Cre/<Emphasis Type="Italic">lox</Emphasis>-mediated marker gene excision in transgenic maize (Zea mays L.) plants

After the initial transformation and tissue culture process is complete, selectable marker genes, which are used in virtually all transformation approaches, are not required for the expression of the gene of interest in the transgenic plants. There are several advantages to removing the selectable marker gene after it is no longer needed, such as enabling the reuse of selectable markers and simplifying transgene arrays. We have tested the Cre/lox system from bacteriophage P1 for its ability to precisely excise stably integrated marker genes from chromosomes in transgenic maize plants. Two strategies, crossing and autoexcision, have been tested and demonstrated. In the crossing strategy, plants expressing the Cre recombinase are crossed with plants bearing a transgene construct in which the selectable marker gene is flanked by directly repeated lox sites. Unlike previous reports in which incomplete somatic and germline excision were common, in our experiments complete somatic and germline marker gene excision occurred in the F₁ plants from most crosses with multiple independent Cre and lox lines. In the autoexcision strategy, the cre gene, under the control of a heat shock-inducible promoter, is excised along with the nptII marker gene. Our results show that a transient heat shock treatment of primary transgenic callus is sufficient for inducing cre and excising the cre and nptII genes. Genetic segregation and molecular analysis confirmed that marker gene removal is precise, complete and stable. The autoexcision strategy provides a way of removing the selectable marker gene from callus or other tissues such as embryos and kernels.Communicated by D. Hoisington     

Removal of the Selectable Marker Gene from Transgenic Tobacco Plants by Expression of Cre Recombinase from a Tobacco Mosaic Virus Vector through Agroinfection

Selection markers are often indispensable during the process of plant transformation, but dispensable once transgenic plants have been established. The Cre/lox site-specific recombination system has been employed to eliminate selectable marker genes from transgenic plants. Here we describe the use of a movement function-improved Tobacco Mosaic Virus (TMV) vector, m30B, to express Cre recombinase for elimination of the selectable marker gene nptII from transgenic tobacco plants. The transgenic tobacco plants were produced by Agrobacterium-mediated transformation with a specially designed binary vector pGNG which contained in its T-DNA region a sequence complex of 35S promoter-lox-the gfp coding sequence-rbcS terminator-Nos promoter-nptII-Nos terminator-lox-the gus coding region-Nos terminator. The expression of the recombinant viral vector m30B:Cre in plant cells was achieved by placing the viral vector under the control of the 35S promoter and through agroinoculation. After co-cultivating the pGNG-leaf discs with agro35S-m30B:Cre followed by shoot regeneration without any selection, plants devoid of the lox-flanked sequences including nptII were obtained with an efficiency of about 34% as revealed by histochemical GUS assay of the regenerants. Three of 11 GUS expressing regenerants, derived from two independent transgenic lines containing single copy of the pGNG T-DNA, proved to be free of the lox-flanked sequences by Southern blot analysis. Excision of the lox-flanked sequences in the three plants could be attributed to transient expression of Cre from the viral vector at the early stage of co-cultivation, since the cre sequence could not be detected in the viral RNA molecules accumulated in the plants, nor in their genomic DNA. The parental marker-free genotype was inherited in their selfed progeny, and all of the progeny were virus-free, apparently because TMV is not seed-transmissible. Therefore, expression of Cre from a TMV-based vector could be used to eliminate selectable marker genes from transgenic tobacco plants without sexual crossing and segregation, and this strategy could be extended to other TMV-infected plant species and applicable to other compatible virus–host plant systems.     

Feasibility of the seed specific cruciferin C promoter in the self excision Cre/<Emphasis Type="Italic">lox</Emphasis>P strategy focused on generation of marker-free transgenic plants

This work is focused on the generation of selectable marker-free transgenic tobacco plants using the self excision Cre/loxP system that is controlled by a strong seed specific Arabidopsis cruciferin C (CRUC) promoter. It involves Agrobacterium-mediated transformation using a binary vector containing the gus reporter gene and one pair of the loxP sites flanking the cre recombinase and selectable nptII marker genes (floxed DNA). Surprisingly, an ectopic activation of CRUC resulting in partial excision of floxed DNA was observed during regeneration of transformed cells already in calli. The regenerated T₀ plants were chimeric, but no ongoing ectopic expression was observed in these one-year-long invitro maintained plants. The process of the nptII removal was expected in the seeds; however, none of the analysed T₀ transgenic lines generated whole progeny sensitive to kanamycin. Detailed analyses of progeny of selected T₀-30 line showed that 10.2% GUS positive plants had completely removed nptII gene while the remaining 86.4% were still chimeras. Repeated activation of the cre gene in T₂ seeds resulted in increased rate of marker-free plants, whereas four out of ten analysed chimeric T₁ plants generated completely marker-free progenies. This work points out the feasibility as well as limits of the CRUC promoter in the Cre/loxP strategy. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Heat-inducible Cre-<Emphasis Type="Italic">lox</Emphasis> system for marker excision in transgenic rice

