ISSN 00954527, Cytology and Genetics, 2015, Vol. 49, No. 6, pp. 397–407. © Allerton Press, Inc., 2015. Original Ukrainian Text © A.S. Sekan, S.V. Isayenkov, Ya.B. Blume, 2015, published in Tsitologiya i Genetika, 2015, Vol. 49, No. 6, pp. 61–72.
Development of MarkerFree Transformants by SiteSpecific Recombinases A. S. Sekan, S. V. Isayenkov, and Ya. B. Blume Institute of Food Biotechnology and Genomics, National Academy of Sciences of Ukraine, Kyiv, Ukraine email:
[email protected] Received March 19, 2015
Abstract—To produce transgenic plants in current biotechnology, selectable marker genes are used that lead to the selectivity of transformants from nontransformed organisms. However, after the transgenic event has been occured, the presence of these genes in transformants genome is generally of no use. Moreover, the con tinued presence of this kind of genes in transgenic plants with their further commercialization may raise pub lic concern. Therefore, various techniques have been developed in recent years to obtain marker free trans genic plants. In the present review are described the main strategies for removal of selective marker DNA sequences that are used in genetic engineering so far. The most popular among them is a sitespecific recom bination technology. The particular attention is paid to sitespecific recombinase system Cre/loxP. The using of a new approach with sitespecific recombinase system Cre/loxP under the control of 35S promoter to gen erate markerfree genetically modified plants is described. Keywords: selectable marker genes, excision of the marker sequences, sitespecific recombination system DOI: 10.3103/S0095452715060080
INTRODUCTION Strategic directions in the development of technol ogies for designing genetically modified (GM) organ isms, as well as the advantages and potential disadvan tages of their practical applications, are a constant subject of discussion not only in scientific environ ments but also on the public level. In parallel to expanding application of GM organisms, in particular plants, in everyday life, the additional questions have rised concerning the effects of transgenic organisms on natural ecosystems, the safety issues of their con sumption, probability of transferring genetic material to untransformed organisms, and improvement of food safety control as well. The big attention is paid to prevent a horizontal transfer of antibioticresistance genes to the animal and human intestinal microflora, as well as exclusion of a vertical transfer of herbicide resistant genes to conventional cultivars and their wild relatives [1, 2]. Development and improvement of techniques for designing novel selectable markerfree transgenic plants would contribute to better public acceptance of genetically modified plants. Therefore, our review describes the main strategies of modern genetic engineering, including the most popular site specific recombinase technology for the removal of selectable marker DNA sequences. Use of Selectable Marker Genes in Genetic Engineering Selectable marker genes (SMG) are DNA sequences that are constituent elements of DNA con
structs, which are used to identify transformed cells [3]. The ready for application developed technologies now comprise nearly 50 different SMGs. However, the plant genetic engineering utilizes only several technol ogies, which are characterized by their inexpensive simple application. In total, the sequences used as genetic markers for the transformation of plant genomes are mainly derived from bacterial or plant origin. These genes can be divided into two types: selectable genes and reporter genes. Selectable genes are thought to be DNA sequences allowing researchers to select transformed cells or tissues during their growth on a nutrient medium in the presence of a cor responding selective agent. These agents may be repre sented by antibiotics or herbicides [1]. Among the most frequently applied selectable markers are the nptII and hpt genes (resistance to aminoglycoside, kanamycin, and hygromycin) and the bar gene (resis tance to phosphinotrycin). The reporter genes (screening markers) are the sequences that allow researchers to identify transgenic plants in the absence of a selective agent. The reporter genes are mainly belong to the sequences encoding enzymes. For exam ple sequences of βglucuronidase (GUS), luciferase, βgalactosidase, et al. are typical reporter genes. The genes that participate in phytohormonal metabolism are also have been used for these needs. For example, the isopentenyl transferase gene (ipt) from the Agro bacterium tumefaciens TDNA was successfully used for the selection of transformants [4, 5]. The applica tion of the dexamethasoneinduced promoter for the
