High-level transgene expression in plant cells: effects of a strong scaffold attachment region from tobacco.

Abstract

We have previously shown that yeast scaffold attachment regions (SARs) flanking a chimeric 0-glucuronidase (GUS) reporter gene increased per-copy expression levels by 24-fold in tobacco suspension cell lines stably transformed by microprojectile bombardment. In this study, we examined the effect of a DNA fragment originally identified in a tobacco genomic clone by its activity in an in vitro binding assay. The tobacco SAR has much greater scaffold binding affinity than does the yeast SAR, and tobacco cell lines stably transformed with constructs containing the tobacco SAR accumulated greater than fivefold more GUS enzyme activity than did lines transformed with the yeast SAR construct. Relative to the control construct, flanking the GUS gene with plant SARs increased overall expression per transgene copy by almost 140-fold.In transient expression assays, the same construct increased expression only approximately threefold relative to a control without SARs, indicating that the full SAR effect requires integration into chromosomal DNA. GUS activity in individual stable transformants was not simply proportional to transgene copy number, and the SAR effect was maximal in cell lines with fewer than 4 0 transgene copies per tobacco genome. Lines with significantly higher copy numbers showed greatly reduced expression relative to the low-copy-number lines. Our results indicate that strong SARs flanking a transgene greatly increase expression without eliminating variation between transformants. We propose that SARs dramatically reduce the severity or likelihood of homology-dependent gene silencing in cells with small numbers of transgenes but do not prevent silencing of transgenes present in many copies.

Flank matrix attachment regions (MARs) from chicken, bean, yeast or tobacco do not prevent homology-dependent trans-silencing in transgenic tobacco plants

“…Plants carrying this construct were then obtained by Agrobacterium-mediated leaf-disc transformation. The p35S-uidA-tNos transgene¯anked by MARs derived from the 3¢ region of the tobacco RB7 gene (Allen et al 1996) was cloned in pBluescript II SK+. Plants carrying this construct were obtained by microprojectile co-bombardment of young seedlings, using a selection plasmid carrying the pNos-Npt-tOcs selectable marker.…”

“…The chicken lyzozyme A MAR resulted in a signi®cant reduction in variation of gene expression, but copy-number dependence was shown to depend on the promoter used (Mlynarova et al 1994(Mlynarova et al , 1995. Yeast ARS-1 or tobacco Rb7 MARs increased the average expression in transgenic tobacco cell lines but the expression level was not proportional to transgene copy number (Allen et al 1993(Allen et al , 1996.…”

Post-transcriptional gene-silencing and RNA interference: genetic immunity, mechanisms and applications

“…29 Matrix attachment regions are AT-rich regions of DNA which are thought to anchor DNA to the nuclear matrix and insulate genes from chromatinmediated repression. 30 Interestingly, the tobacco MAR did not overcome co-suppression induced by multi-copy transgenes, indicating that the most important feature of a single copy transgene locus that determines its ability to trigger co-suppression is its insertional position within the genome (position effect).…”

“…30 Interestingly, the tobacco MAR did not overcome co-suppression induced by multi-copy transgenes, indicating that the most important feature of a single copy transgene locus that determines its ability to trigger co-suppression is its insertional position within the genome (position effect). 29 In turn, this indicates that co-suppression may be a consequence of a high rate of transcription from a heterochromatic template (Fig 4). How transgene transcription from such unfavourable genomic positions results in co-suppression is unclear, but Silenced transgenes can act in trans and inactivate distant, genetically unlinked loci that share homology.…”

The matrix attachment regions (MARs) associated with the Heat Shock Cognate 80 gene (HSC80) of tomato represent specific regulatory elements

“…Early experiments in animal cells suggested that the addition of MARs to transgenes could alleviate the problems associated with transgene regulation and variability of expression (Stief et al 1989;Bonifer et al 1990;Phi-Van et al 1990). Although several studies have investigated the eects of MARs on transgene expression in plants (Breyne et al 1992;Allen et al 1993Allen et al , 1996Mlyna rova et al 1994Mlyna rova et al , 1995Mlyna rova et al , 1996van der Geest et al 1994;Han et al 1997;van der Geest and Hall 1997;Liu and Tabe 1998;Vaucheret et al 1998;Levee et al 1999;Ulker et al 1999;Vain et al 1999), it is dicult to derive general rules for the application of MARs. For example, the results of these studies varied from a two-fold decrease in expression with a ®fteen-fold decrease in expression variation (Breyne et al 1992) to a 140-fold increase in expression with no change in transgene variation (Allen et al 1996).…”