Stoichiometry of the Cre recombinase bound to the lox recombining site.

The site-specific recombinase Cre from bacteriophage P1 binds and carries out recombination at a 34 bp lox site. The lox site consists of two 13 bp inverted repeats, separated by an 8 bp spacer region. Both the palindromic nature of the site and the results of footprinting and band shift experiments suggest that a minimum of two Cre molecules bind to a lox site. We report here experiments that demonstrate the absolute stoichiometry of the Cre-lox complex to be one molecule of Cre bound per inverted repeat, or two molecules per lox site.     

Site-specific recombination of asymmetric lox sites mediated by a heterotetrameric Cre recombinase complex

Previous reports have demonstrated that new Cre recombinase specificities can be developed for symmetrically designed lox mutants through directed evolution. The development of Cre variants that allow the recombination of true asymmetric lox mutant sites has not yet been addressed, however. In the present study, we demonstrate that a mixture of two different site-specific Cre recombinase molecules (wt Cre and a mutant Cre) catalyzes efficient recombination between two asymmetric lox sites in vitro, presumably via formation of a functionally active heterotetrameric complex. The results may broaden the application of site-specific recombination in basic and applied research, including the custom-design of recombinases for natural, asymmetric, and lox-related target sequences present in the genome. Future applications may potentially include genomic manipulations, for example, site-specific integrations, deletions or substitutions within precise regions of the genomes of mammalians and other organisms.     

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.     

Functional expression of the cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae.

The procaryotic cre-lox site-specific recombination system of coliphage P1 was shown to function in an efficient manner in a eucaryote, the yeast Saccharomyces cerevisiae. The cre gene, which codes for a site-specific recombinase, was placed under control of the yeast GALI promoter. lox sites flanking the LEU2 gene were integrated into two different chromosomes in both orientations. Excisive recombination at the lox sites (as measured by loss of the LEU2 gene) was promoted efficiently and accurately by the Cre protein and was dependent upon induction by galactose. These results demonstrate that a procaryotic recombinase can enter a eucaryotic nucleus and, moreover, that the ability of the Cre recombinase to perform precise recombination events on the chromosomes of S. cerevisiae is unimpaired by chromatin structure.     

DNA specificity of the Cre recombinase resides in the 25 kDa carboxyl domain of the protein

The Cre protein of bacteriophage P1 is a 38.5 kDa site-specific recombinase that belongs to the Int family of recombination proteins. Cre acts by binding specifically to a 34 base-pair sequence, lox, where it carries out recombination. A limited chymotryptic digest of Cre resulted in two fragments of sizes 25 and 13.5 kDa, respectively. The sequence of the amino terminus of the purified 25 kDa peptide demonstrates that this peptide represents the carboxyl-terminal portion of the Cre protein. A truncated version of the cre gene was constructed which produces only the 25 kDa peptide. The 25 kDa peptide is capable of specific binding to the lox site, but binds at lower affinity than does wild-type Cre. Footprinting with Fe-EDTA indicates that the 25 kDa peptide protects the inverted repeats of the lox site but shows only partial protection of the spacer region. This is in contrast to the footprint obtained with wild-type Cre which protects the entire spacer region.     

Balancer-Cre transgenic mouse germ cells direct the incomplete resolution of a tri-loxP-targeted Cyp1a1 allele, producing a conditional knockout allele

To generate conditional alleles, genes are commonly engineered to contain recognition sites for bacteriophage recombinases, such as Cre recombinase. When such motifs (lox sites) flank essential gene sequences, and provided that Cre recombinase is expressed, Cre recombinase will excise the flanked sequence-creating a conditional knockout allele. Targeted conditional alleles contain a minimum of three lox sites. It would be desirable to have Cre recombinase perform partial resolution (i.e., recombination some of the time between only the two lox sites flanking the marker gene). Here we report use of the commercially available Balancer2-Cre transgenic mouse line to carry out this function from a tri-loxP-site-containing cytochrome p450 1A1 (Cyp1a1) targeted allele. Such incomplete resolution of this complex locus occurred progressively with age in germ cells of male mice; the conditional Cyp1a1 gene was recovered in offspring from mice containing the targeted Cyp1a1 allele and the Cre recombinase transgene. Removal of the marker gene resulted in a conditional Cyp1a1 allele whose expression was indistinguishable from that of the wild-type allele.     

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.     

Turbo cloning: a fast, efficient method for cloning PCR products and other blunt-ended DNA fragments into plasmids.

The method uses a novel plasmid vector, p9lox5, containing a site-specific recombination sequence lox from the lox/Cre recombinase system of bacteriophage P1. There are two distinct stages. Firstly, vector and fragment DNAs are ligated intermolecularly under conditions of macromolecular crowding (15% polyethylene glycol 6000) which accelerate blunt-end joining a thousandfold. Secondly, circular recombinant molecules are efficiently excised from the ligation products by Cre recombinase acting on pairs of lox sites within directly repeated vector molecules flanking insert DNA. Recombinants are introduced into cells conventionally by transformation or electroporation. In both a model system and the cloning of PCR products, yields approaching those obtainable in cohesive-end cloning were achieved. Applications of the technique to cDNA library generation and recovery of DNA from archive material are discussed.     

