Cre/lox-mediated Site-specific Integration of Agrobacterium T-DNA in Arabidopsis Thaliana by Transient Expression of Cre

Overview

Journal Plant Mol Biol

Date 1998 Sep 25

PMID 9747847

Citations 31

Authors

A C Vergunst

P J Hooykaas

Affiliations

Soon will be listed here.

Abstract

The Cre/lox system was used to obtain targeted integration of an Agrobacterium T-DNA at a lox site in the genome of Arabidopsis thaliana. Site-specific recombinants, and not random events, were preferentially selected by activation of a silent lox-neomycin phosphotransferase (nptII) target gene. To analyse the effectiveness of Agrobacterium-mediated transfer we used T-DNA vectors harbouring a single lox sequence (this vector had to circularize at the T-DNA left- and right-border sequences prior to site-specific integration) or two lox sequences (this vector allowed circularization at the lox sequences within the T-DNA either prior to or after random integration, followed by targeting of the circularized vector), respectively. Furthermore, to control the reversibility of the integration reaction, Cre recombinase was provided transiently by using a cotransformation approach. One precise stable integrant was found amongst the recombinant calli obtained after transformation with a double-lox T-DNA vector. The results indicate that Agrobacterium-mediated transformation can be used as a tool to obtain site-specific integration.

Citing Articles

Unclasping potentials of genomics and gene editing in chickpea to fight climate change and global hunger threat.

Singh C, Kumar R, Sehgal H, Bhati S, Singhal T, Gayacharan Front Genet. 2023; 14:1085024.

PMID: 37144131 PMC: 10153629. DOI: 10.3389/fgene.2023.1085024.

Targeted DNA insertion in plants.

Dong O, Ronald P Proc Natl Acad Sci U S A. 2021; 118(22).

PMID: 34050013 PMC: 8179203. DOI: 10.1073/pnas.2004834117.

Site-specific recombinase genome engineering toolkit in maize.

Cody J, Graham N, Zhao C, Swyers N, Birchler J Plant Direct. 2020; 4(3):e00209.

PMID: 32166212 PMC: 7061458. DOI: 10.1002/pld3.209.

High efficiency Agrobacterium-mediated site-specific gene integration in maize utilizing the FLP-FRT recombination system.

Anand A, Wu E, Li Z, TeRonde S, Arling M, Lenderts B Plant Biotechnol J. 2019; 17(8):1636-1645.

PMID: 30706638 PMC: 6662307. DOI: 10.1111/pbi.13089.

Progress of targeted genome modification approaches in higher plants.

Cardi T, Stewart Jr C Plant Cell Rep. 2016; 35(7):1401-16.

PMID: 27025856 DOI: 10.1007/s00299-016-1975-1.

References

Zheng Z, Hayashimoto A, Li Z, Murai N . Hygromycin resistance gene cassettes for vector construction and selection of transformed rice protoplasts. Plant Physiol. 1991; 97(2):832-5. PMC: 1081085. DOI: 10.1104/pp.97.2.832. View

Yusibov V, Steck T, Gupta V, Gelvin S . Association of single-stranded transferred DNA from Agrobacterium tumefaciens with tobacco cells. Proc Natl Acad Sci U S A. 1994; 91(8):2994-8. PMC: 43501. DOI: 10.1073/pnas.91.8.2994. View

Sanger M, Daubert S, Goodman R . Characteristics of a strong promoter from figwort mosaic virus: comparison with the analogous 35S promoter from cauliflower mosaic virus and the regulated mannopine synthase promoter. Plant Mol Biol. 1990; 14(3):433-43. DOI: 10.1007/BF00028779. View

Hooykaas P, Schilperoort R . Agrobacterium and plant genetic engineering. Plant Mol Biol. 1992; 19(1):15-38. DOI: 10.1007/BF00015604. View

Mozo T, Hooykaas P . Factors affecting the rate of T-DNA transfer from Agrobacterium tumefaciens to Nicotiana glauca plant cells. Plant Mol Biol. 1992; 19(6):1019-30. DOI: 10.1007/BF00040533. View

