Simple and straightforward construction of a mouse gene targeting vector using in vitro transposition reactions

In a gene targeting experiment, the generation of a targeting construct often requires complex DNA manipulations. We developed a set of cassettes and plasmids useful for creating targeting vectors to modify the mammalian genome. A positive selection marker cassette (PGK/EM7p-npt), which included dual prokaryotic and eukaryotic promoters to permit consecutive selection for recombination in Escherichia coli and then in mouse embryonic stem cells, was flanked by two FRT-loxP sequences. The PGK/EM7p-npt cassette was placed between the minimum regions of a Tn7 transposable element for insertion into another DNA by means of Tn7 transposase in vitro. We also constructed a plasmid having a loxP-Zeo-loxP cassette between the modified Tn5 outer elements. These cassettes can be integrated randomly into a given genomic DNA through the in vitro transposition reaction, thus producing a collection of genomic segments flanked by loxP sites (floxed) at various positions without the use of restriction enzymes and DNA ligase. We confirmed that this system remarkably reduced the time and labor for the construction of complex gene targeting vectors.     

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.     

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.     

A genetic screen identifies novel non-compatible loxP sites

The ability of the Cre/lox system to make precise genomic modifications is a tremendous accomplishment. However, recombination between cis-linked heterospecific lox sites limits the use of Cre- mediated exchange of DNA to systems where genetic selection can be applied. To circumvent this problem we carried out a genetic screen designed to identify novel mutant spacer-containing lox sites displaying enhanced incompatibility with the canonical loxP site. One of the mutant sites recovered appears to be completely stable in HEK293 cells constitutively expressing Cre recombinase and supports recombinase-mediated cassette exchange (RMCE) in bacteria and mammalian cell culture. By preventing undesirable recombination, these novel lox sites could improve the efficiency of in vivo gene transfer.     

Site-specific integration of transgene targeting an endogenous <Emphasis Type="Italic">lox-</Emphasis>like site in early mouse embryos

Functional lox-like sequences have been identified within the yeast and mammalian genome. These hetero-specific lox sites also allow Cre recombinase to specifically target efficient integration of exogenous DNA into the endogenous pseudo-lox (ψlox) sequences that occur naturally in the host genome using a Cre/loxP integrative recombination system. We investigated whether the Cre/ψlox system is useful for site-specific integration of transgenes and for improving the production efficiency of transgenic animals. This is the first report on Cre-mediated integrative recombination targeting an endogenous lox-like sequence termed pseudo-loxm5 (ψloxm5) in early mouse embryos. We characterized the Cre/ψloxm5 system in embryonic environment. Cre-expressing plasmid and a transgene (CMV/LacZ gene) flanked by ψloxm5 and ψloxcorem5 sites were co-microinjected into the pronucleus of fertilized mouse oocytes. The injected eggs were transferred into foster mothers, and the recombination products were investigated. The results show that the ψloxm5 site is an active substrate for Cre-mediated recombination in the mouse embryonic environment. The transgenesis efficiency was up to 27% (6/22). The site-specific integration of the transgene into the endogenous ψloxm5 site was found in 50 % of the transgenic pups. Our findings demonstrated that the Cre/ψloxm5 integrative recombination system is an efficient and simple strategy for targeting an endogenous lox-like site in mammalian embryos.     

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     

Bacteriophage P1 site-specific recombination: I. Recombination between loxP sites

We have studied P1 site-specific recombination by cloning a 6·5 × 10³ base EcoRI fragment (fragment 7) of P1 DNA into a λ vector and then asking whether that fragment can promote efficient recombination for λ markers that flank the fragment. Our results indicate that fragment 7 can reassort these markers very efficiently, and that this recombination can occur in the absence of the bacterial recA and recBC functions. The fragment 7 recombination system has been dissected by an analysis of deletion mutations into two components, a site (called loxP) that must be present in both partners in the recombination in order for recombination to occur, and a P1 gene (called cre), whose product is necessary for recombination. The location of the loxP site at the end of the P1 genetic map suggests that this site-specific recombination system is responsible for the lack of linkage between terminal P1 markers and therefore for the linearity of that map.     

