Advanced search
1 file | 413.75 KB Add to list

Site-specific T-DNA integration in Arabidopsis thaliana mediated by the combined action of CRE recombinase and φC31 integrase

Annelies De Paepe (UGent) , Sylvie De Buck (UGent) , Jonah Nolf (UGent) , Els Van Lerberge (UGent) and Anna Depicker (UGent)
(2013) PLANT JOURNAL. 75(1). p.172-184
Author
Organization
Abstract
Random T-DNA integration into the plant host genome can be problematic for a variety of reasons, including potentially variable transgene expression as a result of different integration positions and multiple T-DNA copies, the risk of mutating the host genome and the difficulty of stacking well-defined traits. Therefore, recombination systems have been proposed to integrate the T-DNA at a pre-selected site in the host genome. Here, we demonstrate the capacity of the phi C31 integrase (INT) for efficient targeted T-DNA integration. Moreover, we show that the iterative site-specific integration system (ISSI), which combines the activities of the CRE recombinase and INT, enables the targeting of genes to a pre-selected site with the concomitant removal of the resident selectable marker. To begin, plants expressing both the CRE and INT recombinase and containing the target attP site were constructed. These plants were supertransformed with a T-DNA vector harboring the loxP site, the attB sites, a selectable marker and an expression cassette encoding a reporter protein. Three out of the 35 transformants obtained (9%) showed transgenerational site-specific integration (SSI) of this T-DNA and removal of the resident selectable marker, as demonstrated by PCR, Southern blot and segregation analysis. In conclusion, our results show the applicability of the ISSI system for precise and targeted Agrobacterium-mediated integration, allowing the serial integration of transgenic DNA sequences in plants.
Keywords
technical advance, ZINC-FINGER NUCLEASES, Arabidopsis, gene stacking, CRE recombinase, phi C31 integrase, site-specific integration, T-DNA, FREE TRANSGENIC PLANTS, DOUBLE-STRAND BREAKS, AGROBACTERIUM-TUMEFACIENS, HIGH-FREQUENCY, FLORAL DIP, ZYGOSACCHAROMYCES-ROUXII, HOMOLOGOUS RECOMBINATION, GENOME MODIFICATION, EFFECTOR NUCLEASES

Downloads

  • (...).pdf
    • full text
    • |
    • UGent only
    • |
    • PDF
    • |
    • 413.75 KB

Citation

Please use this url to cite or link to this publication:

MLA
De Paepe, Annelies, Sylvie De Buck, Jonah Nolf, et al. “Site-specific T-DNA Integration in Arabidopsis Thaliana Mediated by the Combined Action of CRE Recombinase and φC31 Integrase.” PLANT JOURNAL 75.1 (2013): 172–184. Print.
APA
De Paepe, Annelies, De Buck, S., Nolf, J., Van Lerberge, E., & Depicker, A. (2013). Site-specific T-DNA integration in Arabidopsis thaliana mediated by the combined action of CRE recombinase and φC31 integrase. PLANT JOURNAL, 75(1), 172–184.
Chicago author-date
De Paepe, Annelies, Sylvie De Buck, Jonah Nolf, Els Van Lerberge, and Anna Depicker. 2013. “Site-specific T-DNA Integration in Arabidopsis Thaliana Mediated by the Combined Action of CRE Recombinase and φC31 Integrase.” Plant Journal 75 (1): 172–184.
Chicago author-date (all authors)
De Paepe, Annelies, Sylvie De Buck, Jonah Nolf, Els Van Lerberge, and Anna Depicker. 2013. “Site-specific T-DNA Integration in Arabidopsis Thaliana Mediated by the Combined Action of CRE Recombinase and φC31 Integrase.” Plant Journal 75 (1): 172–184.
Vancouver
1.
De Paepe A, De Buck S, Nolf J, Van Lerberge E, Depicker A. Site-specific T-DNA integration in Arabidopsis thaliana mediated by the combined action of CRE recombinase and φC31 integrase. PLANT JOURNAL. 2013;75(1):172–84.
IEEE
[1]
A. De Paepe, S. De Buck, J. Nolf, E. Van Lerberge, and A. Depicker, “Site-specific T-DNA integration in Arabidopsis thaliana mediated by the combined action of CRE recombinase and φC31 integrase,” PLANT JOURNAL, vol. 75, no. 1, pp. 172–184, 2013.
@article{4110382,
  abstract     = {{Random T-DNA integration into the plant host genome can be problematic for a variety of reasons, including potentially variable transgene expression as a result of different integration positions and multiple T-DNA copies, the risk of mutating the host genome and the difficulty of stacking well-defined traits. Therefore, recombination systems have been proposed to integrate the T-DNA at a pre-selected site in the host genome. Here, we demonstrate the capacity of the phi C31 integrase (INT) for efficient targeted T-DNA integration. Moreover, we show that the iterative site-specific integration system (ISSI), which combines the activities of the CRE recombinase and INT, enables the targeting of genes to a pre-selected site with the concomitant removal of the resident selectable marker. To begin, plants expressing both the CRE and INT recombinase and containing the target attP site were constructed. These plants were supertransformed with a T-DNA vector harboring the loxP site, the attB sites, a selectable marker and an expression cassette encoding a reporter protein. Three out of the 35 transformants obtained (9%) showed transgenerational site-specific integration (SSI) of this T-DNA and removal of the resident selectable marker, as demonstrated by PCR, Southern blot and segregation analysis. In conclusion, our results show the applicability of the ISSI system for precise and targeted Agrobacterium-mediated integration, allowing the serial integration of transgenic DNA sequences in plants.}},
  author       = {{De Paepe, Annelies and De Buck, Sylvie and Nolf, Jonah and Van Lerberge, Els and Depicker, Anna}},
  issn         = {{0960-7412}},
  journal      = {{PLANT JOURNAL}},
  keywords     = {{technical advance,ZINC-FINGER NUCLEASES,Arabidopsis,gene stacking,CRE recombinase,phi C31 integrase,site-specific integration,T-DNA,FREE TRANSGENIC PLANTS,DOUBLE-STRAND BREAKS,AGROBACTERIUM-TUMEFACIENS,HIGH-FREQUENCY,FLORAL DIP,ZYGOSACCHAROMYCES-ROUXII,HOMOLOGOUS RECOMBINATION,GENOME MODIFICATION,EFFECTOR NUCLEASES}},
  language     = {{eng}},
  number       = {{1}},
  pages        = {{172--184}},
  title        = {{Site-specific T-DNA integration in Arabidopsis thaliana mediated by the combined action of CRE recombinase and φC31 integrase}},
  url          = {{http://dx.doi.org/10.1111/tpj.12202}},
  volume       = {{75}},
  year         = {{2013}},
}

Altmetric
View in Altmetric
Web of Science
Times cited: