Exchangeable gene trap using the Cre/mutated lox system.

The gene trap technique is a powerful approach for characterizing and mutating genes involved in mouse development. However, one shortcoming of gene trapping is the relative inability to induce subtle mutations. This problem can be overcome by introducing a knock-in system into the gene trap strategy. Here, we have constructed a new gene trap vector, pU-Hachi, employing the Cre-mutated lox system (Araki et al., 1997), in which a pair of mutant lox, lox71 and lox66, was used to promote targeted integrative reaction by Cre recombinase. The pU-Hachi carries splicing acceptor (SA)-lox71-internal ribosomal entry site (IRES)-beta-geo-pA-loxP-pA-pUC. By using this vector, we can carry out random insertional mutagenesis as the first step, and then we can replace the beta-geo gene with any gene of interest through Cre-mediated integration. We have isolated 109 trap clones electroporated with pU-Hachi, and analyzed their integration patterns by Southern blotting to select those carrying a single copy of the trap vector. By use of some of these clones, we have succeeded in exchanging the reporter gene at high efficiency, ranging between 20-80%. This integration system is also quite useful for plasmid rescue to recover flanking genomic sequences, because a plasmid vector sequence can be introduced even when the pUC sequence of the trap vector is lost through integration into the genome. Thus, this method, termed exchangeable gene trapping, has many advantages as the trapped clones can be utilized to express genes with any type of mutation.     

Seed-specific gene activation mediated by the Cre/lox site-specific recombination system.

The Cre/lox site-specific recombination system was used to activate a transgene in a tissue-specific manner. Cre-mediated activation of a beta-glucuronidase marker gene, by removal of a lox-bounded blocking fragment, allowed the visualization of the activation process. By using seed-specific promoters, the timing and efficiency of gene activation could be followed within the developing tobacco (Nicotiana tabacum) embryo. To serve as a basis for analyzing gene expression after-Cre-mediated activation, the timing and patterns of expression of the promoters of the genes encoding French bean (Phaseolus vulgaris) beta-phaseolin and the alpha' subunit of soybean (Glycine max) beta-conglycinin, as well as the cauliflower mosaic virus 35S promoter, were studied in developing transgenic tobacco embryos using the same visual marker. These seed-specific promoters were expressed earlier than anticipated. The 35S promoter was expressed earlier than the seed-specific promoters, but not in globular-stage embryos. Cre-mediated gene activation occurred approximately 1 d after promoter activation, based on developmental staging, and spread progressively throughout the embryo. The timing of gene activation was varied by altering Cre expression. Efficient Cre expression ultimately directed gene activation throughout the model tissue, whereas inefficient Cre expression resulted in mosaic tissue. Limited gene activation provides a system for cell lineage and developmental analyses.     

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.     

Cre/lox位点特异性重组系统在高等真核生物中的研究进展

来自于P1噬菌体的Cre/lox系统通过位点特异性重组可以迅速而有效地实现各种生理环境下的基因定点插入、删除、替换和倒位等操作。Cre/lox系统作为目前基因打靶技术的核心工具,已被广泛应用于拟南芥、水稻、小鼠、果蝇、斑马鱼等高等真核模式生物。文章较为全面地介绍了Cre/lox系统的基本概况及其在高等真核生物中的应用,讨论了Cre/lox系统在研究中存在的主要问题和今后的发展方向,为利用该系统在不同高等生物中进行基因操作提供有用的参考。     

Effect of lox-sites of the Cre lox recombination system on promoterless bar gene expression in transgenic plants

We demonstrate that localization of lox site between the right border of T-DNA and promoterless bar gene (RB-lox-bar-) led to its highly efficient expression in transgenic plants of Nicotiana tabacum and N. africana. Plasmid vectors used in gene integration experiments contained neomycin phosphotransferase II (npt II) gene under nos promoter as well. Transgenic plants were selected according to their capacity to grow on the medium with kanamycin and then they were tested on the selective medium containing phosphinothricin. 80% of transgenic plants expressed bar gene at the level similar to that in plants transformed with the bar gene under widely used constitutive promoter. Transformation of plants with the plasmid vector containing only promoterless bar gene near T-DNA right border (RB-bar-) and with the vector containing lox site and promoterless bar gene in the middle of the construction (-lox-bar-) led to obtaining no more than 4.5% of transgenic plants resistant to phosphinothricin. PCR analyses confirmed both the absence of tandem repeats and of plasmid recombination resulting in transference of bar gene under promoter in plasmid vector. Nos-terminator situated between the lox site and the right border of T-DNA did not decrease bar gene expression.     

