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.     

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.     

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.     

Self-excising retroviral vectors encoding the Cre recombinase overcome Cre-mediated cellular toxicity

The Cre-lox system is often used to manipulate sequences in mammalian genomes. We have observed that continuous expression of the Cre recombinase in cultured cells lacking exogenous lox sites caused decreased growth, cytopathic effects, and chromosomal aberrations. Cre mutants defective in DNA cleavage were not geno- or cytotoxic. A self-excising retroviral vector that incorporates a negative feedback loop to limit the duration and intensity of Cre expression avoided measurable toxicity, retained the ability to excise a target sequence flanked by lox sites, and may provide the basis of a less toxic strategy for the use of Cre or similar recombinases.     

Ectopic gene targeting exhibits a bimodal distribution of integration in murine cells, indicating that both intra- and interchromosomal sites are accessible to the targeting vector.

Ectopic gene targeting is an alternative outcome of the gene targeting process in which the targeting vector acquires sequences from the genomic target but proceeds to integrate elsewhere in the genome. Using two-color fluorescent in situ hybridization analysis, we have determined the integration sites of the gene targeting vector with respect to the target locus in a murine fibroblast line (LTA). We found that for ectopic gene targeting the distribution of integration sites was bimodal, being either within 3 Mb of the target or on chromosomes distinct from the chromosome carrying the target locus. Inter- and intrachromosomal sites appeared to be equally accessible to the targeting vector, with site-specific variations. Interestingly, interphase analysis indicated that vector sequences which had integrated ectopically in chromosomes other than the target colocalized with the target locus at a significant frequency compared to that of colocalization to random unlinked loci. We propose that ectopic gene targeting could be used to determine which chromosomal domains within the genome are accessible to a given genetic locus. Thus, recombination access mapping may present a new paradigm for the analysis of DNA accessibility and interaction within the genome.     

Cre/lox-mediated site-specific integration of Agrobacterium T-DNA in Arabidopsis thaliana by transient expression of cre

The Cre/lox system was used to obtain targeted integration of an Agrobacterium T-DNA at a lox site in the genome of Arabidopsis thaliana. Site-specific recombinants, and not random events, were preferentially selected by activation of a silent lox-neomycin phosphotransferase (nptII) target gene. To analyse the effectiveness of Agrobacterium-mediated transfer we used T-DNA vectors harbouring a single lox sequence (this vector had to circularize at the T-DNA left- and right-border sequences prior to site-specific integration) or two lox sequences (this vector allowed circularization at the lox sequences within the T-DNA either prior to or after random integration, followed by targeting of the circularized vector), respectively. Furthermore, to control the reversibility of the integration reaction, Cre recombinase was provided transiently by using a cotransformation approach. One precise stable integrant was found amongst the recombinant calli obtained after transformation with a double-lox T-DNA vector. The results indicate that Agrobacterium-mediated transformation can be used as a tool to obtain site-specific integration.     

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.     

Site-Directed Gene Integration in Transgenic Zebrafish Mediated by Cre Recombinase Using a Combination of Mutant <Emphasis Type="Italic">Lox</Emphasis> Sites

With current gene-transfer techniques in fish, insertion of DNA into the genome occurs randomly and in many instances at multiple sites. Associated position effects, copy number differences, and multiple gene interactions make gene expression experiments difficult to interpret and fish phenotype less predictable. To meet different fish engineering needs, we describe here a gene targeting model in zebrafish. At first, four target zebrafish lines, each harboring a single genomic lox71 target site, were generated by zebrafish transgenesis. The zygotes of transgenic zebrafish lines were coinjected with capped Cre mRNA and a knockin vector pZklox66RFP. Site-specific integration event happened from one target zebrafish line. In this line two integrant zebrafish were obtained from more than 80,000 targeted embryos (integrating efficiency about 10^-4 to 10^-5) and confirmed to have a sole copy of the integrating DNA at the target genome site. Genomic polymerase chain reaction analysis and DNA sequencing verified the correct gene target events where lox71 and lox66 have accurately recombined into double mutant lox72 and wild-type loxP. Each integrant zebrafish chosen for analysis harbored the transgene rfp at the designated egfp concatenates. Although the Cre-mediated recombination is site specific, it is dependent on a randomly placed target site. That is, a genomic target cannot be preselected for integration based solely on its sequence. Conclusively, an rfp reporter gene was successfully inserted into the egfp target locus of zebrafish genome by Cre-lox-mediated recombination. This site-directed knockin system using the lox71/lox66 combination should be a promising gene-targeting platform serving various purposes in fish genetic engineering. Wei-yi Liu and Yun Wang contributed equally to this article     

Turbo cloning: a fast, efficient method for cloning PCR products and other blunt-ended DNA fragments into plasmids.

