A set of loxP marker cassettes for Cre-mediated multiple gene disruption in Schizosaccharomyces pombe

For functional analysis, the presence of gene families and isoenzymes often makes it necessary to delete more than one gene, while the number of marker genes is limited in Schizosaccharomyces pombe. Here we describe a loxP-flanked ura4(+) cassette and Cre recombinase vector for a Cre-loxP-mediated marker removal procedure in S. pombe. This loxP-ura4-loxP cassette can be used for disruption of hmt1(+) as a model target gene. We have constructed two vectors which express Cre recombinase under the control of the nmt1 or nmt41 promoter. Excisive recombination at loxP sites in the chromosome was promoted efficiently and accurately when the Cre recombinase was expressed under the control of the nmt41 promoter. In addition, ura4(+) could be excised from the genome by Cre recombinase, when a single loxP site was adjacent to ura4. The use of the Cre-loxP system proved to be a practical strategy to excise a marker gene for repeated use in S. pombe.     

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.     

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.     

Altered directionality in the Cre-LoxP site-specific recombination pathway

The site-specific recombinase Cre must employ control mechanisms to impose directionality on recombination. When two recombination sites (locus of crossing over in phage P1, loxP) are placed as direct repeats on the same DNA molecule, collision between loxP-bound Cre dimers leads to excision of intervening DNA. If two sites are placed as inverted repeats, the intervening segment is flipped around. Cre catalyzes these reactions in the absence of protein co-factors. Current models suggest that directionality is controlled at two steps in the recombination pathway: the juxtaposition of loxP sites and the single-strand-transfer reactions within the synaptic complex. Here, we show that in Escherichia coli strain 294-Cre, directionality for recombination is altered when the expression of Cre is increased. This leads to deletion instead of inversion on substrates carrying two loxP sites as inverted repeats. The nucleotide sequence composition of loxP sites remaining in aberrant products indicates that site alignment and/or DNA strand transfer in the in vivo Cre-loxP recombination pathway are not always tightly controlled.     

A new efficient gene disruption cassette for repeated use in budding yeast.

The dominant kanr marker gene plays an important role in gene disruption experiments in budding yeast, as this marker can be used in a variety of yeast strains lacking the conventional yeast markers. We have developed a loxP-kanMX-loxP gene disruption cassette, which combines the advantages of the heterologous kanr marker with those from the Cre-lox P recombination system. This disruption cassette integrates with high efficiency via homologous integration at the correct genomic locus (routinely 70%). Upon expression of the Cre recombinase the kanMX module is excised by an efficient recombination between the loxP sites, leaving behind a single loxP site at the chromosomal locus. This system allows repeated use of the kanr marker gene and will be of great advantage for the functional analysis of gene families.     

Heterogeneous populations of ES cells in the generation of a floxed Presenilin-1 allele

Generation of a floxed Presenilin-1 (PS1) allele involved two recombination events in the embryonic stem (ES) cells. First, a targeting vector containing a loxP site in intron 1 and a floxed CMV-HYG/TK double selection cassette in intron 3 was integrated into the PS1 locus by homologous recombination. The use of a negative selection cassette, PGK-DTA, dramatically increased the recombination efficiency within the targeted locus (75-fold). Second, an expression vector encoding Cre recombinase was introduced to excise the floxed CMV-HYG/TK cassette via site-specific recombination. However, all five ES cell clones testing positive for the proper removal of the CMV-HYG/TK cassette also contained a proportion of ES cells in which recombination had occurred between the distal loxP sites in introns 1 and 3, resulting in excision of the entire floxed region. It is therefore critical to screen for possible recombination events involving all 3 loxP sites, in order to identify ES cells clones bearing high proportions of the desired ES cells. genesis 26:5-8, 2000.     

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.     

转基因植物高效删除标记基因的实用型双元转化载体

利用雌激素诱导的Cre/loxPDNA重组系统,构建用于转基因植物删除标记基因的实用新型载体,将所有非目的基因放于两同向loxP位点间,多克隆位点位于两loxP位点外,可插入目的基因,载体设计非常实用。在插入目的基因GUS后转化烟草,雌激素诱导,分析诱导前后DNA的重组情况、不同时间基因转录水平,以及目的基因在诱导前后的表达。结果显示,诱导后染色体DNA实现了高效重组,非目的基因片段消失,目的基因GUS在诱导后表达正常。     

Development of high-titer retroviral producer cell lines by using Cre-mediated recombination.

Retroviral gene transfer is widely used in experimental and human gene therapy applications. We have devised a novel method of generating high-titer retroviral producer cell lines based on the P1 bacteriophage recombinase system Cre-loxP. Incorporation of loxP sites flanking a Neo(r)-SVTK cassette in the proviral DNA allows excision of these selectable markers through expression of Cre recombinase after production of a high-titer producer cell line. The resultant producer line contains a single loxP site flanked by the viral long terminal repeats. Retransfection of this line with the Cre expression vector and a plasmid containing a gene of interest flanked by loxP sites allows insertional recombination of the gene into the favorable preexisting site in the genome and the generation of a new line with a titer equivalent to that of the parental producer cell line. The efficiency of the process is sufficient to allow the generation of multiple new producer lines without the addition of antibiotic resistance genes. We have successfully generated retroviral vectors carrying different genes by using this approach and discuss the potential applications of this method in gene therapy.     