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ipt gene gave the possibility to increase the ability to regenerate of salad and tobacco transformants [5–7]. A successful commercialization of GM cultivars having marker genes in their genomes requires signif icant attention to potential emergence of certain risks in case of their release into the natural environment. The presence of SMGs, such as antibioticresistance genes, is prohibited for commercialization in EU countries, since it is assumed that there may be a risk for a horizontal gene transfer (a transfer of foreing DNA from a transformed organism to an untrans formed one with its future expression) [1]. Another problem associated with the presence of SMGs in transformed plants is the effect of gene silencing. The presence of marker genes in the genome may provoke the suppression or full silence of native or introduced genes during a plant’s growth and development. In other cases, as a result of simultaneous integration of multiple DNA constructs into the genome (multiple integration), enhanced expression of marker sequences is observed, which is also unwanted [4]. The aplication of several different DNA constructs with similar selectable markers for the transformation of the same plant may cause the duplication of promoters and poly(A) signals. The effect of gene silencing is also observed in these cases. Another occurrence of the gene silencing effect may be caused by repeated use of the same promoter and polyadenylation signal for dif ferent SMGs [8]. One of the most important tasks of genetic engi neering is to develop and improvement of novel strate gies for removing SMGs from transformant genomes, which allow researchers to avoid both the effect of gene silencing and the appearence of other problems associated with genetic transformation. There are sev eral main approaches for solving these problems [6]: the use of SMGs that do not carry potential threats; to block completely the use of SMGs; the removal of SMGs after a successful selection, using sitespecific recombination, transposition, or homologous recom bination. The one of best SMGs example without potential threats may be the application of plant DNA sequences. For example, the antibioticresistance genes can be exchanged by some constituent protein genes of the plant cell. This strategic approach could be illustrated by application of mutant tubulin gene responsible for resistance to antimicrotubular herbi cides. A successful application of this gene as a select able marker for obtaining transgenic plants has been described earlier [10]. The use of transformation technology without application of SMGs is sufficiently uncomfortable in work. It is theoretically possible to identify trans formed cells among untransformed using molecular methods [9]. At the same time, the use of sitespecific recombination, transposition, or homologous recom
bination for the removal of SMGs after a successful selection getting more popular. Techniques for the Removal of DNA Marker Sequences One of the ways to exclude SMGs from genes of interest is a sequential use of DNA constructs contain ing genes of interest and marker genes at different stages of transformation. The agrobacteriummedi ated transformation is usually used for this purpose, since the percentage of successful events in separate integration is the highest in this case. When carrying out this cotransformation, the gene of interest and the marker gene can be constructed into two different vec tor constructs within the same bacterium [11, 12] or used in different agrobacterium strains [13]. However, the number of integration events for both the construct with the gene of interest and the construct with the marker is significantly higher than required in this case, and, therefore, the probability is high for the presence of both TDNAs in a plant’s genome. In order to avoid this effect the procedure of continued selection is employed for transformants. This approach was succesfully applied for tobacco and rice plants [14]. This procedure is long, labor intensive, and cannot be applied for the transformation of trees having a considerable period of development. Marker genes can also be eliminated using trans posable elements. This approach is based on binding a certain transposable sequence to selectable genes within the DNA construct composition. In addition, markers and transposable sequences can be segregated between one another after transformation and selec tion. For example, a mobile element that is bound to a marker sequence can be destroyed after transposition [15], which was described in the studies about the transformation of tobacco and aspen [4]. The other variation of this approach is based on relocating the gene of interest at a considerable distance from the ini tial site of DNA construct integration [16]. This approach allows the expression of introduced DNA sequences on different segments of the genome to be provided in order to minimize the effect of overexpres sion in its particular regions. However, instability in TDNA integration is noted if the effect of transposi tion or transposable elements themselves is used, which not infrequently leads to the future shifts along the genome. The selection of plants with stable inser tions is quite complicated and timeconsuming pro cess in this case. Another variation of SMG removal is the applica tion of homologous recombination. The removal of DNA sequences located between direct repeats in the genome was demonstrated in somatic cells. The pro cess of removal occurred at quite a low frequency [17]. Therefore, the use of homologous recombination for plant transformation has been considered ineffective for a long time. However, the expediency of using this CYTOLOGY AND GENETICS