Hobbs S, Kpodar P, Delong C . The effect of T-DNA copy number, position and methylation on reporter gene expression in tobacco transformants. Plant Mol Biol. 1990; 15(6):851-64. DOI: 10.1007/BF00039425. View

Marsh J, Erfle M, Wykes E . The pIC plasmid and phage vectors with versatile cloning sites for recombinant selection by insertional inactivation. Gene. 1984; 32(3):481-5. DOI: 10.1016/0378-1119(84)90022-2. View

Peach C, Velten J . Transgene expression variability (position effect) of CAT and GUS reporter genes driven by linked divergent T-DNA promoters. Plant Mol Biol. 1991; 17(1):49-60. DOI: 10.1007/BF00036805. View

Onouchi H, Nishihama R, Kudo M, Machida Y, Machida C . Visualization of site-specific recombination catalyzed by a recombinase from Zygosaccharomyces rouxii in Arabidopsis thaliana. Mol Gen Genet. 1995; 247(6):653-60. DOI: 10.1007/BF00290396. View

10.

Hoess R, Abremski K . Mechanism of strand cleavage and exchange in the Cre-lox site-specific recombination system. J Mol Biol. 1985; 181(3):351-62. DOI: 10.1016/0022-2836(85)90224-4. View

11.

Comai L, Moran P, Maslyar D . Novel and useful properties of a chimeric plant promoter combining CaMV 35S and MAS elements. Plant Mol Biol. 1990; 15(3):373-81. DOI: 10.1007/BF00019155. View

12.

Qin M, Lee E, Zankel T, Ow D . Site-specific cleavage of chromosomes in vitro through Cre-lox recombination. Nucleic Acids Res. 1995; 23(11):1923-7. PMC: 306964. DOI: 10.1093/nar/23.11.1923. View

13.

Pansegrau W, Schoumacher F, Hohn B, LANKA E . Site-specific cleavage and joining of single-stranded DNA by VirD2 protein of Agrobacterium tumefaciens Ti plasmids: analogy to bacterial conjugation. Proc Natl Acad Sci U S A. 1993; 90(24):11538-42. PMC: 48019. DOI: 10.1073/pnas.90.24.11538. View

14.

Becker D, Kemper E, Schell J, Masterson R . New plant binary vectors with selectable markers located proximal to the left T-DNA border. Plant Mol Biol. 1992; 20(6):1195-7. DOI: 10.1007/BF00028908. View

15.

Bundock P, Beijersbergen A, Hooykaas P . Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. EMBO J. 1995; 14(13):3206-14. PMC: 394382. DOI: 10.1002/j.1460-2075.1995.tb07323.x. View

16.

Sauer B . Multiplex Cre/lox recombination permits selective site-specific DNA targeting to both a natural and an engineered site in the yeast genome. Nucleic Acids Res. 1996; 24(23):4608-13. PMC: 146311. DOI: 10.1093/nar/24.23.4608. View

17.

Fukushige S, Sauer B . Genomic targeting with a positive-selection lox integration vector allows highly reproducible gene expression in mammalian cells. Proc Natl Acad Sci U S A. 1992; 89(17):7905-9. PMC: 49823. DOI: 10.1073/pnas.89.17.7905. View

18.

Citovsky V, Wong M, Zambryski P . Cooperative interaction of Agrobacterium VirE2 protein with single-stranded DNA: implications for the T-DNA transfer process. Proc Natl Acad Sci U S A. 1989; 86(4):1193-7. PMC: 286652. DOI: 10.1073/pnas.86.4.1193. View

19.

Halfter U, Morris P, Willmitzer L . Gene targeting in Arabidopsis thaliana. Mol Gen Genet. 1992; 231(2):186-93. DOI: 10.1007/BF00279790. View

20.

Gietl C, Hohn B . Mobilization of T-DNA from Agrobacterium to plant cells involves a protein that binds single-stranded DNA. Proc Natl Acad Sci U S A. 1987; 84(24):9006-10. PMC: 299680. DOI: 10.1073/pnas.84.24.9006. View