Cre/lox-mediated site-specific integration of Agrobacterium T-DNA in Arabidopsis thaliana by transient expression of cre

The Cre/lox system was used to obtain targeted integration of an Agrobacterium T-DNA at a lox site in the genome of Arabidopsis thaliana. Site-specific recombinants, and not random events, were preferentially selected by activation of a silent lox-neomycin phosphotransferase (nptII) target gene. To analyse the effectiveness of Agrobacterium-mediated transfer we used T-DNA vectors harbouring a single lox sequence (this vector had to circularize at the T-DNA left- and right-border sequences prior to site-specific integration) or two lox sequences (this vector allowed circularization at the lox sequences within the T-DNA either prior to or after random integration, followed by targeting of the circularized vector), respectively. Furthermore, to control the reversibility of the integration reaction, Cre recombinase was provided transiently by using a cotransformation approach. One precise stable integrant was found amongst the recombinant calli obtained after transformation with a double-lox T-DNA vector. The results indicate that Agrobacterium-mediated transformation can be used as a tool to obtain site-specific integration.     

The promiscuity of heterospecific lox sites increases dramatically in the presence of palindromic DNA

Heterospecific lox sites are mutated lox sites that in the presence of Cre recombinase recombine with themselves but not or much less with wildtype loxP. We here show that in Escherichia coli both lox511 and lox2272 sites become highly promiscuous with respect to loxP when in the presence of Cre one of the recombination partners is present in a larger stretch of an inverted repeat of non-lox DNA. In such a palindromic DNA configuration, also the occurrence of other DNA repeat-mediated recombination events is somewhat increased in the presence of Cre. The results indicate that in recombinase mediated cassette exchange or other double lox applications based on the exclusivity of heterospecific lox sites, or in research combining Cre-lox approaches with hairpin RNA for gene silencing, the presence of duplicated DNA around lox sites has to be taken into account. It is proposed that the presence of palindromic non-lox DNA interferes with the homology search of the Cre enzyme prior to the actual recombination event.     

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.     

Excision of specific DNA-sequences from integrated retroviral vectors via site-specific recombination.

Vectors for gene transfer and gene therapy were developed which combine the advantages of the integrase and recombinase systems. This was achieved by inserting two loxP sites for specific DNA excision into an MESV based retroviral vector. We show that this 'retroviral lox system' allows the infection of cells and the expression of transferred genes. In addition, we constructed an efficient retrovirus-based expression system for a modified Cre recombinase. Functional tests for DNA excision from integrated retroviral lox vectors were performed by the use of a negative selectable marker gene (thymidine kinase). Cre expression in cells infected with retroviral lox vectors and subsequent BrdU selection for cells in which site-specific recombination has occurred results in large numbers of independent cell clones. These results were confirmed by detailed molecular analysis. In addition we developed retroviral suicide vectors in which the enhancer/promoter elements of both LTRs were replaced by lox sequences. We show that lox-sequences located in the LTRs of retroviral vectors are stable during retroviral replication. Potential applications of this system would be the establishment of revertants of retrovirus-infected cells by controlled excision of nearly the complete proviral DNA.     

Site-specific modification of the bovine genome using Cre recombinase-mediated gene targeting.

Cre recombinase (Cre)-mediated targeted insertion of a transgene is a powerful technique that can be used to tailor genomes. When combined with somatic cell nuclear transfer it could offer an efficient way to generate transgenic livestock with site-specific genetic modifications that are free of antibiotic selection markers. We have engineered primary bovine fibroblasts to contain a chromosomal acceptor site with incompatible loxP/lox2272 sites for Cre-mediated cassette exchange and show for the first time that Cre-mediated targeting can be applied in these acceptor cells. Molecular characterization of the resulting cell clones revealed Cre-mediated transgene insertion efficiencies of up to 98% when antibiotic selection was used to identify transgene containing cell clones. Most clonal lines also contained random insertions of the targeting and Cre expression plasmids with only about 10% of the clones being exclusively modified by the intended targeted insertion. This targeting efficiency was sufficient to enable the isolation of correctly targeted clones without the help of antibiotic selection. Therefore, this recombinase-mediated insertion strategy has the potential to produce transgenic cattle from antibiotic selection marker-free somatic cells with transgenes inserted into proven genomic loci ensuring reliable expression levels.     

Genomic targeting with purified Cre recombinase.

Purified Cre recombinase protein introduced directly into cultured mammalian cells by lipofection catalyzes both site-specific chromosomal integration of a co-transfected lox targeting vector and precise excision of genomic DNA flanked by directly repeated lox sites. This procedure eliminates the need to transfect cre expression plasmids to activate recombination at lox sites. We used this simplified procedure to investigate the effect on targeting efficiency of both lox vector design and chromosomal position of the lox target. We show that such chromosomal position effects can exert at least a 50-fold per lox target difference in targeting efficiency in a human osteosarcoma cell line.     