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phenomenon has been proven later in tobacco trans formants [18]. The kanamycin gene was placed between two sites of excision (attP sites) and further removed along with these sites at high frequency dur ing the plant’s growth. Due to using the proposed methods, the process of obtaining markerfree trans formants was accelerated, compared with the use of repeated transformation or cross pollination. The dis covery of systems with attP sites and bacteriophage λ helped solve this problem. This system is applied for the integration of specific bacteriophage DNA seg ment between the attB sites of E. coli. The reaction will require the presence of two proteins: integrase of the bacteriophage (int, phageencoded integrase) and the bacterial factor of integration (IHF, integration host factor) [19]. The recombination event induced by double strand breaks in DNA occurs due to the further repair of their integrity. Specific sequences in the genome can be activated through the route of doublestrand breaks in DNA, which will intensify the process of recombination in plant cells [20]. For example, the doublestrand breaks in DNA trigger the transient expression of the ISceI restriction enzyme. When a break occurs between the introduced in vivo restric tion sites, a homologous recombination was enhanced. The efficiency of this approach has been demonstrated in tobacco plants, where the marker gene in the used construction was flanked by the restriction sites for the ISceI protein. After expression of the gene encoding this enzyme, the marker gene was removed using a homologous recombination in a third of transformants [21]. This approach can also be applied for the elimination of multiple TDNA inte gration into the genome of transformants. Homolo gous recombination in somatic cells can also be inten sified by expression of proteins, such as, recA, ruvC, et al. [22]. But this approach implies that other homologous sequences will be activated along with the introduced genes, which might lead to total plant genome destabilization. Therefore, the use of the DNA doublestrand breaks event for providing homologous recombination is not quite efficient for the removal of marker genes. The sitespecific recombination technology is based on the possibility to construct DNA sequences at certain sites in the genome. This approach was ini tially demonstrated for the removal of selectable and marker genes from the genome of transformants in tobacco plants [23]. The kanamycinresistance gene was constructed between two loxP sites of vector DNAcassette. After introducing the construct into the plant genome, the gene of selection along with loxP sites were removed. The excision was performed using the Cre recombinase. Apart from Cre, either the FLP/ftr sitespecific system from S. cerevisiae [24] or the RRS system from the pSR1 plasmid isolated from Zygosaccharomyces rouxii [25] are used today for implementing this strategy. In contrast to the majority CYTOLOGY AND GENETICS
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of recombinases, Cre, Flp, and R do not require addi tional structure modifications in their application or the presence of specific factors during their work. The sitespecific recombinase system is also used for the transformation of chloroplast DNA, which allows to avoid a threat of interspecies cross between transgenic and wildtype plants [26]. As has also been revealed, it is undesirable to use marker genes for transforming the plastid genome, since thousands of copies of the same SMG sequence are at once repre sented in plastids. The sitespecific Cre/loxP system, which successfully works in the nuclear genome, was used to bypass the accumulation of marker gene prod ucts in cells and successfully eliminate transplastome lines of plants [27]. However, it has also been estab lished that the Cre recombinase can catalyze the reac tion of elimination among reliable native “pseudo lox” sequences that significantly differ from the lox sites in the construct [28]. A result of the Cre activity in this case is, for example, the occurence of sterility in plants [29]. Therefore, a homologous recombination was suggested for the SMG removal