Plant tissue-specific promoters can drive gene expression in Escherichia coli

Plant transgenesis often requires the use of tissue-specific promoters to drive the transgene expression exclusively in targeted tissues. Although the eukaryotic promoters are expected to stay silent in Escherichia coli, when the promoter-transgene units within the plant transformation vectors are constructed and propagated, some eukaryotic promoters have been reported to be active in prokaryotes. The potential activity of plant promoter in E. coli cells should be considered in cases of expression of proteins that are toxic for host cells, environmental risk assessment or the stability in E. coli of plant vectors for specific Cre/loxP applications. In this study, DNA fragments harbouring four embryo- and/or pollen-specific Arabidopsis thaliana promoters were investigated for their ability to drive heterologous gene expression in E. coli cells. For this, they were fused to gfp:gus reporter genes in the pCAMBIA1304 vector. Although BPROM, bacterial sigma70 promoter recognition program identified several sequences with characteristics similar to bacterial promoters including -10 and -35 sequences in each of tested fragments, the experimental approach showed that only one promoter fragment was able to drive relatively strong- and one promoter fragment relatively weak-GUS expression in E. coli cells. Remaining two tested promoters did not drive any transgene expression in bacteria. Our results also showed that cloning of a shorter plant promoter sequence into vectors containing lacZ α-complementation system can increase the probability of gene expression driven by upstream located lac promoter. This should be considered when cloning of plant expression units, the expression of which is unwanted in E. coli.     

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.     

PCR-based gene targeting of the inducible nitric oxide synthase (NOS2) locus in murine ES cells,a new and more cost-effective approach

Gene targeting by double homologous recombination in murine embryonic stem (ES) cells is a powerful tool used to study the cellular consequences of specific genetic mutations. A typical targeting construct consists of a neomycin phosphotransferase (neo) gene flanked by genomic DNA fragments that are homologous to sequences in the target chromosomal locus. Homologous DNA fragments are typically cloned from a murine genomic DNA library. Here we describe an alternative approach whereby the inducible nitric oxide synthase (NOS2) gene locus is partially mapped and homologous DNA sequences obtained using a long-range PCR method. A 7 kb NOS2 amplicon is used to construct a targeting vector where theneo gene is flanked by PCR-derived homologous DNA sequences. The vector also includes a thymidine kinase (tk) negative-selectable marker gene. Following transfection into ES cells, the PCR-based targeting vector undergoes efficient homologous recombination into the NOS2 locus. Thus, PCR-based gene targeting can be a valuable alternative to the conventional cloning approach. It expedites the acquisition of homologous genomic DNA sequences and simplifies the construction of targeting plasmids by making use of defined cloning sites. This approach should result in substantial time and cost savings for appropriate homologous recombination projects.     

Crystal structure of a wild-type Cre recombinase-loxP synapse reveals a novel spacer conformation suggesting an alternative mechanism for DNA cleavage activation

Escherichia coli phage P1 Cre recombinase catalyzes the site-specific recombination of DNA containing loxP sites. We report here two crystal structures of a wild-type Cre recombinase–loxP synaptic complex corresponding to two distinct reaction states: an initial pre-cleavage complex, trapped using a phosphorothioate modification at the cleavable scissile bond that prevents the recombination reaction, and a 3′-phosphotyrosine protein–DNA intermediate resulting from the first strand cleavage. In contrast to previously determined Cre complexes, both structures contain a full tetrameric complex in the asymmetric unit, unequivocally showing that the anti-parallel arrangement of the loxP sites is an intrinsic property of the Cre–loxP recombination synapse. The conformation of the spacer is different to the one observed for the symmetrized loxS site: a kink next to the scissile phosphate in the top strand of the pre-cleavage complex leads to unstacking of the TpG step and a widening of the minor groove. This side of the spacer is interacting with a ‘cleavage-competent’ Cre subunit, suggesting that the first cleavage occurs at the ApT step in the top strand. This is further confirmed by the structure of the 3′-phosphotyrosine intermediate, where the DNA is cleaved in the top strands and covalently linked to the ‘cleavage-competent’ subunits. The cleavage is followed by a movement of the C-terminal part containing the attacking Y324 and the helix N interacting with the ‘non-cleaving’ subunit. This rearrangement could be responsible for the interconversion of Cre subunits. Our results also suggest that the Cre-induced kink next to the scissile phosphodiester activates the DNA for cleavage at this position and facilitates strand transfer.     

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.     

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.     

Mutually exclusive recombination of wild-type and mutant loxP sites in vivo facilitates transposon-mediated deletions from both ends of genomic DNA in PACs

Recombination of wild-type and mutant loxP sites mediated by wild-type Cre protein was analyzed in vivo using a sensitive phage P1 transduction assay. Contrary to some earlier reports, recombination between loxP sites was found to be highly specific: a loxP site recombined in vivo only with another of identical sequence, with no crossover recombination either between a wild-type and mutant site; or between two different mutant sites tested. Mutant loxP sites of identical sequence recombined as efficiently as wild-type. The highly specific and efficient recombination of mutant loxP sites in vivo helped in developing a procedure to progressively truncate DNA from either end of large genomic inserts in P1-derived artificial chromosomes (PACs) using transposons that carry either a wild-type or mutant loxP sequence. PAC libraries of human DNA were constructed with inserts flanked by a wild-type and one of the two mutant loxP sites, and deletions from both ends generated in clones using newly constructed wild-type and mutant loxP transposons. Analysis of the results provides new insight into the very large co-integrates formed during P1 transduction of plasmids with loxP sites: a model with tri- and possibly multimeric co-integrates comprising the PAC plasmid, phage DNA, and transposon plasmid(s) as intermediates in the cell appears best to fit the data. The ability to truncate a large piece of DNA from both ends is likely to facilitate functionally mapping gene boundaries more efficiently, and make available precisely trimmed genes in their chromosomal contexts for therapeutic applications.     

