title: Tackling heterogeneity: a leaf disc-based assay for the high-throughput screening of transient gene expression in tobacco.
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.
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Aachen, Germany.
authors.forename: Natalia
authors.initials: N
authors.lastname: Piotrzkowski
authors.forename: Stefan
authors.initials: S
authors.lastname: Schillberg
authors.forename: Stefan
authors.initials: S
authors.lastname: Rasche
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics; D000485:Allergens / Q000096:biosynthesis / Q000235:genetics; D000704:Analysis of Variance; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards; D015870:Gene Expression; D005822:Genetic Vectors; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D009928:Organ Specificity; D018515:Plant Leaves / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000378:metabolism; D055550:Protein Stability; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012015:Reference Standards; D057127:Single-Chain Antibodies / Q000096:biosynthesis / Q000235:genetics; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism; D059467:Transcriptome
chemical_list: D000485:Allergens; D008164:Luminescent Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; C413663:fluorescent protein 583; C090396:PHLPI protein, Phleum pratense