from the chloro plast DNA as the alternative to the sitespecific recombinase system. It has been revealed that this approach is more efficient for the transformation of chloroplasts and bacteria than for nuclear DNA in higher eukaryotes [30]. The efficiency of the men tioned approach was proved in tobacco plants trans formed by different marker sequences flanked by homologous 174–418 bps segments [31]. Use of Recombinases in Plant Biotechnology The application of recombinases in genetic engi neering is quite a novel and promising technology. The sitespecific recombinase system was discovered for the first time in bacteria and yeasts. Its multiple func tionality has now been characterized [32–33]. The sitespecific recombination is constructed into the regions of specific sequence either at the recognition site, which leads to the cleavage (or binding) in target sequences as a result of integration and deletion, or through the inversion of DNA fragments without acquisition or loss of nucleotides [34–36]. Their action mechanism has been successfully described by crystallo graphic analysis of some sitespecific recombinases and complexes with DNA [37]. The recognition sites for these recombinases are palindromes containing 6–8 bps. Segments are asymmetric relative to one another and flanked by inverted repeats with 12–13 bps for binding the recombinase. The asymmetry of inverted segments serves for orientation in the direction of recombinase route. For example, the inversion of plasmid segments limited by the sites in the reverse direction drives to the reverse orientation of the replication fork. An enzyme binds to DNA only through one sub unit, thus creating a recombinasebinding element. The incisions at sites are done at the boundary between a recombinasebinding element and a target DNA
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loxP
Cre
SMG
loxP
GI
rb
GI Fig. 1. Schematic image of the removal of selectable marker sequences from the genome using a sitespecific recombinase system. The DNA sequence is limited by the excision sites loxP (Cre recombinase, selectable or marker gene, SMG) which is flanked by the left (lb) and right (rb) boundaries of the genome. As a result of Cre recombinase work, only the gene of interest (GI) remains in the genome.
sequence (Fig. 1). The Cre recombinase isolated from bacteriophage P1 is responsible for the transformation of two plasmids into a monomeric unit in the Escheri chia coli genome by the route of recombinations between two loxP sites. In contrast to the Cre recom binase, the FlP and R yeast recombinases are repli cated not so efficiently by the plasmids containing the FRT and RS sites in their structure. The superfamily of recombinases could be divided into two families: the tyrosine recombinases and the serine recombinases. This division is based on the type of active amino acid (Tyr or Ser) inside the catalytic domain of the enzyme (Fig. 2). The two families are in turn divided into special subfamilies according to the size of enzymes or the type of their activity. Today, the most characterized are tyrosine recombinases (the Cre–loxP [38], FLP–FRT [39], and R–RS [40] sys tems, where Cre, FLP, and R are bidirectional tyrosine recombinases, while loxP, FRT, and RS are DNA rec
ognition sites for the enzymes). The function of bidi rectional tyrosine recombinase systems is targeted to the recognition of identical sequences at two DNA sites. On the contrary, the work of unidirectional tyrosine recombinases is directed to the recognition of nonidentical sites known as attB (attachment site bac teria) and attP (attachment site phage). In this case, the irreversible recombination is implemented in the absence of an auxiliary protein—excisionase. Unidi rectional tyrosine recombinases are used for manipu lations with the genomes of bacteriophage HK022 [41–42] and bacteriophage λ [43]. The family of serine recombinases also have two special subfamilies differing between themselves in the size of enzymes. One serine subfamily includes the βsix [44], γβrec [45], CinHRS2 [46, 47], and ParA MRS [48, 49] recombinase systems, where β, γδ, CinH, and ParA are small serine recombinases, while six, rec, RS2, and MRS are their recognition sites. Small serine recombinases, also known as resolvases, recognize only clearly distinguishable DNA sites. It has been established that small serine recombinases cannot influence intermolecular integration, due to conformational deformations [50]. Thus, the removal of DNA sequences, using small serine recombinases, is an irreversible process. The members of large serine recombinases subfamily are PhiC31 [51, 52], TP9011 [53], R4 [54], and Bxb1 [55]. These enzymes interact with sites of different sequences attB and attP, as well as with hybrid sequences attL and attR. The process of removal or inversion can occur if attB and attP are replaced, respectively, for attL and attR in the con struction. In the case of a reverse order, the reaction of replacement or removal will not occur. The presence
