Negative selection with the Diphtheria toxin A fragment gene improves frequency of Cre-mediated cassette exchange in ES cells

The Cre-lox system is an important tool for genetic manipulation in embryonic stem cells. We previously reported that the cassette exchange strategy using the mutant lox66/71 and lox2272 combination showed high recombination efficiency and stability. However, the efficiency was strongly affected by the position of chromosomal target lox sites. To enrich successful cassette exchange events, even in clones showing lower recombination efficiency, we have improved exchange vector. The Diphtheria toxin A fragment gene was placed in the un-exchanged region for negative selection and the puromycin N-acetyltransferase gene, instead of the neomycin phosphotransferase gene, was used for positive selection. By reducing random integration, the frequency of successful cassette exchange increased up to 2-4 fold. Furthermore, by adding the third lox site to induce intrarmolecular recombination, the recombination efficiency of cassette exchange itself was improved, and the frequency increased to maximum 5 fold, in which the percentage of exchanged clones reached to 50-70%. This strategy should be useful for other recombinase-mediated cassette exchanges.     

Selection-marker-free modification of the murine beta-casein gene using a lox2272 [correction of lox2722] site

Gene targeting and site-specific recombination strategies allow the precise modification of the eukaryotic genome. Many of the recombination strategies currently used, however, will introduce a selection marker gene at the modified site. DNA sequences of prokaryotic origin like vector sequences, selection marker, and reporter genes have been shown to markedly influence the regulation of the modified genomic loci. In order to avoid the insertion of excess sequences, a biphasic recombination strategy involving homologous recombination and Cre-recombinase-mediated cassette exchange (RMCE) was devised and used to insert a foreign gene into the beta-casein gene in murine embryonic stem cells. The incompatibility of the heterospecific lox sites used for the recombinase-mediated cassette exchange was found to be critical for the success of the strategy. The frequently used mutant site lox511, which differs from the natural loxP site by a single point mutation, proved unsuitable for this approach. A mutant lox site carrying two point mutations, however, was highly effective and 90% of the selected cell clones carried the desired modification. This biphasic recombination strategy allows for the efficient and precise modification of gene loci without the concomitant introduction of a selectable marker gene.     

Recombinase-mediated cassette exchange (RMCE) and BAC engineering via VCre/VloxP and SCre/SloxP systems

Site-specific recombination is a powerful biotechnological tool for genome engineering. We previously reported two novel site-specific recombination systems, VCre/VloxP and SCre/SloxP, that do not cross-react with Cre/loxP and Flp/FRT in culture cells and mouse embryonic stem (ES) cells. In this study, a site-specific recombination assay in Escherichia coli was used to examine the activity of mutant VCre (H314L and Y349F) and mutant SCre (H317L and Y352F), in which both mutated residues lie within the active center of Cre recombination. The site-specific recombination activity of both mutants was significantly decreased. Recombinase-mediated cassette exchange (RMCE) using VloxP and the Vlox2272 mutant site was performed in E. coli by introducing a cassette bearing VloxP and Vlox2272 into a recipient plasmid bearing the same sites. RMCE using SloxP and Slox2272 was also performed by SCre recombinase. Moreover, BAC engineering via Red recombination and VCre/VloxP were demonstrated. First, the DNA cassette for modification was introduced into a BAC clone via Red recombination; second, the antibiotics resistance gene flanked by VloxP was removed from the BAC clone by induction of VCre recombinase. Such site-specific recombination systems may effectively be used in combination with other site-specific recombination systems or engineering tools (e.g., Red recombination).     

Gene tagging and gene replacement using recombinase-mediated cassette exchange in Schizosaccharomyces pombe

Cre/lox site-specific recombination systems provide important tools for genetic manipulation. Here we present an efficient method for gene tagging and gene replacement using Cre recombinase-mediated cassette exchange (RMCE). The cassette consists of the S. pombe ura4(+) selectable marker flanked by a wild-type loxP site at one end and by a modified heterospecific lox site (loxM3) at the other. The cassette is stable because the flanking lox sites cannot recombine with each other. Following integration of the cassette at the chosen chromosomal locus, exchange is achieved by introducing a Cre-expression plasmid containing an equivalent cassette containing the required tag or gene sequence. Recombinants are selected by uracil prototrophy using the reagent 5-fluoroorotic acid (5-FOA). The cassette exchange system provides for repetitive integrations at the same locus, allowing different protein tags or gene sequences to be integrated quickly and efficiently. We have established a range of reagents and verified utility by C-terminally tagging the S. pombe rad4 and swi1 genes with yEGFP and the yEGFP derivatives yECFP and yECitrine and by transferring the coding sequence for both genes.     

