Cre/lox位点特异重组系统在转基因植物中的应用

位点特异重组系统由重组酶和相应的重组酶识别位点组成,通过两者间的相互作用,实现外源基因精确整合与切除等一系列遗传操作.主要可分为Cre/lox系统、FLP/frt系统、R/RS系统和Gin/gix系统.目前,研究最充分应用最广泛的位点特异重组系统为Cre/lox系统.此系统为位点特异重组系统家族中的一员,由38.5kDCre重组酶和34bplox位点组成,最早被应用于动物转基因研究,包括基因敲除、基因激活、基因易位等.近年来,随着研究的深入,Cre/lox系统被逐步应用到植物研究中,并在诸多领域取得重大进展.本文总结归纳了Cre/lox系统在定点整合、定点切除以及叶绿体转化等方面的最新研究成果,旨在为利用Cre/lox系统构建环境安全和高效表达的植物遗传转化体系提供参考.     

Exchange of gene activity in transgenic plants catalyzed by the Cre-lox site-specific recombination system

The Cre-lox site-specific recombination system of bacteriophage P1 was used to excise a firefly luciferase (luc) gene which had previously been incorporated into the tobacco genome. The excision event was due to site-specific DNA recombination between two lox sequences flanking the luc gene and was catalyzed by the Cre recombinase introduced by cross-fertilization. Recombination resulted in the fusion of a promoter with a distally located hygromycin phosphotransferase (hpt) coding sequence and the excision event was monitored as a phenotypic change from expression of luc to expression of hpt. The efficiency of recombination was estimated from the exchange of gene activity and confirmed by molecular analysis. The relevance to potential applications of site-specific deletion-fusion events for chromosome engineering are discussed.     

Cre/lox介导剔除转双价抗虫sck+cryIAc基因籼稻恢复系明恢86材料的选择标记基因

Cre/lox系统通过其Cre重组酶对lox序列进行切割和重新连接,介导lox序列发生特异性重组.利用重组报告基因系统Pactin-lox-hpt-lox-gusA,对Cre/lox系统在水稻(Oryzasativa L.)中介导转基因的剔除进行了研究.Pactin-lox-hpt-lox-gusA系统中选择标记hpt基因侧翼含两个同向lox位点,并位于水稻actinl启动子和gusA基因之间.当hpt在Cre酶作用下被剔除时,actinl启动子可以和gusA基因融合在一起从而驱动GUS表达.通过农杆菌介导获得了分别转cre基因、Pactin-lox-hpt-lox-gusA结构和双价抗虫基因lox-hpt-lox-sck-cryIAc结构的水稻.利用有性杂交方法将cre基因导入到转化lox结构的植株中.在4个转Pactin-lox-hpt-lox-gusA T0植株×转cre T0植株所配组合的30个杂交F1植株中,12个植株表达GUS活性,9个表现潮霉素敏感,表明hpt基因被剔除.研究进一步通过Cre/lox介导剔除转双价抗虫sck cryIAc基因籼稻恢复系明恢86材料基因组中的选择标记hpt基因.在9个转lox-hpt-lox-sck-cryIAcT2代纯合植株×转creT2代纯合植株所配组合的77个杂交F1植株中,56个植株表现潮霉素敏感.分子分析证实在这些对潮霉素敏感的植株中hpt基因已经被剔除.     

