Activity of yeast FLP recombinase in maize and rice protoplasts.

We have demonstrated that a yeast FLP/FRT site-specific recombination system functions in maize and rice protoplasts. FLP recombinase activity was monitored by reactivation of beta-glucuronidase (GUS) expression from vectors containing the gusA gene inactivated by insertion of two FRTs (FLP recombination targets) and a 1.31 kb DNA fragment. The stimulation of GUS activity in protoplasts cotransformed with vectors containing FRT inactivated gusA gene and a chimeric FLP gene depended on both the expression of the FLP recombinase and the presence and structure of the FRT sites. The FLP enzyme could mediate inter- and intramolecular recombination in plant protoplasts. These results provide evidence that a yeast recombination system can function efficiently in plant cells, and that its performance can be manipulated by structural modification of the FRT sites.     

FLP-mediated recombination of FRT sites in the maize genome.

Molecular evidence is provided for genomic recombinations in maize cells induced by the yeast FLP/FRT site-specific recombination system. The FLP protein recombined FRT sites previously integrated into the maize genome leading to excision of a selectable marker, the neo gene. NPTII activity was not observed after the successful recombination process; instead, the gusA gene was activated by the removal of the blocking DNA fragment. Genomic sequencing in the region of the FRT site (following the recombination reaction) indicated that a precise rearrangement of genomic DNA sequences had taken place. The functional FLP gene could be either expressed transiently or after stable integration into the maize genome. The efficiency of genomic recombinations was high enough that a selection for recombination products, or for FLP expression, was not required. The results presented here establish the FLP/FRT site-specific recombination system as an important tool for controlled modifications of maize genomic DNA.     

Heat-shock-mediated elimination of the nptII marker gene in transgenic apple (Malus×domestica Borkh.)

Production of marker-free genetically modified (GM) plants is one of the major challenges of molecular fruit breeding. Employing clean vector technologies, allowing the removal of undesired DNA sequences from GM plants, this goal can be achieved. The present study describes the establishment of a clean vector system in apple Malus×domestica Borkh., which is based on the use of the neomycin phosphotransferase II gene (nptII) as selectable marker gene and kanamycin/paramomycin as selective agent. The nptII gene can be removed after selection of GM shoots via site-specific excision mediated by heat-shock-inducible expression of the budding yeast FLP recombinase driven by the soybean Gmhsp17.5-E promoter. We created a monitoring vector containing the nptII and the flp gene as a box flanked by two direct repeats of the flp recognition target (FRT) sites. The FRT-flanked box separates the gusA reporter gene from the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter. Consequently, GUS expression does only occur after elimination of the FRT-flanked box. Transformation experiments using the monitoring vector resulted in a total of nine transgenic lines. These lines were investigated for transgenicity by PCR, RT-PCR and Southern hybridization. Among different temperature regimes tested, exposure to 42 °C for 3.5 to 4h led to efficient induction of FLP-mediated recombination and removal of the nptII marker gene. A second round of shoot regeneration from leaf explants led to GM apple plants completely free of the nptII gene.     

FLP recombinase-mediated site-specific recombination in rice

The feasibility of using the FLP/ FRT site-specific recombination system in rice for genome engineering was evaluated. Transgenic rice plants expressing the FLP recombinase were crossed with plants harbouring the kanamycin resistance gene ( neomycin phosphotransferase II , nptII ) flanked by FRT sites, which also served to separate the corn ubiquitin promoter from a promoterless gusA . Hybrid progeny were tested for excision of the nptII gene and the positioning of the ubiquitin promoter proximal to gusA . While the hybrid progeny from various crosses exhibited β-glucuronidase (GUS) expression, the progeny of selfed parental rice plants did not show detectable GUS activity. Despite the variable GUS expression and incomplete recombination displayed in hybrids from some crosses, uniform GUS staining and complete recombination were observed in hybrids from other crosses. The recombined locus was shown to be stably inherited by the progeny. These data demonstrate the operation of FLP recombinase in catalysing excisional DNA recombination in rice, and confirm that the FLP/ FRT recombination system functions effectively in the cereal crop rice. Transgenic rice lines expressing active FLP recombinase generated in this study provide foundational stock material, thus facilitating the future application and development of the FLP/ FRT system in rice genetic improvement.     