An Alternative Approach in Gateway® Cloning when the Bacterial Antibiotic Selection Cassettes of the Entry Clone and Destination Vector are the Same

The Gateway^® recombination technology has revolutionized the method of gene cloning for functional analyses and high-throughput ORFeome projects. In general, Gateway cloning is highly efficient because after LR recombination and bacterial transformation, only cells containing the recombinant destination clone are selected on an antibiotic selection plate. However, when the antibiotic resistance gene for bacterial selection is the same in the entry and destination vectors, the direct selection of recombinant destination clones on an antibiotic plate is difficult. Here, we demonstrate an efficient and comprehensive approach to obtain positive destination clones directly on an antibiotic selection plate in this situation. The strategy involves polymerase chain reaction (PCR)-mediated amplification of the entry clone using entry vector-specific primers that bind outside the attL sequences and the subsequent use of this purified PCR product for LR recombination with the destination vector. Our results suggest that cloning of linear DNA fragments into circular destination vectors through LR recombination is an efficient method for inserts up to 7 kb in size. Using this approach, the yield of colony PCR positive destination clones was 100 % for genes of various sizes tested in our experiments.     

Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation

Lox sites of the Cre/lox recombination system from bacteriophage P1 were analyzed for their ability to affect on transgene expression when inserted upstream from a gene coding sequence adjacent to the right border (RB) of T-DNA. Wild and mutated types of lox sites were tested for their effect upon bar gene expression in plants obtained via Agrobacterium-mediated and biolistic transformation methods. Lox-mediated expression of bar gene, recognized by resistance of transgenic plants to PPT, occurred only in plants obtained via Agrobacterium-mediated transformation. RT-PCR analysis confirms that PPT-resistant phenotype of transgenic plants obtained via Agrobacterium-mediated transformation was caused by activation of bar gene. The plasmid with promoterless gus gene together with the lox site adjacent to the RB was constructed and transferred to Nicotiana tabacum as well. Transgenic plants exhibited GUS activity and expression of gus gene was detected in plant leaves. Expression of bar gene from the vectors containing lox site near RB allowed recovery of numerous PPT-resistant transformants of such important crops as Beta vulgaris, Brassica napus, Lactuca sativa and Solanum tuberosum. Our results demonstrate that the lox site sequence adjacent to the RB can be used to control bar gene expression in transgenic plants.     

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.     

Efficient mouse transgenesis using Gateway-compatible ROSA26 locus targeting vectors and F1 hybrid ES cells

The ability to rapidly and efficiently generate reliable Cre/loxP conditional transgenic mice would greatly complement global high-throughput gene targeting initiatives aimed at identifying gene function in the mouse. We report here the generation of Cre/loxP conditional ROSA26-targeted ES cells within 3–4 weeks by using Gateway® cloning to build the target vectors. The cDNA of the gene of interest can be expressed either directly by the ROSA26 promoter providing a moderate level of expression or by a CAGG promoter placed in the ROSA26 locus providing higher transgene expression. Utilization of F1 hybrid ES cells with exceptional developmental potential allows the production of germ line transmitting, fully or highly ES cell-derived mice by aggregation of cells with diploid embryos. The presented streamlined procedures accelerate the examination of phenotypical consequences of transgene expression. It also provides a unique tool for comparing the biological activity of polymorphic or splice variants of a gene, or products of different genes functioning in the same or parallel pathways in an overlapping manner.     

New vector system for induction of gene expression in dicotyledonous plants

A system of two vectors, pEnLox and pCre, was developed. The pEnLox vector is used to induce insertional mutations, while pCre is used to obtain transgenic plants expressing the Cre recombinase gene. The T-DNA enhancer is flanked by the loxP sites in the pEnLox vector. As an Arabidopsis thaliana insertional mutant obtained by transformation with pEnLox is crossed with another transgenic line carrying the cre gene, the enhancer sequence is eliminated. The vector system makes it possible to induce insertional mutations and to determine whether the mutant phenotype is caused by the loss-of-function insertional mutation or by overespression of nearby genes in response to the enhancer contained in the insert.     

Cloning Streptomyces genes for antibiotic production

The cloning and recombination of the genes of Streptomyces bacteria offer a method of increasing antibiotic yields and generating new antibiotics. Novel vectors, both plasmids and phages, have been developed for use with Streptomyces. This article describes some of these vectors and relevant cloning and screening techniques.