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          Institute: MPI für molekulare Zellbiologie und Genetik     Collection: MPI-CBG Publications 2013 (arch)     Display Documents



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ID: 688450.0, MPI für molekulare Zellbiologie und Genetik / MPI-CBG Publications 2013 (arch)
Direct cloning of isogenic murine DNA in yeast and relevance of isogenicity for targeting in embryonic stem cells
Authors:Andréasson, Claes; Schick, Anna J; Pfeiffer, Susanne M; Sarov, Mihail; Stewart, Francis; Wurst, Wolfgang; Schick, Joel A
Date of Publication (YYYY-MM-DD):2013
Title of Journal:PLoS ONE
Volume:8
Issue / Number:9
Sequence Number of Article:74207
Copyright:not available
Audience:Experts Only
Intended Educational Use:No
Abstract / 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.
External Publication Status:published
Document Type:Article
Version Comment:Automatic journal name synchronization
Communicated by:Lib
Affiliations:MPI für molekulare Zellbiologie und Genetik
Identifiers:LOCALID:5512
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