Transposon-Mediated Single-Copy Gene Delivery Leads to Increased Transgene Expression Stability in Barley

• Published in: Plant physiology (Bethesda) 2001-03, Vol.125 (3), p.1354-1362
• Main Authors: Thomas Koprek, Sergio Rangel, McElroy, David, Jeanine D. Louwerse, Rosalind E. Williams-Carrier, Lemaux, Peggy G.
• Format: Article
• Language: English
• Subjects: Agronomy. Soil science and plant productions; bar gene; Barley; Base Sequence; Basta; Biological and medical sciences; Biotechnology; DNA; DNA Methylation; DNA Primers; DNA probes; DNA Transposable Elements; Fundamental and applied biological sciences. Psychology; Genetic engineering; Genetic engineering applications; Genetic technics; Genetic transposition; Genetics and breeding of economic plants; Genetics, Molecular Biology, and Gene Expression; Genomics; Hordeum - genetics; Hordeum vulgare; Methods. Procedures. Technologies; Methylation; Plant breeding: fundamental aspects and methodology; Plants; Plants, Genetically Modified - genetics; Promoter regions; Promoter Regions, Genetic; Transgenes; Transgenic animals and transgenic plants; Transgenic plants; transposon Ac; transposon Ds
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.125.3.1354

Instability of transgene expression in plants is often associated with complex multicopy patterns of transgene integration at the same locus, as well as position effects due to random integration. Based on maize transposable elements Activator (Ac) and Dissociation (Ds), we developed a method to generate large numbers of transgenic barley (Hordeum vulgare var Golden Promise) plants, each carrying a single transgene copy at different locations. Plants expressing Ac transposase (AcTPase) were crossed with plants containing one or more copies of bar, a selectable herbicide (Basta) resistance gene, located between inverted-repeat Ds ends (Ds-bar). F1 plants were self-pollinated and the F2 generation was analyzed to identify plants segregating for transposed Ds-bar elements. Of Ds-bar transpositions, 25% were in unlinked sites that segregated from vector sequences, other Ds-bar copies, and the AcTPase gene, resulting in numerous single-copy Ds-bar plants carrying the transgene at different locations. Transgene expression in F2 plants with transposed Ds-bar was 100% stable, whereas only 23% of F2 plants carrying Ds-bar at the original site expressed the transgene product stably. In F3 and $\text{F}_{4}$ populations, transgene expression in 81.5% of plants from progeny of F2 plants with single-copy, transposed Ds-bar remained completely stable. Analysis of the integration site in single-copy plants showed that transposed Ds-bar inserted into single- or low-copy regions of the genome, whereas silenced Ds-bar elements at their original location were inserted into redundant or highly repetitive genomic regions. Methylation of the non-transposed transgene and its promoter, as well as a higher condensation of the chromatin around the original integration site, was associated with plants exhibiting transgene silencing.