Excision of selectable marker gene from transgenic tobacco using the GM-gene-deletor system regulated by a heat-inducible promoter

To excise a selectable marker gene from transgenic plants, a new binary expression vector based on the 'genetically modified (GM)-gene-deletor' system was constructed. In this vector, the gene coding for FLP site-specific recombinase under the control of a heat shock-inducible promoter HSP18.2 from Arabidopsis thaliana and isopentenyltransferase gene (ipt), as a selectable marker gene under the control of the cauliflower mosaic virus 35S (CaMV 35S) promoter, were flanked by two loxP/FRT fusion sequences as recombination sites in direct orientation. Histochemical staining for GUS activity showed that, upon induction by heat shock, all exogenous DNA, including the selectable marker gene ipt, beta-glucuronidase (gusA) gene and the FLP recombinase gene, between two loxP/FRT sites was eliminated efficiently from primary transgenic tobacco plants. Molecular analysis further confirmed that excision of the marker gene (ipt) was heritable and stable. Our approach provides a reliable strategy for auto-excising a selectable marker gene from calli, shoots or other tissues of transgenic plants after transformation and producing marker-free transgenic plants.     

Elimination of marker genes and targeted integration via FLP/<Emphasis Type="BoldItalic">FRT</Emphasis> recombination system from yeast in hybrid aspen (<Emphasis Type="BoldItalic">Populus tremula</Emphasis> L. × <Emphasis Type="Italic">P. tremuloides</Emphasis> Michx.)

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.     

FLP-mediated site-specific recombination in microinjected murine zygotes

The FLP recombinase of yeast catalyses site-specific recombination between repeated FLP recombinase target (FRT) elements in yeast and in heterologous system (Escherichia coli, Drosophila, mosquito and cultured mammalian cells). In this report, it is shown that transient FLP recombinase expression can recombine and activate an extrachromosomal silent reporter gene following coinjection into fertilized one-cell mouse eggs. Furthermore, it is demonstrated that introduction of a FLP-recombinase expression vector into transgenic one-cell fertilized mouse eggs induces a recombination event at a chromosomal FRT target locus. The resulting event occured at the one-cell stage and deleted a chromosomal tandem array of a FRT containinglacZ expression cassette down to one or two copies. These results demonstrate that the FLP recombinase can be utilized to manipulate the genome of transgenic animals and suggest that FLP recombinase-mediated plasmid-to-chromosome targeting is feasible in microinjected eggs.     

FLP-mediated recombination in the vector mosquito, Aedes aegypti.

The activity of a yeast recombinase, FLP, on specific target DNA sequences, FRT, has been demonstrated in embryos of the vector mosquito, Aedes aegypti. In a series of experiments, plasmids containing the FLP recombinase under control of a heterologous heat-shock gene promoter were co-injected with target plasmids containing FRT sites into preblastoderm stage mosquito embryos. FLP-mediated recombination was detected between (i) tandem repeats of FRT sites leading to the excision of specific DNA sequences and (ii) FRT sites located on separate plasmids resulting in the formation of heterodimeric or higher order multimeric plasmids. In addition to FRT sites originally isolated from the yeast 2 microns plasmid, a number of synthetic FRT sites were also used. The synthetic sites were fully functional as target sites for recombination and gave results similar to those derived from the yeast 2 microns plasmid. This successful demonstration of yeast FLP recombinase activity in the mosquito embryo suggests a possible future application of this system in establishing transformed lines of mosquitoes for use in vector control strategies and basic studies.     

