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.     

Cre/lox system and PCR-based genome engineering in Bacillus subtilis

We have developed a fast and accurate method to engineer the Bacillus subtilis genome that involves fusing by PCR two flanking homology regions with an antibiotic resistance gene cassette bordered by two mutant lox sites (lox71 and lox66). The resulting PCR products were used directly to transform B. subtilis, and then transient Cre recombinase expression in the transformants was used to recombine lox71 and lox66 into a double-mutant lox72 site, thereby excising the marker gene. The mutation process could also be accomplished in 2 days by using a strain containing a cre isopropyl-beta-d-thiogalactopyranoside (IPTG)-inducible expression cassette in the chromosome as the recipient or using the lox site-flanked cassette containing both the cre IPTG-inducible expression cassette and resistance marker. The in vivo recombination efficiencies of different lox pairs were compared; the lox72 site that remains in the chromosome after Cre recombination had a low affinity for Cre and did not interfere with subsequent rounds of Cre/lox mutagenesis. We used this method to inactivate a specific gene, to delete a long fragment, to realize the in-frame deletion of a target gene, to introduce a gene of interest, and to carry out multiple manipulations in the same background. Furthermore, it should also be applicable to large genome rearrangement.     

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.     

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.     

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.     

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.     

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.     

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.     

Requirement for 3-phosphoinositide-kependent dinase-1 (PDK-1) in insulin-induced glucose uptake in immortalized brown adipocytes

To provide insight into the physiological importance of 3-phosphoinositide-dependent kinase-1 (PDK-1) in the metabolic actions of insulin, we have generated mice that harbor a PDK-1 gene containing LoxP sites (PDK-1(lox/lox) mice) and established immortalized brown preadipocyte cell lines both from these animals and from wild-type mice. Exposure to appropriate hormonal inducers resulted in the differentiation of >80% of the immortalized brown preadipocytes derived from both types of mice into mature adipocytes. Introduction of the Cre recombinase with the use of adenovirus-mediated gene transfer induced a dose-dependent decrease in the abundance of PDK-1 in PDK-1(lox/lox) adipocytes but not in the wild-type cells. In Cre-expressing PDK-1(lox/lox) adipocytes in which the abundance of PDK-1 was reduced by approximately 85%, the insulin-induced phosphorylation both of Akt on threonine 308 and of p70 S6 kinase on threonine-389 was markedly inhibited. The phosphorylation both of Akt on serine 473 and of p42 and p44 isoforms of mitogen-activated protein kinase induced by insulin was not affected by Cre expression, indicating that the latter specifically inhibits PDK-1-dependent signaling. Both glucose uptake and the translocation of glucose transporter 4 to the plasma membrane induced by insulin as well as glucose uptake induced by a constitutively active form of phosphoinositide 3-kinase were also greatly inhibited by Cre expression in PDK-1(lox/lox) adipocytes. Phosphorylation of AMP-activated protein kinase and glucose uptake induced by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) were not affected by Cre expression in PDK-1(lox/lox) adipocytes. These results indicate that PDK-1 is essential for insulin-induced glucose uptake in adipocytes.     

Unidirectional Cre-mediated genetic inversion in mice using the mutant loxP pair lox66/lox71

The Cre/loxP recombination system is a commonly used tool to alter the mouse genome in a conditional manner by deletion or inversion of loxP-flanked DNA segments. While Cre-mediated deletion is essentially unidirectional, inversion is reversible and therefore does not allow the stable alteration of gene function in cells that constitutively express Cre. Site-directed mutagenesis yielded a pair of asymmetric loxP sites (lox66 and lox71) that display a favorable forward reaction equilibrium. Here, we demonstrate that lox66/lox71 mediates efficient and predominantly unidirectional inversion of a switch substrate targeted to the mouse genome in combination with either inducible or cell type-specific cre-transgenes in vivo.     