The method uses a novel plasmid vector, p9lox5, containing a site-specific recombination sequence lox from the lox/Cre recombinase system of bacteriophage P1. There are two distinct stages. Firstly, vector and fragment DNAs are ligated intermolecularly under conditions of macromolecular crowding (15% polyethylene glycol 6000) which accelerate blunt-end joining a thousandfold. Secondly, circular recombinant molecules are efficiently excised from the ligation products by Cre recombinase acting on pairs of lox sites within directly repeated vector molecules flanking insert DNA. Recombinants are introduced into cells conventionally by transformation or electroporation. In both a model system and the cloning of PCR products, yields approaching those obtainable in cohesive-end cloning were achieved. Applications of the technique to cDNA library generation and recovery of DNA from archive material are discussed.     

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

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

选择标记可去除的植物高效表达载体的构建

在常用的植物组成型表达载体pBI121的选择标记基因NPTII两侧插入同向的lox位点并用多克隆位点(MCS)取代了GUS基因序列,构建了NPTII基因可被去除的和可插入目的基因的通用植物表达载体pBI121-lox-MCS。替换pBI121-lox-MCS中驱动目的基因表达的35S启动子,可构建成一系列具有其他表达特性的植物表达载体,如本文描述的韧皮部特异表达载体pBdENP-lox-MCS。为方便地筛选去除选择标记基因的转基因植物,还构建了绿色荧光蛋白(GFP)表达框与NPTII表达框连锁的pBI121-gfp-lox-MCS载体。上述植物表达载体可广泛应用于培育选择标记可去除的转基因植物。     

Target frequency and integration pattern for insertion and replacement vectors in embryonic stem cells.

Gene targeting has been used to direct mutations into specific chromosomal loci in murine embryonic stem (ES) cells. The altered locus can be studied in vivo with chimeras and, if the mutated cells contribute to the germ line, in their offspring. Although homologous recombination is the basis for the widely used gene targeting techniques, to date, the mechanism of homologous recombination between a vector and the chromosomal target in mammalian cells is essentially unknown. Here we look at the nature of gene targeting in ES cells by comparing an insertion vector with replacement vectors that target hprt. We found that the insertion vector targeted up to ninefold more frequently than a replacement vector with the same length of homologous sequence. We also observed that the majority of clones targeted with replacement vectors did not recombine as predicted. Analysis of the recombinant structures showed that the external heterologous sequences were often incorporated into the target locus. This observation can be explained by either single reciprocal recombination (vector insertion) of a recircularized vector or double reciprocal recombination/gene conversion (gene replacement) of a vector concatemer. Thus, single reciprocal recombination of an insertion vector occurs 92-fold more frequently than double reciprocal recombination of a replacement vector with crossover junctions on both the long and short arms.     

Stable recombinase-mediated cassette exchange in Arabidopsis using Agrobacterium tumefaciens

Site-specific integration is an attractive method for the improvement of current transformation technologies aimed at the production of stable transgenic plants. Here, we present a Cre-based targeting strategy in Arabidopsis (Arabidopsis thaliana) using recombinase-mediated cassette exchange (RMCE) of transferred DNA (T-DNA) delivered by Agrobacterium tumefaciens. The rationale for effective RMCE is the precise exchange of a genomic and a replacement cassette both flanked by two heterospecific lox sites that are incompatible with each other to prevent unwanted cassette deletion. We designed a strategy in which the coding region of a loxP/lox5171-flanked bialaphos resistance (bar) gene is exchanged for a loxP/lox5171-flanked T-DNA replacement cassette containing the neomycin phosphotransferase (nptII) coding region via loxP/loxP and lox5171/lox5171 directed recombination. The bar gene is driven by the strong 35S promoter, which is located outside the target cassette. This placement ensures preferential selection of RMCE events and not random integration events by expression of nptII from this same promoter. Using root transformation, during which Cre was provided on a cotransformed T-DNA, 50 kanamycin-resistant calli were selected. Forty-four percent contained a correctly exchanged cassette based on PCR analysis, indicating the stringency of the selection system. This was confirmed for the offspring of five analyzed events by Southern-blot analysis. In four of the five analyzed RMCE events, there were no additional T-DNA insertions or they easily segregated, resulting in high-efficiency single-copy RMCE events. Our approach enables simple and efficient selection of targeting events using the advantages of Agrobacterium-mediated transformation.     

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.     

Identification of cryptic lox sites in the yeast genome by selection for Cre-mediated chromosome translocations that confer multiple drug resistance.

The Cre recombinase efficiently causes site-specific DNA recombination at loxP sites placed into the eukaryotic genome. Since the loxP site of phage P1 is 34 base-pairs in size, the natural occurrence of this exact sequence is unlikely in any eukaryotic genome. However, related sequences may exist in eukaryotic genomes that could recombine at low efficiency with an authentic loxP site. This work identifies such cryptic lox sites in the yeast genome using a positive selection procedure that allows the detection of events occurring at a frequency of less than 1 x 10(-7). The selection is based on the disruption/reconstruction of the yeast gene YGL022. Disruption of YGL022 confers multiple drug sensitivity. Recombination events at a loxP site 5' to the structural gene restore expression of YGL022 and result in a multiple drug resistant phenotype. These drug resistant mutants all display chromosomal rearrangements resulting from low-frequency Cre-mediated recombination with an endogenous cryptic lox site. Ten such sites have been found and they have been mapped physically to a number of different yeast chromosomes. Although the efficiency of Cre-mediated recombination between loxP and such endogenous sites is quite low, it may be possible to redesign recombination substrates to improve recombination efficiency. Because of the greater complexity of the human and mouse genomes compared with yeast, an analogous situation is likely to exist in these organisms. The availability of such sites would be quite useful in the development of alternative strategies for gene therapy and in the generation of transgenic animals.     

