 The achievements can be grouped in scientific results (summarized in sections A to D), translational research (see section E) and academic contributions (section F).
A. Seed based molecular farming of antibodies
A seed specific expression cassette was exploited to produce several antibody formats. Several fusions of single chain Fv antibody fragments with the Fc part of a human IgG1 were produced to levels of 1 % of seed weight and more. Those bivalent ScFv-Fc fusions were fully functional and equivalent to CHO cell produced antibodies in a virus neutralization assay (Van Droogenbroeck et al., 2007).
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| Immunogold detection of other proteins in the PS of imbibed scFv-Fc-producing Arabidopsis seeds. (A) Presence of the 11S storage globulin cruciferin in both the protein storage vacuole (PSV) and the periplasmic space (PS). (B) Calreticulin. (C) BiP. Gold particles of 10 nm were used throughout. Arrows mark the plasma membrane. (Bars: 100 nm.) |
Our group  was involved in the PHARMA-PLANTA EU project which aimed at developing efficient and safe strategies for the production of clinical-grade protein pharmaceuticals in plants, and to define procedures and methods for the production of these proteins in compliance with all appropriate regulations. Within this project we were carrying out a detailed characterisation of A. thaliana seed-produced recombinant antibodies in collaboration with different Pharma-Planta partners. N-glycan analysis was carried out in collaboration with the group of Prof. Dr. Friedrich Altmann (BOKU University, Vienna, Austria), while a subcellular localisation study was carried out in collaboration with the group of Prof. Dr. David Robinson (Heidelberg University, Heidelberg, Germany). These collaborations have led to a paper recently published in PNAS, describing the high-level accumulation of scFv-Fc antibodies in Arabidopsis seeds and their detailed characterisation (Van Droogenbroeck et al. 2007). An international Pharma-Planta PhD student Andreas Loos evaluated the accumulation of full-length antibodies in wild type and mutant Arabidopsis plants as collaboration between Gent and Prof. Dr. Herta Steinkellner, BOKU University, Vienna, Austria).
B. Control of T-DNA copy number in transformants
Cotransformation experiments revealed that upon floral dip transformation, a significant portion of the transformants contain concatemers of replicated T-DNAs, while this is much less the case after root transformation (De Buck et al., 2009).
Further, because single-copy T-DNA transformants display stable and high transgene expression, we developed several approaches to increase the frequency of single-copy transformants by using the site-specific Cre/loxP recombination system (De Buck et al., 2007; Marjanac et al., 2008; De Paepe et al., 2009).
C. Transgene silencing
Detailed analysis of the posttranscriptional silencing efficiency showed that it is determined by the tissue type, nature of the silencing inducer locus and the differential expression of the targeted gene (Marjanac et al., 2009).
Analysis of several silencing loci demonstrated that posttranscriptional silencing spreads along the 35S driven target pre mRNA, is a time dependent gradual process and results in silencing of genes not homologous with the silencing inducing locus (Bleys et al, 2006a; Bleys et al 2006b; Vermeersch et al., 2010)
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| Figure: Proof of concept of transitivity as an alternative silencing technology. Transitively silenced plants (X21X21/Y11Y11/CATCAT) clearly showed necrosis after exposition to high-light irradiation, whereas in wild-type control plants this phenotype did not occur. |
D. Mutagenesis
| Five missense mutants and one recombination substrate of the ß-glucuronidase (GUS)-encoding gene of Escherichia coli were developed as a tool for detecting mutation and recombination events in transgenic Arabidopsis plants by reactivation of GUS activity in clonal sectors. The missense mutants were designed to find C:G to- T:A transitions in a symmetrical sequence context. Small peptide tags (hemagglutinin tag and Strep tag II) and green fluorescent protein were translationally fused to GUS, which offers possibilities to check for mutant GUS production levels. Because all GUS missense mutants were cloned in a bacterial expression vector, they can also be used to score mutation events in E. coli (Vanderauwera et al., 2008). |
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| Glycosylases are known to be involved in mismatch repair. Because methylated cytosines are hotspots of mutation and in view of our interest in epigenetics and methylation, an in depth analysis of glycosylases and methylbinding proteins was made and published as a review (Baute and Depicker, 2008). |
E. Industrial collaboration for veterinary vaccines production
An industrial collaboration was set up with Intervet/Sheringh Plough Animal Health as part of an IWT-supported postdoctoral research project (01/03/2008 – 13/06/2010). This company is a global leader in the research, development, manufacturing and sale of veterinary medicines. The goal was to evaluate the production of several subunit vaccines in seeds.
F. Education accomplishments
Prof Depicker has combined her research activities with responsibilities such as Chair of the UGent department Plant biotechnology and Genetics and coordination of the teaching, President of the examination commission of the bachelor and master students in Biochemistry and Biotechnology (about 300 students), and lecturing of 60 hours genetics, epigenetics and plant experimental biotechnology courses to about 140 students in total.
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Gene reg projects