BACKGROUND: Plant transformation via Agrobacterium tumefaciens is characterized by integration of commonly low number of T-DNAs at random positions in the genome. When integrated into an active gene region, promoterless reporter genes placed near the T-DNA border sequence are frequently transcribed and even translated to reporter proteins, which is the principle of promoter- and gene-trap lines. RESULTS: Here we show that even internal promotorless regions of T-DNAs are often transcribed. Such spontaneous transcription was observed in the majority of independently transformed tobacco BY-2 lines (over 65%) and it could effectively induce silencing if an inverted repeat was present within the T-DNA. We documented that the transcription often occurred in both directions. It was not directly connected with any regulatory elements present within the T-DNAs and at least some of the transcripts were initiated outside of the T-DNA. The likeliness of this read-through transcription seemed to increase in lines with higher T-DNA copy number. Splicing and presence of a polyA tail in the transcripts indicated involvement of Pol II, but surprisingly, the transcription was able to run across two transcription terminators present within the T-DNA. Such pervasive transcription was observed with three different T-DNAs in BY-2 cells and with lower frequency was also detected in Arabidopsis thaliana. CONCLUSIONS: Our results demonstrate unexpected pervasive read-through transcription of T-DNAs. We hypothesize that it was connected with a specific chromatin state of newly integrated DNA, possibly affected by the adjacent genomic region. Although this phenomenon can be easily overlooked, it can have significant consequences when working with highly sensitive systems like RNAi induction using an inverted repeat construct, so it should be generally considered when interpreting results obtained with the transgenic technology.

• Klíčová slova
• GFP, Inverted repeat, Promoterless, RNAi, Read-through transcription, T-DNA, Tobacco BY-2 cell line,
• MeSH
• Agrobacterium tumefaciens genetika   MeSH
• Arabidopsis genetika   MeSH
• buněčné linie MeSH
• DNA bakterií genetika   MeSH
• genetická transkripce * MeSH
• geneticky modifikované rostliny MeSH
• messenger RNA genetika   MeSH
• obrácené repetice genetika   MeSH
• promotorové oblasti (genetika) genetika   MeSH
• reportérové geny MeSH
• tabák genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA bakterií MeSH
• messenger RNA MeSH
• T-DNA MeSH Prohlížeč

Transient 5-azacytidine treatment of leaf explants from potato plants with transcriptionally silenced transgenes allows de novo regeneration of plants with restored transgene expression at the whole plant level. Transgenes introduced into plant genomes frequently become silenced either at the transcriptional or the posttranscriptional level. Transcriptional silencing is usually associated with DNA methylation in the promoter region. Treatments with inhibitors of maintenance DNA methylation were previously shown to allow reactivation of transcriptionally silenced transgenes in single cells or tissues, but not at the whole plant level. Here we analyzed the effect of DNA methylation inhibitor 5-azacytidine (AzaC) on the expression of two silenced reporter genes encoding green fluorescent protein (GFP) and neomycin phosphotransferase (NPTII) in potato plants. Whereas no obvious reactivation was observed in AzaC-treated stem cuttings, transient treatment of leaf segments with 10 μM AzaC and subsequent de novo regeneration of shoots on the selective medium with kanamycin resulted in the production of whole plants with clearly reactivated expression of previously silenced transgenes. Reactivation of nptII expression was accompanied by a decrease in cytosine methylation in the promoter region of the gene. Using the plants with reactivated GFP expression, we found that re-silencing of this transgene can be accidentally triggered by de novo regeneration. Thus, testing the incidence of transgene silencing during de novo regeneration could be a suitable procedure for negative selection of transgenic lines (insertion events) which have an inclination to be silenced. Based on our analysis of non-specific inhibitory effects of AzaC on growth of potato shoots in vitro, we estimated that AzaC half-life in the culture media is approximately 2 days.

