Advertisement

Plant Molecular Biology

, Volume 35, Issue 4, pp 523–530 | Cite as

Gene targeting approaches using positive-negative selection and large flanking regions

  • Thomas Thykjær
  • Jørgen Finnemann
  • Leif Schauser
  • Liselotte Christensen
  • Carsten Poulsen
  • Jens Stougaard*
Article

Abstract

We report here on strategies aimed at improving the frequency of detectable recombination in plants by increasing the efficiency of selecting double-recombinants in transgenic calli. Gene targeting was approached on the Gln1 and the Pzf loci of Lotus japonicus, using Agrobacterium tumefaciens T-DNA replacement vectors. Large flanking regions, up to 22.9 kb, surrounding a positive selection marker were presented as substrates for homologous recombination. For easier detection of putative recombinants the negative selectable marker cytosine deaminase was inserted at the outside borders of the flanking regions offered for cross-over. A combination of positive and negative selection allowing double-recombinants to grow, while counter-selecting random insertions, was used to select putative targeting events. The more than 1000-fold enrichment observed with replacement vectors designed to minimize gene silencing demonstrated the efficiency of the negative selection. Using five different replacement vectors an estimated total of 18974 transformation events were taken through the positive-negative selection procedure and 185 resistant calli obtained. Targeting events could not be verified in the survivors by PCR screening and Southern blot analysis. With this approach the frequency of detectable gene targeting in L. japonicus was below 5.3×10−5, despite the large flanking sequences offered for recombination.

