It should be explicitly men tioned that the T3 generation seeds were not collected from exactly the same plants used here as T2 generation. Both seed generations had been collected beforehand and both generations were grown simultaneously Brefeldin A ATPase adja cent Inhibitors,Modulators,Libraries to each other in the glasshouse. Regardless, the in tensity of the methylation increase was highly reproducible and the patterns from both generations matched perfectly. Among all analyzed clones certain asymmetric positions were only methylated at very low frequencies. In particular, the cytosines at the 14th and 160th position showed, for instance, 0% methyla tion in both generations. We grouped the asymmetric CHH sites into low, medium and high meth ylated positions and found that the overall methylation preference was nucleotide specific with higher probability at certain positions compared to others.
These findings were similar to the site specific preferences of asymmetric positions found in a genome wide analysis of the epigenome in Arabidopsis. The epigenetic status of the transgene was equally inherited by parental lines Since we commonly combine phenotypes Inhibitors,Modulators,Libraries of transgenic plants by crossing, we wanted to determine whether the heredity of a silenced transgene might be parent of ori gin specific. We performed reciprocal crosses between wild type and transgenic lines and tested the hemizygous offspring for hygromycin resistance. The crosses with the silencing affected lines all showed high levels of hygromycin sensitivity, independ ent of the direction of the cross revealing equal inherit ance of the silenced allele through both female and male gametes.
The crossings with the stable expressing control lines always retained their hygromycin resistance. Although crossings had, in certain cases, the potential to reduce silencing, we did not observe a reduction compared to plants Inhibitors,Modulators,Libraries produced from self pollinations. Equivalent transgene inactivation in IR lines Unwanted transgene inactivation Inhibitors,Modulators,Libraries is not restricted to sense expression lines Inhibitors,Modulators,Libraries and has been reported frequently for inverted repeat constructs, which can also lose their in trans silencing ability. In the process of producing several hundred IR lines for the targeted si lencing of endogenous N. attenuata genes involved in plant defense against herbivores, we have observed several incidents of resistance marker loss in several IR lines over the past decade.
Most recently, this was ob served in the T3 generation Perifosine FDA of the ir ACX1 line, which normally shows a reduced ability to accumulate jasmonic acid after wounding due to the in trans silencing of the endogenous acx1 gene. Consistently with our previous observations, the T3 seedlings of ir ACX1 also lost the ability to grow on hygromycin containing media. To test the general applic ability of a cell culture induced transgene reactivation we included this IR line as a candidate for the second ary regeneration process.