Skip to main content
SpringerLink
Log in

Elimination of marker genes and targeted integration via FLP/FRT recombination system from yeast in hybrid aspen (Populus tremula L. × P. tremuloides Michx.)

  • Original Paper
  • Published:
Tree Genetics & Genomes Aims and scope Submit manuscript

Abstract

Marker gene elimination was investigated in hybrid aspen (Populus tremula L. × Populus tremuloides Michx.) using the FLP/FRT recombination system. The construct contained the FLP recombinase under control of a heat inducible promoter, the antibiotic resistance gene nptII driven by the CaMV 35S promoter, and a promoterless uidA gene. The construct was integrated into poplar via Agrobacterium-mediated transformation. The active FLP recombinase excised the nptII marker gene and combined the promoterless uidA gene with the CaMV 35S promoter to form an active uidA gene. For targeted transgene integration, two constructs were used. The first one carried FLP under control of the heat-inducible Gmhsp17.5-E promoter from soybean as well as an active nptII gene flanked by two FRT sites; the second contained the promoterless bar selection marker gene also flanked by two FRT sites. Following transformation and induction of FLP, the enzyme mediated a site-specific recombination at the FRT sites of both constructs. This recombination leads to an excision of the FLP and nptII gene from the first as well as an excision of the promoterless bar gene from the second construct. The promoterless bar gene reintegrated exactly at the former position of the FLP and nptII genes in the first construct to form an active bar gene. The FLP/FRT recombination system from yeast forms a promising basis for the production of antibiotic-free transgenic plants and a useful tool for directed integration of transgenes into plant genomes.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Adang MJ, Brody MS, Cardineau G, Eagan N, Roush RT (1993) The reconstruction and expression of a Bacillus thuringiensis cryIIIA gene in protoplasts and potato plants. Plant Mol Biol 21:1131–1145

    Article  PubMed  CAS  Google Scholar 

  • Ambros PF, Matzke AJ, Matzke MA (1986) Localization of Agrobacterium rhizogenes T-DNA in plant chromosomes by in situ hybridization. EMBO J 5:2073–2077

    PubMed  CAS  Google Scholar 

  • Araki K, Araki M, Yamamura K (1997) Targeted integration of DNA using mutant lox sites in embryonic stem cells. Nucleic Acids Res 25:868–872

    Article  PubMed  CAS  Google Scholar 

  • Araki K, Araki M, Yamamura K (2002) Site-directed integration of the cre gene mediated by Cre recombinase using a combination of mutant lox sites. Nucleic Acids Res 30:e103

    Article  PubMed  Google Scholar 

  • Ballester A, Cervera M, Pena L (2007) Efficient production of transgenic citrus plants using isopentenyl transferase positive selection and removal of the marker gene by site-specific recombination. Plant Cell Rep 26:39–45

    Article  PubMed  CAS  Google Scholar 

  • Calderone TL, Stevens RD, Oas TG (1996) High-level misincorporation of lysine for arginine at AGA codons in a fusion protein expressed in Escherichia coli. J Mol Biol 4(262):407–412

    Article  Google Scholar 

  • Caplan A, Herrera-Estrella L, Inzé D, Van Haute E, Van Montagu M, Schell J, Zambryski P (1983) Introduction of genetic material into plant cells. Science 222:815–821

    Article  PubMed  CAS  Google Scholar 

  • Chakraborti D, Sarkar A, Mondal HA, Schuermann D, Hohn B, Sarmah BK, Das S (2008) Cre/lox system to develop selectable marker free transgenic tobacco plants conferring resistance against sap sucking homopteran insect. Plant Cell Rep 27:1623–1633

    Article  PubMed  CAS  Google Scholar 

  • Chawla R, Ariza-Nieto M, Wilson AJ, Moore SK, Srivastava V (2006) Transgene expression produced by biolistic-mediated, site-specific gene integration is consistently inherited by the subsequent generations. Plant Biotechnol J 4:209–218

    Article  PubMed  CAS  Google Scholar 

  • Chen S, Jin W, Wang M, Zhang F, Zhou J, Jia Q, Wu Y, Liu F, Wu P (2003) Distribution and characterization of over 1000 T-DNA tags in rice genome. Plant J 36:105–113

    Article  PubMed  CAS  Google Scholar 

  • Craig W, Tepfer M, Degrassi G, Ripandelli D (2008) An overview of general features of risk assessments of genetically modified crops. Euphytica 164:853–880