cytosine deaminase homologous recombination Lotus japonicus negative selection plants 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Barnes W: PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda. Bacteriophage templates. Proc Natl Acad Sci USA 91: 2216-2220 (1994).PubMedGoogle Scholar
  2. 2.
    Borg S, Brandstrup B, Jensen TJ, Poulsen C: Identification of newprotein species among 33 different small GTP binding proteins encoded by cDNAs from Lotus japonicus and expression of corresponding mRNAs in developing root nodules. Plant J 11: 237-250 (1997).PubMedGoogle Scholar
  3. 3.
    Bradley A, Hasty P, Davis A, Ramirez-Solis R: Modifying the mouse: design and desire. Bio/technology 10: 534-539 (1992).PubMedGoogle Scholar
  4. 4.
    Cheng S, Fockler C, Barnes WM, Higuchi R: Effective amplification of long targets from cloned inserts and human genomic DNA. Proc Natl Acad Sci USA 91: 5695-5699 (1994).PubMedGoogle Scholar
  5. 5.
    Czako M, Marton L: The herpes simplex virus thymidine kinase gene as a conditional negative selection marker gene in Arabidopsis thaliana. Plant Physiol 104: 1067-1071 (1994).PubMedGoogle Scholar
  6. 6.
    Dellaporta SL, Wood J, Hicks JB: A plant DNA minpreparation: Version II. Plant Mol Biol Rep 1: 19-21 (1983).Google Scholar
  7. 7.
    Deng C, Capecchi M: Reexamination of gene targeting frequency as a function of the extend of homology between the targeting vector and the target locus. Mol Cell Biol 12: 3365-3371 (1992).PubMedGoogle Scholar
  8. 8.
    Halfter U, Morris PC, Willmitzer L: Gene targeting in Arabidopsis thaliana. Mol Gen Genet 231: 186-193 (1992).PubMedGoogle Scholar
  9. 9.
    Handberg K, Stiller J, Thykjaer T, Stougaard J: Transgenic plants: Agrobacterium mediated transformation of the diploid legume Lotus japonicus. In: Celis JE (ed) Cell Biology: A Laboratory Handbook, pp. 119-127. Academic Press, New York (1994).Google Scholar
  10. 10.
    Handberg K, Stougaard J: Lotus japonicus, an autogamous, diploid legume species for classical and molecular genetics. Plant J 2: 487-496 (1992).Google Scholar
  11. 11.
    Hasty P, Rivera-Perez J, Bradley A: The length of homology required for gene targeting in embryonic stem cells. Mol Cell Biol 11: 5586-5591 (1991).PubMedGoogle Scholar
  12. 12.
    Jones JDG, Schlumukov L, Carland F, English J, Scofield SR, Bishop GJ, Harrison K: Effective vectors for transformation, expression of heterologous genes, and assaying transposon excision in transgenic plants. Transgen Res 1: 285-297 (1992).Google Scholar
  13. 13.
    Kobayashi T, Hisajima S, Stougaard J, Ichikawa H: A conditional negative selection for Arabidopsis expressing a bacterial cytosine deaminase gene. Jpn J Genet 70: 409-422 (1995).PubMedGoogle Scholar
  14. 14.
    Lazo GR, Stein PA, Ludwig RA: A DNA transformationcompetent Arabidopsis genomic library in Agrobacterium. Bio/technology 9: 963-967 (1991).PubMedGoogle Scholar
  15. 15.
    Miao ZH, Lam E: Targeted disruption of the TGA3 locus in Arabidopsis thaliana. Plant J 7: 359-365 (1995).PubMedGoogle Scholar
  16. 16.
    Offringa R, de Groot MJ, Haagsman HJ, Does MP, van den Elzen PJ, Hooykaas PJ: Extrachromosomal homologous recombination and gene targeting in plant cells after Agrobacterium mediated transformation. EMBO J 9: 3077-3084 (1990).PubMedGoogle Scholar
  17. 17.
    Offringa R, Franke-van Dijk ME, de Groot MJ, van den Elzen PJ, Hooykaas PJ: Nonreciprocal homologous recombination between Agrobacterium transferred DNA and a plant chromosomal locus. Proc Natl Acad Sci USA 90: 7346-7350 (1993).PubMedGoogle Scholar
  18. 18.
    O'Keefe DP, Tepperman JM, Dean C, Leto KJ, Erbes DL, Odell JT: Plant expression of a bacterial cytochrome P450 that catalyzes activation of a sulfonylurea pro-herbicide. Plant Physiol 105: 473-482 (1994).PubMedGoogle Scholar
  19. 19.
    Paszkowski J, Baur M, Bogucki A, Potrykus I: Gene targeting in plants. EMBO J 7: 4021-4026 (1988).Google Scholar
  20. 20.
    Perera RJ, Linard CG, Signer ER: Cytosine deaminase as a negative selection marker for Arabidopsis. Plant Mol Biol 23: 793-799 (1993).PubMedGoogle Scholar
  21. 21.
    Reiss B, Klemm M, Kosak H, Schell J: RecA protein stimulates homologous recombination in plants. Proc Natl Acad Sci USA 93: 3094-3098 (1996).PubMedGoogle Scholar
  22. 22.
    Hourda M, Paszkowski J: High fidelity extrachromosomal recombination and gene targeting plants. Mol Gen Genet 243: 106-111 (1994).PubMedGoogle Scholar
  23. 23.
    Risseeuw E, Offringa R, Franke-van Dijk ME, Hooykaas PJ: Targeted recombination in plant using Agrobacterium coincides with additional rearrangements at the target locus. Plant J 7: 109-119 (1995).PubMedGoogle Scholar
  24. 24.
    Schauser L, Christensen L, Borg S, Poulsen C: PZF, a cDNA isolated from Lotus japonicus and soybean root nodule libraries, encodes a new plant member of the RING-finger family of zinc-binding proteins. Plant Physiol 107: 1457-1458 (1995).PubMedGoogle Scholar
  25. 25.
    Stougaard J: Substrate-dependent negative selection in plants using a bacterial cytosine deaminase gene. Plant J 3: 755-761 (1993).Google Scholar
  26. 26.
    Stougaard J, Beuselinck PR: Registration of GIFU B-129-S9 Lotus japonicus germplasm. Crop Sci 36: 476 (1996).Google Scholar
  27. 27.
    Thorsners MK, Kandasamy M, Nasrallah ME, Nasrallah JB: A Brassica S-locus gene promoter targets toxic gene expression and cell death to the pistil and pollen of transgenic Nicotiana. Devel Biol 143: 173-184 (1991).Google Scholar
  28. 28.
    Thykjaer T, Danielsen D, She Q, Stougaard J: Organization and expression of genes in the genomic region surrounding a glutamine synthetase gene. Mol Gen Genet, in press (1997).Google Scholar
  29. 29.
    Viney JL: Transgenic and gene knockout mice in cancer research. Cancer Metastasis Rev 14: 77-90 (1995).PubMedGoogle Scholar
  30. 30.
    Zhang H, Hasty P, Bradley A: Targeting frequency for deletion vectors in embryonic stem cells. Mol Cell Biol 14: 2404-10 (1994).PubMedGoogle Scholar

Copyright information

© Kluwer Academic Publishers 1997

Authors and Affiliations

  • Thomas Thykjær
    • 1
  • Jørgen Finnemann
    • 2
  • Leif Schauser
    • 1
  • Liselotte Christensen
    • 1
  • Carsten Poulsen
    • 1
  • Jens Stougaard*
    • 1
  1. 1.Laboratory of Gene Expression, Department of Molecular and Structural BiologyUniversity of AarhusAarhus CDenmark
  2. 2.Plant Nutrition Laboratory, Dep. of Agricultural SciencesThe Royal Veterinary and Agricultural UniversityCopenhagenDenmark

Personalised recommendations