Ann Depicker - Academia.edu (original) (raw)

Papers by Ann Depicker

Research paper thumbnail of Optimized t-dna transfer and vectors therefor

Research paper thumbnail of Plants as bioreactors for protein production: avoiding the problem of transgene silencing

Plant Gene Silencing, 2000

Research paper thumbnail of Production of monoclonal antibodies with a controlled N-glycosylation pattern in seeds of Arabidopsis thaliana

Plant biotechnology journal, 2011

Seed-specific expression is an appealing alternative technology for the production of recombinant... more Seed-specific expression is an appealing alternative technology for the production of recombinant proteins in transgenic plants. Whereas attractive yields of recombinant proteins have been achieved by this method, little attention has been paid to the intracellular deposition and the quality of such products. Here, we demonstrate a comparative study of two antiviral monoclonal antibodies (mAbs) (HA78 against Hepatitis A virus; 2G12 against HIV) expressed in seeds of Arabidopsis wild-type (wt) plants and glycosylation mutants lacking plant specific N-glycan residues. We demonstrate that 2G12 is produced with complex N-glycans at great uniformity in the wt as well as in the glycosylation mutant, carrying a single dominant glycosylation species, GnGnXF and GnGn, respectively. HA78 in contrast, contains additionally to complex N-glycans significant amounts of oligo-mannosidic structures, which are typical for endoplasmic reticulum (ER)-retained proteins. A detailed subcellular localizat...

Research paper thumbnail of Callus-induced epiallelism of an invertedly repeated transgene locus influences its trans-silencing abilities

GBM Annual Spring meeting Mosbach 2008, 2008

Research paper thumbnail of Transgene Expression in Plants, Control of

Sustainable Food Production, 2013

Research paper thumbnail of Production of Camel-Like Antibodies in Plants

Methods in Molecular Biology, 2012

Transgenic plants for the production of high-value recombinant complex and/or glycosylated protei... more Transgenic plants for the production of high-value recombinant complex and/or glycosylated proteins are a promising alternative for conventional systems, such as mammalian cells and bacteria. Many groups use plants as production platform for antibodies and antibody fragments. Here, we describe how bivalent camel-like antibodies can be produced in leaves and seeds. Camel-like antibodies are fusions of the antigen-binding domain of heavy chain camel antibodies (VHH) with an Fc fragment of choice. Transient expression in Nicotiana benthamiana leaves allows the production of VHH-Fc antibodies within a few days after the expression plasmid has been obtained. Generation of stable Arabidopsis thaliana transformants allows production of scalable amounts of VHH-Fc antibodies in seeds within a year. Further, we describe how the in planta-produced VHH-Fc antibodies can be quantified by Western blot analysis with Fc-specific antibodies.

Research paper thumbnail of Nanobody-based products as research and diagnostic tools

Trends in Biotechnology, 2014

Research paper thumbnail of Stable high-level transgene expression inArabidopsis thalianausing gene silencing mutants and matrix attachment regions

The Plant Journal, 2004

Basic and applied research involving transgenic plants often requires consistent high-level expre... more Basic and applied research involving transgenic plants often requires consistent high-level expression of transgenes. However, high inter-transformant variability of transgene expression caused by various phenomena, including gene silencing, is frequently observed. Here, we show that stable, high-level transgene expression is obtained using Arabidopsis thaliana post-transcriptional gene silencing (PTGS) sgs2 and sgs3 mutants. In populations of first generation (T1) A. thaliana plants transformed with a beta-glucuronidase (GUS) gene (uidA) driven by the 35S cauliflower mosaic virus promoter (p35S), the incidence of highly expressing transformants shifted from 20% in wild type background to 100% in sgs2 and sgs3 backgrounds. Likewise, when sgs2 mutants were transformed with a cyclin-dependent kinase inhibitor 6 gene under control of p35S, all transformants showed a clear phenotype typified by serrated leaves, whereas such phenotype was only observed in about one of five wild type transformants. p35S-driven uidA expression remained high and steady in T2 sgs2 and sgs3 transformants, in marked contrast to the variable expression patterns observed in wild type T2 populations. We further show that T-DNA constructs flanked by matrix attachment regions of the chicken lysozyme gene (chiMARs) cause a boost in GUS activity by fivefold in sgs2 and 12-fold in sgs3 plants, reaching up to 10% of the total soluble proteins, whereas no such boost is observed in the wild type background. MAR-based plant transformation vectors used in a PTGS mutant background might be of high value for efficient high-throughput screening of transgene-based phenotypes as well as for obtaining extremely high transgene expression in plants.