Rapid induction of large chromosomal deletions by a Cre/inverted loxP system in mouse ES Cell hybrids

Loss of heterozygosity by whole or partial loss of chromosomal regions is crucial to genetic disorders, cancers and diseases. It is difficult to analyze the mechanisms of pathogenesis caused by large-scale chromosomal abnormalities due to the extreme rarity of this mutagenesis. Using a Cre/inverted loxP system, we have generated a chromosome elimination cassette (CEC) that induces a selective loss of embryonic-stem-cell-derived chromosomes in undifferentiated embryonic stem cell-somatic cell hybrids. Here, due to the increased expression of Cre, rapid formation of Cre recombination products and immediate loss of CEC-tagged chromosomes were detected by fluorescence in situ hybridization. Cre also initiated intrachromosomal recombination between identical short sequences outside loxP, leading to large chromosomal deletions of CEC-tagged regions. The Cre-mediated antiparallel synapses likely act as a scaffold to bring the identical short sequences into close proximity for recombination. This CEC technology might allow better understanding of the modulator sequences responsible for the tangled structure formation and its solution mechanism, inducing mitotic recombination leading to chromosomal deletions.     

The role of the loxP spacer region in P1 site-specific recombination.

The lox-Cre site-specific recombination system of bacteriophage P1 is comprised of a site on the DNA where recombination occurs called loxP, and a protein, Cre, which mediates the reaction. The loxP site is 34 base pairs (bp) in length and consists of two 13 bp inverted repeats separated by an 8 bp spacer region. Previously it has been shown that the cleavage and strand exchange of recombining loxP sites occurs within this spacer region. We report here an analysis of various base substitution mutations within the spacer region of loxP, and conclude the following: Homology is a requirement for efficient recombination between recombining loxP sites. There is at least one position within the spacer where a base change drastically reduces recombination even when there is homology between the two recombining loxP sites. When two loxP sites containing symmetric spacer regions undergo Cre-mediated recombination in vitro, the DNA between the sites undergoes both excision and inversion with equal frequency.     

Spontaneous ectopic recombination in cell-type-specific Cre mice removes loxP-flanked marker cassettes in vivo

Conditional gene targeting using the Cre/loxP technology generally includes integration of a selection marker cassette flanked by loxP recognition sites (floxed) in the target gene locus. Subsequent marker removal avoids possible impairment of gene expression or mosaicism due to partial and total deletions after Cre-mediated recombination in vivo. The use of deleter Cre mice for in vivo marker removal in floxed connexin43 mice revealed considerable mosaicism, but no selective marker removal. In addition, we noted that several Cre transgenic lines displayed spontaneous ectopic activity, reminiscent of deleter Cre mice, and required the confirmation of cell type-specific deletion in every individual mouse. When we used myosin heavy chain promoter Cre (alphaMyHC-Cre) mice for cardiomyocyte specific deletion, we observed, in addition to cardiomyocyte-restricted or complete excision, selective marker removal in a subgroup of mice as well. Thus, selective marker removal can be achieved as a byproduct of cell-type restricted deletion.     

PCR- and ligation-mediated synthesis of split-marker cassettes with long flanking homology regions for gene disruption in Candida albicans

Because Candida albicans is a diploid organism, two consecutive steps of gene disruption are required to generate a gene knock-out. The same marker (URA3) is often used for disruption of both copies of the gene. This is possible because, after the first round of disruption, homologous recombination between direct repeats flanking the URA3 marker and the subsequent counterselection allow for the efficient recovery of Ura- revertants. Unfortunately, the URA-blaster disruption cassette cannot be used in a PCR-based disruption approach. The hisG repeats flanking the URA3 gene in the disruption cassette anneal to one another during PCR and thereby prevent amplification of the complete cassette. We explored the use of transformation based on split-marker recombination to circumvent this problem. To avoid any cloning steps and to retain the advantage of long flanking regions for disruption, we combined this with a PCR- and ligation-mediated approach for generating marker cassettes. We used this approach to disrupt the C. albicans FAL1 (ATP-dependent RNA helicase) gene. Long 5' and 3' FAL1-specific regions were amplified by PCR and individually ligated to a URA-blaster cassette. The resulting ligation reactions were used separately as templates to generate two FAL1 disruption cassettes with overlapping URA3 marker regions. Simultaneous transformation with both overlapping disruption cassettes yielded efficient disruption of one FAL1 allele.     

