TY - JOUR
T1 - Resources for targeted insertional and deletional mutagenesis in Arabidopsis
AU - Zhang, Sen
AU - Raina, Surabhi
AU - Li, Hong
AU - Li, Jun
AU - Dec, Ewa
AU - Ma, Hong
AU - Huang, Hai
AU - Fedoroff, Nina V.
N1 - Funding Information:
We would like to thank M. Zeng for seed stock organization and maintenance, L. Xu for technical support, and the Shanghai Scientific Committee for partial financial support. This work was supported by a grant from the State Key Program of Basic Research (973), and a grant from the Chinese Ministry of Science and Technology (863), to H.H. H.M. gratefully acknowledges the support of K.C. Wong Educational Foundation, the Foreign Distinguished Young Scholar Award, and a grant from the Shanghai Municipal Office for Introduction of Foreign Experts for supporting collaborations with H.H. This work was also supported by the endowment associated with the Willaman Chair in Life Sciences at the Pennsylvania State University.
PY - 2003/9
Y1 - 2003/9
N2 - The maize transposons Activator (Ac) and Dissociation (Ds) are active in many monocots and dicots, including Arabidopsis. We describe a new Ac-derived transposon construct, designated the Ds-loxP T-DNA, which can be used for both insertional and deletional mutagenesis. There are loxP sites in both orientations on both the transposon and the donor site T-DNA and an arrangement of marker genes that permits selection of transposition events, as well as deletions and inversions extending from the donor site to a transposon reinserted on either side of it. We show that Cre-mediated deletions and inversions occur at a high frequency. The tendency of Ac-Ds transposons to reinsert near the donor site can be used to target both insertional and deletional mutagenesis, but efficient exploitation of this property requires a library of mapped marked donor sites distributed in the genome. We have created a population of independent Ds T-DNA transformants and we have mapped an initial set of 75 Ds T-DNA integration sites. We assessed the potential efficiency of targeted mutagenesis by detecting Ds reinsertion events at several loci over a 400 kb interval from each of two donor sites with different Ds T-DNA constructs. The distribution of reinsertion sites is similar around the two tested loci, with roughly 10, 4, and ca. 1% of reinsertions detected within 1-2 kb of sites 10, 100, and 200-400 kb from the donor site, respectively. To facilitate the use of this targeted mutagenesis system, we have constructed a searchable database of the mapped Ds T-DNA integration sites.
AB - The maize transposons Activator (Ac) and Dissociation (Ds) are active in many monocots and dicots, including Arabidopsis. We describe a new Ac-derived transposon construct, designated the Ds-loxP T-DNA, which can be used for both insertional and deletional mutagenesis. There are loxP sites in both orientations on both the transposon and the donor site T-DNA and an arrangement of marker genes that permits selection of transposition events, as well as deletions and inversions extending from the donor site to a transposon reinserted on either side of it. We show that Cre-mediated deletions and inversions occur at a high frequency. The tendency of Ac-Ds transposons to reinsert near the donor site can be used to target both insertional and deletional mutagenesis, but efficient exploitation of this property requires a library of mapped marked donor sites distributed in the genome. We have created a population of independent Ds T-DNA transformants and we have mapped an initial set of 75 Ds T-DNA integration sites. We assessed the potential efficiency of targeted mutagenesis by detecting Ds reinsertion events at several loci over a 400 kb interval from each of two donor sites with different Ds T-DNA constructs. The distribution of reinsertion sites is similar around the two tested loci, with roughly 10, 4, and ca. 1% of reinsertions detected within 1-2 kb of sites 10, 100, and 200-400 kb from the donor site, respectively. To facilitate the use of this targeted mutagenesis system, we have constructed a searchable database of the mapped Ds T-DNA integration sites.
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U2 - 10.1023/B:PLAN.0000009271.08420.d9
DO - 10.1023/B:PLAN.0000009271.08420.d9
M3 - Article
C2 - 14756312
AN - SCOPUS:1642506197
SN - 0167-4412
VL - 53
SP - 133
EP - 150
JO - Plant molecular biology
JF - Plant molecular biology
IS - 1-2
ER -