New multiple-deletion method for the Corynebacterium glutamicum genome, using a mutant lox sequence

Due to the difficulty of multiple deletions using the Cre/loxP system, a simple, markerless multiple-deletion method based on a Cre/mutant lox system combining a right-element (RE) mutant lox site with a left-element (LE) mutant lox site was employed for large-scale genome rearrangements in Corynebacterium glutamicum. Eight distinct genomic regions that had been identified previously by comparative analysis of C. glutamicum R and C. glutamicum 13032 genomes were targeted for deletion. By homologous recombination, LE and RE mutant lox sites were integrated at each end of a target region. Highly efficient and accurate deletions between the two chromosomal mutant lox sites in the presence of Cre recombinase were realized. A deletion mutant lacking 190 kb of chromosomal regions, encoding a total of 188 open reading frames (ORFs), was obtained. These deletions represent the largest genomic excisions in C. glutamicum reported to date. Despite the loss of numerous predicted ORFs, the mutant exhibited normal growth under standard laboratory conditions. The Cre/loxP system using a pair of mutant lox sites provides a new, efficient genome rearrangement technique for C. glutamicum. It should facilitate the understanding of genome functions of microorganisms.     

A new approach for the identification and cloning of genes: the pBACwich system using Cre/lox site-specific recombination

With current plant transformation methods ( Agrobacterium, biolistics and protoplast fusion), insertion of DNA into the genome occurs randomly and in many instances at multiple sites. Associated position effects, copy number differences and multigene interactions can make gene expression experiments difficult to interpret and plant phenotypes less predictable. An alternative approach to random integration of large DNA fragments into plants is to utilize one of several site-specific recombination (SSR) systems, such as Cre/ lox. Cre has been shown in numerous instances to mediate lox site-specific recombination in animal and plant cells. By incorporating the Cre/ lox SSR system into a bacterial artificial chromosome (BAC) vector, a more precise evaluation of large DNA inserts for genetic complementation should be possible. Site-specific insertion of DNA into predefined sites in the genome may eliminate unwanted 'position effects' caused by the random integration of exogenously introduced DNA. In an effort to make the Cre/ lox system an effective tool for site-directed integration of large DNAs, we constructed and tested a new vector potentially capable of integrating large DNA inserts into plant and fungal genomes. In this study, we present the construction of a new BAC vector, pBACwich, for the system and the use of this vector to demonstrate SSR of large DNA inserts (up to 230 kb) into plant and fungal genomes.     

Targeted integration of DNA using mutant lox sites in embryonic stem cells.

Site-directed DNA integration has been achieved by using a pair of mutant lox sites, a right element (RE) mutant lox site and a left element (LE) mutant lox site [Albertet al. (1995)Plant J., 7, 649-659], in mouse embryonic stem (ES) cells. We established ES cell lines carrying a single copy of the wild-type lox Por LE mutant lox site as a target and examined the frequency of site-specific integration of a targeting vector carrying a loxP or RE mutant lox site induced by Cre transient expression. Since our targeting vector contains a complete neo gene, random integrants can form colonies as in the case of a gene targeting event through homologous recombination. With our system, the frequency of site-specific integration via the mutant lox sites reached a maximum of 16%. In contrast, the wild-type loxP sites yielded very low frequencies (<0.5%) of site-specific integration events. This mutatedloxsystem will be useful for 'knock-in' integration of DNA in ES cells.     

利用Cre/loxP系统删除转Bt基因水稻中的选择标记基因

来源于噬菌体P1的Cre/loxP位点特异性重组系统是目前在植物遗传转化中应用较多,较成熟的一个标记基因删除系统。在这个系统中,Cre酶可以特异性的识别和切割位于两个lox位点之间的标记基因,整个系统重组仅需Cre和lox识别位点即可完成而无需其它辅因子的参加。利用农杆菌介导法成功地将cre基因导入供试材料"皖粳97",得到转hpt-cre基因水稻植株;将其与先期转基因育成的携带loxp-hpt-loxp-bt基因的"皖粳97"株系进行田间杂交,通过PCR分析,Cre/loxP重组系统定向删除了潮霉素抗性筛选标记基因。     