Bacteriophage P1 Cre-loxP site-specific recombination. Site-specific DNA topoisomerase activity of the Cre recombination protein

Site-specific recombination in bacteriophage P1 occurs between two loxP sites in the presence of the Cre recombination protein. The structure of the 34-base pair loxP site consists of two 13-base pair inverted repeats separated by an 8-base pair spacer region. A mutation in the loxP site has been constructed which deletes one of the internal bases of the spacer region at the axis of dyad symmetry. This mutant loxP site shows a 10-fold reduction in recombination activity with a wild-type site both in vivo and in vitro. This low level of intramolecular recombination between a wild-type loxP site and the mutant loxP501 site is observed in vitro only when the DNA substrate is supercoiled. The majority of the supercoiled substrate is relaxed by the Cre protein, and on longer incubations, single-stranded nicks accumulate in the DNA. We have determined that these nicks occur in both the wild-type and the mutant sites. The positions of these nicks correspond to the positions of cleavage found during recombination of two wild-type sites, suggesting that the Cre protein is attempting to carry out recombination with the mutant site but most of the time this reaction is abortive. We have determined that the Cre protein relaxes a supercoiled topoisomer of a DNA substrate containing one wild-type site and one mutant site to yield a distribution of topoisomers whose linking numbers differ by steps of one, indicating that Cre can act as a type I topoisomerase.     

Simultaneous on/off regulation of transgenes located on a mammalian chromosome with Cre-expressing adenovirus and a mutant loxP

The site-specific recombinase Cre has often been used for on/off regulation of expression of transgenes introduced into the mammalian chromosome. However, this method is only applicable to the regulation of a single gene and cannot be used to simultaneously regulate two genes, because site-specific recombination occurs from the target loxP sequence of one regulating unit to the loxP sequence of any other unit and would eventually disrupt the structure of both regulating units. We previously reported a mutant loxP sequence with a two base substitution called loxP V (previously called loxP 2272), with which wild-type loxP cannot recombine but with which the identical mutant loxP recombines efficiently. In the present study we isolated cell lines bearing two regulating units on a chromosome containing a pair of wild-type loxP sequences or mutant loxP V sequences. After infection with Cre-expressing recombinant adenovirus AxCANCre, expression of a gene in each regulating unit was simultaneously turned on and off. Southern analyses showed that both regulating units were processed simultaneously and independently, even after infection with a limited amount of AxCANCre. The results showed that simultaneous regulation of gene expression on a mammalian chromosome mediated by Cre can be achieved by using mutant loxP V and wild-type loxP. This method may be a useful approach for conditional transgenic/knockout animals and investigation of gene function involving two genes simultaneously. Another possible application is for preparation of a new packaging cell line of viral vectors through simultaneous production of toxic viral genes.     

IntI2 integron integrase in Tn7

Integrons can insert and excise antibiotic resistance genes on plasmids in bacteria by site-specific recombination. Class 1 integrons code for an integrase, IntI1 (337 amino acids in length), and are generally borne on elements derived from Tn5090, such as that found in the central part of Tn21. A second class of integron is found on transposon Tn7 and its relatives. We have completed the sequence of the Tn7 integrase gene, intI2, which contains an internal stop codon. This codon was found to be conserved among intI2 genes on three other Tn7-like transposons harboring different cassettes. The predicted peptide sequence (IntI2*) is 325 amino acids long and is 46% identical to IntI1. In order to detect recombination activity, the internal stop codon at position 179 in the parental allele was changed to a triplet coding for glutamic acid. The sequences flanking the cassette arrays in the class 1 and 2 integrons are not closely related, but a common pool of mobile cassettes is used by the different integron classes; two of the three antibiotic resistance cassettes on Tn7 and its close relatives are also found in various class 1 integrons. We also observed a fourth excisable cassette downstream of those described previously in Tn7. The fourth cassette encodes a 165-amino-acid protein of unknown function with 6.5 contiguous repeats of a sequence coding for 7 amino acids. IntI2*179E promoted site-specific excision of each of the cassettes in Tn7 at different frequencies. The integrases from Tn21 and Tn7 showed limited cross-specificity in that IntI1 could excise all cassettes from both Tn21 and Tn7. However, we did not observe a corresponding excision of the aadA1 cassette from Tn21 by IntI2*179E.     

Insertion of a foreign gene into the β-casein locus by Cre-mediated site-specific recombination

The expression of foreign genes in transgenic animals is generally unpredictable as transgenes are integrated at random after pro-nuclear injection into fertilized oocytes. In many cases, transgene expression is inhibited by neighbouring chromatin structures or by the repeated nature of the multiple transgene copies present at the integration site. A strategy involving homologous and site-specific recombination has been devised by which single copies of a foreign gene can be inserted specifically into the locus of a highly expressed gene. As a first step, a loxP recombination target site is introduced by homologous recombination into a predetermined gene locus such that the loxP sequence is placed next to the promoter region and replaces the translational initiation signal. In a subsequent site-specific recombination reaction, a gene of interest can be integrated into the pre-existing loxP site. This biphasic recombination strategy was used to integrate a luciferase reporter gene into the locus of the murine β-casein gene in embryonic stem cells.