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An improved plant toolset for high-throughput recombineering

View ORCID ProfileJ. Brumos, View ORCID ProfileC. Zhao, View ORCID ProfileY. Gong, D. Soriano, A.P. Patel, View ORCID ProfileM.A. Perez-Amador, View ORCID ProfileA.N. Stepanova, View ORCID ProfileJ.M Alonso
doi: https://doi.org/10.1101/659276
J. Brumos
aDepartment of Plant and Microbial Biology, Program in Genetics, North Carolina State University, Raleigh, NC, USA
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C. Zhao
aDepartment of Plant and Microbial Biology, Program in Genetics, North Carolina State University, Raleigh, NC, USA
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Y. Gong
aDepartment of Plant and Microbial Biology, Program in Genetics, North Carolina State University, Raleigh, NC, USA
bDepartment of Biology, Stanford University, Stanford, CA, USA
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D. Soriano
aDepartment of Plant and Microbial Biology, Program in Genetics, North Carolina State University, Raleigh, NC, USA
cDepartment of Biomedical Engineering, Duke University, Durham, NC, USA
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A.P. Patel
aDepartment of Plant and Microbial Biology, Program in Genetics, North Carolina State University, Raleigh, NC, USA
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M.A. Perez-Amador
aDepartment of Plant and Microbial Biology, Program in Genetics, North Carolina State University, Raleigh, NC, USA
dInstituto de Biología Molecular y Celular de Plantas (IBMCP), Universidad Politécnica de Valencia (UPV)-Consejo Superior de Investigaciones Científicas (CSIC), Valencia, Spain
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A.N. Stepanova
aDepartment of Plant and Microbial Biology, Program in Genetics, North Carolina State University, Raleigh, NC, USA
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J.M Alonso
aDepartment of Plant and Microbial Biology, Program in Genetics, North Carolina State University, Raleigh, NC, USA
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  • For correspondence: jmalonso@ncsu.edu
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Abstract

Gene functional studies often rely on the expression of a gene of interest as transcriptional and translational fusions with specialized tags. Ideally, this is done in the native chromosomal contexts to avoid potential misexpression artifacts. Although recent improvements in genome editing make it possible to directly modify the target genes in their native chromosomal location, classical transgenesis is still the preferred experimental approach chosen in most gene tagging studies because of its time efficiency and accessibility. We have developed a recombineering-based tagging system that brings together the convenience of the classical transgenic approaches and the high degree of confidence in the obtained results provided by the direct chromosomal tagging achievable by genome editing strategies. These simple and customizable recombineering toolsets and protocols allow for high-throughput generation of a variety of genetic modifications. In addition, a highly efficient recombinase-mediated cassette exchange system has been developed to facilitate the transfer of the desired sequences from a BAC clone to a transformation-compatible binary vector, expanding the use of the recombineering approaches beyond Arabidopsis. The utility of this system is demonstrated by the generation of over 250 whole-gene translational fusions and 123 Arabidopsis transgenic lines corresponding to 62 auxin-related genes, and the characterization of the translational reporter expression patterns for 14 auxin biosynthesis genes.

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Posted June 03, 2019.
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An improved plant toolset for high-throughput recombineering
J. Brumos, C. Zhao, Y. Gong, D. Soriano, A.P. Patel, M.A. Perez-Amador, A.N. Stepanova, J.M Alonso
bioRxiv 659276; doi: https://doi.org/10.1101/659276
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An improved plant toolset for high-throughput recombineering
J. Brumos, C. Zhao, Y. Gong, D. Soriano, A.P. Patel, M.A. Perez-Amador, A.N. Stepanova, J.M Alonso
bioRxiv 659276; doi: https://doi.org/10.1101/659276

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