The Southern blot analysis of the silencing affected lines ICE 4. 4. 1, PNA 1. 2. 1 and PNA 10. 1. 1 indicated in the XbaI digest no learn more evi dence for abnormalities, but the digest with EcoRV indi cated two T DNA insertions Inhibitors,Modulators,Libraries for all three lines. Unusually, the second Inhibitors,Modulators,Libraries T DNA fragment showed nearly the same size in all three independently transformed lines. Since the fragment size resembles the size of the entire transgenic cassette from left to right border this indicates the integration of two T DNA copies adjacent to each other, which could be responsible for the observed transgene silencing in these lines. However, multiple T DNA copies at two independent loci can be also identified much earlier in the screening process by their unusual segregation rate.
In our dataset, only a very small portion of lines showed a segregation rate around 6. 25% in the T1 stage and were considered as harboring transgenes at two independent loci. This en ables an early exclusion of these lines from the further screening process. The Southern blot indicated for most of the analyzed lines only single T DNA insertions, includ Inhibitors,Modulators,Libraries ing the stable control lines. Sensitive Inhibitors,Modulators,Libraries seedlings showed increased NOS promoter methylation Unwanted or unintended transgene silencing was com monly associated with an increase in methylation within the promoter region of the transgene. Since we found evidence for epigenetic gene silencing, we analyzed promoter methylation levels in the transgenic cassette by bisulfite sequencing. Seed lings from line ICE 10.
1 showed a transitional loss of hygromycin resistance and we separated hygromycin sensitive and hygromycin resistant seedlings to compare NOS promoter methylation Inhibitors,Modulators,Libraries levels within a 294 bp fragment. Among these isogenic seedlings, the resistant pheno types were consistent with the methylation levels and sensitive seedlings had increased methylation levels, par ticular in the CHG and CHH sites. Interestingly, the CTG at the 84th position was entirely methylation free in resistant seedlings, but to 100% methylated in sensitive seedlings. Since this site is located dir ectly downstream of a CCAAT box it appears to be particularly important for the transcription process. Hypermethylation of the 35S promoter For new the methylation analysis of the 35S promoter, indi vidual reverse primers were designed for the two differ ent expression cassettes which allowed amplification of nearly the entire 35S promoter sequence. Within the 346 bp fragment a total of 14 CG, 7 CHG and 65 CHH sites were found as potential targets for methylation. To allow the direct comparison of promoter methylation differences in T2 and T3 seedlings, all seeds were germinated on hygromycin free media. The analysis of line PNA 1.