title: 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 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 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 approximately 10 transgene copies per tobacco genome. Lines with significantly higher copy numbers showed greatly greatly reduced expression relative to the low-copy-number lines. Our results indicate that strong SARs flanking a transgene greatly increases 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.
authors.affiliation: Department of Botany, North Carolina State University, Raleigh 27695, USA.
authors.forename: G C
authors.initials: GC
authors.lastname: Allen
authors.forename: G
authors.initials: G
authors.lastname: Hall
authors.forename: S
authors.initials: S
authors.lastname: Michalowski
authors.forename: W
authors.initials: W
authors.lastname: Newman
authors.forename: S
authors.initials: S
authors.lastname: Spiker
authors.forename: A K
authors.initials: AK
authors.lastname: Weissinger
authors.forename: W F
authors.initials: WF
authors.lastname: Thompson
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis; D001483:Base Sequence; D002460:Cell Line; D002843:Chromatin / Q000648:ultrastructure; D003001:Cloning, Molecular; D017931:DNA Primers; D004271:DNA, Fungal; D015870:Gene Expression; D005966:Glucuronidase / Q000096:biosynthesis; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000096:biosynthesis; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014026:Nicotiana / Q000235:genetics*; D014162:Transfection
chemical_list: D002843:Chromatin; D017931:DNA Primers; D004271:DNA, Fungal; D011993:Recombinant Fusion Proteins; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; C021883:nopaline synthase; D005966:Glucuronidase