Family of recombinases
Tyrosine recombinases
Serine recombinases (resolvases)
Bidirectional (Cre, Flp, R)
Unidirectional (λ, HK101, pSAM2)
Small (λsix, CinH, ParA, γδ)
Large (Bxb1, PhiC31, TP901)
Reversible excision, inversion, and integration
Irreversible excision, inversion, and integration
Only irreversible excision
Irreversible excision, inversion, and integration
Identical recognition sites
Nonidentical recognition sites
Identical recognition sites
Nonidentical recognition sites
Fig. 2. Scheme for the classification of sitespecific recombinases on the basisc of active amino acid of the catalytic domain [36]. The family of tyrosine recombinases can be divided between subfamilies that use identical and nonidentical recognition sites, as well as the families of large (~60 kDa) and small (~23 kDa) recombinases according to the size of the enzyme molecule. CYTOLOGY AND GENETICS
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of another protein, excisionase, is required for a replacement in this case [56, 57]. The sitespecific recombination is one of the first technologies used for obtaining transgenic SMGfree plants [23, 36, 58]. The application of this approach for transformation allows geneticists to avoid the risk of penetration of selectable marker genes into the nat ural environment. A series of DNA constructs with sitespecific recombinase systems were designed for removing SMGs from plant genomes. The efficiency of using this strategy was demonstrated both in model plants [23, 49, 59–63] and cultivars [25, 65–69]. The LY038 maize line produced by Monsanto Company was the United States’ first officially per mitted (by US Department of Agriculture) genetically modified cultivar obtained using the Cre/loxP recom binase system and permitted for commercialization in the consumer market [70]. This maize line contains increased lysine and was designed for poultry and live stock farming [71]. To design the line, geneticists used a plasmid containing the cordapA sequence (the encoding region of the dihydrodipicolinate synthase gene from Corynebacterium glutamicum) and the selectable kanamycinresistance gene (nptII). The nptII gene is flanked by the loxP sites in the DNA con struct. After transformation, the regenerants were crossed with a different transformed maize line expressing the Cre recombinase. As a result, the nptII gene was removed, while the cordapA sequence alone remained. Thus, to design a maize line that would not contain any SMG, two previously transformed maize lines were used with their subsequent cross between them. The process of obtaining a GM cultivar free of DNA marker sequence proved to be quite compli cated, as well as time and costconsuming. Subse quent generations of transformed maize lines were used in this case to design transgenic plants. This strat egy can be considered efficient when the time of obtaining a subsequent generation of transformants is short. But this approach is low efficient if the technol ogies are applied for modifying various species of trees or other cultivars. The sitespecific recombinase technology can be used for providing the presence of only a single foreign DNA copy in a specified genome fragment [72]. The presence of one DNA insertion copy per genome causes many adverse effects due to the process of transformation, such as elimination of the position effect [73], mosaicism [74], genomic instability [75], genetic diversity [76], and gene silencing [77]. Due to the presence of only a single transgenic DNA copy, the level of its expression is higher compared with that of multiple TDNAs in the genome [78]. This was described for the first time by Srivasta et al. in [68]. Using vector constructs with the sitespecific Cre/loxP recombinase system, we have also obtained trans formed plants of Arabidopsis thaliana Columbia ecotype and analyzed the work of recombinase in the CYTOLOGY AND GENETICS
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T1 generation transformants [70]. It became clear in the course of experiments that the event of the removal of DNA segments limited by the loxP sites occurred approximately in 25% of studied lines of transfor mants. By application of Southern blot analysis it was revealed that, using recombinase excision, we reduced the number of integrated TDNAs to the minimum in some plants. This technology also allows geneticists to design transgenic plants with the known location of reporter genes in the genome, which was demon strated in plants of tobacco [64], maize [69], rice [80], Arabidopsis [81], aspen [82], and the tobacco plastid genome [83]. The integration efficiensy of an foreign insertion was 33% [84] in the tobacco and 50% in the rice genomes [85]. Interestingly, the distribution of inser tions in the rice genome was not uniform