Research paper thumbnail of Site‐specific T–DNA integration in Arabidopsis thaliana mediated by the combined action of CRE recombinase and ϕC31 integrase

The Plant Journal, 2013

SummaryRandom T–DNA integration into the plant host genome can be problematic for a variety of re... more SummaryRandom T–DNA integration into the plant host genome can be problematic for a variety of reasons, including potentially variable transgene expression as a result of different integration positions and multiple T–DNA copies, the risk of mutating the host genome and the difficulty of stacking well‐defined traits. Therefore, recombination systems have been proposed to integrate the T–DNA at a pre‐selected site in the host genome. Here, we demonstrate the capacity of the ϕC31 integrase (INT) for efficient targeted T–DNA integration. Moreover, we show that the iterative site‐specific integration system (ISSI), which combines the activities of the CRE recombinase and INT, enables the targeting of genes to a pre‐selected site with the concomitant removal of the resident selectable marker. To begin, plants expressing both the CRE and INT recombinase and containing the target attP site were constructed. These plants were supertransformed with a T–DNA vector harboring the loxP site, the...

Research paper thumbnail of Transitive RNA silencing signals induce cytosine methylation of a transgenic but not an endogenous target

The Plant Journal, 2013

SummaryPost‐transcriptional gene silencing of a primary target gene in plants can coincide with t... more SummaryPost‐transcriptional gene silencing of a primary target gene in plants can coincide with the production of secondary small interfering RNAs (siRNAs) of coding sequences adjacent to the target region and with de novo RNA‐directed DNA methylation (RdDM) thereof. Here, we analyzed the susceptibility of transgenic and endogenous targets to RdDM induced by primary and secondary silencing signals. In three different configurations, primary silencing signals were able to direct in trans methylation of chimeric transgenes and the CATALASE2 (CAT2) endogene; however, extensive spreading of methylation occurred only in the transgene, resulting in the methylation of the flanking CAT2 sequence, whereas methylation of the CAT2 endogene was restricted to the target region and the enclosed introns. The secondary silencing signals arising from this transgenic primary target simultaneously silenced a secondary transgene target and the CAT2 endogene, but were only capable of directing RdDM to t...

Research paper thumbnail of Introns reduce transitivity proportionally to their length, suggesting that silencing spreads along the pre-mRNA

Research paper thumbnail of T-DNA transfer and T-DNA integration efficiencies upon Arabidopsis thaliana root explant cocultivation and floral dip transformation

Research paper thumbnail of Cell Culture-Induced Gradual and Frequent Epigenetic Reprogramming of Invertedly Repeated Tobacco Transgene Epialleles  

Plant Physiology, 2009

Using a two-component transgene system involving two epiallelic variants of the invertedly repeat... more Using a two-component transgene system involving two epiallelic variants of the invertedly repeated transgenes in locus 1 (Lo1) and a homologous single-copy transgene locus 2 (Lo2), we have studied the stability of the methylation patterns and trans-silencing interactions in cell culture and regenerated tobacco (Nicotiana tabacum) plants. The posttranscriptionally silenced (PTGS) epiallele of the Lo1 trans-silences and trans-methylates the target Lo2 in a hybrid (Lo1/Lo2 line), while its transcriptionally silenced variant (Lo1E) does not. This pattern was stable over several generations in plants. However, in early Lo1E/Lo2 callus, decreased transgene expression and partial loss of Lo1E promoter methylation compared with leaf tissue in the parental plant were observed. Analysis of small RNA species and coding region methylation suggested that the transgenes were silenced by a PTGS mechanism. The Lo1/Lo2 line remained silenced, but the nonmethylated Lo1 promoter acquired partial meth...

Research paper thumbnail of The influence of matrix attachment regions on transgene expression in Arabidopsis thaliana wild type and gene silencing mutants

Plant Molecular Biology, 2006

Research paper thumbnail of Stability of the T-DNA flanking regions in transgenic Arabidopsis thaliana plants under influence of abiotic stress and cultivation practices

Research paper thumbnail of Non‐food/feed seeds as biofactories for the high‐yield production of recombinant pharmaceuticals