一种新的用于删除选择标记基因的Cre／lox系统

设计了一种新的诱导型Cre/lox系统,并在转基因烟草(NicotianatabacumL.)中进行了验证。在诱导剂的作用下,位于同向lox位点之间的选择标记基因(hpt)和重组酶基因(Cre)在烟草愈伤组织中被删除。在该系统中,Cre基因在玉米乙酰苯胺类化合物诱导启动子(In5-2)的控制下表达。对转基因后代的分子检测结果表明,不论是否加入了诱导剂,目的基因(gus)均被整合到烟草基因组中;在诱导剂处理的48株转基因烟草T0代中,45株的hpt基因被删除了。该系统只使用一个载体,克服了二次转化系统带来的问题。     

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.     

一种新的用于删除选择标记基因的Cre/lox系统

设计了一种新的诱导型Cre/lox系统,并在转基因烟草(Nicotianatabacum L.)中进行了验证.在诱导剂的作用下,位于同向lox位点之间的选择标记基因(hpt)和重组酶基因(Cre)在烟草愈伤组织中被删除.在该系统中,Cre基因在玉米乙酰苯胺类化合物诱导启动子(In5-2)的控制下表达.对转基因后代的分子检测结果表明,不论是否加入了诱导剂,目的基因(gus)均被整合到烟草基因组中;在诱导剂处理的48株转基因烟草To代中,45株的hpt基因被删除了.该系统只使用一个载体,克服了二次转化系统带来的问题.     

A test for ectopic exchange catalyzed by Cre recombinase in maize

A maize line expressing Cre recombinase as well as the recipient line without the transgene were assayed for evidence of ectopic recombination within the maize genome. Such a test is valuable for understanding the action of Cre as well as for its use to recombine two target lox sites present in the chromosomes. Pollen examination and seed set tests of material expressing Cre provided no evidence of ectopic recombination, which would be manifested in the production of translocations or inversions and result in pollen abortion and reduced seed set. Root-tip chromosome karyotype analysis was also performed on material with and without Cre expression. Chromosomal aberrations in Cre+ material were not observed above the background level.     

Cre-lox-based system for multiple gene deletions and selectable-marker removal in Lactobacillus plantarum

The classic strategy to achieve gene deletion variants is based on double-crossover integration of nonreplicating vectors into the genome. In addition, recombination systems such as Cre-lox have been used extensively, mainly for eukaryotic organisms. This study presents the construction of a Cre-lox-based system for multiple gene deletions in Lactobacillus plantarum that could be adapted for use on gram-positive bacteria. First, an effective mutagenesis vector (pNZ5319) was constructed that allows direct cloning of blunt-end PCR products representing homologous recombination target regions. Using this mutagenesis vector, double-crossover gene replacement mutants could be readily selected based on their antibiotic resistance phenotype. In the resulting mutants, the target gene is replaced by a lox66-P(32)-cat-lox71 cassette, where lox66 and lox71 are mutant variants of loxP and P(32)-cat is a chloramphenicol resistance cassette. The lox sites serve as recognition sites for the Cre enzyme, a protein that belongs to the integrase family of site-specific recombinases. Thus, transient Cre recombinase expression in double-crossover mutants leads to recombination of the lox66-P(32)-cat-lox71 cassette into a double-mutant loxP site, called lox72, which displays strongly reduced recognition by Cre. The effectiveness of the Cre-lox-based strategy for multiple gene deletions was demonstrated by construction of both single and double gene deletions at the melA and bsh1 loci on the chromosome of the gram-positive model organism Lactobacillus plantarum WCFS1. Furthermore, the efficiency of the Cre-lox-based system in multiple gene replacements was determined by successive mutagenesis of the genetically closely linked loci melA and lacS2 in L. plantarum WCFS1. The fact that 99.4% of the clones that were analyzed had undergone correct Cre-lox resolution emphasizes the suitability of the system described here for multiple gene replacement and deletion strategies in a single genetic background.