In vivo modification of a maize engineered minichromosome

Engineered minichromosomes provide efficient platforms for stacking transgenes in crop plants. Methods for modifying these chromosomes in vivo are essential for the development of customizable systems for the removal of selection genes or other sequences and for the addition of new genes. Previous studies have demonstrated that Cre, a site-specific recombinase, could be used to modify lox sites on transgenes on maize minichromosomes; however, these studies demonstrated somatic recombination only, and modified minichromosomes could not be recovered. We describe the recovery of an engineered chromosome composed of little more than a centromere plus transgene that was derived by telomere-mediated truncation. We used the fiber fluorescence in situ hybridization technique and detected a transgene on the minichromosome inserted among stretches of CentC centromere repeats, and this insertion was large enough to suggest a tandem insertion. By crossing the minichromosome to a plant expressing Cre-recombinase, the Bar selection gene was removed, leaving behind a single loxP site. This study demonstrates that engineered chromosomes can be modified in vivo using site-specific recombinases, a demonstration essential to the development of amendable chromosome platforms in plants.     

Sequence of the loxP site determines the order of strand exchange by the Cre recombinase

Conservative site-specific recombinases of the integrase family carry out recombination via a Holliday intermediate. The Cre recombinase, a member of the integrase family, was previously shown to initiate recombination by cleaving and exchanging preferentially on the bottom strand of its loxP target sequence. We have confirmed this strand bias for an intermolecular recombination reaction that used wild-type loxP sites and Cre protein. We have examined the sequence determinants for this strand preference by selectively mutating the two asymmetric scissile base-pairs in the lox site (those immediately adjacent to the sites of cleavage by Cre). We found that the initial strand exchange occurs preferentially next to the scissile G residue. Resolution of the Holliday intermediate thus formed takes place preferentially next to the scissile A residue. Lys86, which contacts the scissile nucleotides in the Cre-lox crystal structures, was important for establishing the strand preference in the resolution of the loxP-Holliday intermediate, but not for the initiation of recombination between loxP sites.     

Multiplex Cre/lox recombination permits selective site-specific DNA targeting to both a natural and an engineered site in the yeast genome.

Variant lox sites having an altered spacer region (heterospecific lox sites) are not proficient for Cre-mediated recombination with the canonical 34 bp loxP site, but can recombine with each other. By placing different heterospecific lox sites at different genomic locations, Cre can catalyze independent DNA recombination events at multiple loci in the same cell without concern that unwanted inter-locus recombination events will be generated. Such heterospecific lox sites also allow Cre to specifically target efficient integration of exogenous DNA to endogenous lox-like sequences that naturally occur in the genome. Specific targeting occurs only with a DNA vector carrying a heterospecific lox site in which the spacer region has been redesigned to match the 'spacer' region of the targeted chromosomal element. Moreover, in cells expressing a catalytically active Cre recombinase, naturally occurring lox-like sequences can exhibit almost 20% mitotic recombination. Thus, in the same cell, heterospecific lox sites can be used independently at multiple loci for integration, for deletion and for enhanced mitotic recombination, thereby increasing the repertoire of genomic manipulations catalyzed by the Cre recombinase.     

Cre/lox系统介导的位点特异性重组技术及其应用

Ｃｒｅ／ｌｏｘ系统是源于Ｐ１噬菌体的一个ＤＮＡ重组体系,它能导致在特定的ＤＮＡ序列（ｌｏｘＰ位点）处发生定点重组。该系统以将外源基因定点整合到染色体上或将特定ＤＮＡ片段删除;这种定位重组系统在遗传操作中发挥了重要的作用。     

In vivo evaluation of PhiC31 recombinase activity using a self-excision cassette

Gene targeting allows precise tailoring of the mouse genome such that desired modifications can be introduced under precise temporal and spatial control. This can be achieved through the use of site-specific recombinases, which mediate deletion or inversion of genomic DNA flanked by recombinase-specific recognition sites, coupled with gene targeting to introduce the recombinase recognition sites at the desired genomic locations within the mouse genome. The introduction of multiple modifications at the same locus often requires use of multiple recombination systems. The most commonly used recombination system is Cre/lox. We here evaluated in vivo the ability of PhiC31 phage integrase to induce a genomic deletion in mouse. We engineered a self-excision cassette, modeled after one previously designed for Cre, containing a positive selection marker and PhiC31 driven by a testis-specific promoter, all flanked by PhiC31 specific attP/B sites. We found in vivo PhiC31 mediated self-excision in 38% of transmitted alleles, although 18% of these showed evidence of imprecise deletion. Furthermore, in the 69% of un-recombined cassettes, sequence analysis revealed that PhiC31 mediated an intra-molecular deletion of the attB site preventing any subsequent recombination. This study demonstrates that PhiC31 can be used to automatically remove Neo, in the male chimera germline, although it is not as efficient or as accurate as Cre.