HIGH-LEVEL TRANSGENE EXPRESSION IN PLANT-CELLS - EFFECTS OF A STRONG SCAFFOLD ATTACHMENT REGION FROM TOBACCO

We have previously shown that yeast scaffold attachment regions (SARs) flanking a chimeric beta-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 tobac co genomic clone by its activity in an in vitro binding assay, The tob acco SAR has much greater scaffold binding affinity than does the yeas t SAR, and tobacco cell lines stably transformed with constructs conta ining the tobacco SAR accumulated greater than fivefold more GUS enzym e activity than did lines transformed with the yeast SAR construct, Re lative to the control construct, flanking the GUS gene with plant SARs increased overall expression per transgene copy by almost 140-fold, I n transient expression assays, the same construct increased expression only approximately threefold relative to a control without SARs, indi cating that the full SAR effect requires integration into chromosomal DNA, GUS activity in individual stable transformants was not simply pr oportional to transgene copy number, and the SAR effect was maximal in cell lines with fewer than similar to 10 transgene copies per tobacco genome. Lines with significantly higher copy numbers showed greatly r educed expression relative to the low copy-number lines. Our results i ndicate that strong SARs flanking a transgene greatly increase express ion without eliminating variation between transformants. We propose th at SARs dramatically reduce the severity or likelihood of homology-dep endent gene silencing in cells with small numbers of transgenes but do not prevent silencing of transgenes present in many copies.