Cre／lox介导剔除转双价抗虫sck＋cryIAc基因籼稻恢复系明恢86材料的选择标记基因

Cre/lox系统通过其Cre重组酶对lox序列进行切割和重新连接,介导lox序列发生特异性重组。利用重组报告基因系统Pactin-lox-hpt-lox-gusA,对Cre/lox系统在水稻(Oryza　sativa　L.)中介导转基因的剔除进行了研究。Pactin-lox-hpt-lox-gusA系统中选择标记hpt基因侧翼含两个同向lox位点,并位于水稻actin1启动子和gusA基因之间。当hpt在Cre酶作用下被剔除时,actin1启动子可以和gusA基因融合在一起从而驱动GUS表达。通过农杆菌介导获得了分别转cre基因、Pactin-lox-hpt-lox-gusA结构和双价抗虫基因lox-hpt-lox-sck-cryIAc结构的水稻。利用有性杂交方法将cre基因导入到转化lox结构的植株中。在4个转Pactin-lox-hpt-lox-gusA　T0植株×转cre　T0植株所配组合的30个杂交F1植株中,12个植株表达GUS活性,9个表现潮霉素敏感,表明hpt基因被剔除。研究进一步通过Cre/lox介导剔除转双价抗虫sck　 cryIAc基因籼稻恢复系明恢86材料基因组中的选择标记hpt基因。在9个转lox-hpt-lox-sck-cryIAc　T2代纯合植株×转creT2代纯合植株所配组合的77个杂交F1植株中,　56个植株表现潮霉素敏感。分子分析证实在这些对潮霉素敏感的植株中hpt基因已经被剔除。     

Directed excision of a transgene from the plant genome

Summary The effectiveness of loxP-Cre directed excision of a transgene was examined using phenotypic and molecular analyses. Two methods of combining the elements of this system, re-transformation and cross pollination, were found to produce different degrees of excision in the resulting plants. Two linked traits, -glucuronidase (GUS) and a gene encoding sulfonylurea-resistant acetolactate synthase (ALS^r), were integrated into the genome of tobacco and Arabidopsis. The ALS^r gene, bounded by loxP sites, was used as the selectable marker for transformation. The directed loss of the ALS^T gene through Cre-mediated excision was demonstrated by the loss of resistance to sulfonylurea herbicides and by Southern blot analysis. The -glucuronidase gene remained active. The excision efficiency varied in F₁ progeny of different lox and Cre parents and was correlated with the Cre parent. Many of the lox × Cre F₁ progeny were chimeric and some F₂ progeny retained resistance to sulfonylureas. Re-transformation of lox/ALS/lox/GUS tobacco plants with cre led to much higher efficiency of excision. Lines of tobacco transformants carrying the GUS gene but producing only sulfonylurea-sensitive progeny were obtained using both approaches for introducing cre. Similarly, Arabidopsis lines with GUS activity but no sulfonylurea resistance were generated using cross pollinations.     

Visual characterization of recombination at FRT-gusA loci in transgenic tobacco mediated by constitutive expression of the native FLP recombinase

FLP/FRT-mediated site-specific recombination was studied with a recombination-reporter gene system which allows visualization of -glucuronidase (GUS) expression after site-specific excisional activation of a silent gusA gene. This system was used for characterization of the functional activity of the Saccharomyces cerevisiae native FLP recombinase driven by the cauliflower mosaic virus (CaMV) 35s promoter [linked to the tobacco mosaic virus (TMV) omega translational leader] in mediating site-specific recombination of chromosomal FRT sites in tobacco FLP x FRT-reporter hybrids. Six hybrids were generated from crosses of lines containing either a stably integrated recombination-reporter or a FLP-expression construct. The activated gusA phenotype was specific to hybrid progenies and was not observed in either parental plants or their selfed progenies. Recombination efficiency in whole seedlings was estimated by the percent of radioactivity on a Southern blot which was incorporated into the recombined DNA product. Estimated efficiency mean values for the six crosses ranged from 5.2 to 52.0%. Histochemical analysis in hybrid plants visualized GUS activity with variable chimeric patterns and intensities. Recombination efficiency and GUS expression varied both among and within crosses, while higher recombination efficiency coincided with larger and more intense patterns of GUS activity. These data suggest that recombination is induced randomly during somatic developmental stages and that the pattern and intensity generated in a given plant are affected by factors imposing varibility not only between but also within crosses. Additionally, while recombination in a population of FLP/FRT hybrids may occur in all plants, recombination efficiency may still be low in any given plant. The activity of the native, as compared to a modified, FLP (Kilby et al. 1995) in the activation of transgenic traits in tobacco is discussed.     