    Article  Google Scholar 

  • Cuellar W, Gaudin A, Solórzano D, Casas A, Nopo L, Chudalayandi P, Medrano G, Kreuze J, Ghislain M (2006) Self-excision of the antibiotic resistance gene nptII using a heat inducible Cre-loxP system from transgenic potato. Plant Mol Biol 62:71–82

    Article  PubMed  CAS  Google Scholar 

  • Dale EC, Ow DW (1990) Intra- and intermolecular site-specific recombination in plant cells mediated by the bacteriophage P1 recombinase. GENE 91:79–85

    Article  PubMed  CAS  Google Scholar 

  • Dale EC, Ow DW (1991) Gene transfer with subsequent removal of the selection gene from the host genome. Proc Natl Acad Sci USA 88:10558–10562

    Article  PubMed  CAS  Google Scholar 

  • Dumolin S, Demesure B, Petit RJ (1995) Inheritance of chloroplast and mitochondrial genomes in pedunculate oak investigated with an efficient PCR method. Theor Appl Genet 91:1253–1256

    Article  CAS  Google Scholar 

  • Ebinuma H, Sugita K, Matsunaga E, Endo S, Yamada-Watanabe K (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. The Hawoth Press, New York, pp 309–338

    Google Scholar 

  • Fedoryshyn M, Petzke L, Welle E, Bechthold A, Luzhetskyy A (2008) Marker removal from actinomycetes genome using Flp recombinase. Gene 419:43–47

    Article  PubMed  CAS  Google Scholar 

  • Feldmann K (1991) T-DNA insertion mutagenesis in Arabidopsis: mutational spectrum. Plant J 1:71–82

    Article  CAS  Google Scholar 

  • FengYQ SJ, Alami R, Eisen A, Westerman KA, Leboulch P, Fiering S, Bouhassira EE (1999) Site-specific chromosomal integration in mammalian cells: highly efficient CRE recombinase-mediated cassette exchange. J Mol Biol 292:779–785

    Article  Google Scholar 

  • Fladung M (1999) Gene stability in transgenic aspen-Populus. I. Flanking DNA sequences and T-DNA structure. Mol Gen Genet 260:574–581

    Article  PubMed  CAS  Google Scholar 

  • Fladung M, Ahuja MR (1995) ‘Sandwich’ method for non-radioactive hybridizations. Biotechniques 18:3–5

    Google Scholar 

  • Fladung M, Kumar S, Ahuja R (1997) Genetic transformation of Populus genotypes with different chimaeric gene constructs: transformation efficiency and molecular analysis. Transgen Res 6:111–121

    Article  CAS  Google Scholar 

  • 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 USA 89:7905–7909

    Article  PubMed  CAS  Google Scholar 

  • Giridhar P, Parimalan R (2004) Genetically engineered crops: myths and canards about their biosafety and benefits—a review. Adv Plant Sci 17:353–360

    Google Scholar 

  • Golic KG, Lindquist S (1989) The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome. Cell 59:499–509

    Article  PubMed  CAS  Google Scholar 

  • Hönicka H, Fladung M (2006) Biosafety in Populus spp. and other forest trees: from non-native species to taxa derived from traditional breeding and genetic engineering. Trees 20:131–144

    Article  Google Scholar 

  • Gustafsson C, Govindarajan S, Minshull J (2004) Codon bias and heterologous protein expression. Trends Biotechnol 22:346–353

    Article  PubMed  CAS  Google Scholar 

  • Jansson S, Douglas CJ (2007) Populus: a model system for plant biology. Ann Rev Plant Biol 58:435–458

    Article  CAS  Google Scholar 

  • Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS-fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907

    PubMed  CAS  Google Scholar 

  • Koncz C, Martini N, Mayerhofer R, Koncz-Kalman Z, Korber H, Redei GP, Schell J (1989) High-frequency T-DNA-mediated gene tagging in plants. Proc Natl Acad Sci USA 86:8467–8471

    Article  PubMed  CAS  Google Scholar 

  • Koncz C, Nemeth K, Redei GP, Schell J (1992) T-DNA insertional mutagenesis in Arabidopsis. Plant Mol Biol 20:963–976

    Article  PubMed  CAS  Google Scholar 

  • Krysan PJ, Young JC, Sussman MR (1999) T-DNA as an insertional mutagen in Arabidopsis. Plant Cell 11:2283–2290

    Article  PubMed  CAS  Google Scholar 

  • Krysan PJ, Young JC, Jester PJ, Monson S, Copenhaver G, Preuss D, Sussman MR (2002) Characterization of T-DNA insertion sites in Arabidopsis thaliana and the implications for saturation mutagenesis. OMICS 6:163–174