A genetic system for direct selection of gene-positive clones during recombinational cloning in yeast

Transformation-associated recombination (TAR) is a cloning technique that allows specific chromosomal regions or genes to be isolated directly from genomic DNA without prior construction of a genomic library. This technique involves homologous recombination during spheroplast transformation between genomic DNA and a TAR vector that has 5′ and 3′ gene targeting sequences (hooks). Typically, TAR cloning produces positive YAC recombinants at a frequency of ~0.5%; the positive clones are identified by PCR or colony hybridization. This paper describes a novel TAR cloning procedure that selects positive clones by positive and negative genetic selection. This system utilizes a TAR vector with two targeting hooks, HIS3 as a positive selectable marker, URA3 as a negative selectable marker and a gene-specific sequence called a loop sequence. The loop sequence lies distal to a targeting hook sequence in the chromosomal target, but proximal to the targeting hook and URA3 in the TAR vector. When this vector recombines with chromosomal DNA at the gene-specific targeting hook, the recombinant YAC product carries two copies of the loop sequence, therefore, the URA3 negative selectable marker becomes mitotically unstable and is lost at high frequency by direct repeat recombination involving the loop sequence. Positive clones are identified by selecting against URA3. This method produces positive YAC recombinants at a frequency of ~40%. This novel TAR cloning method provides a powerful tool for structural and functional analysis of complex genomes.     

Parameters controlling the rate of gene targeting frequency in the protozoan parasite Leishmania.

In this study we investigated the role of several parameters governing the efficiency of gene targeting mediated by homologous recombination in the protozoan parasite Leishmania. We evaluated the relative targeting frequencies of different replacement vectors designed to target several sequences within the parasite genome. We found that a decrease in the length of homologous sequences <1 kb on one arm of the vector linearly influences the targeting frequency. No homologous recombination was detected, however, when the flanking homologous regions were <180 bp. A requirement for a very high degree of homology between donor and target sequences was found necessary for efficient gene targeting in Leishmania , as targeted recombination was strongly affected by base pair mismatches. Targeting frequency increased proportionally with copy number of the target only when the target was part of a linear amplicon, but remained unchanged when it was present on circles. Different chromosomal locations were found to be targeted with significantly variable levels of efficiency. Finally, different strains of the same species showed differences in gene targeting frequency. Overall, gene targeting mediated by homologous recombination in Leishmania shares similarities to both the yeast and the mammalian recombination systems.     

The molecular basis of multiple vector insertion by gene targeting in mammalian cells

Gene targeting using sequence insertion vectors generally results in integration of one copy of the targeting vector generating a tandem duplication of the cognate chromosomal region of homology. However, occasionally the target locus is found to contain >1 copy of the integrated vector. The mechanism by which the latter recombinants arise is not known. In the present study, we investigated the molecular basis by which multiple vectors become integrated at the chromosomal immunoglobulin mu locus in a murine hybridoma. To accomplish this, specially designed insertion vectors were constructed that included six diagnostic restriction enzyme markers in the Cmu region of homology to the target chromosomal mu locus. This enabled contributions by the vector-borne and chromosomal Cmu sequences at the recombinant locus to be ascertained. Targeted recombinants were isolated and analyzed to determine the number of vector copies integrated at the chromosomal immunoglobulin mu locus. Targeted recombinants identified as bearing >1 copy of the integrated vector resulted from a Cmu triplication formed by two vector copies in tandem. Examination of the fate of the Cmu region markers suggested that this class of recombinant was generated predominantly, if not exclusively, by two targeted vector integration events, each involving insertion of a single copy of the vector. Both vector insertion events into the chromosomal mu locus were consistent with the double-strand-break repair mechanism of homologous recombination. We interpret our results, taken together, to mean that a proportion of recipient cells is in a predetermined state that is amenable to targeted but not random vector integration.     

Gene conversion during vector insertion in embryonic stem cells.

Recombination of an insertion vector into its chromosomal homologue is a conservative event in that both the chromosomal and the vector sequences are preserved. However, gene conversion may accompany homologous recombination of an insertion vector. To examine gene conversion in more detail we have determined the targeting frequencies and the structure of the recombinant alleles generated with a series of vectors which target the hprt gene in embryonic stem cells. We demonstrate that gene conversion of the introduced mutation does not significantly limit homologous recombination and that gene conversion occurs without a sequence specific bias for five different mutations. The frequency of the loss of a vector mutation and the gain of a chromosomal sequence is inversely proportional to the distance between the vector mutation and the double-strand break. The loss of a chromosomal sequence and the gain of a vector mutation occurs at a low frequency.