Advertisement

Marker-Free Targeted Transformation

  • Hiroyasu Ebinuma
  • Kazuya Nanto
Chapter

Abstract

There are many transformation methods available for stable integration of a desirable gene into plant cells. In transformation, variable numbers of desired genes together with marker genes are randomly inserted into the plant genome. Therefore, cumbersome screening procedures are required to identify transgenic plants with a single copy of transgenes at appropriate expression levels. However, the lack of reproducibility of expression levels limits studies of both the gene expression and physiological effects of transgenes. And remaining of marker genes precludes retransformation with the same marker system and can raise safety and public concerns. The targeting approach is the best way to solve this problem. In this chapter, we focus on the application of site-specific recombination systems for introducing a desirable gene into a predefined site in a plant genome. Furthermore, we discuss an approach for removing a marker gene from targeted transgenic plants.

Keywords

Transgenic Plant Marker Gene Target Site Plant Genome Recognition Sequence 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. Albert H, Dale E-C, Lee E et al. (1995) Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome. Plant J 7:649–659CrossRefPubMedGoogle Scholar
  2. Birch R-G (1997) Plant transformation: problems and strategies for practical application. Annu Rev Plant Physiol Plant Mol Biol 48:297–326CrossRefPubMedGoogle Scholar
  3. Chawla C, Ariza-Nieto M, Wilson A-J et al. (2006) Transgene expression produced by biolistic-mediated site-specific gene integration is consistently inherited by the subsequent generations. Plant Biotechnol J 4:209–218CrossRefPubMedGoogle Scholar
  4. Chilton M-D, Que Q (2003) Targeted integration of T-DNA into the tobacco genome at double-strand breaks: new insights on the mechanism of T-DNA integration. Plant Physiol 133:956–965CrossRefPubMedGoogle Scholar
  5. Choi S, Begum D, Koshinsky H et al. (2000) A new approach for the identification and cloning of genes: the pBACwich system using Cre/lox site-specific recombination. Nucleic Acids Res 28:E19–E26CrossRefGoogle Scholar
  6. Day C-D, Lee E, Kobayashi J et al. (2000) Transgene integration into the same chromosome location can produce alleles that express at a predictable level, or alleles that are differentially silenced. Genes Dev 14:2869–2880CrossRefPubMedGoogle Scholar
  7. De Buck S, Windels P, De Loose M et al. (2004) Single-copy T-DNAs integrated at different positions in the Arabidopsis genome display uniform and comparable B-glucuronidase accumulation levels. CMLS Cell Mol Life Sci 61:2632–2645CrossRefGoogle Scholar
  8. De Wilde C, Podevin N, Windels P et al. (2001) Silencing of antibody genes in plants with single-copy transgene inserts as a result of gene dosage effects. Mol Genet Genom 265:647–653CrossRefGoogle Scholar
  9. Ebinuma H, Nanto K (2007) Marker-free transgenic plants with a site-specific integrated copy produced by recombinase-mediated cassette exchange (RMCE). International Conference – Plant Tansformation Technologies (Abstract: pp 41). Vienna, AustriaGoogle Scholar
  10. Ebinuma H, Sugita K, Matsunaga E et al. (1997) Selection of marker-free transgenic plants using the isopentenyl transferase gene. Proc Natl Acad Sci U S A 94:2117–2121CrossRefPubMedGoogle Scholar
  11. Ebinuma H, Sugita K, Matsunaga E et al. (2001) Systems for the removal of a selection marker and their combination with a positive marker. Plant Cell Rep 20:383–392CrossRefGoogle Scholar
  12. Ebinuma H, Sugita K, Matsunaga E et al. (2004) Asexual production of marker-free transgenic aspen using MAT vector systems. In: Kumar S, Fladung M (eds) Molecular genetics and breeding of forest trees. Food Products, New York, pp 309–338Google Scholar
  13. Ebinuma H, Sugita K, Endo S et al. (2005) Elimination of marker genes from transgenic plants using MAT vector systems. In: Pena L (ed) Transgenic plants. Humana, New Jersey, pp 237–253Google Scholar
  14. Eszterhas S-K, Bouhassira E-E, Martin D-I et al. (2002) Transcriptional interference by independently regulated genes occurs in any relative arrangement of the genes and is influenced by chromosomal integration position. Mol Cell Biol 22(2):469–479CrossRefPubMedGoogle Scholar
  15. Feng Y-Q, Seibler J, Alami R et al. (1999) Site-specific chromosomal integration in mammalian cells: highly efficient Cre recombinase-mediated cassette exchange. J Mol Biol 292:779–785CrossRefPubMedGoogle Scholar
  16. Fukushige S, Sauer B (1992) Genomic targeting with a positive-selection lox integration vector allows highly reproducible gene expression in mammalian cells. Proc Natl Acad Sci U S A 89:7905–7909CrossRefPubMedGoogle Scholar
  17. Feng Y-Q, Lorincz M-C, Fiering S et al. (2001) Position effects are influenced by the orientation of a transgene with respect to flanking chromatin. Mol Cell Biol 21(1):298–309CrossRefPubMedGoogle Scholar
