel showed a clear dependence on isogenicity for targeting, but for three genes isogenic DNA was found to be inhibitory. In summary, this study provides a straightforward methodological approach for the direct generation of isogenic gene targeting vectors.
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spelling pubmed-37728852013-09-20 Direct Cloning of Isogenic Murine DNA in Yeast and Relevance of Isogenicity for Targeting in Embryonic Stem Cells Andréasson, Claes Schick, Anna J. Pfeiffer, Susanne M. Sarov, Mihail Stewart, Francis Wurst, Wolfgang Schick, Joel A. PLoS One Research Article Efficient gene targeting in embryonic stem cells requires that modifying DNA sequences are identical to those in the targeted chromosomal locus. Yet, there is a paucity of isogenic genomic clones for human cell lines and PCR amplification cannot be used in many mutation-sensitive applications. Here, we describe a novel method for the direct cloning of genomic DNA into a targeting vector, pRTVIR, using oligonucleotide-directed homologous recombination in yeast. We demonstrate the applicability of the method by constructing functional targeting vectors for mammalian genes Uhrf1 and Gfap. Whereas the isogenic targeting of the gene Uhrf1 showed a substantial increase in targeting efficiency compared to non-isogenic DNA in mouse E14 cells, E14-derived DNA performed better than the isogenic DNA in JM8 cells for both Uhrf1 and Gfap. Analysis of 70 C57BL/6-derived targeting vectors electroporated in JM8 and E14 cell lines in parallel showed a clear dependence on isogenicity for targeting, but for three genes isogenic DNA was found to be inhibitory. In summary, this study provides a straightforward methodological approach for the direct generation of isogenic gene targeting vectors. Public Library of Science 2013-09-13 /pmc/articles/PMC3772885/ /pubmed/24058528 http://dx.doi.org/10.1371/journal.pone.0074207 Text en © 2013 Andréasson et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Andréasson, Claes
Schick, Anna J.
Pfeiffer, Susanne M.
Sarov, Mihail
Stewart, Francis
Wurst, Wolfgang
Schick, Joel A.
Direct Cloning of Isogenic Murine DNA in Yeast and Relevance of Isogenicity for Targeting in Embryonic Stem Cells
title Direct Cloning of Isogenic Murine DNA in Yeast and Relevance of Isogenicity for Targeting in Embryonic Stem Cells
title_full Direct Cloning of Isogenic Murine DNA in Yeast and Relevance of Isogenicity for Targeting in Embryonic Stem Cells
title_fullStr Direct Cloning of Isogenic Murine DNA in Yeast and Relevance of Isogenicity for Targeting in Embryonic Stem Cells
title_full_unstemmed Direct Cloning of Isogenic Murine DNA in Yeast and Relevance of Isogenicity for Targeting in Embryonic Stem Cells
title_short Direct Cloning of Isogenic Murine DNA in Yeast and Relevance of Isogenicity for Targeting in Embryonic Stem Cells
title_sort direct cloning of isogenic murine dna in yeast and relevance of isogenicity for targeting in embryonic stem cells
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3772885/
https://www.ncbi.nlm.nih.gov/pubmed/24058528
http://dx.doi.org/10.1371/journal.pone.0074207
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