Stable high‐level transgene expression in Arabidopsis thaliana using gene silencing mutants and matrix attachment regions

Abstract

SummaryBasic 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 b-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.

Targeted Degradation of the Cyclin-Dependent Kinase Inhibitor ICK4/KRP6 by RING-Type E3 Ligases Is Essential for Mitotic Cell Cycle Progression duringArabidopsisGametogenesis

“…However, in the rhf1a rhf2a background, although the transcription level was similar or even lower, ICK4/KRP6-myc was significantly accumulated, in the two independent transgenic lines ( Figure 5E). Moreover, the accumulation of ICK4/KRP6-myc in the rhf1a rhf2a background led to serrated leaves ( Figure 5E), a previously reported phenotype that was observed in ICK4/KRP6-overexpressing plants (Zhou et al, 2002;Butaye et al, 2004). These results, together Alexander's staining analysis was performed to assess pollen grain development of wild-type plants and rhf1a rhf2a double mutants.…”

Analysis of chromatin condensation states by DNase I sensitivity assay at 500-base resolution in Arabidopsis

“…Variability of transgene expression is thus likely to be caused by factors other than chromatin condensation, such as sequence-specific gene silencing mediated by small interfering RNA and/or by increasing copy number (Butaye et al 2004;Schubert et al 2004). Approaches to eliminating these factors intrinsic to transgenes should contribute to minimizing variability of transgene expression in plants.…”

The gypsy Insulator of Drosophila melanogaster, Together With Its Binding Protein Suppressor of Hairy-Wing, Facilitate High and Precise Expression of Transgenes in Arabidopsis thaliana

“…Screening for single-copy T-DNA transformants greatly enriches for stable and high transgene expression because PTGS is thought to be particularly triggered by multiple, complexly arranged copies of transgenes (De Buck et al 2004;Nagaya et al 2005). In addition, the use of PTGS mutant backgrounds as the target of transformation has been proven to be of high value for a stable and high expression level of transgenes (Butaye et al 2004;De Bolle et al 2007). Recently, it has been shown that the CRE/loxP recombination system is capable of resolving complex T-DNA loci into single T-DNA inserts and producing a high frequency of single-copy T-DNA transformants (De Buck et al 2007;De Paepe et al 2009).…”

“…In plant transgenesis, the transgene is usually integrated randomly into the host genome. The expression level of the transgene may vary among independent lines due to a number of factors including the transgene copy number, RNA silencing, and transgene insertion site (Butaye et al 2004;De Bolle et al 2007;De Paepe et al 2009). The variation of transgene expression has complicated phenotype characterization and necessitates the analysis of multiple transgenic lines.…”