• Klíčová slova
• 5-Azacytidine, De novo regeneration, Methylation, Reactivation, TGS, Transgene silencing,
• MeSH
• azacytidin farmakologie   MeSH
• geneticky modifikované rostliny účinky léků   genetika   metabolismus   MeSH
• metylace DNA účinky léků   genetika   MeSH
• regulace genové exprese u rostlin účinky léků   genetika   MeSH
• Solanum tuberosum účinky léků   genetika   metabolismus   MeSH
• transgeny účinky léků   genetika   MeSH
• umlčování genů MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• azacytidin MeSH
• zelené fluorescenční proteiny MeSH

BACKGROUND AND AIMS: Transgenic plants represent an excellent tool for experimental plant biology and are an important component of modern agriculture. Fully understanding the stability of transgene expression is critical in this regard. Most changes in transgene expression occur soon after transformation and thus unwanted lines can be discarded easily; however, transgenes can be silenced long after their integration. METHODS: To study the long-term changes in transgene expression in potato (Solanum tuberosum), the activity of two reporter genes, encoding green fluorescent protein (GFP) and neomycin phosphotransferase (NPTII), was monitored in a set of 17 transgenic lines over 5 years of vegetative propagation in vitro. KEY RESULTS: A decrease in transgene expression was observed mainly in lines with higher initial GFP expression and a greater number of T-DNA insertions. Complete silencing of the reporter genes was observed in four lines (nearly 25 %), all of which successively silenced the two reporter genes, indicating an interconnection between their silencing. The loss of GFP fluorescence always preceded the loss of kanamycin resistance. Treatment with the demethylation drug 5-azacytidine indicated that silencing of the NPTII gene, but probably not of GFP, occurred directly at the transcriptional level. Successive silencing of the two reporter genes was also reproduced in lines with reactivated expression of previously silenced transgenes. CONCLUSIONS: We suggest a hypothetical mechanism involving the successive silencing of the two reporter genes that involves the switch of GFP silencing from the post-transcriptional to transcriptional level and subsequent spreading of methylation to the NPTII gene.

• MeSH
• geneticky modifikované rostliny genetika   růst a vývoj   MeSH
• kanamycinkinasa genetika   MeSH
• metylace DNA MeSH
• regulace genové exprese u rostlin genetika   MeSH
• Solanum tuberosum genetika   růst a vývoj   MeSH
• transgeny genetika   MeSH
• umlčování genů fyziologie   MeSH
• zelené fluorescenční proteiny genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kanamycinkinasa MeSH
• zelené fluorescenční proteiny MeSH

BACKGROUND: Phenotypic characterization of transgenic cell lines, frequently used in plant biology studies, is complicated because transgene expression in individual cells is often heterogeneous and unstable. To identify the sources and to reduce this heterogeneity, we transformed tobacco (Nicotiana tabacum L.) BY-2 cells with a gene encoding green fluorescent protein (GFP) using Agrobacterium tumefaciens, and then introduced a simple cloning procedure to generate cell lines derived from the individual transformed cells. Expression of the transgene was monitored by analysing GFP fluorescence in the cloned lines and also in lines obtained directly after transformation. RESULTS: The majority ( approximately 90%) of suspension culture lines derived from calli that were obtained directly from transformation consisted of cells with various levels of GFP fluorescence. In contrast, nearly 50% of lines generated by cloning cells from the primary heterogeneous suspensions consisted of cells with homogenous GFP fluorescence. The rest of the lines exhibited "permanent heterogeneity" that could not be resolved by cloning. The extent of fluorescence heterogeneity often varied, even among genetically identical clones derived from the primary transformed lines. In contrast, the offspring of subsequent cloning of the cloned lines was uniform, showing GFP fluorescence intensity and heterogeneity that corresponded to the original clone. CONCLUSION: The results demonstrate that, besides genetic heterogeneity detected in some lines, the primary lines often contained a mixture of epigenetically different cells that could be separated by cloning. This indicates that a single integration event frequently results in various heritable expression patterns, which are probably accidental and become stabilized in the offspring of the primary transformed cells early after the integration event. Because heterogeneity in transgene expression has proven to be a serious problem, it is highly advisable to use transgenes tagged with a visual marker for BY-2 transformation. The cloning procedure can be used not only for efficient reduction of expression heterogeneity of such transgenes, but also as a useful tool for studies of transgene expression and other purposes.

• MeSH
• buněčné klony MeSH
• buněčné kultury MeSH
• buněčné linie MeSH
• geneticky modifikované rostliny genetika   MeSH
• regulace genové exprese u rostlin * MeSH
• tabák genetika   MeSH
• technika přenosu genů * MeSH
• transformace genetická MeSH
• transgeny * MeSH
• zelené fluorescenční proteiny genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• zelené fluorescenční proteiny MeSH