Cryptic loxP sites in mammalian genomes: genome-wide distribution and relevance for the efficiency of BAC/PAC recombineering techniques

Cre is widely used for DNA tailoring and, in combination with recombineering techniques, to modify BAC/PAC sequences for generating transgenic animals. However, mammalian genomes contain recombinase recognition sites (cryptic loxP sites) that can promote illegitimate DNA recombination and damage when cells express the Cre recombinase gene. We have created a new bioinformatic tool, FuzznucComparator, which searches for cryptic loxP sites and we have applied it to the analysis of the whole mouse genome. We found that cryptic loxP sites occur frequently and are homogeneously distributed in the genome. Given the mammalian nature of BAC/PAC genomic inserts, we hypothesised that the presence of cryptic loxP sites may affect the ability to grow and modify BAC and PAC clones in E. coli expressing Cre recombinase. We have observed a defect in bacterial growth when some BACs and PACs were transformed into EL350, a DH10B-derived bacterial strain that expresses Cre recombinase under the control of an arabinose-inducible promoter. In this study, we have demonstrated that Cre recombinase expression is leaky in un-induced EL350 cells and that some BAC/PAC sequences contain cryptic loxP sites, which are active and mediate the introduction of single-strand nicks in BAC/PAC genomic inserts.     

Development of a simple and efficient system for excising selectable markers in Arabidopsis using a minimal promoter::Cre fusion construct

The development of rapid and efficient strategies to generate selectable marker-free transgenic plants could help increase the consumer acceptance of genetically modified (GM) plants. To produce marker-free transgenic plants without conditional treatment or the genetic crossing of offspring, we have developed a rapid and convenient DNA excision method mediated by the Cre/loxP recombination system under the control of a −46 minimal CaMV 35S promoter. The results of a transient expression assay showed that −46 minimal promoter::Cre recombinase (−46::Cre) can cause the loxP-specific excision of a selectable marker, thereby connecting the 35S promoter and β-glucuronidase (GUS) reporter gene. Analysis of stable transgenic Arabidopsis plants indicated a positive correlation between loxP-specific DNA excision and GUS expression. PCR and DNA gel-blot analysis further revealed that nine of the 10 tested T₁ transgenic lines carried both excised and nonexcised constructs in their genomes. In the subsequent T₂ generation plants, over 30% of the individuals for each line were marker-free plants harboring the excised construct only. These results demonstrate that the −46::Cre fusion construct can be efficiently and easily utilized for producing marker-free transgenic plants.     

Incomplete cre‐mediated excision leads to phenotypic differences between Stra8‐iCre; Mov10l1lox/lox and Stra8‐iCre; Mov10l1lox/Δ mice

In the Cre–loxp system, expression level and activity of Cre recombinase in a Cre deleter line are critical because these determine not only the cell specificity of gene knockout (KO), but also the efficiency of Cre‐mediated excision in a specific cell lineage. Although the spatiotemporal expression pattern of a Cre transgene is usually defined upon the generation of the mouse line, the Cre excision efficiency in a specific targeted cell lineage is rarely evaluated and often assumed to be 100%. Incomplete excision can lead to highly variable phenotypes owing to mosaicism (i.e., coexistence of cells with the flox or the recombined flox allele) and this problem has long been overlooked. Here, we report that Stra8‐codon‐improved Cre recombinase (iCre), a transgenic allele expressing iCre under the control of the male germ cell‐specific Stra8 promoter, could efficiently delete one Mov10l1 flox allele in spermatogenic cells, whereas the excision was incomplete when two Mov10l1 flox alleles were present. The incomplete Cre‐mediated excision led to a testicular phenotype that was much less severe than that in the true conditional KO (inactivation, 100%) mice. Our findings suggest that it is essential to determine the efficiency of Cre excision when Cre–loxp system is used for deleting genes in a specific cell lineage and the Cre; gene^lox/Δ genotype should be used to evaluate phenotypes instead of Cre; gene^lox/lox owing to the fact that the latter usually bears incomplete deletion of the flox allele(s). genesis 51:481–490. © 2013 Wiley Periodicals, Inc.     

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.     

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.     

