Inducible Excision of Selectable Marker Gene from Transgenic Plants by the Cre/<Emphasis Type="Italic">lox</Emphasis> 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.     

A transformation vector for the production of marker-free transgenic plants containing a single copy transgene at high frequency

We represent here the GST-MAT vector system. The R recombinase gene of the site-specific recombination system R/RS from Zygosaccharomyces rouxii was fused to the chemical inducible promoter of the glutathione-S-transferase (GST-II-27) gene from Zea mays. Upon excision, the isopentenyltransferase (ipt) gene that is used as a selectable marker gene is removed. When the cauliflower mosaic virus 35S promoter (CaMV 35S) was used to express R recombinase, 67% of the marker-free transgenic plants had more than three transgene copies. Because the CaMV 35S promoter transiently and efficiently excised the ipt gene before callus and adventitious bud formation, the frequency of emergence of the ipt-shooty explants with a single T-DNA copy might be reduced. In this study we show that the GST-MAT vector efficiently produced transgenic ipt-shooty explants from 37 (88%) out of 42 differentiated adventitious buds and marker-free transgenic plants containing the GUS gene from five (14%) out of 37 ipt-shooty lines. Furthermore, the GST-MAT vector also induced two marker-free transgenic plants without the production of ipt-shooty intermediates. Southern blot analysis showed that six (86%) out of seven marker-free transgenic plants had a single GUS gene. This result suggests that the GST-MAT vector is useful to generate high frequency, marker-free transgenic plants containing a single transgene.     

利用FLP/frt重组系统产生无选择标记的转基因烟草植株

在植物转基因植株产生过程中,对转化细胞进行抗性筛选是通用程序,转化细胞的抗性一般是抗生素抗性或除草剂抗性,将赋予转化细胞抗性的选择标记基因删除是提高转基因植物生物安全性的重要措施。来自于啤酒酵母的FLP/frt位点特异性重组系统可有效删除同向定点重组位点frt之间的基因。通过多步骤重组,建立了可在植物中广泛应用的FLP/frt位点特异性重组系统。该系统包括含有frt位点的植物表达载体pCAMBIA1300-betA-frt-als-frt和含有由热诱导启动子hsp启动的FLP重组酶基因的植物表达载体pCAMBIA1300-hsp-FLP-hpt。利用二次转化的方式将二者先后转入烟草植株,热激处理后,热诱导型启动子hsp调控的重组酶FLP基因的表达催化位于选择标记基因als两侧同向frt位点间的重组反应,有效地删除了选择标记基因als。41%的经热激处理的二次转化植株发生了选择标记基因的删除,表明该系统在获得无选择标记基因的转基因植株中有很好的应用价值。     

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.     

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.     

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.     

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.     

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.     

Production of proinsulin in marker-free transgenic tobacco plants using <Emphasis Type="Italic">CRE</Emphasis>/loxP system

The demand for INSULIN is increasing rapidly along with the increased number of diabetic patients. Using the CRE/loxP system, we developed a selective marker-free system without crossing to produce PROINSULIN in transgenic plant. In frame of this approach, the induced promoter pRD29A was isolated from Arabidopsis. The CRE recombinase gene was placed under the control of pRD29A between two loxP recombination sites together with the selective NPTII gene. Furthermore, the binary vector with CRE recombinase and PROINSULIN was constructed and introduced into tobacco (Nicotiana tabacum L.) by Agrobacterium-mediated transformation. Gene excision was used to remove the sequence between the two loxP sites at the presence of 200 mM NaCl. PCR analysis showed that self-excision occurred in several T₀ transgenic plants. Transgenic plants without any marker gene successfully expressed PROINSULIN. This auto-excision strategy provides efficient means of removing the selectable marker gene from transgenic plants. It is an efficient method for producing bio-safe recombinant protein and other valuable substances in plants.     

Heat shock induced excision of selectable marker genes in transgenic banana by the Cre-lox site-specific recombination system

Selectable marker genes are indispensable for efficient production of transgenic events, but are no longer needed after the selection process and may cause public concern and technological problems. Although several gene excision systems exist, few have been optimized for vegetatively propagated crops. Using a Cre-loxP auto-excision strategy, we obtained transgenic banana plants cv. Grande Naine (Musa AAA) devoid of the marker gene used for selection. We used T-DNA vectors with the cre recombinase gene under control of a heat shock promoter and selectable marker gene cassettes placed between two loxP sites in direct orientation, and a gene of interest inserted outside of the loxP sites. Heat shock promoters pGmHSP17.6-L and pHSP18.2, from soybean and Arabidopsis respectively, were tested. A transient heat shock treatment of primary transgenic embryos was sufficient for inducing cre and excising cre and the marker genes. Excision efficiency, as determined by PCR and Southern hybridization was 59.7 and 40.0% for the GmHSP17.6-L and HSP18.2 promoters, respectively. Spontaneous excision was not observed in 50 plants derived from untreated transgenic embryos. To our knowledge this is the first report describing an efficient marker gene removal system for banana. The method described is simple and might be generally applicable for the production of marker-free transgenic plants of many crop species.     