and more concentrated in special pools. The integrated DNA sequences were uniformly distributed along the genome, but a part of DNA insertions was methylated, which caused gene silencing [86]. The application of recombinase excision causes the presence of at least one recognition site for the enzyme in the transforming construction, and at least two sites that flank TDNA is the optimal variation for the work of the recombinase. The whole pools of transforming sequences might be simultaneously removed from the genome during the process of excision, due to the mul tiple recognition sites. The process of removal will continue until the last recognition site remained. In addition, there is the probability for a partial location of TDNA beyond the recognition sites, as can be revealed by molecular genetic analysis. The excision of TDNA segments limited by loxP sites during the work of recombinase can occur heterogeneously, which is detected by a histological analysis. As a result of GUS test, we can reveal a partial removal of the gus marker from the genome of special cell or tissue pools, which points to a chimerical nature of plants [87]. Gene silencing may show a different form of spec ificity after genetic transformation events. For exam ple, the efficiency of sitespecific recombinase has been studied in the rice plant genome with the effect of gene silencing as a sequence of multiple integration of TDNA [88]. No TDNA expression was initially observed, but the activated expression of the gene of interest was detected after the removal of the main part of introduced sequences from the genome by recombi nase. The work of recombinases is not limited by the dis tance between the recognition sites along the genome, although its extension can decrease the working effi ciency of the enzyme. It has become possible in med ical practice to study genetic diseases, such as Down syndrome, Lejeune syndrome, and CharcotMarie Tooth disease type 1A during studying the extension of large deletions, duplications or chromosomal translo cations [89, 90]. The phenomenon of Creinduced
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sitespecific translocation is identified in embryonic stem cells [91] and plants [92]. The work of enzymes could face the difficulties by possible location of exci sion sites on different chromosomes. In contrast to eukaryotes, the development of events associated with the removal of specific DNA sequence in prokaryotic organisms is predictable. In this case, the reaction takes place in the presence of two loxP sites located on the same chromosome. The duplication of events is observed in eukaryotic cells, which was described pre viously [93, 94]. The event of deletion/duplication simultaneously takes place between unpaired chroma tids, which leads to the occurrence of balanced and, at the same time, unbalanced types of chromatids. Thus, the difference in the interactions between chromo somes (for example, intrachromosomal interactions between homologous or nonhomologous chromo somes) and the loxP sites located on the chromosomes can be used for the frequency control of recombina tion events [95]. During deletion or duplication that is under enhanced control, the specific SYCP1 promoter (synaptonemal complex protein 1) is utilized. With the loxP sites located on the same chromosome, the event of inversion is observed in the reversible orientation. This type of rearrangement in chromosome can be used genetic deviations study etc. [96]. The irregular orientation of the loxP sites may also cause uneven recombination between daughter chromatids [94]. Different factors affect the stability and expression efficiency of a transgenic DNA, for example, the inte gration complexity of the transforming construct and its location on the genome (position effect). The transgenic expression, depending on the surrounding TDNA genomic elements, can be enhanced, sup pressed or incompletely regulated. The process of integration depends on such aspects as structural con figuration of the construct, its copying ability (the number of integration events per genome), etc. The position of the loxP sites in a similar orientation on a nonhomologous chromosome may lead to both even and uneven chromosomal translocation. This effect was used for the study of spatial and temporal expres sion regulation in the genome, which causes, in turn, the occurrence of different forms of tumors, abnor malities, and genetic diseases [97]. The process of translocations in tobacco plants was studied within chromosomal rearrangements. For example, the event of recombination in a chromosome with one open reading frame for the loxP/higromycin sequence can take place only in the presence of the 35S/loxP pro moter. For example, 2.5% hygromycinresistant plants have been revealed from the total number of transfor mants in studying the Cre protein expression in an in vivo system [92].