    Article  PubMed  CAS  Google Scholar 

  • Kumar S, Fladung M (2001) Controlling transgene integration in plants. Trends Plant Sci 6:155–159

    Article  PubMed  CAS  Google Scholar 

  • Kumar S, Fladung M (2002a) Gene targeting in plants. In: Jain SM, Brar DS, Ahloowalia BS (eds) Molecular techniques in crop improvement. Kluwer Academic Press, Dordrecht, pp 481–500

    Google Scholar 

  • Kumar S, Fladung M (2002b) Transgene integration in aspen: structures of integration sites and mechanism of T-DNA integration. Plant J 31:543–551

    Article  CAS  Google Scholar 

  • Lakso M, Sauer B, Mosinger B Jr, Lee EJ, Manning RW, Yu SH, Mulder KL, Westphal H (1992) Targeted oncogene activation by site-specific recombination in transgenic mice. Proc Natl Acad Sci USA 89:6232–6236

    Article  PubMed  CAS  Google Scholar 

  • Lee KY, Lund P, Lowe K, Dunsmuir P (1990) Homologous recombination in plant cells after Agrobacterium-mediated transformation. Plant Cell 2:415–425

    Article  PubMed  CAS  Google Scholar 

  • Lee WK, Kim SH, Park YD (2007) Marker-free transgenic plants produced by autoexcised Cre-lox system. Horticult Environ Biotechnol 48:265–269

    CAS  Google Scholar 

  • Li Y, Rosso MG, Ulker B, Weisshaar B (2006) Analysis of T-DNA insertion site distribution patterns in Arabidopsis thaliana reveals special features of genes without insertions. Genomics 87:645–652

    Article  PubMed  CAS  Google Scholar 

  • Logie C, Stewart AF (1995) Ligand-regulated site-specific recombination. Proc Natl Acad Sci USA 92:5940–5944

    Article  PubMed  CAS  Google Scholar 

  • Louwerse JD, van Lier MC, van der Steen DM, de Vlaam CM, Hooykaas PJ, Vergunst AC (2007) Stable recombinase-mediated cassette exchange in Arabidopsis using Agrobacterium tumefaciens. Plant Physiol 145:1282–93

    Article  PubMed  CAS  Google Scholar 

  • Liu YG, Mitsukawa N, Oosumi T, Whittier RF (1995) Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J 8:457–463

    Article  PubMed  CAS  Google Scholar 

  • Luo K, Duan H, Zhao D, Zheng X, Deng W et al (2007) ‘GM-gene-deletor’: fused loxP-FRT recognition sequences dramatically improve the efficiency of FLP or CRE recombinase on transgene excision from pollen and seed of tobacco plants. Plant Biotechnol J 5:263–74

    Article  PubMed  CAS  Google Scholar 

  • Luo KM, Sun M, Deng W, Xu S (2008) Excision of selectable marker gene from transgenic tobacco using the GM-gene-deletor system regulated by a heat-inducible promoter. Biotechnol Lett 30:1295–1302

    Article  PubMed  CAS  Google Scholar 

  • Lutz KA, Svab Z, Maliga P (2006) Construction of marker-free transplastomic tobacco using the Cre-loxP site-specific recombination system. Nat Protocols 1:900–910

    Article  CAS  Google Scholar 

  • Lyznik LA, Ryan RD, Ritchie SW, Hodges TK (1989) Stable co-transformation of maize protoplasts with gusA and neo genes. Plant Mol Biol 13:151–161

    Article  PubMed  CAS  Google Scholar 

  • Lyznik LA, Gordon-Kamm WJ, Tao Y (2003) Site-specific recombination for genetic engineering in plants. Plant Cell Rep 21:925–932

    Article  PubMed  CAS  Google Scholar 

  • Marillonnet S, Giritch A, Gils M, Kandzia R, Klimyuk V, Gleba Y (2004) In planta engineering of viral RNA replicons: efficient assembly by recombination of DNA modules delivered by Agrobacterium. Proc Natl Acad Sci USA 101:6852–6857

    Article  PubMed  CAS  Google Scholar 

  • Matzke AJM, Matzke MA (1998) Position effects and epigenetic silencing of plant transgenes. Curr Opin Plant Biol 1:142–148

    Article  PubMed  CAS  Google Scholar 

  • Mullins ED, Kang S (2001) Transformation: a tool for studying fungal pathogens of plants. Cell Mol Life Sci 58:2043–52

    Article  PubMed  CAS  Google Scholar 

  • Miki B, McHugh S (2004) Selectable marker genes in transgenic plants: applications, alternatives and biosafety. J Biotechnol 107:193–232

