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          Institute: MPI für molekulare Zellbiologie und Genetik     Collection: Publikationen MPI-CBG 2011-arch     Display Documents

ID: 585257.0, MPI für molekulare Zellbiologie und Genetik / Publikationen MPI-CBG 2011-arch
Recombination-mediated genetic engineering of large genomic DNA transgenes.
Authors:Ejsmont, Radoslaw K; Ahlfeld, Peter; Pozniakovsky, Andrei I.; Stewart, A Francis; Tomancak, Pavel; Sarov, Mihail
Date of Publication (YYYY-MM-DD):2011
Title of Journal:Methods in Molecular Biology (Clifton, N.J.)
Start Page:445
End Page:458
Copyright:not available
Audience:Experts Only
Intended Educational Use:No
Abstract / Description:Faithful gene activity reporters are a useful tool for evo-devo studies enabling selective introduction of specific loci between species and assaying the activity of large gene regulatory sequences. The use of large genomic constructs such as BACs and fosmids provides an efficient platform for exploration of gene function under endogenous regulatory control. Despite their large size they can be easily engineered using in vivo homologous recombination in Escherichia coli (recombineering). We have previously demonstrated that the efficiency and fidelity of recombineering are sufficient to allow high-throughput transgene engineering in liquid culture, and have successfully applied this approach in several model systems. Here, we present a detailed protocol for recombineering of BAC/fosmid transgenes for expression of fluorescent or affinity tagged proteins in Drosophila under endogenous in vivo regulatory control. The tag coding sequence is seamlessly recombineered into the genomic region contained in the BAC/fosmid clone, which is then integrated into the fly genome using ?C31 recombination. This protocol can be easily adapted to other recombineering projects.
External Publication Status:published
Document Type:Article
Version Comment:Automatic journal name synchronization
Communicated by:thuem
Affiliations:MPI für molekulare Zellbiologie und Genetik
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