Strategies for the Use of SiteSpecific Recombinase Systems The efficiency of using sitespecific recombinase systems was demonstrated on different plant species [23, 60, 61, 65, 66] and cultivars [25, 67, 68]. Researchers have created a DNA construct that can activate the selfexcision of unwanted DNA segments for one step, thus simplifying the process of removal of selectable genes and accelerating the procedure. The gene of interest, marker gene, and recombinase were put in this construction under the control of inducible promoter, such as the heat shock gene promoter HSP811 [86, 98, 99]. The marker gene was flanked by recognition sites for recombinase, whereas the gene of interest was located beyond the region of excision sites. Some other systems of induction have the sequence that encodes the Cre recombinase bound to ligands that were linked, in turn, with the estradiol receptor gene [100, 101]. In the absence of this hor mone, as an auxiliary agent, the limited penetration of recombinasechimera inside the nucleus took place and the activity of recombinase was thus inhibited [102]. The strategy of selfexcision, similar to hor monedependent recombination systems, was applied for plants of Arabidopsis [7], maize [68], tomato [103], rice, and aspen [25], as well as tobacco [104]. Removing the marker gene, using recombinase, will require, in this case, a heat or chemical treatment that will limit a circle of plants for future transformation [105]. Besides inducible promoters, the tissuespecific promoters are used as TDNA controlling elements. The activation of such promoters and the recombinase expression take place only in certain organs or tissues of transformants [106, 107]. In comparison with the strategy of application external activation agents, this approach is easily applicable due to a lesser number of stages. The further improvent of the recombinase sys tem activity regulation is directed to reduce the risk for the penetration of alien DNAs into external environ ment via pollination. It could e achived by recombi nase expression activation and removal of the specified transformed DNA sequence segment during the pol len maturation period of GM plants [106, 108]. For example, the efficient application of the sitespecific recombinase DNA removal method for the transfor mation of soya plants in the absence of activating agents was demonstrated in [105]. In this case, the embryospecific promoter was used for the transient expression of the Cre recombinase in the DNA con struct. The other approach to the applied sitespecific recombinase strategy foresaw reaching shortterm enzyme expression. In order to achive this the geneti cists designed a DNA vector where recombinase was highly expressed without additional activation after integration into the genome [33, 59]. In this case, the sequence that encoded the enzyme was unstably inte CYTOLOGY AND GENETICS
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grated into the cell genome. Two special expressing vectors were designed to overcome this difficulty. One of them was based on specific sequences of A. tumefa ciens [109], while the other was designed on the basis of viral vector and contained the cre gene [110]. In other cases of application this strategy for transforma tion, the recombinase mRNA gene was directly intro duced [111, 112]. Thus synthesized enzyme molecules remove DNA segments for one cycle. Another approach without the additional stage for treating transformants by external agents or via cross pollination between subsequent generations was sug gested to obtain plants free of marker sequences [113]. In this case, geneticists used the DNA constructs where the Cre/loxP recombinase system was under the control of the –46 minimal promoter 35S and the ter minal nos sequence. The marker gus gene sequence was divided and removed beyond the boundaries of the loxP excision sites, while the gene parts were joined together during the removal of DNA fragment limited by excision sites. Thus, the presence of the gus gene expression evidenced the activity of the enzyme. The issue of stability in these transformants was analyzed in several Arabidopsis generations. However, the applica tion of this approach for obtaining SMGfree trans formants is complicated becuse the question of further presence of the marker gene in plant genome remains open. As a result of breaks in the encoding DNA sequence, the probability of the effect of gene silenc