    Article  PubMed  CAS  Google Scholar 

  • Nanto K, Ebinuma H (2008) Marker-free site-specific integration plants. Transgen Res 17:337–344

    Article  CAS  Google Scholar 

  • Nanto K, Yamada-Watanabe K, Ebinuma H (2005) Agrobacterium-mediated RMCE approach for gene replacement. Plant Biotechnol J 3:203–214

    Article  PubMed  CAS  Google Scholar 

  • Offringa R (1992) Gene targeting in plants using Agrobacterium vector system. Ph.D. thesis, Leiden University, Netherlands

  • Offringa R, de Groot MJ, Haagsman HJ, Does MP, van den Elzen PJ, Hooykaas PJ (1990) Extrachromosomal homologous recombination and gene targeting in plant cells after Agrobacterium mediated transformation. EMBO J 9:3077–3084

    PubMed  CAS  Google Scholar 

  • Ow DW (2002) Recombinase-directed plant transformation for the post-genomic era. Plant Mol Biol 48:183–200

    Article  PubMed  CAS  Google Scholar 

  • Pakull B, Groppe K, Meyer M, Markussen T, Fladung M (2009) Genetic linkage mapping in aspen (Populus tremula L. and P. tremuloides Michx.). Tree Gen Genom 5:505–515

    Article  Google Scholar 

  • Puchta H (2000) Removing selectable marker genes: taking the shortcut. Trend Plant Sci 5:273–274

    Article  CAS  Google Scholar 

  • Puchta H (2003) Marker-free transgenic plants. Plant Cell Tiss Org Cult 74:123–134

    Article  CAS  Google Scholar 

  • Robischon M (2006) Field trials with transgenic trees—state of the art and developments. In: Fladung M, Ewald D (eds) Tree transgenesis: recent developments. Springer, Berlin, pp 3–23

    Google Scholar 

  • Rosso MG, Li Y, Strizhov N, Reiss B, Dekker K, Weisshaar B (2003) An Arabidopsis thaliana T-DNA mutagenized population (GABI-Kat) for flanking sequence tag-based reverse genetics. Plant Mol Biol 53:247–259

    Article  PubMed  CAS  Google Scholar 

  • Schaefer DG, Zryd JP (1997) Efficient gene targeting in the moss Physcomitrella patens. Plant J 11:1195–206

    Article  PubMed  CAS  Google Scholar 

  • Sallaud C, Gay C, Larmande P, Bès M, Piffanelli P, Piégu B, Droc G, Regad F, Bourgeois E, Meynard D, Périn C, Sabau X, Ghesquière A, Glaszmann JC, Delseny M, Guiderdoni E (2004) High throughput T-DNA insertion mutagenesis in rice: a first step towards in silico reverse genetics. Plant J 39:450–464

    Article  PubMed  CAS  Google Scholar 

  • Schneeberger RG, Zhang K, Tatarinova T, Troukhan M, Kwok SF, Drais J, Klinger K, Orejudos F, Macy K, Bhakta A, Burns J, Subramanian G, Donson J, Flavell R, Feldmann KA (2005) Agrobacterium T-DNA integration in Arabidopsis is correlated with DNA sequence compositions that occur frequently in gene promoter regions. Funct Integr Genomics 5:240–253

    Article  PubMed  CAS  Google Scholar 

  • Shaw CH, Leemans J, Shaw CH, van Montagu M, Schell J (1983) A general method for the transfer of cloned genes to plant cells. Gene 23:315–330

    Article  PubMed  CAS  Google Scholar 

  • Song H, Niederweis M (2007) Functional expression of the Flp recombinase in Mycobacterium bovis BCG. Gene 399:112–119

    Article  PubMed  CAS  Google Scholar 

  • Soukharev S, Miller JL, Sauer B (1999) Segmental genomic replacement in embryonic stem cells by double lox targeting. Nucleic Acids Res 27:e21

    Article  PubMed  CAS  Google Scholar 

  • Srivastava V, Ow DW (2004) Marker-free site-specific gene integration in plants. Trends Biotechnol 22:627–629

    Article  PubMed  CAS  Google Scholar 

  • Szabados L, Kovács I, Oberschall A, Abrahám E, Kerekes I, Zsigmond L, Nagy R, Alvarado M, Krasovskaja I, Gál M, Berente A, Rédei GP, Haim AB, Koncz C (2002) Distribution of 1000 sequenced T-DNA tags in the Arabidopsis genome. Plant J 32:233–242

    Article  PubMed  CAS  Google Scholar 

  • Tzfira T, White C (2005) Towards targeted mutagenesis and gene replacement in plants. Trends Biotechnol 23:567–569

