Inducible Excision of Selectable Marker Gene from Transgenic Plants by the Cre/lox Site-specific Recombination System
In a plant transformation process, it is necessary to use marker genes that allow the selection of regenerated transgenic plants. However, selectable marker genes are generally superfluous once an intact transgenic plant has been established. Furthermore, they may cause regulatory difficulties for approving transgenic crop release and commercialization. We constructed a binary expression vector with the Cre/lox system with a view to eliminating a marker gene from transgenic plants conveniently. In the vector, recombinase gene cre under the control of heat shock promoter and selectable marker gene nptII under the control of CaMV35S promoter were placed between two lox P sites in direct orientation, while the gene of interest was inserted outside of the lox P sites. By using this vector, both cre and nptII genes were eliminated from most of the regenerated plants of primary transformed tobacco through heat shock treatment, while the gene of interest was retained and stably inherited. This autoexcision strategy, mediated by the Cre/lox system and subjected to heat shock treatment to eliminate a selectable marker gene, is easy to adopt and provides a promising approach to generate marker-free transgenic plants.
Keywordsautoexcision Cre/lox recombination system heat shock treatment plant expression vector selectable marker gene transgenic tobacco
Ainley, WM, Key, JL 1990Development of a heat-shock inducible expression cassette for plants: Characterization of parameters for its use in transient expression assaysPlant Mol Biol14949967CrossRefPubMedGoogle Scholar Bayley, CC, Morgan, M, Dale, EC, Ow, DW 1992Exchange of gene activity in transgenic plants catalysed by the Cre-lox site specific recombination systemPlant Mol Biol18353361CrossRefPubMedGoogle Scholar Branda, CS, Dymecki, SM 2004Talking about a revolution: the impact of site-specific recombinases on genetic analyses in miceDev Cell6728CrossRefPubMedGoogle Scholar Corneille, S, Lutz, K, Svab, Z, Maliga, P 2001Efficient elimination of selectable marker genes from the plastid genome by the CRE-lox site-specific recombination systemPlant J27171178CrossRefPubMedGoogle Scholar Czarnecka, E, Gurley, WB, Nagao, RT, Mosquera, LA, Key, JL 1985DNA sequence and transcript mapping of a soybean gene encoding a small heat-shock proteinProc Natl Acad Sci USA8237263730Google Scholar Dale, EC, Ow, DW 1990Intra- and inter site-specific recombination in plant cells mediated by bacteriophage P1 recombinaseGene917985CrossRefPubMedGoogle Scholar Dale, EC, Ow, DW 1991Gene transfer with subsequent removal of the selection gene from the host genomeProc Natl Acad Sci USA881055810562PubMedGoogle Scholar Daley, M, Knauf, V, Summerfelt, KR, Turner, JC 1998Cotransformation with one Agrobacterium tumefaciens strain containing two binary plasmids as a methods for producing marker-free transgenic plantsPlant Cell Rep17489496CrossRefGoogle Scholar Block, M, Debrouwer, D 1991Two T-DNA's co-transformed into Brassica napus by a double Agrobacterium tumefacien infection are mainly integrated at the same locusTheor Appl Genet82257263CrossRefGoogle Scholar Depicker, A, Herman, L, Jacobs, A, Schell, J, Montagu, M 1985Frequencies of simultaneous transformation with different T-DNAs and their relevance to the Agrobacterium/plant cell interactionMol Gen Genet201477484CrossRefGoogle Scholar Foster, E, Hattori, J, Labbe, H, Ouellet, T, Fobert, PR, James, LE., et al. 1999A tobacco cryptic constitutive promoter, tCUP, revealed by T-DNA taggingPlant Mol Biol414555CrossRefPubMedGoogle Scholar Foster, E, Hattori, J, Zhang, P, Labbe, H, Martin-Heller, T, Li-Pook-Than, J., et al. 2003The new RENT family of repetitive elements in Nicotiana species harbors gene regulatory elements related to the tCUP cryptic promoterGenome46146155CrossRefPubMedGoogle Scholar Gao, YF, Zhu, Z, Xiao, GF, Zhu, Y, Wu, Q, Li, XH 1998Isolation of soybean kunitz trypsin inhibitor gene and its application in plant insect-resistant genetic engineeringActa Bot Sin40405411Google Scholar Gelvin, SB 2003Agrobacterium-mediated plant transformation: the biology behind the “Gene-Jockeying” toolMicrobiol