Site-specific targeting of exogenous DNA into the genome of Candida albicans using the FLP recombinase

We have created a system that utilizes the FLP recombinase of Saccharomyces cerevisiae to reversibly introduce exogenous cloned DNA at defined locations into the Candida albicans genome. Recombination target (FRT) sites and the FLP gene can be introduced permanently at defined locations using homologous recombination. FLP recombinase is provided as needed through the regulated expression of its gene using the MAL2 promoter. Exogenous DNA is introduced on a cloning vector that is unable to replicate in C. albicans, and contains an FRT site and a selectable marker (URA3). Transformation by the lithium acetate or electroporation procedure is sufficient to obtain site-specific integration. This system permits rapid and precise excision of the introduced DNA when needed. It should facilitate studies on C. albicans genome structure and function, simplifying a wide range of chromosomal cloning applications, and generally enhancing the utility of C. albicans as a model organism for the study of fungal pathogenicity.     

选择标记可去除的植物高效表达载体的构建

在常用的植物组成型表达载体pBI121的选择标记基因NPTII两侧插入同向的lox位点并用多克隆位点(MCS)取代了GUS基因序列,构建了NPTII基因可被去除的和可插入目的基因的通用植物表达载体pBI121-lox-MCS。替换pBI121-lox-MCS中驱动目的基因表达的35S启动子,可构建成一系列具有其他表达特性的植物表达载体,如本文描述的韧皮部特异表达载体pBdENP-lox-MCS。为方便地筛选去除选择标记基因的转基因植物,还构建了绿色荧光蛋白(GFP)表达框与NPTII表达框连锁的pBI121-gfp-lox-MCS载体。上述植物表达载体可广泛应用于培育选择标记可去除的转基因植物。     

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.     

The 35S promoter used in a selectable marker gene of a plant transformation vector affects the expression of the transgene

Positive selection of transgenic plants is essential during plant transformation. Thus, strong promoters are often used in selectable marker genes to ensure successful selection. Many plant transformation vectors, including pPZP family vectors, use the 35S promoter as a regulatory sequence for their selectable marker genes. We found that the 35S promoter used in a selectable marker gene affected the expression pattern of a transgene, possibly leading to a misinterpretation of the result obtained from transgenic plants. It is likely that the 35S enhancer sequence in the 35S promoter is responsible for the interference, as in the activation tagging screen. This affected expression mostly disappeared in transgenic plants generated using vectors without the 35S sequences within their T-DNA region. Therefore, we suggest that caution should be used in selecting a plant transformation vector and in the interpretation of the results obtained from transgenic approaches using vectors carrying the 35S promoter sequences within their T-DNA regions.     

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 recombinase transgenic mice of C57BL/6 strain for conditional gene targeting

We constructed an expression vector of Flp recombinase modified by adding a nuclear localization signal. Injection of the expression vector into fertilized eggs of the C57BL/6 strain yielded transgenic mouse lines expressing the Flp recombinase transgene in the testis. We crossed the transgenic mice to reporter mice carrying the neomycin phosphotransferase gene flanked by target sites of Flp recombinase. Examination of the deletion of the neomycin phosphotransferase gene in the progeny showed that Flp-mediated recombination took place efficiently in vivo in FLP66 transgenic mouse line. These results suggest that the Flp recombinase system is effective in mice and in combination with the Cre recombinase system extends the potentials of gene manipulation in mice. One of the useful applications of FLP66 transgenic mouse line is the removal of marker genes from mice manipulated for the conditional gene targeting with the Cre/loxP system in the pure C57BL/6 genetic background.     

Heat-inducible expression of FLP gene in maize cells

The soybean heat-shock gene promoter ( Gmhsp 17.5-E ) has been used to direct expression of gusA and FLP genes in maize cells. At inducible temperatures, in transient expression assays, gusA gene expression controlled by the heat-shock promoter is about 10-fold higher than the expression directed by the CaMV 35S promoter. The Gmhsp 17.5-E promoter preserves its regulatory functions in heterologous maize cells after random integration into genomic DNA.
Heat-shock inducible expression of the FLP gene was investigated by co-transformation of the FLP expression vector (pHsFLP) and a recombination test vector (pUFNeo-FmG) into maize protoplasts. Co-transformed protoplasts were incubated at 42°C for 2 h. This treatment induced recombination of 20–25% of the available FRT sites in transient assays. As a result of heat-shock treatment of stably co-transformed maize cells, activation of gusA gene expression and an associated decrease or elimination of NPT-II activity in transgenic maize lines was observed. Molecular evidence was obtained of the expected DNA excision process catalyzed by the FLP protein in maize transgenic cells. Thus, the experiments presented in this paper indicate that the FLP protein can recognize and subsequently recombine the FRT target sites that had integrated into plant genomic DNA, and that regulated expression of the FLP gene is possible in maize cells using the soybean heat-shock promoter.     

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.