    Article  PubMed  CAS  Google Scholar 

  • Tzfira T, Frankman LR, Vaidya M, Citovsky V (2003) Site-specific integration of Agrobacterium tumefaciens T-DNA via double-stranded intermediates. Plant Physiol 133:1011–1023

    Article  PubMed  CAS  Google Scholar 

  • Thomsen JG, Yau YY, Blanvillain R, Chiniquy D, Thilmony R, Ow DW (2008) ParA resolvase catalyzes site-specific excision of DNA from the Arabidopsis genome. Transgen Res. doi:10.1007/s11248-008-9213-4

    Google Scholar 

  • Tuskan GA, DiFazio S, Jansson S, Bohlmann J et al (2006) The genome of black cottonwood, Populus trichocarpa (Torr. & Gray). Science 313:1596–1604

    Article  PubMed  CAS  Google Scholar 

  • Vergunst AC, Hooykaas PJ (1998) Cre/lox-mediated site-specific integration of Agrobacterium T-DNA in Arabidopsis thaliana by transient expression of cre. Plant Mol Biol 38:393–406

    Article  PubMed  CAS  Google Scholar 

  • Vergunst AC, Hooykaas PJ (1999) Recombination in the plant genome and its application in biotechnology. Crit Rev Plant Sci 18:1–31

    Article  CAS  Google Scholar 

  • Vergunst AC, Jansen LE, Hooykaas PJ (1998) Site-specific integration of Agrobacterium T-DNA in Arabidopsis thaliana mediated by Cre recombinase. Nucl Acids Res 26:2729–2734

    Article  PubMed  CAS  Google Scholar 

  • de Vetten N, Wolters AM, Raemakers K, van der Meer I, ter Stege R, Heeres E, Heeres P, Visser R (2003) A transformation method for obtaining marker-free plants of a cross-pollinating and vegetatively propagated crop. Nat Biotechnol 21:439–442

    Article  PubMed  CAS  Google Scholar 

  • Wang J, Lewis ME, Whallon JH, Sink KC (1995) Chromosomal mapping of T-DNA inserts in transgenic Petunia by in situ hybridization. Transgen Res 4:241–246

    Article  CAS  Google Scholar 

  • Wohlleben W, Arnold W, Broer I, Hillemann D, Strauch E, Puhler A (1988) Nucleotide sequence of the phosphinothricin N-acetyltransferase gene from Streptomyces viridochromogenes Tu494 and its expression in Nicotiana tabacum. Gene 70:25–37

    Article  PubMed  CAS  Google Scholar 

  • Yoder JI, Goldsbrough AP (1994) Transformation systems for generating marker-free transgenic plants. Bio-Technol 12:263–267

    CAS  Google Scholar 

  • Zelasco S, Ressegotti V, Confalonieri M, Carbonera D, Calligari P, Bonadei M, Bisoffi S, Yamada K, Balestrazzi A (2007) Evaluation of MAT-vector system in white poplar (Populus alba L.) and production of ipt marker-free transgenic plants by ‘single-step transformation’. Plant Cell Tiss Org Cult 91:61–72

    Article  CAS  Google Scholar 

  • Zubko E, Scutt C, Meyer P (2000) Intrachromosomal recombination between attP regions as a tool to remove selectable marker genes from tobacco transgenes. Nat Biotechnol 18:442–445

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgment

This research was funded by the Bundesministerium für Bildung und Forschung, Germany (Projektnummer PTJ-Bio/0313264T).

Author information

Authors and Affiliations

  1. Johann Heinrich von Thünen-Institut, Federal Research Institute for Rural Areas, Forestry and Fisheries, Institute of Forest Genetics, Grosshansdorf, Germany

    Matthias Fladung

  2. Biocentre Klein Flottbek, Developmental Biology and Biotechnology, University of Hamburg, Hamburg, Germany

    Tobias M. H. Schenk, Olaf Polak & Dirk Becker

Authors
  1. Matthias Fladung
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tobias M. H. Schenk
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Olaf Polak
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dirk Becker
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Matthias Fladung or Dirk Becker.

Additional information

Communicated by R. Sederoff

Matthias Fladung and Tobias M. H. Schenk contributed equally to this work.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fladung, M., Schenk, T.M.H., Polak, O. et al. Elimination of marker genes and targeted integration via FLP/FRT recombination system from yeast in hybrid aspen (Populus tremula L. × P. tremuloides Michx.). Tree Genetics & Genomes 6, 205–217 (2010). https://doi.org/10.1007/s11295-009-0241-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11295-009-0241-x

Keywords

Search

Navigation