FLP-mediated recombination for use in hybrid plant production

We have studied the feasibility in Arabidopsis of using a site-specific recombination system FLP/FRT, from the 2 microm plasmid of yeast, for making plant hybrids. Initially, Arabidopsis plants expressing the FLP site-specific recombinase were crossed with plants transformed with a vector containing kanamycin-resistance gene (npt) flanked by FRT sites, which also served to separate the CaMV35S promoter from a promoterless gusA. Hybrid progeny were tested for excision of the npt gene and the positioning of 35S promoter proximal to gusA. GUS activity was observed in the progeny of all crosses, but not in the progeny derived from the self-pollinated homozygous parents. We then induced male sterility in Arabidopsis plants using the antisense expression of a pollen- and tapetum-specific gene, bcp1, flanked by FRT sites. Upon cross-pollination of flowers on the same male-sterile plants with pollen from FLP-containing plants, viable seeds were produced and the progeny hybrid plants developed normally. Molecular analyses revealed that the antisense expression cassette of bcp1 had been excised in these plants. These results show for the first time that a site-specific recombinase can be used to restore fertility in male-sterile plants, providing an alternative method for the production of hybrid seeds and plants.     

Site-specific gene integration in rice genome mediated by the FLP-FRT recombination system

Plant transformation based on random integration of foreign DNA often generates complex integration structures. Precision in the integration process is necessary to ensure the formation of full-length, single-copy integration. Site-specific recombination systems are versatile tools for precise genomic manipulations such as DNA excision, inversion or integration. The yeast FLP-FRT recombination system has been widely used for DNA excision in higher plants. Here, we report the use of FLP-FRT system for efficient targeting of foreign gene into the engineered genomic site in rice. The transgene vector containing a pair of directly oriented FRT sites was introduced by particle bombardment into the cells containing the target locus. FLP activity generated by the co-bombarded FLP gene efficiently separated the transgene construct from the vector-backbone and integrated the backbone-free construct into the target site. Strong FLP activity, derived from the enhanced FLP protein, FLPe, was important for the successful site-specific integration (SSI). The majority of the transgenic events contained a precise integration and expressed the transgene. Interestingly, each transgenic event lacked the co-bombarded FLPe gene, suggesting reversion of the integration structure in the presence of the constitutive FLPe expression. Progeny of the precise transgenic lines inherited the stable SSI locus and expressed the transgene. This work demonstrates the application of FLP-FRT system for site-specific gene integration in plants using rice as a model.     

A Cre::FLP fusion protein recombines FRT or loxP sites in transgenic maize plants

SUMMARY: The coding sequences of Cre (site-specific recombinase from bacteriophage P1) and FLP (yeast 2-microm plasmid site-specific recombinase) were fused in frame to produce a novel, dual-function, site-specific recombinase gene. Transgenic maize plants containing the Cre::FLP fusion expression vector were crossed to transgenic plants containing either the loxP or FRT excision substrate. Complete and precise excisions of chromosomal fragments flanked by the respective target sites were observed in the F1 and F2 progeny plants. The episomal DNA recombination products were frequently lost. Non-recombined FRT substrates found in the F1 plants were recovered in the F2 generation after the Cre::FLP gene segregated out. They produced the recombination products in the F3 generation when crossed back to the FLP-expressing plants. These observations may indicate that the efficiency of site-specific recombination is affected by the plant developmental stage, with site-specific recombination being more prevalent in developing embryos. The Cre::FLP fusion protein was also tested for excisions catalysed by Cre. Excisions were identified in the F1 plants and verified in the F2 plants by polymerase chain reaction and Southern blotting. Both components of the fusion protein (FLP and Cre) were functional and acted with similar efficiency. The crossing strategy proved to be suitable for the genetic engineering of maize using the FLP or Cre site-specific recombination system.     

T-DNA recombination and replication in maize cells

T-DNA recombination and replication was analyzed in 'black mexican sweet' (BMS) cells transformed with T-DNAs containing the replication system from wheat dwarf virus (WDV). Upon recombination between the T-DNA ends, a promoterless marker gene (gusA) was activated. Activation of the recombination marker gene was delayed and increased exponentially over time, suggesting that recombination and amplification of the T-DNA occurred in maize cells. Mutant versions of the viral initiator gene (rep), known to be defective in the replication function, failed to generate recoverable recombinant T-DNA molecules. Circularization of T-DNA by the FLP/FRT site-specific recombination system and/or homologous recombination was not necessary to recover circular T-DNAs. However, replicating T-DNAs appeared to be suitable substrates for site-specific and homologous recombination. Among 33 T-DNA border junctions sequenced, only one pair of identical junction sites was found implying that the population of circular T-DNAs was highly heterogenous. Since no circular T-DNA molecules were detected in treatments without rep, it suggested that T-DNA recombination was linked to replication and might have been stimulated by this process. The border junctions observed in recombinant T-DNA molecules were indicative of illegitimate recombination and were similar to left-border recombination of T-DNA into the genome after Agro-mediated plant transformation. However, recombination between T-DNA molecules differed from T-DNA/genomic DNA junction sites in that few intact right borders were observed. The replicating T-DNA molecules did not enhance genomic random integration of T-DNA in the experimental configuration used for this study.     