  18. Kim S-I, Veena, Gelvin S-B (2007) Genome-wide analysis of Agrobacterium T-DNA integration sites in the Arabidopsis genome generated under nonselective conditions. Plant J 51:779–791CrossRefPubMedGoogle Scholar
  19. Kumar S, Fladung M (2001) Gene stability in transgenic aspen (Populus). II. Molecular characterization of variable expression of transgene in wild and hybrid aspen. Planta 213:731–740CrossRefPubMedGoogle Scholar
  20. Kohli A, Twyman R-M, Abranches W et al. (2003) Transgene integration, organization and interaction in plants. Plant Mol Biol 52:247–258CrossRefPubMedGoogle Scholar
  21. Louwerse J-D, Van Lier M-C-M, Van Der Steen et al. (2007) Stable recombinase-mediated cassette exchange in Arabidopsis using Agrobacterium tumefaciens. Plant Physiol 145:1282–1293CrossRefPubMedGoogle Scholar
  22. Lyznik L-A, Gordon-Kamm W-J, Tao Y (2003) Site-specific recombination for genetic engineering in plants. Plant Cell Rep 21:925–932CrossRefPubMedGoogle Scholar
  23. Maqbool S-B, Christou P (1999) Multiple traits of agronomic importance in transgenic indica rice plants analysis of transgene integration patterns, expression levels and stability. Mol Breed 5:471–480CrossRefGoogle Scholar
  24. Matsuzaki H, Nakajima R, Nishiyama J et al. (1990) Chromosome engineering in Saccharomyces cerevisiae by using a site-specific recombination system of a yeast plasmid. J Bact 172:610–618PubMedGoogle Scholar
  25. Matzke M-A, Matzke A-J-M (1991) Differential inactivation and methylation of a transgene in plants by two suppressor loci containing homologous sequences. Plant Mol Biol 16:821–830CrossRefPubMedGoogle Scholar
  26. Matzke M-A, Mette M-F (2000) Transgene silencing by the host genome defense: implications for the evolution of epigenetic control mechanism in plants and vertebrates. Plant Mol Biol 43:401–415CrossRefPubMedGoogle Scholar
  27. Meyer P (2000) Transcriptional transgene silencing and chromatin component. Plant Mol Biol 43:221–234CrossRefPubMedGoogle Scholar
  28. Nanto K, Ebinuma H (2007) Expression of a transgene exchanged by the recombinase-mediated cassette exchange (RMCE) method in plants. International Conference - Plant Tansformation Technologies (Abstract: pp 71) Vienna, AustriaGoogle Scholar
  29. Nanto K, Ebinuma H (2008, on line: 2007) Marker-free site-specific integration plants. Transgenic Res 17:337–344CrossRefPubMedGoogle Scholar
  30. Nanto K, Yamada-Watanabe K, Ebinuma H (2005) Agrobacterium-mediated RMCE approach for gene replacement. Plant Biotechnol J 3:203–214CrossRefPubMedGoogle Scholar
  31. Nanto K, Sato K, Katayama Y, Ebinuma H (2009) Expression of a transgene exchanged by the recombinase-mediated cassette exchange (RMCE) method in plants. Plant Cell Rep 28:777–785CrossRefPubMedGoogle Scholar
  32. O’Gorman S, Fox D-T, Wahl G-M (1991) Recombinase-mediated gene activation and site-specific integration in mammalian cells. Science 251:1351–1355CrossRefPubMedGoogle Scholar
  33. Onouchi H, Yokoi K, Machida C et al. (1991) Operation of an efficient site-specific recombination system of Zygosaccharomyces rouxii in tobacco cells. Nucleic Acids Res 19:6373–6378CrossRefPubMedGoogle Scholar
  34. Ow D-W (2002) Recombinase-directed plant transformation for the post-genomic era. Plant Mol Biol 48:183–200CrossRefPubMedGoogle Scholar
  35. Schlaman H-R-M, Hooykaas P-J-J (1997) Effectiveness of the bacterial gene codA encoding cytosine deaminase as a negative selectable marker in Agrobacterium-mediated plant transformation. Plant J 11:1377–1385CrossRefGoogle Scholar
  36. Srivastava V, Ow D-W (2001) Biolistic mediated site-specific integration in rice. Mol Breed 8:345–350CrossRefGoogle Scholar
  37. Srivastava V, Ow D-W (2004) Marker-free site-specific gene integration in plants. Trends Biotech 22:627–629CrossRefGoogle Scholar
  38. Srivastava V, Ariza-Nieto M, Wilson A-J (2004) Cre-mediated site-specific gene integration for consistent transgene expression in rice. Plant Biotechnol J 2(2):169–179CrossRefPubMedGoogle Scholar
  39. Sugita K, Matsunaga E, Ebinuma H (1999) Effective selection system for generating marker-free transgenic plants independent of sexual crossing. Plant Cell Rep 18:941–947CrossRefGoogle Scholar
  40. Tzfira T, Frankman L-R, Vaidya M et al. (2003) Site-specific integration of Agrobacterium tumefaciens T-DNA via doublestranded intermediates. Plant Physiol 133:1011–1023CrossRefPubMedGoogle Scholar
  41. Vain P, James V-A, Worland B et al. (2002) Transgene behaviour across two generations in a large random population of transgenic rice plants produced by particle bombardment. Theor Appl Genet 105:878–889CrossRefPubMedGoogle Scholar
  42. Vergunst A-C, Hooykaas P-J (1998) Cre/lox-mediated site-specific integration of Agrobacterium T-DNA in Arabidopsis thaliana by transient expression of cre. Plant Mol Biol 38:393–406CrossRefPubMedGoogle Scholar
  43. Vergunst A-C, Jansen L-E, Hooykaas P-J (1998) Site-specific integration of Agrobacterium T-DNA in Arabidopsis thaliana mediated by Cre recombinase. Nucleic Acids Res 26:2729–2734CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  1. 1.R&D DivisionNippon Paper Industries Co. LtdTokyoJapan

Personalised recommendations