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     

细胞可透过性Cre重组酶表达、纯化及生物活性检测(英)

Cre/lox P系统由Cre位点特异重组酶和可被Cre特异性识别的lox P位点构成,该系统广泛用于条件性基因敲除和表达,以研究基因功能.为了表达和纯化一种细胞可透过性Cre重组酶(即His6-NLS-Cre-MTS);经IPTG诱导,在BL21(DE3)宿主菌成功表达His6-NLS-Cre-MTS融合蛋白,通过His-Bond Ni-NTA树脂分离并纯化了该蛋白质,随后借助细胞和非细胞的重组系统成功检测了His6-NLS-Cre-MTS的生物活性.细胞可透过性Cre重组酶提供了一种快捷而高效的在细胞和在体水平进行遗传操作的新工具.     

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.     

Selectable antibiotic resistance marker gene-free transgenic rice harbouring the garlic leaf lectin gene exhibits resistance to sap-sucking planthoppers

Rice, the major food crop of world is severely affected by homopteran sucking pests. We introduced coding sequence of Allium sativum leaf agglutinin, ASAL, in rice cultivar IR64 to develop sustainable resistance against sap-sucking planthoppers as well as eliminated the selectable antibiotic-resistant marker gene hygromycin phosphotransferase (hpt) exploiting cre/lox site-specific recombination system. An expression vector was constructed containing the coding sequence of ASAL, a potent controlling agent against green leafhoppers (GLH, Nephotettix virescens) and brown planthopper (BPH, Nilaparvata lugens). The selectable marker (hpt) gene cassette was cloned within two lox sites of the same vector. Alongside, another vector was developed with chimeric cre recombinase gene cassette. Reciprocal crosses were performed between three single-copy T₀ plants with ASAL- lox-hpt-lox T-DNA and three single-copy T₀ plants with cre-bar T-DNA. Marker gene excisions were detected in T₁ hybrids through hygromycin sensitivity assay. Molecular analysis of T₁ plants exhibited 27.4% recombination efficiency. T₂ progenies of L03C04(1) hybrid parent showed 25% cre negative ASAL-expressing plants. Northern blot, western blot and ELISA showed significant level of ASAL expression in five marker-free T₂ progeny plants. In planta bioassay of GLH and BPH performed on these T₂ progenies exhibited radical reduction in survivability and fecundity compared with the untransformed control plants.     

Site-specific recombination in Zea mays

The elimination of marker genes after selection is recommended for the commercial use of genetically modified plants. We compared the applicability of the two site-specific recombination systems Cre/lox and Flp/FRT for marker gene elimination in maize plants. The selection marker gene pat surrounded by two identically directed lox or FRT sites was introduced into maize. Sexual crossing with plants harboring the corresponding constitutively expressed recombinase led to the precise and complete excision of the lox-flanked marker gene in the F₁ progeny, whereas Flp-mediated recombination of FRT sequences occurred rarely. Further examination of site-specific integration was done by biolistic bombardment of immature embryos harboring only one lox site with a lox.uidA sequence with results indicating directed integration.     

利用Cre/lox重组系统建立反义基因工程雄性不育恢复系

利用Cre/lox重组系统中的Cre重组酶能特异性识别并介导两个同向lox位点之间DNA序列发生重组删除的特点,将TA29驱动下的反义豌豆卷须肌动蛋白基因置于两个同向lox位点之间并与Bar基因连锁,转化烟草Wisconsin 38后获得抗除草剂Basta的转基因植株.将Cre基因导入烟草Wisconsin 38建立雄性不育工程恢复系.反义Actin转基因植株与Cre转基因植株杂交获得F1,通过Cre重组酶将F1中的反义肌动蛋白基因表达盒删除实现育性的恢复.结果显示:来自豌豆卷须的肌动蛋白基因在Wisconsin 38烟草绒毡层中反义表达但未能导致明显的雄性不育,转基因植株在花器官形态、花粉形状、活力、结实、结籽等方面与野生型植株间无明显的差异.而获得的烟草Cre转基因工程恢复系除少量植株出现叶片褪绿、结果少等异常外,绝大多数植株形态结构及开花结果习性与野生型一致;其中3个Cre转基因工程恢复系与Actin反义肌动蛋白转基因植株TAA-3杂交后,杂交后代中的绝大多数反义肌动蛋白基因表达盒均被精确删除,表明将Cre/lox重组系统用于建立基于反义基因工程雄性不育的恢复系是可行的.     

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.