原创力文档tackling heterogeneity a leaf disc-based assay for the high-throughput screening of transient gene expression in tobacco解决异质性的叶子对于测定瞬时基因表达的大规模筛选烟草.pdfVIP
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tackling heterogeneity a leaf disc-based assay for the high-throughput screening of transient gene expression in tobacco解决异质性的叶子对于测定瞬时基因表达的大规模筛选烟草
Tackling Heterogeneity: A Leaf Disc-Based Assay for the
High-Throughput Screening of Transient Gene
Expression in Tobacco
Natalia Piotrzkowski, Stefan Schillberg, Stefan Rasche*
Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Aachen, Germany
Abstract
Transient Agrobacterium-mediated gene expression assays for Nicotiana tabacum (N. tabacum) are frequently used because
they facilitate the comparison of multiple expression constructs regarding their capacity for maximum recombinant protein
production. However, for three model proteins, we found that recombinant protein accumulation (rpa) was significantly
influenced by leaf age and leaf position effects. The ratio between the highest and lowest amount of protein accumulation
(max/min ratio) was found to be as high as 11. Therefore, construct-based impacts on the rpa level that are less than 11-fold
will be masked by background noise. To address this problem, we developed a leaf disc-based screening assay and
infiltration device that allows the rpa level in a whole tobacco plant to be reliably and reproducibly determined. The
prototype of the leaf disc infiltration device allows 14 Agrobacterium-mediated infiltration events to be conducted in
parallel. As shown for three model proteins, the average max/min rpa ratio was reduced to 1.4 using this method, which
allows for a sensitive comparison of different genetic elements affecting recombinant protein expression.
Citation: Piotrzkowski N, Schillberg S, Rasche S (2012) Tackling Heterogeneity: A Leaf Disc-Based Assay for the High-Throughput Screening of Transient Gene
Expression in Tobacco. PLoS ONE 7(9): e45803. doi:10.1371/journal.pone.0045803
Editor: Peter Meyer, University of Leeds, United Kingdom
Received December 28, 2011; Accepted August 24, 2012; Published September 21, 2012
Copyright: 2012 Pi
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