Mol Biol Rev671637CrossRefPubMedGoogle Scholar Gilbertson, L 2003Cre-lox recombination: Cre-active tools for plant biotechnologyTrends Biotechnol21550555CrossRefPubMedGoogle Scholar Gleave, AP, Mitra, DS, Mudhe, SR, Morris, Bret AM 1999Selectable marker-free transgenic plants without sexual crossing: transient expression of cre recombinase and use of a conditional lethal dominant genePlant Mol Biol40223235CrossRefPubMedGoogle Scholar Gurley, WB, Czarnecka, E, Nagao, RT, Key, JL 1986Upstream sequences required for efficient expression of a soybean heat-shock geneMol Cell Biol6559565PubMedGoogle Scholar Hajdukiewica, Peter TJ, Gilbertson, L, Staub, JM 2001Multiple pathways for Cre/lox-mediated recombination in plastidsPlant J27161170CrossRefPubMedGoogle Scholar Hoff, T, Schnorr, KM, Mundy, J 2001A recombinase-mediated transcriptional induction system in transgenic plantsPlant Mol Biol454149CrossRefPubMedGoogle Scholar Horsch, RB, Fry, JE, Hoffmana, NL, Eichholtz, D, Rogers, SG, Fraley, RT. 1985A simple and general method for transferring genes into plantsScience22712291231Google Scholar Jefferson, RA 1987Assaying chimeric genes in plants: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plantsEMBO J639013907PubMedGoogle Scholar Klaus, SMJ, Huang, F-C, Golds, TJ, Koop, H-U 2004Generation of marker-free plastid transformants using a transiently cointegrated selection geneNat Biotechnol1815Google Scholar Komari, T, Hiei, Y, Saito, Y, Murai, N, Kumashiro, T 1996Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefacien and segregation of transformants free from selection markersPlant J10165174CrossRefPubMedGoogle Scholar
Lixia W (2002) Cre recombinase modification and its regulation in plants. Report on postdoctoral research. (pp. 67–73) Peking University.Google Scholar
Lichtenstein, CPFHB, Pazkowski, J, Hohn, B 1994Intrachromosomal recombination between genomic repeatsPaszkowski, J. eds. Homologous Recombination and Gene Silencing in PlantsKluwer Academic PublishersDordrecht95122Google Scholar Lyznik, LA, Hirayama, L, Rao, KV, Abad, A, Hodges, TK 1995Heat-inducible expression of FLP gene in maize cellsPlant J8177186CrossRefPubMedGoogle Scholar McCormac, AC, Fowler, MR, Chen, DF, Elliott, MC 2001Efficient co-transformation of Nicotiana tabacum by two independent T-DNAs, the effect of T-DNA size and implications for genetic separationTransgenic Res10143155CrossRefPubMedGoogle Scholar Mlynarova, L, Nap, J-P 2003A self-excising Cre recombinase allows efficient recombination of multiple ectopic heterospecific lox sites in transgenic tobaccoTransgenic Res124557CrossRefPubMedGoogle Scholar Odell, J, Caimi, P, Sauer, B, Russell, S 1990Site-directed recombination in the genome of transgenic tobaccoMol Gen Genet223369378CrossRefPubMedGoogle Scholar Ow, DW 2001The right chemistry for marker gene removalNat Biotechnol19115116CrossRefPubMedGoogle Scholar Russell, SH, Hoopes, JL, Odell, JT 1992Directed excision of a transgene from the plant genomeMol Gen Genet2344959PubMedGoogle Scholar Sugita, K, Kasahara, T, Matsunaga, E, Ebinuma, H 2000A transformation vector for the production of marker-free transgenic plants containing a single copy transgene at high frequencyPlant J22461469CrossRefPubMedGoogle Scholar Tronche, F, Casanova, E, Turiault, M, Sahly, I, Kellendonk, C 2002When reverse genetics meets physiology: the use of site-specific recombinases in miceFEBS Letters529116121CrossRefPubMedGoogle Scholar Wu, K, Malik, K, Tian, L, Martin, T, Foster, E, Brown, D., et al. 2001Enhancers and core promoter elements are essential for the activity of a cryptic gene activation sequence from tobaccoMol Genet Genomics265763770CrossRefPubMedGoogle Scholar Xing, A, Zhang, Z, Sato, S, Staswick, P, Clemente, T 2000The use of the two T-DNA binary system to derive marker-free transgenic soybeansIn Vitro Cell Dev Biol–Plant36453456Google Scholar Zubko, E, Scutt, C, Meyer, P 2000Intrachromosomal recombination between attP regions as a tool to remove selectable marker genes from tobacco transgenesNat Biotechnol18442445CrossRefPubMedGoogle Scholar Zuo, J, Niu, Q-W, Moller, SG, Chua, N-H 2001Chemical-regulated, site-specific DNA excision in transgenic plantsNat Biotechnol19157161CrossRefPubMedGoogle Scholar