Selection-Marker-Free Modification of the Murine β-Casein Gene Using a lox2722 Site

Gene targeting and site-specific recombination strategies allow the precise modification of the eukaryotic genome. Many of the recombination strategies currently used, however, will introduce a selection marker gene at the modified site. DNA sequences of prokaryotic origin like vector sequences, selection marker, and reporter genes have been shown to markedly influence the regulation of the modified genomic loci. In order to avoid the insertion of excess sequences, a biphasic recombination strategy involving homologous recombination and Cre-recombinase-mediated cassette exchange (RMCE) was devised and used to insert a foreign gene into the β-casein gene in murine embryonic stem cells. The incompatibility of the heterospecific lox sites used for the recombinase-mediated cassette exchange was found to be critical for the success of the strategy. The frequently used mutant site lox511, which differs from the natural loxP site by a single point mutation, proved unsuitable for this approach. A mutant lox site carrying two point mutations, however, was highly effective and 90% of the selected cell clones carried the desired modification. This biphasic recombination strategy allows for the efficient and precise modification of gene loci without the concomitant introduction of a selectable marker gene.     

Site-specific cassette exchange and germline transmission with mouse ES cells expressing phiC31 integrase

Currently two site-specific recombinases are available for engineering the mouse genome: Cre from P1 phage and Flp from yeast. Both enzymes catalyze recombination between two 34-base pair recognition sites, lox and FRT, respectively, resulting in excision, inversion, or translocation of DNA sequences depending upon the location and the orientation of the recognition sites. Furthermore, strategies have been designed to achieve site-specific insertion or cassette exchange. The problem with both recombinase systems is that when they insert a circular DNA into the genome (trans event), two cis-positioned recognition sites are created, which are immediate substrates for excision. To stabilize the trans event, functional mutant recognition sites had to be identified. None of the systems, however, allowed efficient selection-free identification of insertion or cassette exchange. Recently, an integrase from Streptomyces phage phiC31 has been shown to function in Schizosaccharomyces pombe and mammalian cells. This enzyme recombines between two heterotypic sites: attB and attP. The product sites of the recombination event (attL and attR) are not substrates for the integrase. Therefore, the phiC31 integrase is ideal to facilitate site-specific insertions into the mammalian genome.     

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.     

Role of nucleotide sequences of loxP spacer region in Cre-mediated recombination

The Cre recombinase mediates precise site-specific recombination between a pair of loxP sequences through an intermediate containing Holiday junction. The recombination junction in the loxP sequence is located within the asymmetric 8-nucleotide spacer region. To examine the role of each nucleotide sequence of the spacer region in the recombination process, we synthesized a complete set of 24 loxP spacer mutants with single-base substitutions and 30 loxP spacer mutants with double-base substitutions. Each synthesized loxP mutant was ligated at both ends of a linear DNA or to one end of a DNA-containing wild-type loxP at the other end and their recombination efficiencies were analyzed with an in vitro system. The sequence identity of the right two nucleotides and left four nucleotides in the central six bases of the spacer region was found to be essential for formation and resolution, respectively, of the intermediate product. Furthermore, even when homology was maintained, the recombination efficiencies were lower than that of wild-type loxP and varied among mutants. Based on this knowledge, we identified two loxP mutants with double-base substitutions, mutants 5171 and 2272, which recombine efficiently with an identical mutant but not with the other mutant or wild-type loxP.     

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.     

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.     

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.     

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.     

A cell-free recombination system for site-specific integration of multigenic shuttle plasmids into the herpes simplex virus type 1 genome.

This report describes a novel method for complementation studies of defective herpes simplex virus (HSV) genes. Viral test gene and nonviral reporter gene cassettes were rapidly integrated into the HSV genome in a site-specific and reversible manner by using the P1 phage-based Cre-lox recombination system. Shuttle plasmids contained a functional loxP recombination site, an expressible form of the bacterial lacZ gene, and a copy of the wild-type glycoprotein B (gB) gene or double mutant gB allele containing both a temperature-sensitive (ts) mutation and a syncytium (syn)-forming mutation. A recipient viral genome, K delta T::lox1, was constructed from the HSV type 1 (syn) gB-deficient mutant virus, K delta T, by marker transfer of the loxP recombination site into the viral thymidine kinase locus. Shuttle plasmids of up to 12.9 kb in length were recombined with high efficiency (11 to 20%) into the K delta T::lox1 genome in cell-free, Cre-mediated recombination reactions. Expression of a functional wild-type or double mutant gB polypeptide complemented the nonfunctional polypeptide expressed from the deleted, normal gB locus and allowed production of either wild-type or Syn- plaques on Vero cells. The latter recombinant virus was also ts for growth. The ability to express viral genes from plasmids which can be shuttled into and out of the HSV genome in cell-free recombination reactions makes this a powerful method for performing genetic studies of the biologic properties of viral gene products.     

