Getting the most out of publicly available T-DNA insertion lines.
In the course of several different projects, we came to realize that there is a significant amount of untapped potential in the publicly available T-DNA insertion lines. In addition to the GABI-Kat lines, which were designed specifically for activation tagging, lines from the SAIL and FLAGdb collections are also useful for this purpose. As well as the 35S promoter chosen for activation tagging in GABI-Kat lines, we found that the 1'2' bidirectional promoter is capable of activating expression of flanking genomic sequences in both GABI-Kat and SAIL lines. Thus these lines have added potential for activation tagging. We also show that these lines are capable of generating antisense transcripts and so have the potential to be used for suppression (loss/reduction of function) studies. By virtue of weak terminator sequences in some T-DNA constructs, transcript read-through from selectable markers is also possible, which again has the potential to be exploited in activation/suppression studies. Finally, we show that, by selecting and characterizing lines in which the T-DNA insertions are present specifically within introns of a target gene, an allelic series of mutants with varying levels of reduced expression can be generated, due to differences in efficiency of intron splicing. Taken together, our analyses demonstrate that there is a wealth of untapped potential within existing insertion lines for studies on gene function, and the effective exploitation of these resources is discussed.
Ulker
Bekir
B
School of Biological and Biomedical Sciences, Durham University, South Road, Durham DH1 3LE, UK.
Peiter
Edgar
E
Dixon
David P
DP
Moffat
Caroline
C
Capper
Richard
R
Bouché
Nicolas
N
Edwards
Robert
R
Sanders
Dale
D
Knight
Heather
H
Knight
Marc R
MR
D016375:Antisense Elements (Genetics); D017360:Arabidopsis / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics*; D018744:DNA, Plant / Q000235:genetics*; D018506:Gene Expression Regulation, Plant; D020868:Gene Silencing; D017343:Genes, Plant; D005822:Genetic Vectors; D018745:Genome, Plant; D016254:Mutagenesis, Insertional / Q000379:methods*; D030821:Plants, Genetically Modified / Q000235:genetics; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
D016375:Antisense Elements (Genetics); D004269:DNA, Bacterial; D018744:DNA, Plant; C036483:T-DNA