利用热诱导的位点专一性重组系统在烟草中控制基因表达

采用热激启动子Gmhsp17.5C控制Cre定位重组酶介导的DNA删除系统.在这个系统中,在热激启动子控制下的Cre重组酶的表达导致两侧带有相同方向loxp位点的CaMV35S-GUS片段从转基因烟草(Nicotiana tabacum L.cv.W38)的基因组中删除.通过定量PCR的方法鉴定这个转基因系统,显示了这个系统的重组效率.结果显示在两个小时热激处理后转基因烟草中有41%的CaMV35S-GUS片段被删除.由于热激诱导的定点重组系统有容易操作、对热敏感和无背景表达等优点,因此有利于采用这个系统在转基因植物中进行可诱导的基因操作.     

FLP recombinase-mediated site-specific recombination in silkworm, Bombyx mori

A comprehensive understanding of gene function and the production of site-specific genetically modified mutants are two major goals of genetic engineering in the post-genomic era. Although site-specific recombination systems have been powerful tools for genome manipulation of many organisms, they have not yet been established for use in the manipulation of the silkworm Bombyx mori genome. In this study, we achieved site-specific excision of a target gene at predefined chromosomal sites in the silkworm using a FLP/FRT site-specific recombination system. We first constructed two stable transgenic target silkworm strains that both contain a single copy of the transgene construct comprising a target gene expression cassette flanked by FRT sites. Using pre-blastoderm microinjection of a FLP recombinase helper expression vector, 32 G3 site-specific recombinant transgenic individuals were isolated from five of 143 broods. The average frequency of FLP recombinase-mediated site-specific excision in the two target strains genome was approximately 3.5%. This study shows that it is feasible to achieve site-specific recombination in silkworms using the FLP/FRT system. We conclude that the FLP/FRT system is a useful tool for genome manipulation in the silkworm. Furthermore, this is the first reported use of the FLP/FRT system for the genetic manipulation of a lepidopteran genome and thus provides a useful reference for the establishment of genome manipulation technologies in other lepidopteran species.     

Production of Marker-free Transgenic Tobacco Plants by FLP/frt Recombination System

Selectable marker genes that usually encode antibiotic or herbicide resistances are widely used for the selection of transgenic plants, but they become unnecessary and undesirable after transformation selection. An important strategy to improve the transgenic plants' biosafety is to eliminate the marker genes after successful selection. In the FLP/frt site-specific system of 2-μm plasmid from Saccharomyces cerevisiae, the FLP enzyme efficiently catalyzes recombination between two directly repeated FLP recombination target (frt) sites, eliminating the sequence between them. By controlled expression of the FLP recombinase and specific allocation of the frt sites within transgenic constructs, the system can be applied to eliminate the marker genes after selection. Through a series of procedures, the plant FLP/frt site-specific recombination system was constructed, which included the frt-containing vector pCAMBIA1300-betA-frt-als-frt and the FLP expression vector pCAMBIA1300-hsp-FLP-hpt. The FLP recombinase gene was introduced into transgenic (betA-frt-als-frt) tobacco plants by re-transformation. In re-transgenic plants, after heat-shock treatment, the marker gene als flanked by two identical orientation frt sites could be excised by the inducible expression of FLP recombinase under the control of hsp promoter. Excision of the als gene was found in 41 % re-transgenic tobacco plants, which indicated that this system could make a great contribution obtaining the marker-free transgenic plants.