Site-directed integration of the cre gene mediated by Cre recombinase using a combination of mutant lox sites

The Cre-lox system is an important tool for genetic manipulation. To promote integrative reactions, two strategies using mutant lox sites have been developed. One is the left element/right element (LE/RE)-mutant strategy and the other is the cassette exchange strategy using heterospecific lox sites such as lox511 or lox2272. We compared the recombination efficiencies using these mutant lox sites in embryonic stem (ES) cells, and found that the combination of the LE/RE mutant and lox2272 showed high recombination efficiency and stability of the recombined structure. Taking advantage of this stability, we successfully integrated the cre gene into the mutant lox sites by Cre-mediated recombination. Germ line chimeric mice were produced from the cre-integrated ES cell clones, and Cre-expressing mouse lines were established. The inserted cre gene was stably maintained through the generations. This cre knock-in system using the LE/RE-lox2272 combination should be useful for the production of various cre mice for gene targeting or gene trapping.     

Directionality in FLP protein-promoted site-specific recombination is mediated by DNA-DNA pairing

The 2 mu plasmid of the yeast Saccharomyces cerevisiae encodes a site-specific recombination system consisting of plasmid-encoded FLP protein and two recombination sites on the plasmid. The recombination site possesses a specific orientation, which is determined by an asymmetric 8-base pair spacer sequence separating two 13-base pair inverted repeats. The outcome or directionality of site-specific recombination is defined by the alignment of two sites in the same orientation during the reaction. Sites containing point mutations or 1-base pair insertions or deletions within the spacer generally undergo recombination with unaltered sites at reduced levels. In contrast, recombination between the two identical mutant sites (where homology is restored) proceeds efficiently in all cases. Sites containing spacer sequences of 10 base pairs or more are nonfunctional under all conditions. A recombination site in which 5 base pairs are changed to yield an entirely symmetrical spacer sequence again recombines efficiently, but only with an identical site. This reaction, in addition, produces a variety of new products which can only result from random alignment of the two sites undergoing recombination, i.e. the reaction no longer exhibits directionality. These and other results demonstrate that both the efficiency and directionality of site-specific recombination is dependent upon homology between spacer sequences of the two recombining sites. This further implies that critical DNA-DNA interactions between the spacer region of the two sites involved in the reaction occur at some stage during site-specific recombination in this system. The specific spacer sequence itself appears to be unimportant as long as homology is maintained; thus, these sequences are probably not involved in recognition by FLP protein.     

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.     

Analysis of spacer regions derived from intramolecular recombination between heterologous loxP sites

In Cre-loxP recombination system, Cre recombinase binds cooperatively to two 13bp inverted repeats in a 34bp loxP and catalyzes strand exchange in the 8bp spacer region. Up to date, spacer sequences within the recombined loxP sites derived from two loxP sties that have different 8bp spacer regions have never been analyzed. In the present study, we analyzed the spacer sequences within the recombined products, resulted from intramolecular recombination between heterologous loxP sites including M2, M3, M7, M11, and 2272 in vivo and in vitro. From the analyses, it was found that loxP sites with aberrant 8bp spacers can be generated from Cre-mediated recombination between heterologous loxP sites at significantly high frequency, proposing the possibility that recombination between heterologous loxP sites would have not undergone typical formula of Cre-loxP recombination.     

Characterization of Cre-mediated cassette exchange after plasmid microinjection in fertilized mouse oocytes

DNA cassette exchange methodologies are powerful tools for rapid modification of defined genomic loci. Until now, most studies have focused on transfected cell lines that were subjected to positive and negative selection to enrich for correctly recombined clones. This is the first report on Cre-mediated cassette exchange (CMCE) in early mouse embryos, a nonselectable biological system. A Cre expression plasmid and a replacement plasmid were introduced into fertilized oocytes by plasmid microinjection and recombination products were investigated. Both, replacement plasmid and oocyte genome carried heterospecific lox sites (lox511 and loxP) to direct CMCE toward integration. Here, we demonstrate the general feasibility of CMCE in early mouse embryos and characterize the system with respect to integration efficiency, competing reactions and parameters affecting recombination rates.     

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.