omb3dyw

IMPRECISION & UNPREDICTABILITY OF S & T OF GENETIC ENGINEERINGnumber, linkage and transmission of two transgenes inserted in the same T-DNA. Thirty-four independent transgenic lines were categorised into three types:type I events (38% of total) where the gusA and bar genes displayed completegenetic linkage, segregating together as a single functional locus at the expectedratio of 3:1; type II events (18%), which possessed two or more transgene locieach containing gusA and bar; and type III events (44%), containing an incompleteT-DNA in which either the gusA or bar gene was lost. Most lines in this last categoryhad lost the bar gene situated near the left T-DNA border. Southern analysisindicated that 30% of all lines possessed a single T-DNA copy containing gusAand bar. However, when data on expression and molecular analysis are combined,only 23% of all lines have single copy T-DNAs in which both gene cassettes arefunctioning. We also report on the presence of plasmid backbone DNA sequencein transgene loci detected using primer pairs outside the left and right T-DNAborders and within the plasmid selectable marker (NptI) gene. Approximately twothirds of the lines contained some vector backbone DNA, more frequently adjacentto the left border. Taken together, these data imply unstable left border functioncausing premature T-strand termination or read-through into vector backbone. Asfar as we are aware, this is the first report revealing near border T-DNA truncationand vector backbone integration in wheat transgenic lines produced byAgrobacterium-mediated transformation.26. Jackson, Aimee L., Burchard, Julja, Schelter, Janell, Chau, B. Nelson, Cleary,Michele, Lim, Lee & Linsley, Peter S. (2006) : Widespread siRNA “offtarget”transcript silencing mediated by seed region sequencecomplementarity. RNA 12 : 1179-1187.Transfected siRNAs and miRNAs regulate numerous transcripts that have onlylimited complementarity to the active strand of the RNA duplex. This processreflects natural target regulation by miRNAs, but is an unintended (“off-target”)consequence of siRNA-mediated silencing. Here we demonstrate that thisunintended off-target silencing is widespread, and occurs in a manner reminiscentof target silencing by miRNAs. A high proportion of unintended transcripts silencedby siRNAs showed 3' UTR sequence complementarity to the seed region of thesiRNA. Base mismatches within the siRNA seed region reduced the set of originaloff-target transcripts but generated new sets of silenced transcripts with sequencecomplementarity to the mismatched seed sequence. An inducible shRNA silenceda subset of transcripts that were silenced by an siRNA of the same sequence,demonstrating that unintended silencing is sequence mediated and isindependent of delivery method. In all cases, off-target transcript silencing wasaccompanied by loss of the corresponding protein and occurred with dependenceon siRNA concentration similar to that of silencing of the target transcript. Thus,short stretches of sequence complementarity to the siRNA or shRNA seed regionare key to the silencing of unintended transcripts.http://rnajournal.cshlp.org/content/12/7/1179.short(15)