Using the Cre/lox system for targeted integration into the human genome: loxFAS-loxP pairing and delayed introduction of Cre DNA improve gene swapping efficiency

Cre recombinase is a commonly-used genome editing tool suitable for site-specific integrations in mammalian genomes; however, the efficiency of transgenic swapping events compared to excision remains limited. Here we sought to identify important parameters and limiting factors that influence swapping propensity in this system, especially when using one wild-type loxP site. To modulate and increase the occurrence of swapping events, we identified two novel parameters. First, we identified the loxFAS-loxP pairing, a sequence never before used in mammalian systems, as the best choice for increasing swapping events in human cell lines. Second, for the first time we implicate the importance of delayed introduction of Cre DNA for optimal swapping efficiency. This same modification could potentially be of use to other systems catalyzing trimolecular reactions such as ΦC31 integrase and FLP recombinase where we hypothesize that transport of the exchange cassette is likewise initially rate limiting. The total number of recombination events, but not the ratio of swapping to excision, was found to be influenced by the quantity of Cre DNA transfected. Through this study, we were able to obtain Cre-mediated swapping frequencies of 8-12% without antibiotic enrichment, which represents nearly an order of magnitude increase over prior reports in the literature.     

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。     

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

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

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.     

Cre/lox位点特异重组系统在植物基因工程中的应用

Cre/lox位点特异重组系统是植物基因工程中的重要工具,利用其可以在转基因植物中对目的基因实现精确删除和定点整合。概述Cre/lox系统的基本结构及作用方式,并以基因删除和定点整合为重点,详细介绍该系统在这两方面的应用。     

Marker Removal in Staphylococci via Cre Recombinase and Different lox Sites

Allelic replacement in staphylococci is frequently aided by antibiotic resistance markers that replace the gene(s) of interest. In multiply modified strains, the number of mutated genes usually correlates with the number of selection markers in the strain's chromosome. Site-specific recombination systems are capable of eliminating such markers, if they are flanked by recombinase recognition sites. In this study, a Cre-lox setting was established that allowed the efficient removal of resistance genes from the genomes of Staphylococcus carnosus and S. aureus. Two cassettes conferring resistance to erythromycin or kanamycin were flanked with wild-type or mutant lox sites, respectively, and used to delete single genes and an entire operon. After transformation of the cells with a newly constructed cre expression plasmid (pRAB1), genomic eviction of the resistance genes was observed in approximately one out of ten candidates analyzed and subsequently verified by PCR. Due to its thermosensitive origin of replication, the plasmid was then easily eliminated at nonpermissive temperatures. We anticipate that the system presented here will prove useful for generating markerless deletion mutants in staphylococci.     

Cre/ lox site-specific recombination controls the excision of a transgene from the rice genome

The Cre/lox site-specific recombination controls the excision of a target DNA segment by recombination between two lox sites flanking it, mediated by the Cre recombinase. We have studied the functional expression of the Cre/lox system to excise a transgene from the rice genome. We developed transgenic plants carrying the target gene, hygromycin phosphotransferase (hpt) flanked by two lox sites and transgenic plants harboring the Cre gene. Each lox plant was crossed with each Cre plant reciprocally. In the Cre/lox hybrid plants, the Cre recombinase mediates recombination between two lox sites, resulting in excision of the hpt gene. The recombination event could be detected because it places the CaMV 35S promoter of the hpt gene adjacent to a promoterless gusA gene; as a result the gusA gene is activated and its expression could be visualized. In 73 Cre/lox hybrid plants from various crosses of T0 transgenic plants, 19 expressed GUS, and in 132 Cre/lox hybrid plants from crosses of T2 transgenic plants, 77 showed GUS expression. Molecular data proved the excision event occurred in all the GUS⁺ plants. Recombination occurred with high efficiency at the early germinal stage, or randomly during somatic development stages. Received. 2 April 2001 / Accepted: 29 June 2001     

A randomized library approach to identifying functional lox site domains for the Cre recombinase

The bacteriophage P1 Cre/loxP site-specific recombination system is a useful tool in a number of genetic engineering processes. The Cre recombinase has been shown to act on DNA sequences that vary considerably from that of its bacteriophage recognition sequence, loxP. However, little is known about the sequence requirements for functional lox-like sequences. In this study, we have implemented a randomized library approach to identify the sequence characteristics of functional lox site domains. We created a randomized spacer library and a randomized arm library, and then tested them for recombination in vivo and in vitro. Results from the spacer library show that, while there is great plasticity, identity between spacer pairs is the most important factor influencing function, especially in in vitro reactions. The presence of one completely randomized arm in a functional loxP recombination reaction revealed that only three wild-type loxP arms are necessary for successful recombination in Cre-expressing bacteria, and that there are nucleotide preferences at the first three and last three positions of the randomized arm for the most efficiently recombined sequences. Finally, we found that in vitro Cre recombination reactions are much more stringent for evaluating which sequences can support efficient recombination compared to the 294-CRE system.     