The present study assessed the efficacy of a heat-inducible cre gene for conditional removal of the marker gene from a rice genome via Cre-lox recombination. A cre gene controlled by the soybean heat-shock promoter was introduced into the rice genome along with the recombination target (lox) construct. Cre-mediated recombination was expected to remove the marker gene and activate the promoter-less GUS gene. Six transgenic lines displayed well-regulated heat-inducible Cre activity in the callus. However, only one line that contained a single copy of the cre gene maintained this property in the regenerated plants and their progeny. Marker-free progeny were obtained from the plant that was heat-treated at the seedling stage, indicating the inheritance of the recombination ‘footprint’. The presence of the ‘footprint’ was verified by polymerase chain reaction and Southern analysis. Therefore, the cre gene controlled by the soybean heat-shock promoter is an effective tool for conditional removal of the marker gene in rice.     

Excision of a selectable marker gene in transgenic banana using a Cre/lox system controlled by an embryo specific promoter

Antibiotic and herbicide resistance genes have been used in transgene technology as powerful selection tools. Nonetheless, once transgenic events have been obtained their presence is no longer needed and can even be undesirable. In this work, we have developed a system to excise the selectable marker and the cre recombinase genes from transgenic banana cv. ‘Grande Naine’ (Musa AAA). To achieve this, the embryo specific REG-2 promoter was isolated from rice and its expression pattern in banana cell clumps, somatic embryos and regenerated plantlets was characterized by using a pREG2::uidA fusion construct. Subsequently, the REG-2 promoter was placed upstream of the cre gene, conferring Cre functionality in somatic embryos and recombination of lox sites resulting in excision of the selectable marker and cre genes. PCR analysis revealed that 41.7 % of the analysed transgenic plants were completely marker free, results that were thereafter confirmed by Southern blot hybridization. These results demonstrate the feasibility of using developmentally controlled promoters to mediate marker excision in banana. This system does not require any extra handling compared to the conventional transformation procedure and might be useful in other species regenerating through somatic embryogenesis.     

Developmentally regulated site-specific marker gene excision in transgenic <Emphasis Type="Italic">B. napus</Emphasis> plants

We have developed a self-excision Cre-vector to remove marker genes from Brassica napus. In this vector cre recombinase gene and bar expression cassette were inserted between two lox sites in direct orientation. These lox-flanked sequences were placed between the seed-specific napin promoter and the gene of interest (vstI). Tissue-specific cre activation resulted in simultaneous excision of the recombinase and marker genes. The vector was introduced into B. napus by Agrobacterium-mediated transformation. F1 progeny of seven lines with single and multiple transgene insertions was subjected to segregation and molecular analysis. Marker-free plants could be detected and confirmed by PCR and Southern blot in all transgenic lines tested. The recombination efficiency expressed as a ratio of plants with complete gene excision to the total number of investigated plants varied from 13 to 81% dependent on the transgene copy number. Potential application of this system would be the establishment of marker-free transgenic plants in generatively propagated species.     

Cre-mediated seed-specific transgene excision in tobacco

Here we report the production of marker-free transgenic plants expressing phenolic compounds with high pharmacological value. Our strategy consisted in simultaneous delivery of lox-target and cre-containing constructs into the plant genome by cotransformation. In the Cre-vector, the cre recombinase gene was controlled by a seed-specific napin promoter. In the lox-target construct the selectable bar gene was placed between two lox sites in direct orientation, while a napin promoter driven vstI gene was inserted outside of the lox sites. Upon seed-specific cre induction the bar expression cassette was excised from the tobacco genome. Genetic and molecular analysis of T1 progeny plants indicated DNA excision in all 10 transgenic lines tested. RP-HPLC analysis demonstrated that the expression of the vstI gene resulted in accumulation of trans-resveratrol and its glycosylated derivative piceid in seeds of all marker free lines. These findings indicate that the seed-specific marker gene excision did not interfere with the expression of the gene of interest. Our data demonstrated the feasi of a developmentally controlled cre gene to mediate site-specific excision in tobacco very efficiently.     