Plant Biotechnology Journal, 2011

SummaryWe describe an attractive cloning system for the seed‐specific expression of recombinant p... more SummaryWe describe an attractive cloning system for the seed‐specific expression of recombinant proteins using three non‐food/feed crops. A vector designed for direct subcloning by Gateway® recombination was developed and tested in Arabidopsis, tobacco and petunia plants for the production of a chimeric form (GAD67/65) of the 65 kDa isoform of glutamic acid decarboxylase (GAD65). GAD65 is one of the major human autoantigens involved in type 1 diabetes (T1D). The murine anti‐inflammatory cytokine interleukin‐10 (IL‐10) was expressed with the described system in Arabidopsis and tobacco, whereas proinsulin, another T1D major autoantigen, was expressed in Arabidopsis. The cost‐effective production of these proteins in plants could allow the development of T1D prevention strategies based on the induction of immunological tolerance. The best yields were achieved in Arabidopsis seeds, where GAD67/65 reached 7.7% of total soluble protein (TSP), the highest levels ever reported for this prot...

Research paper thumbnail of Expression of chimaeric genes transferred into plant cells using a Ti-plasmid-derived vector

Research paper thumbnail of Effect of T-DNA configuration on transgene expression

Molecular and General Genetics MGG, 1992

Research paper thumbnail of Expression of antibodies and Fab fragments in transgenic potato plants: a case study for bulk production in crop plants

Molecular Breeding, 2002

To use transgenic potato tubers (Solanum tuberosum cv. Désirée) for bulk production of recombinan... more To use transgenic potato tubers (Solanum tuberosum cv. Désirée) for bulk production of recombinant antibodies, constructs were engineered for accumulating full-size IgGs and Fab fragments in the plant cell apoplast or endoplasmic reticulum (ER). An in-house transformation protocol was worked out for the efficient co-transformation of potato root explants. Accumulation levels in tubers of up to 0.5% of total soluble

Research paper thumbnail of Different 5′ leader sequences modulate β-glucuronidase accumulation levels in transgenic Nicotiana tabacum plants

Euphytica, 1995

Summary Three random synthetic leaders and three naturally-occurring leaders, the tobacco mosaic... more Summary Three random synthetic leaders and three naturally-occurring leaders, the tobacco mosaic virus (TMV) coat protein, the satellite tobacco necrosis virus (STNV) and the plant chlorophyll a/b-binding protein (Cab22L), were shown to modulate the β-glucuronidase reporter protein accumulation levels in transient expression experiments. The same chimeric constructs also confer differential distribution patterns of reporter protein accumulation in stably-transformed tobacco calli or

Research paper thumbnail of Optimized t-dna transfer and vectors therefor

Research paper thumbnail of Plants as bioreactors for protein production: avoiding the problem of transgene silencing

Plant Gene Silencing, 2000

Research paper thumbnail of Production of monoclonal antibodies with a controlled N-glycosylation pattern in seeds of Arabidopsis thaliana

Plant biotechnology journal, 2011

Seed-specific expression is an appealing alternative technology for the production of recombinant... more Seed-specific expression is an appealing alternative technology for the production of recombinant proteins in transgenic plants. Whereas attractive yields of recombinant proteins have been achieved by this method, little attention has been paid to the intracellular deposition and the quality of such products. Here, we demonstrate a comparative study of two antiviral monoclonal antibodies (mAbs) (HA78 against Hepatitis A virus; 2G12 against HIV) expressed in seeds of Arabidopsis wild-type (wt) plants and glycosylation mutants lacking plant specific N-glycan residues. We demonstrate that 2G12 is produced with complex N-glycans at great uniformity in the wt as well as in the glycosylation mutant, carrying a single dominant glycosylation species, GnGnXF and GnGn, respectively. HA78 in contrast, contains additionally to complex N-glycans significant amounts of oligo-mannosidic structures, which are typical for endoplasmic reticulum (ER)-retained proteins. A detailed subcellular localizat...

Research paper thumbnail of Callus-induced epiallelism of an invertedly repeated transgene locus influences its trans-silencing abilities

GBM Annual Spring meeting Mosbach 2008, 2008

Research paper thumbnail of Transgene Expression in Plants, Control of

Sustainable Food Production, 2013

Research paper thumbnail of Production of Camel-Like Antibodies in Plants

Methods in Molecular Biology, 2012

Transgenic plants for the production of high-value recombinant complex and/or glycosylated protei... more Transgenic plants for the production of high-value recombinant complex and/or glycosylated proteins are a promising alternative for conventional systems, such as mammalian cells and bacteria. Many groups use plants as production platform for antibodies and antibody fragments. Here, we describe how bivalent camel-like antibodies can be produced in leaves and seeds. Camel-like antibodies are fusions of the antigen-binding domain of heavy chain camel antibodies (VHH) with an Fc fragment of choice. Transient expression in Nicotiana benthamiana leaves allows the production of VHH-Fc antibodies within a few days after the expression plasmid has been obtained. Generation of stable Arabidopsis thaliana transformants allows production of scalable amounts of VHH-Fc antibodies in seeds within a year. Further, we describe how the in planta-produced VHH-Fc antibodies can be quantified by Western blot analysis with Fc-specific antibodies.

