The Distribution of Transgene Insertion Sites in Barley Determined by Physical and Genetic Mapping

Abstract

The exact site of transgene insertion into a plant host genome is one feature of the genetic transformation process that cannot, at present, be controlled and is often poorly understood. The site of transgene insertion may have implications for transgene stability and for potential unintended effects of the transgene on plant metabolism. To increase our understanding of transgene insertion sites in barley, a detailed analysis of transgene integration in independently derived transgenic barley lines was carried out. Fluorescence in situ hybridization (FISH) was used to physically map 23 transgene integration sites from 19 independent barley lines. Genetic mapping further confirmed the location of the transgenes in 11 of these lines. Transgene integration sites were present only on five of the seven barley chromosomes. The pattern of transgene integration appeared to be nonrandom and there was evidence of clustering of independent transgene insertion events within the barley genome. In addition, barley genomic regions flanking the transgene insertion site were isolated for seven independent lines. The data from the transgene flanking regions indicated that transgene insertions were preferentially located in gene-rich areas of the genome. These results are discussed in relation to the structure of the barley genome.

Mammary Specific Transgenic Over-expression of Insulin-like Growth Factor-I (IGF-I) Increases Pig Milk IGF-I and IGF Binding Proteins, with no Effect on Milk Composition or Yield

“…One possibility may be due to the site of insertion of the IGF-I transgenes between the studies. The site of transgene insertion, or its genomic environment, influences its expression and may have implications for transgene stability and potential unintended effects (Salvo-Garrido et al, 2004). The transgene insertion site was not determined herein or in previous reports (Hadsell et al, 1996;Wolfe et al, 1997).…”

Unintended consequences of plant transformation: A molecular insight

“…However, these analyses were usually done on a rather low number of transgenic lines with limited sequence information on the surrounding DNA (e.g. Salvo-Garrido et al 2004;Tagashira et al 2005). In both large scale experiments on Arabidopsis cited above promoters and UTRs were significantly more often targeted by T-DNA Alonso et al 2003).…”

Detection of transgenes in three genetically modified rice lines by fluorescence in situ hybridization

“…If molecular cytogenetic data is available in addition to the molecular evidence, it would give more accurate information for the GM plant and that will be helpful to explain the safety problems such as uncertian or unstable gene functions. FISH has been used to complement molecular data, especially to visualize single copy transgenes on chromosomes (Abranches et al, 2000;Dong et al, 2001;Jackson et al, 2001;Salvo-Garrido et al, 2001, 2004Choi et al, 2002;Svitashev et al, 2000;Romano et al 2003;Harwood et al, 2005;Travella et al, 2005;Santos et al, 2006). This technique makes it possible to map transgenes on a specific chromosome accurately.…”