Cre recombinase-mediated inversion using lox66 and lox71: method to introduce conditional point mutations into the CREB-binding protein

CREB-binding protein (CBP) is a multifunctional cofactor implicated in many intracellular signal transduction pathways. We aimed to investigate the involvement of CBP in the cAMP response element-binding protein (CREB)-mediated pathway. The point mutation Tyr658Ala in the CREB-binding domain (CBD) was shown to abolish the binding activity of CBP to phospho-CREB, the activated form of CREB. By using a mutant Cre/loxP recombination system, this point mutation was aimed to be generated in the mouse genome in a tissue- and time-specific manner. A targeting construct in which CBD exon 5 and inverted exon 5* containing the point mutation flanked by two mutant loxP sites (lox66 and lox71) oriented in a head-to-head position was generated. When Cre recombinase is present, the DNA flanked by the two mutant loxP sites is inverted, forming one loxP and one double mutated loxP site. As the double mutated loxP site shows low affinity for Cre recombinase, the favorable reaction leads to a product where the mutated exon 5* is placed into the position to be correctly transcribed and spliced. Inversion was observed to be complete in both bacteria and mouse embryonic stem cells. Our results indicate that this Cre- mediated inversion method is a valuable tool to introduce point mutations in the mouse genome in a regulatable manner.     

Use of lambda Red-mediated recombineering and Cre/lox for generation of markerless chromosomal deletions in avian pathogenic Escherichia coli

Avian pathogenic Escherichia coli (APEC) are bacteria associated with extraintestinal diseases in poultry. A method to generate markerless deletions of APEC genome is described. Lambda Red recombination is used to introduce a LoxP cassette (loxP-rpsL-neo-loxP) containing the rpsL gene for streptomycin sensitivity and the neo gene for kanamycin/neomycin resistance into the APEC genome, with attendant deletion of a desired chromosomal gene. The loxP sites are incorporated into primers used to amplify the rpsL-neo marker during the construction of the LoxP cassette, making the method rapid and efficient. The cassette is specifically integrated into the fiu gene or intergenic region 2051-52, and the Cre/lox system is used to remove the marker, hence deletion of the drug-resistance genes. The results demonstrate that the Cre/lox system can successfully be used to generate markerless deletions in APEC, and rpsL counter-selection can be used to select the deletions so that one does not have to pick and test to find the desired product.     

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。     

Reversible gene knockdown in mice using a tight, inducible shRNA expression system

RNA interference through expression of short hairpin (sh)RNAs provides an efficient approach for gene function analysis in mouse genetics. Techniques allowing to control time and degree of gene silencing in vivo, however, are still lacking. Here we provide a generally applicable system for the temporal control of ubiquitous shRNA expression in mice. Depending on the dose of the inductor doxycycline, the knockdown efficiency reaches up to 90%. To demonstrate the feasibility of our tool, a mouse model of reversible insulin resistance was generated by expression of an insulin receptor (Insr)-specific shRNA. Upon induction, mice develop severe hyperglycemia within seven days. The onset and progression of the disease correlates with the concentration of doxycycline, and the phenotype returns to baseline shortly after withdrawal of the inductor. On a broad basis, this approach will enable new insights into gene function and molecular disease mechanisms.     

Arabidopsis lox3 lox4 double mutants are male sterile and defective in global proliferative arrest

Fertility and flower development are both controlled in part by jasmonates, fatty acid-derived mediators produced via the activity of 13-lipoxygenases (13-LOXs). The Arabidopsis thaliana Columbia-0 reference genome is predicted to encode four of these enzymes and it is already known that one of these, LOX2, is dispensable for fertility. In this study, the roles of the other three 13-LOXs (LOX3, LOX4 and LOX6) were investigated in single and double mutants. Four independent lox3 lox4 double mutants assembled with different mutated lox3 and lox4 alleles had fully penetrant floral phenotypes, displaying abnormal anther maturation and defective dehiscence. The plants were no longer self-fertile and pollen was not viable. Fertility in the double mutant was restored genetically by complementation with either the LOX3 or the LOX4 cDNAs and biochemically with exogenous jasmonic acid. Furthermore, deficiency in LOX3 and LOX4 causes developmental dysfunctions, compared to wild type; lox3 lox4 double mutants are taller and develop more inflorescence shoots and flowers. Further analysis revealed that developmental arrest in the lox3 lox4 inflorescence occurs with the production of an abnormal carpelloid flower. This distinguishes lox3 lox4 mutants from the wild type where developmentally typical flower buds are the terminal inflorescence structures observed in both the laboratory and in nature. Our studies of lox3 lox4 as well as other jasmonic acid biosynthesis and perception mutants show that this plant hormone is not only required for male fertility but also involved in global proliferative arrest.