Molecular and cytogenetic analyses of stably and unstably expressed transgene loci in tobacco.

Abstract

To study the influence of genomic context on transgene expression, we have determined the T-DNA structure, flanking DNA sequences, and chromosomal location of four independent transgene loci in tobacco. Two of these loci were stably expressed in the homozygous condition over many generations, whereas the other two loci became unstable after several generations of homozygosity. The stably expressed loci comprised relatively simple T-DNA arrangements that were flanked on at least one side by plant DNA containing AT-rich regions that bind to nuclear matrices in vitro. Of the unstably expressed loci, one consisted of multiple incomplete T-DNA copies, and the second contained a single intact T-DNA; in both cases, however, binary vector sequences were directly contiguous to a right T-DNA border. Fluorescence in situ hybridiration demonstrated that the two stably expressed inserts were present in the vicinity of telomeres.The two unstably expressed inserts occupied intercalary and paracentromeric locations, respectively. Results on the stability of transgene expression in F, progeny obtained by intercrossing the four lines and the sensitivity of the four transgene loci to inactivation in the presence of an unlinked " frans-silencing" locus are also presented. The findings are discussed in the context of repetitive DNA sequences and the allotetraploid nature of the tobacco genome.

Stability of transgene expression in poplar: A model forest tree species

“…Molecular analyses revealed that this line contained an elevated number of transgene copies (> 10) and extra-border vector sequences. While both of these conditions are often associated with gene-silencing events [22,23,27], the fact that 25 lines (out of the 65 transformed lines) contained extra-border sequences but still expressed the transgene would suggest that, in poplar, the presence of Ti-plasmid sequences is not a major factor in influencing transgene expression. RT-PCR experiments performed on the silent line revealed that both the uidA gene and the selection gene nptII were transcribed suggesting that silencing occurs at the posttranscriptional level.…”

Real-time PCR for the detection of precise transgene copy number in durum wheat

“…Genetic engineering of plants sometimes results in transgene silencing after integration into the host genome, which may be related to a defence mechanism against foreign DNA expression [28]. In fact, when analysing the effects of the surrounding genomic environment on transgene expression, one must also consider the potentially broader impact of the genome organization of the host plant with respect to the chromosomal distribution of structural genes, abundance, diversity, arrangement of repetitive DNA and the constitution of sub-genomes in polyploids [24]. Transgene silencing has been reported for diploid, autopolyploids and allopolyploids [25,29].…”

“…Reliable and stable expression of transgenes as well as the characterization and field adaptation of transgenic lines are prerequisites for the successful application of gene technology. Many papers have reported significant variability in the behaviour of the same transgene in different lines [24]. Loci that appear to be stably expressed initially can become progressively silenced over several generations [25].…”

Generation of transgenic sugar beet (Beta vulgarism L.) overexpressing the polygalacturonase inhibiting protein 1 of Phaseolus vulgaris (PvPGIP1) through Agrobacterium-mediated transformation

“…In comparison with the previous systems reported for sugar beet transformation (Joersbo et al, 1998;Ivic-Haymes and Smigocki, 2005), our method was characterized by higher transformation efficiency, a lower transgene copy number in plants, and a shorter period to recover transgenic plants. Furthermore, low transgene copies reduce the possibility of gene silencing and increase the stability of the transgene (Iglesias et al, 1997;Li, 2008).…”