Agrobacterium-mediated transformation of maize (Zea mays) with Cre-lox site specific recombination cassettes in BIBAC vectors

The Cre/loxP site-specific recombination system has been applied in various plant species including maize (Zea mays) for marker gene removal, gene targeting, and functional genomics. A BIBAC vector system was adapted for maize transformation with a large fragment of genetic material including a herbicide resistance marker gene, a 30 kb yeast genomic fragment as a marker for fluorescence in situ hybridization (FISH), and a 35S-lox-cre recombination cassette. Seventy-five transgenic lines were generated from Agrobacterium-mediated transformation of a maize Hi II line with multiple B chromosomes. Eighty-four inserts have been localized among all 10 A chromosome pairs by FISH using the yeast DNA probe together with a karyotyping cocktail. No inserts were found on the B chromosomes; thus a bias against the B chromosomes by the Agrobacterium-mediated transformation was revealed. The expression of a cre gene was confirmed in 68 of the 75 transgenic lines by a reporter construct for cre/lox mediated recombination. The placement of the cre/lox site-specific recombination system in many locations in the maize genome will be valuable materials for gene targeting and chromosome engineering.     

Salicylic-Acid-Induced Self-excision of the Marker Gene <Emphasis Type="Italic">npt</Emphasis>II from Transgenic Tomato Using the Cre–<Emphasis Type="Italic">loxP</Emphasis> System

The presence of antibiotic-resistant genes in genetically engineered crops together with the target gene has generated a number of environmental and consumer concerns. In order to alleviate public concerns over the safety of food derived from transgenic crops, marker gene elimination is desirable. Marker-free transgenic tomato plants were obtained by using a salicylic-acid-regulated Cre–loxP-mediated site-specific DNA recombination system in which the selectable marker neomycin phosphotransferase nptII and cre genes were flanked by two directly oriented loxP sites. Upon induction by salicylic acid, the cre gene produced a recombinase that eliminated sequences encoding nptII and cre genes, sandwiched by two loxP sites from the tomato genome. Regenerant plants with the Cre–loxP system were obtained by selection on kanamycin media and polymerase chain reaction (PCR) screening. Transgenic plants were screened for excision by PCR using nptII, cre, and PR-1a promoter primers following treatment with salicylic acid. The footprint of the excision was determined by sequencing the T-DNA borders after a perfect recombination event. The excision efficiency was 38.7%. A new plant transformation vector, pBLNSC (Genbank accession number EU327497), was developed, containing six cloning sites and the self-excision system. This provided an effective approach to eliminate the selectable marker gene from transgenic tomato, thus expediting public acceptance of genetically modified tomato.     

Generation of marker free salt tolerant transgenic plants of Arabidopsis thaliana using the gly I gene and cre gene under inducible promoters

Despite the advances in transgenesis, transformation technologies still rely on the introduction of a selectable marker gene to identify cells and tissues that have integrated the gene of interest in their genome. The continuous presence of the marker genes in the transgenics is often controversial as it can potentially have multiple undesirable impacts. The present study employed the self-excising Cre-loxP system to generate marker-free Arabidopsis thaliana expressing the agronomically important glyoxalase I (glyI) gene from Brassica juncea to confer salt stress tolerance. A binary vector was constructed wherein the salt-inducible rd29A promoter was used to drive the expression of the glyI gene and the transformants of A. thaliana were recovered using kanamycin resistance as the selectable marker. The neomycin phosphotransferase II (nptII) gene was flanked by the loxP sites followed by the introduction of a heat-inducible Cre-recombinase in between the loxP sites. The kanamycin-resistant transgenic lines of A. thaliana using this vector showed an ability to withstand stress imposed by 150 mM NaCl. The exposure of the T2 transgenic lines to a mild heat shock (37°C) resulted in the recovery of salt-tolerant, kanamycin-sensitive T3 progeny. Molecular analyses of the T3 transgenic lines following the heat shock treatment confirmed the excision of the nptII gene and the completion of their life cycle in the presence of 150 mM NaCl-induced stress.     

Excision of a selective marker in transgenic rice using a novel Cre/<Emphasis Type="Italic">loxP</Emphasis> system controlled by a floral specific promoter

Based on the Cre/loxP system, we have developed a novel marker-free system mediating a direct auto-excision of loxP-flanked marker genes from T₁ transgenic rice without any treatment or further offspring crossing. To achieve this, the floral-specific promoter OsMADS45 was isolated from rice and the expression patterns of OsMADS45 promoter was characterised by using the pOs45:GUS transgenic plants. Furthermore, the binary vector with Cre recombinase under the control of OsMADS45 promoter was constructed and introduced into rice by Agrobacterium-mediated transformation and transgenic rice plants were generated. Southern blot analysis showed that auto-excision of the selective markers occurred in some T₁ progeny of the transgenic plants, suggesting that a high auto-excision frequency can be achieved with our Cre/loxP system. This auto-excision strategy provides an efficient way of removing the selectable marker gene from transgenic rice. Xianquan Bai and Qiuyun Wang contributed equally to the work.     