Research paper thumbnail of Nanobody-based products as research and diagnostic tools

Trends in Biotechnology, 2014

Research paper thumbnail of Stable high-level transgene expression inArabidopsis thalianausing gene silencing mutants and matrix attachment regions

The Plant Journal, 2004

Basic and applied research involving transgenic plants often requires consistent high-level expre... more Basic and applied research involving transgenic plants often requires consistent high-level expression of transgenes. However, high inter-transformant variability of transgene expression caused by various phenomena, including gene silencing, is frequently observed. Here, we show that stable, high-level transgene expression is obtained using Arabidopsis thaliana post-transcriptional gene silencing (PTGS) sgs2 and sgs3 mutants. In populations of first generation (T1) A. thaliana plants transformed with a beta-glucuronidase (GUS) gene (uidA) driven by the 35S cauliflower mosaic virus promoter (p35S), the incidence of highly expressing transformants shifted from 20% in wild type background to 100% in sgs2 and sgs3 backgrounds. Likewise, when sgs2 mutants were transformed with a cyclin-dependent kinase inhibitor 6 gene under control of p35S, all transformants showed a clear phenotype typified by serrated leaves, whereas such phenotype was only observed in about one of five wild type transformants. p35S-driven uidA expression remained high and steady in T2 sgs2 and sgs3 transformants, in marked contrast to the variable expression patterns observed in wild type T2 populations. We further show that T-DNA constructs flanked by matrix attachment regions of the chicken lysozyme gene (chiMARs) cause a boost in GUS activity by fivefold in sgs2 and 12-fold in sgs3 plants, reaching up to 10% of the total soluble proteins, whereas no such boost is observed in the wild type background. MAR-based plant transformation vectors used in a PTGS mutant background might be of high value for efficient high-throughput screening of transgene-based phenotypes as well as for obtaining extremely high transgene expression in plants.

Research paper thumbnail of Site‐specific T–DNA integration in Arabidopsis thaliana mediated by the combined action of CRE recombinase and ϕC31 integrase

The Plant Journal, 2013

SummaryRandom T–DNA integration into the plant host genome can be problematic for a variety of re... more SummaryRandom T–DNA integration into the plant host genome can be problematic for a variety of reasons, including potentially variable transgene expression as a result of different integration positions and multiple T–DNA copies, the risk of mutating the host genome and the difficulty of stacking well‐defined traits. Therefore, recombination systems have been proposed to integrate the T–DNA at a pre‐selected site in the host genome. Here, we demonstrate the capacity of the ϕC31 integrase (INT) for efficient targeted T–DNA integration. Moreover, we show that the iterative site‐specific integration system (ISSI), which combines the activities of the CRE recombinase and INT, enables the targeting of genes to a pre‐selected site with the concomitant removal of the resident selectable marker. To begin, plants expressing both the CRE and INT recombinase and containing the target attP site were constructed. These plants were supertransformed with a T–DNA vector harboring the loxP site, the...

Research paper thumbnail of Transitive RNA silencing signals induce cytosine methylation of a transgenic but not an endogenous target

The Plant Journal, 2013

SummaryPost‐transcriptional gene silencing of a primary target gene in plants can coincide with t... more SummaryPost‐transcriptional gene silencing of a primary target gene in plants can coincide with the production of secondary small interfering RNAs (siRNAs) of coding sequences adjacent to the target region and with de novo RNA‐directed DNA methylation (RdDM) thereof. Here, we analyzed the susceptibility of transgenic and endogenous targets to RdDM induced by primary and secondary silencing signals. In three different configurations, primary silencing signals were able to direct in trans methylation of chimeric transgenes and the CATALASE2 (CAT2) endogene; however, extensive spreading of methylation occurred only in the transgene, resulting in the methylation of the flanking CAT2 sequence, whereas methylation of the CAT2 endogene was restricted to the target region and the enclosed introns. The secondary silencing signals arising from this transgenic primary target simultaneously silenced a secondary transgene target and the CAT2 endogene, but were only capable of directing RdDM to t...

