Neither inverted repeat T‐DNA configurations nor arrangements of tandemly repeated transgenes are sufficient to trigger transgene silencing

Lechtenberg, Berthold; Schubert, Daniel; Forsbach, Alexandra; Gils, Mario; Schmidt, Renate

doi:10.1046/j.1365-313x.2003.01746.x

Cited by 106 publications

(85 citation statements)

References 55 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Alternatively, it may be envisioned that these lines have a duplicated T-DNA insertion in the respective locus so that the right border of the second insertion is oriented such that productive LUC1 fusions can be formed. Such more complex T-DNA insertion events have frequently been reported for T-DNA insertions (De Neve et al, 1997;Forsbach et al, 2003;Lechtenberg et al, 2003;Windels et al, 2003). In summary, we suggest that LucTrap-2 can be used as a gene trap vector that will allow generation of random LUC fusion proteins.…”

Section: Characterization Of a Luctrap-2 Collectionsupporting

confidence: 67%

LucTrap Vectors Are Tools to Generate Luciferase Fusions for the Quantification of Transcript and Protein Abundance in Vivo

Calderon-Villalobos

Kuhnle

et al. 2006

Plant Physiology

View full text Add to dashboard Cite

(M.R., B.W.)Proper plant growth and development strongly rely on the plant's ability to respond dynamically to signals and cues from the intra-and extracellular environment. Whereas many of these responses require specific changes at the level of gene expression, in recent years it has become increasingly clear that many plant responses are at least in part also controlled at the level of protein turnover. It is a challenge for signal transduction research to understand how distinct incoming signals are integrated to generate specific changes at the transcript or protein level. The activity of luciferase (LUC) reporters can be detected in nondestructive qualitative and quantitative assays in vivo. Therefore, LUC reporters are particularly well suited for the detection of changes at the transcript and protein level. To the best of our knowledge, the number of plant transformation vectors for LUC fusions is very limited. In this article, we describe the LucTrap plant transformation vectors that allow generation of targeted and random transcriptional and translational fusions with the modified firefly LUC reporter LUC1. We demonstrate that LucTrap-based fusions can be used to monitor rapid changes in gene expression and protein abundance in vivo.

show abstract

Section: Characterization Of a Luctrap-2 Collectionsupporting

confidence: 67%

LucTrap Vectors Are Tools to Generate Luciferase Fusions for the Quantification of Transcript and Protein Abundance in Vivo

Calderon-Villalobos

Kuhnle

et al. 2006

Plant Physiology

View full text Add to dashboard Cite

show abstract

“…The different T-DNA vectors were used for Agrobacterium tumefaciensmediated transformation of Arabidopsis Columbia-0 and single-copy T-DNA lines were identified as described Lechtenberg et al, 2003). Integrity of the reporter gene cassettes was evaluated for plants of different generations using DNA gel blot and PCR analyses.…”

Section: Molecular-genetic Analysis Of Transgenic Plantsmentioning

confidence: 99%

Silencing in Arabidopsis T-DNA Transformants: The Predominant Role of a Gene-Specific RNA Sensing Mechanism versus Position Effects

et al. 2004

Self Cite

View full text Add to dashboard Cite

Pronounced variability of transgene expression and transgene silencing are commonly observed among independent plant lines transformed with the same construct. Single-copy T-DNA lines harboring reporter genes of various kind and number under the control of a strong promoter were established in Arabidopsis thaliana for a comprehensive analysis of transgene expression. Characterization of 132 independent transgenic lines revealed no case of silencing as a result of site of T-DNA integration. Below a certain number of identical transgenes in the genome, gene copy number and expression were positively correlated. Expression was high, stable over all generations analyzed, and of a comparable level among independent lines harboring the same copy number of a particular transgene. Conversely, RNA silencing was triggered if the transcript level of a transgene surpassed a gene-specific threshold. Transcript level–mediated silencing effectively accounts for the pronounced transgene expression variability seen among transformants. It is proposed that the RNA sensing mechanism described is a genome surveillance system that eliminates RNA corresponding to excessively transcribed genes, including transgenes, and so plays an important role in genome defense

show abstract

“…While more recent studies indicated that position effects, inverted repeat T-DNA configurations, and arrangements of tandemly repeated transgenes may not be sufficient to trigger transgene silencing (Lechtenberg et al, 2003), there is strong evidence that expression levels of transgenes affect the frequencies of transgene silencing. It has been observed that highly expressing transgenes are often associated with high frequencies of transgene silencing (Lindbo et al, 1993;Vaucheret et al, 1998;Schubert et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

Improperly Terminated, Unpolyadenylated mRNA of Sense Transgenes Is Targeted by RDR6-Mediated RNA Silencing in Arabidopsis

2007

View full text Add to dashboard Cite

RNA silencing can be induced by highly transcribed transgenes through a pathway dependent on RNA-DEPENDENT RNA POLYMERASE6 (RDR6) and may function as a genome protection mechanism against excessively expressed genes. Whether all transcripts or just aberrant transcripts activate this protection mechanism is unclear. Consistent RNA silencing induced by a transgene with three direct repeats of the b-glucuronidase (GUS) open reading frame (ORF) is associated with high levels of truncated, unpolyadenylated transcripts, probably from abortive transcription elongation. Truncated, unpolyadenylated transcripts from triple GUS ORF repeats were degraded in the wild type but accumulated in an rdr6 mutant, suggesting targeting for degradation by RDR6-mediated RNA silencing. A GUS transgene without a 39 transcription terminator produced unpolyadenylated readthrough mRNA and consistent RDR6-dependent RNA silencing. Both GUS triple repeats and terminator-less GUS transgenes silenced an expressed GUS transgene in trans in the wild type but not in the rdr6 mutant. Placing two 39 terminators in the GUS transgene 39 reduced mRNA 39 readthrough, decreased GUS-specific small interfering RNA accumulation, and enhanced GUS gene expression. Moreover, RDR6 was localized in the nucleus. We propose that improperly terminated, unpolyadenylated mRNA from transgene transcription is subject to RDR6-mediated RNA silencing, probably by acting as templates for the RNA polymerase, in Arabidopsis thaliana.

show abstract

Neither inverted repeat T‐DNA configurations nor arrangements of tandemly repeated transgenes are sufficient to trigger transgene silencing

Cited by 106 publications

References 55 publications

LucTrap Vectors Are Tools to Generate Luciferase Fusions for the Quantification of Transcript and Protein Abundance in Vivo

LucTrap Vectors Are Tools to Generate Luciferase Fusions for the Quantification of Transcript and Protein Abundance in Vivo

Silencing in Arabidopsis T-DNA Transformants: The Predominant Role of a Gene-Specific RNA Sensing Mechanism versus Position Effects

Improperly Terminated, Unpolyadenylated mRNA of Sense Transgenes Is Targeted by RDR6-Mediated RNA Silencing in Arabidopsis

Contact Info

Product

Resources

About