ing increases. Therefore, the similar approach has been devel oped to simplify the process of transformation using the Cre/loxP system under the control of 35S pro moter [114]. In this case, we used the DNA construct where the gus marker gene was located between the loxP sites of excision and was simultaneously removed along with all marker sequences. The efficiency of the Cre recombinase work was studied in two A. thaliana generations. The constructs having the cre gene, reporter gus gene under the control of 35S promoter, and the hptII hygromycinresistance gene have been created to transform Arabidopsis. By aplication of sev eral variants of TDNA the efficiency of recombinase was analyzed for every variant of construct (60 samples in total). The recombinasemediated activity was studied using the GUS test and the expression of the βglucuronidase gene in plants grown in a selective medium. Since there was the probability for the gene silencing effect during seed germination in a selective medium, geneticists repeated the GUS test for the plants sown to the soil. The activity of a recombinase system in the genome of plants was confirmed by molecular genetic analysis. It became clear that the efficiency of recombinase increased by 14–17% in each subsequent generation of plants. Thus, the sug gested methods for producing transformants are sim ple and require no additional stages for activating the recombinases. The used approach is an efficient way to obtain transformed plants free of marker sequences. CYTOLOGY AND GENETICS
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CONCLUSIONS The presence of SMGs in the genome, in particu lar, in antibioticresistance genomes, is necessary only at the stage of initial selection of transformed cells or tissues. The later considerations about the threat of a horizontal transfer of SMGs to wildgrowing species cannot be excluded after releasing transformed plants into the natural environment. At the same time, the problem of localization of introduced DNA sequence occured during the transformation of genomes. The impossibility of controlling the localization of an for eign DNA in the genome may lead to the risk of unwanted effects, such as mutagenesis and unexpected expression variations. A multiple integration of a con structs may lead to both the effect of overexpression of target and marker sequence and the appearence of gene silencing. Therefore, it is quite important to solve the problem of transformation process control and the removal of DNA marker sequences at the time of pro ducing regenerants. The application of sitespecific recombination allows researchers to overcome the main complica tions related to the process of plant genome transfor mation. The application efficiency of recombinases in genetic manipulations has already been demonstrated on both the monocotyledons and the dicotyledons. There is a vide range of approaches in genetic engi neering to the application of sitespecific recombina tion systems, which are used in the development of novel strategies for the optimizing of transformation. The most popular technology is the Cre/loxP recom binase system. With this technology, the selfexcision of required segments of constructions along with sites of excision (in this case this is loxP) occurs. Thus, the sitespecific recombinase technology can be consid ered as the main tool for obtaining plants free of marker genes. The further use of this strategy in genetic engineering will allow researchers to solve a series of problems related to the creation and cultiva tion of genetically modified cultivars. REFERENCES 1. Tuteja, N., Verma, S., Sahoo, R.K., et al., Recent advances in development of markerfree transgenic plants: regulation and biosafety concern, J. Biosci., 2012, vol. 37, no. 1, pp. 167–197. 2. Dale, P.J., Clarke, B., and Fontes, E.M.G., Potential for the environmental impact of transgenic crops, Nat. Biotechnol., 2002, vol. 20, pp. 567–574. 3. Lee, L.Y. and Gelvin, S.B., TDNA binary vectors and systems, Plant. Physiol., 2008, vol. 146, pp. 325–332. 4. Ebinuma, H., Sugita, K., Matsunaga, E., and Yamakado, M., Selection of markerfree transgenic plants using the isopentenyl transferase gene, Proc. Nat. Acad. Sci. U.S.A., 1997, vol. 94, pp. 2117–2121. 5. Puchta, H., Markerfree transgenic plants, Plant Cell, Tissue Org. Cult., 2003, vol. 74, pp. 123–134.
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Translated by N. Tarasyuk
