These selection criteria are inde pendent of the mechanism responsible for the trans gene silencing process, whether it be by kinase inhibitor Gefitinib TGS or PTGS. As long as the selected plant lines show uni form levels Inhibitors,Modulators,Libraries of gene expression and Mendelian pattern of inheritance for the resistance marker, they could be considered as stable and used for further experiments. Sense transgene silencing in Nicotiana attenuata The intensity of transgene silencing can vary greatly among different plant species. In transgenic gentian plants the 35S enhancer This study summarizes our experience in the overall oc currence of transgene silencing during the screening of N. attenuata plants and provides guidance in identifying and avoiding unstable plant lines.
Erratic occurrence and variegated phenotypes are commonly reported phenom ena of transgene silencing and have been shown in many different plant species. This was recently illustrated for N. benthamiana plants, transformed with a 35S GFP construct. These plants showed erratic and non uniform gfp expression Inhibitors,Modulators,Libraries phenotypes, which dif fered strongly among isogenic sibling plants, but also among tissues from the same plant. If no visual marker is used, as in our case, the accurate selection based on the resistance marker turns out to be extremely import ant. Here, the miscellaneous inactivation pattern could be found in the intermediate resistance stages of seed lings or so called gradual silencing. We fre quently found intermediate resistant seedlings together with a non Mendelian distribution.
We hypothesize that the gene silencing starts in the 35S promoter and then gradually spreads into the NOS promoter of the re sistance marker, as discussed in Mishiba et al. Here the advantage of a head to head orientation of both pro moters becomes clear, as it places Inhibitors,Modulators,Libraries them in close vicinity and a loss of the resistance marker would provide an ac curate harbinger of the forthcoming silencing within the expression cassette. The following three indicators were our major criteria for the early detection of plant lines affected Inhibitors,Modulators,Libraries by un wanted gene silencing unusual segregation rates with 50% of sensitive seedlings, intermediate phe notypes of seedlings with unclear levels of resistance sequence showed progressive methylation, independently of copy number and position of insertion. All tested gentian lines showed strong de novo methylation, whereas the same construct was methylated with much lower rates in N.
Inhibitors,Modulators,Libraries tabacum. Even among closely re lated Nicotiana species the spontaneous silencing of a transgene was associated with higher methylation levels in N. benthamiana more information than in N. tabacum. Similar ob servations were made with unstable transgene expres sion in N. plumbaginifolia. These reports are consistent with the hypothesis of a more rigorous gene silencing machinery in wild diploid plant species, than in the cultivated tetraploid crop.