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Single-site integration of foreign DNA into Arabidopsis showed rearrangements and deletions of both plant DNA and foreign DNA.

Manipulated Organism: Arabidopsis thaliana (Thale Cress; mustard family).

Inserted Transgenes: Various gene constructs containing in each case a neomycin phosphotransferase marker gene and one or more different reporter genes. Agrobacterium was used as a vector.

Goal of This Study: Investigate how a transgenic gene construct is integrated into the plant's genome.

Results of This Study: Initial screening indicated that in 80% of the transgenic lines the gene construct had been inserted at a single site (locus). About 22% of these contained a single intact copy of the gene construct. From this group, 122 lines were selected for further investigation, since the gene construct was of "sufficient length and quality to determine unambiguously the position of integration in the Arabidopsis genome" (p. 165). In connection with these 122 lines, the researchers found:
  • The gene construct was identified in all five of the plant's chromosomes; there was no apparent bias toward any particular chromosome.
  • Over half of the insertions were directly upstream or downstream from plant genes (where regulatory DNA sequences usually lie) or within plant genes. The rest were in non-coding regions.
  • Only three of 71 lines investigated contained a gene construct that corresponded precisely to the full-length strand of DNA in the originally inserted construct; in all the others there were small deletions at one or both ends of the sequence.
  • In 88.7% of the lines, the integration of the gene construct resulted in deletions of plant DNA at the site of integration. Only 2.7% "inserted exactly into the target site" (p. 168).
  • In over half the lines, short "filler sequences" of DNA were found between the borders of the construct and plant DNA that flanked those borders. In those cases in which the origin of this filler DNA could be identified, it was found to consist of DNA fragments from either the plant or the transgenic construct.
  • In two of the lines "extensive chromosomal rearrangements associated with the integration" (p. 169) of the gene construct were found. Chromosomal fragments were swapped between different chromosomes (reciprocal translocation).
The researchers summarize: "In the majority of cases, single-copy T-DNA [that is, the transgenic construct] were associated with small or large rearrangements such as deletions and/or duplications of target site sequences, deletions and/or duplications of T-DNA sequences and gross chromosomal rearrangements such as translocations" (p. 161).

Additional Comments: The authors point out that the frequency of DNA rearrangements "is likely to be even higher if transgenic lines containing multiple T-DNA inserts are analyzed" (p. 173), and in this regard refer to other research.

Source: Forsbach, A., D. Schubert, B. Lechtenberg, M. Gils et al. (2003). "A Comprehensive Characterization of Single-Copy T-DNA Insertions in the Arabidopsis thaliana Genome," Plant Molecular Biology vol. 52, pp. 161-76.

Author Affiliations: Max-Planck Institut für Molekulare Pflanzenphysiologie, Golm, Germany; Coley Pharmaceutical, Langenfeld, Germany; Icon Genetics, Halle (Saale), Germany.

Funding: German Ministry for Education and Research (BMBF) and fellowships from the Max Planck Society.

Product Status: basic research; not related to the development of a potential commercialized product.

Copyright 2008 The Nature Institute.

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