Abstract
Site-specific gene integration is a powerful technique for ensuring stable transgene expression. Transgenic plants produced by conventional transformation techniques often display highly variable transgene expression, which is mostly attributed to integration patterns consisting of multiple copies of transgene constructs. Therefore, it is desirable to generate single-copy integrations, preferably in a characterized genomic position. Precise integration of foreign genes into a selected genomic position can be obtained by employing site-specific recombination systems derived from bacteria or yeast. P1 bacteriophage Cre-lox system has been particularly successful in directing precise integration of foreign genes into “previously engineered” genomic sites. The resulting transgenic plants display stable expression through successive generations. Therefore, site-specific integration approach is useful for streamlining production of transgenic plants.
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References
Breyne P, Gheysen G, Jacobs A, Van Montagu M, Depicker A (1992) Effect of T-DNA configuration on transgene expression. Mol Gen Genet 235:389–396
Iglesias VA, Moscone EA, Papp I, Neuhuber F, Michalowski S, Phelan T, Spiker S, Matzke M, Matzke AJ (1997) Molecular and cytogenetic analyses of stably and unstably expressed transgene loci in tobacco. Plant Cell 9:1251–1264
Kumpatla SP, Hall TC (1998) Recurrent onset of epigenetic silencing in rice harboring a multi-copy transgene. Plant J 14:129–135
De Wilde C, Podevin N, Windels P, Depicker A (2001) Silencing of antibody genes in plants with single-copy transgene inserts as a result of gene dosage effects. Mol Genet Genomics 265:647–653
Elmayan T, Vaucheret H (1996) Expression of single copies of a strongly expressed 35S transgene can be silenced post-transcriptionally. Plant J 9:787–797
Jorgensen R, Snyder C, Jones JDG (1987) T-DNA is organized predominantly in inverted repeat structures in plants transformed with Agrobacterium tumefaciens C58 derivatives. Mol Gen Genet 207:471–477
Mayerhofer R, Koncz-Kalman Z, Nawrath C, Bakkeren G, Crameri A et al (1991) T-DNA integration: a mode of illegitimate recombination in plants. EMBO J 10:697–704
Kohli A, Leech M, Vain P, Laurie DA, Christou P (1998) Transgene organization in rice engineered through direct DNA transfer supports a two-phase integration mechanism mediated by the establishment of integration hot spots. Proc Natl Acad Sci USA 95:7203–7208
Pawlowski WP, Somers DA (1998) Transgenic DNA integrated into the oat genome is frequently interspersed by host DNA. Proc Natl Acad Sci USA 95:12106–12110
Cheng ZQ, Huang XQ, Ray W (2001) Comparison of biolistic and Agrobacterium-mediated transformation methods on transgene copy number and rearrangement frequency in rice. Acta Botanica Sinica 43:826–833
Shrawat AK, Lörz H (2006) Agrobacterium-mediated transformation of cereals: a promising approach crossing barriers. Plant Biotechnol J 4:575–603
Kononov ME, Bassuner B, Gelvin SB (1997) Integration of T-DNA binary vector ‘backbone’ sequences into the tobacco genome: evidence for multiple complex patterns of integration. Plant J 11:945–957
De Buck S, De Wilde C, Van Montagu M, Depicker A (2000) T-DNA vector backbone sequences are frequently integrated into the genome of transgenic plants obtained by Agrobacterium-mediated transformation. Mol Breed 6:459–468
Ulker B, Li Y, Rosso MG, Logemann E, Somssich IE, Weisshaar B (2008) T-DNA-mediated transfer of Agrobacterium tumefaciens chromosomal DNA into plants. Nat Biotechnol 26:1015–1017
Wright DA, Townsend JA, Winfrey RJ Jr, Irwin PA, Rajagopal J, Lonosky PM, Hall BD, Jondle MD, Voytas DF (2005) High-frequency homologous recombination in plants mediated by zinc-finger nucleases. Plant J 44:693–705
Ow DW (2002) Recombinase-directed plant transformation for the post-genomic era. Plant Mol Biol 48:183–200
Srivastava V, Ariza-Nieto M, Wilson AJ (2004) Cre-mediated site-specific gene integration for consistent transgene expression in rice. Plant Biotechnol J 2:169–179
Srivastava V, Ow DW (2002) Biolistic-mediated site-specific integration in rice. Mol Breed 8:345–350
Albert H, Dale EC, Lee E, Ow DW (1995) Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome. Plant J 7:649–659
Vergunst AC, Jansen LE, Hooykaas PJJ (1998) Site-specific integration of Agrobacterium T-DNA in Arabidopsis thaliana mediated by Cre recombinase. Nucleic Acids Res 26:2729–2734
Hajdukiewicz P, Svab Z, Maliga P (1994) The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989–994
Odell JT, Nagy F, Chua NH (1985) Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313:810–812
Mei C, Zhou X, Yang Y (2007) Use of RNA interference to dissect defense-signaling pathways in rice. Methods Mol Biol 354:161–171
Christensen AH, Sharrock RA, Quail PH (1992) Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Mol Biol 18:675–689
Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321–4325
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Srivastava, V. (2013). Site-Specific Gene Integration in Rice. In: Yang, Y. (eds) Rice Protocols. Methods in Molecular Biology, vol 956. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-194-3_7
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DOI: https://doi.org/10.1007/978-1-62703-194-3_7
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