Research paper thumbnail of Introns reduce transitivity proportionally to their length, suggesting that silencing spreads along the pre-mRNA

Research paper thumbnail of T-DNA transfer and T-DNA integration efficiencies upon Arabidopsis thaliana root explant cocultivation and floral dip transformation

Research paper thumbnail of Cell Culture-Induced Gradual and Frequent Epigenetic Reprogramming of Invertedly Repeated Tobacco Transgene Epialleles  

Plant Physiology, 2009

Using a two-component transgene system involving two epiallelic variants of the invertedly repeat... more Using a two-component transgene system involving two epiallelic variants of the invertedly repeated transgenes in locus 1 (Lo1) and a homologous single-copy transgene locus 2 (Lo2), we have studied the stability of the methylation patterns and trans-silencing interactions in cell culture and regenerated tobacco (Nicotiana tabacum) plants. The posttranscriptionally silenced (PTGS) epiallele of the Lo1 trans-silences and trans-methylates the target Lo2 in a hybrid (Lo1/Lo2 line), while its transcriptionally silenced variant (Lo1E) does not. This pattern was stable over several generations in plants. However, in early Lo1E/Lo2 callus, decreased transgene expression and partial loss of Lo1E promoter methylation compared with leaf tissue in the parental plant were observed. Analysis of small RNA species and coding region methylation suggested that the transgenes were silenced by a PTGS mechanism. The Lo1/Lo2 line remained silenced, but the nonmethylated Lo1 promoter acquired partial meth...

Research paper thumbnail of The influence of matrix attachment regions on transgene expression in Arabidopsis thaliana wild type and gene silencing mutants

Plant Molecular Biology, 2006

Research paper thumbnail of Stability of the T-DNA flanking regions in transgenic Arabidopsis thaliana plants under influence of abiotic stress and cultivation practices

Research paper thumbnail of Non‐food/feed seeds as biofactories for the high‐yield production of recombinant pharmaceuticals

Plant Biotechnology Journal, 2011

SummaryWe describe an attractive cloning system for the seed‐specific expression of recombinant p... more SummaryWe describe an attractive cloning system for the seed‐specific expression of recombinant proteins using three non‐food/feed crops. A vector designed for direct subcloning by Gateway® recombination was developed and tested in Arabidopsis, tobacco and petunia plants for the production of a chimeric form (GAD67/65) of the 65 kDa isoform of glutamic acid decarboxylase (GAD65). GAD65 is one of the major human autoantigens involved in type 1 diabetes (T1D). The murine anti‐inflammatory cytokine interleukin‐10 (IL‐10) was expressed with the described system in Arabidopsis and tobacco, whereas proinsulin, another T1D major autoantigen, was expressed in Arabidopsis. The cost‐effective production of these proteins in plants could allow the development of T1D prevention strategies based on the induction of immunological tolerance. The best yields were achieved in Arabidopsis seeds, where GAD67/65 reached 7.7% of total soluble protein (TSP), the highest levels ever reported for this prot...

Research paper thumbnail of Expression of chimaeric genes transferred into plant cells using a Ti-plasmid-derived vector

Research paper thumbnail of Effect of T-DNA configuration on transgene expression

Molecular and General Genetics MGG, 1992

Research paper thumbnail of Expression of antibodies and Fab fragments in transgenic potato plants: a case study for bulk production in crop plants

Molecular Breeding, 2002

To use transgenic potato tubers (Solanum tuberosum cv. Désirée) for bulk production of recombinan... more To use transgenic potato tubers (Solanum tuberosum cv. Désirée) for bulk production of recombinant antibodies, constructs were engineered for accumulating full-size IgGs and Fab fragments in the plant cell apoplast or endoplasmic reticulum (ER). An in-house transformation protocol was worked out for the efficient co-transformation of potato root explants. Accumulation levels in tubers of up to 0.5% of total soluble

Research paper thumbnail of Different 5′ leader sequences modulate β-glucuronidase accumulation levels in transgenic Nicotiana tabacum plants

Euphytica, 1995

Summary Three random synthetic leaders and three naturally-occurring leaders, the tobacco mosaic... more Summary Three random synthetic leaders and three naturally-occurring leaders, the tobacco mosaic virus (TMV) coat protein, the satellite tobacco necrosis virus (STNV) and the plant chlorophyll a/b-binding protein (Cab22L), were shown to modulate the β-glucuronidase reporter protein accumulation levels in transient expression experiments. The same chimeric constructs also confer differential distribution patterns of reporter protein accumulation in stably-transformed tobacco calli or