A Self-Excising Cre Recombinase Allows Efficient Recombination of Multiple Ectopic Heterospecific Lox Sites in Transgenic Tobacco

To study the impact of different DNA configurations on the stability of transgene expression, a variant of the cre gene was developed. This variant allows for the highly efficient in planta removal of its own loxP-flanked coding sequence as well as other DNAs flanked by ectopic heterospecific lox sites, either lox511 or lox2272 or both, in trans. The plant intron-containing cre gene, cre ^INT , was configured in such a way that self-excision generated an intact hygromycin resistance selectable marker gene. In this combination, all selected transformants showed highly efficient excision. Plants obtained showed no indication of any chimerism, indicating a cell autonomous nature of the hygromycin selection during transformation and regeneration. The highly efficient concomitant removal of wildtype and heterospecific lox site-flanked DNA demonstrated that upon retransformation with the self-excising cre ^INT , sufficient amounts of Cre enzyme were produced prior to its removal. Plants obtained with cre ^INT showed much less frequently the Cre-associated phenomenon of reduced fertility than plants obtained with a continuous presence of Cre recombinase. The cre ^INT system has therefore advantages over systems with a continuously present Cre. The cre ^INT system was successfully used for removal of two chromatin boundary elements from transgene cassettes in tobacco. Analysis of plants with and without boundary elements on the same chromosomal location will contribute to a better evaluation of the role of such elements in the regulation of transgene expression in plants.     

Chemical-induced autoexcision of selectable markers in elite tomato plants transformed with a gene conferring resistance to lepidopteran insects

Marker-free transgenic tomato plants harboring a synthetic Bacillus thuringiensis endotoxin gene, cryIAc, were obtained by using a chemically regulated, Cre/loxP-mediated site-specific DNA recombination system, in which the selectable marker neomycin phosphotransferase gene flanked by two directly oriented loxP sites was located between the cauliflower mosaic virus 35S promoter and a promoterless cryIAc. Upon induction by 2 μM β-estradiol, sequences encoding the selectable marker and cre sandwiched by two loxP sites were excised from the tomato genome, leading to activation of the downstream endotoxin gene cryIAc with high expression levels as shown by Northern blot and ELISA assay (250–790 ng g⁻¹ fresh wt) in T₁ generation. For transgenic line with single transgenic loci, 15% of T₁ progenies were revealed marker-free. This autoexcision strategy provides an effective approach to eliminate a selectable marker gene from transgenic tomato, thus expediting the public acceptance of genetically modified crop.     

A co-transformation system to produce transgenic grapevines free of marker genes

A co-transformation system was developed to produce grapevines free of selectable marker genes. This was achieved by transforming Vitis vinifera L. ‘Thompson Seedless’ somatic embryos with a mixture of two Agrobacterium strains. The first strain contained a binary plasmid with an egfp gene of interest between the T-DNA borders. The second strain harbored the neomycin phosphotransferase (nptII) gene for positive selection and the cytosine deaminase (codA) gene for negative selection, linked together by a bi-directional dual promoter complex. Our technique included a short positive selection phase on medium containing 100 mg l⁻¹ kanamycin before subjecting cultures to prolonged negative selection on medium containing 250 mg l⁻¹ 5-fluorocytosine. We regenerated 25 stable EGFP expressing transgenic lines. PCR analysis confirmed 18 lines contained only the egfp gene, whereas the remaining contained both egfp and codA/nptII genes. Presumably, the 18 monogenic lines arose through cross protection by being in close proximity to cells that expressed nptII and thus detoxified kanamycin in the immediate vicinity. This is the first report for grapevine using a combination of positive and negative selection to produce transgenic plants that do not contain marker genes.     

Self-excision of the antibiotic resistance gene nptII using a heat inducible Cre-loxP system from transgenic potato

Resistance to antibiotics mediated by selectable marker genes remains a powerful selection tool for transgenic event production. However, regulatory agencies and consumer concerns favor these to be eliminated from food crops. Several excision systems exist but none have been optimized or shown to be functional for clonally propagated crops. The excision of the nptII gene conferring resistance to kanamycin has been achieved here using a gene construct based on a heat-inducible cre gene producing a recombinase that eliminates cre and nptII genes flanked by two loxP sites. First-generation regenerants with the Cre-loxP system were obtained by selection on kanamycin media. Following a heat treatment, second generation regenerants were screened for excision by PCR using nptII, cre, and T-DNA borders primers. Excision efficiency appeared to be at 4.7% depending on the heat treatment. The footprint of the excision was shown by sequencing between T-DNA borders to correspond to a perfect recombination event. Selectable marker-free sprouts were also obtained from tubers of transgenic events when submitted to similar heat treatment at 4% frequency. Spontaneous excision was not observed out of 196 regenerants from untreated transgenic explants. Biosafety concerns are minimized because the expression of cre gene driven by the hsp70 promoter of Drosophila melanogaster was remarkably low even under heat activation and no functional loxP site were found in published Solanum sequence database. A new plant transformation vector pCIP54/55 was developed including a multiple cloning site and the self-excision system which should be a useful tool not only for marker genes in potato but for any gene or sequence removal in any plant.     

Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome

The bacteriophage P1 Cre—lox site-specific recombination system has been used to integrate DNA specifically at lox sites previously placed in the tobacco genome. As integrated molecules flanked by wild-type lox sites can readily excise in the presence of Cre recombinase, screening for mutant lox sites that can resist excisional recombination was performed. In gene integration experiments, wild-type and mutant lox sites were used in conjunction with two strategies for abolishing post-integration Cre activity: (i) promoter displacement of a cre-expression construct present in the target genome; and (ii) transient expression of cre. When the promoter displacement strategy was used, integrant plants were recovered after transformation with constructs containing mutant lox sequences, but not with constructs containing wild-type lox sites. When cre was transiently expressed, integrant plants were obtained after transformation with either mutant or wild-type lox sites. DNA rearrangements at the target locus were less frequent when mutant lox sites were used. DNA integration at the genomic lox site was usually without additional insertions in the genome. Thus, the Cre—lox site-specific recombination system is useful for the single-copy integration of DNA into a chromosomal lox site.     

Expression of stilbene synthase gene in transgenic tomato using salicylic acid-inducible Cre/loxP recombination system with self-excision of selectable marker

A plant transformation vector, pCLKSCLA25 (EU327498), was developed to contain eight cloning sites and the inducible self-excision system which provided an effective approach to eliminate the selectable marker gene(s) from transgenic plants. Upon induction by salicylic acid, the cre gene produced a recombinase that eliminated sequences encoding the selectable marker neomycin phosphotransferase and cre itself. The excision efficiency was 41% in transgenic tomato regenarants. The stilbene synthase gene (vst1) from Vitis vinifera L. was cloned into pCLKSCLA25. The expression of vst1 gene contributed to the accumulation of trans-reveratrol from 3.4 to 8.7 μg/g fresh wt in different marker-free transgenic tomato lines. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A passage through in vitro culture leads to efficient production of marker-free transgenic plants in <Emphasis Type="Italic">Brassica juncea</Emphasis> using the Cre–<Emphasis Type="Italic">loxP</Emphasis> system

The Cre–loxP site-specific recombination system was deployed for removal of marker genes from Brassica juncea (Indian mustard). Excision frequencies, monitored by removal of nptII or gfp genes in F₁ plants of crosses between LOX and CRE lines, were high in quiescent, differentiated somatic tissues but extremely poor in the meristematic regions (and consequently the germinal cells) thus preventing identification and selection of marker-free transgenic events which are devoid of both the marker gene as well as the cre gene, in F₂ progeny. We show that a passage through in vitro culture of F₁ leaf explants allows efficient development of marker-free transgenics in the F₂ generation addressing current limitations associated with efficient use of the Cre/loxP technology for marker gene removal. N. Arumugam and Vibha Gupta have contributed equally to this work.     

Co-transformation of gene expression cassettes via particle bombardment to generate safe transgenic plant without any unwanted DNA

The presence of resistant selectable marker genes and other added DNAs such as the vector backbone sequence in transgenic plant might be an unpredictable hazard to the ecosystem as well as to human health, which have affected the safe assessment of transgenic plants seriously. Using minimal gene expression cassette (containing the promoter, coding region, and terminator) without vector backbone sequence for particle bombardment is the new trend of plant genetic transformation. In the present paper, we co-transformed the selectable marker bar gene cassette and non-selected cecropinB gene cassette into rice (Oryza sativa L.) by particle bombardment, then eliminated the selectable marker bar gene in R₁ generation applying the hereditary segregation strategy and attained two safe transgenic plants only harboring cecropinB gene cassettes without any superfluous DNA. This is the fist report indicating that the combination of minimal gene cassettes transformation with the co-transformation and segregation strategy can generate selectable marker-free transgenic plants, which will promote the advancement in plant genetic engineering greatly.