A Cre::FLP fusion protein recombines FRT or loxP sites in transgenic maize plants

SUMMARY: The coding sequences of Cre (site-specific recombinase from bacteriophage P1) and FLP (yeast 2-microm plasmid site-specific recombinase) were fused in frame to produce a novel, dual-function, site-specific recombinase gene. Transgenic maize plants containing the Cre::FLP fusion expression vector were crossed to transgenic plants containing either the loxP or FRT excision substrate. Complete and precise excisions of chromosomal fragments flanked by the respective target sites were observed in the F1 and F2 progeny plants. The episomal DNA recombination products were frequently lost. Non-recombined FRT substrates found in the F1 plants were recovered in the F2 generation after the Cre::FLP gene segregated out. They produced the recombination products in the F3 generation when crossed back to the FLP-expressing plants. These observations may indicate that the efficiency of site-specific recombination is affected by the plant developmental stage, with site-specific recombination being more prevalent in developing embryos. The Cre::FLP fusion protein was also tested for excisions catalysed by Cre. Excisions were identified in the F1 plants and verified in the F2 plants by polymerase chain reaction and Southern blotting. Both components of the fusion protein (FLP and Cre) were functional and acted with similar efficiency. The crossing strategy proved to be suitable for the genetic engineering of maize using the FLP or Cre site-specific recombination system.     

Activity of yeast FLP recombinase in maize and rice protoplasts.

We have demonstrated that a yeast FLP/FRT site-specific recombination system functions in maize and rice protoplasts. FLP recombinase activity was monitored by reactivation of beta-glucuronidase (GUS) expression from vectors containing the gusA gene inactivated by insertion of two FRTs (FLP recombination targets) and a 1.31 kb DNA fragment. The stimulation of GUS activity in protoplasts cotransformed with vectors containing FRT inactivated gusA gene and a chimeric FLP gene depended on both the expression of the FLP recombinase and the presence and structure of the FRT sites. The FLP enzyme could mediate inter- and intramolecular recombination in plant protoplasts. These results provide evidence that a yeast recombination system can function efficiently in plant cells, and that its performance can be manipulated by structural modification of the FRT sites.     

FLP-mediated site-specific recombination in microinjected murine zygotes

The FLP recombinase of yeast catalyses site-specific recombination between repeated FLP recombinase target (FRT) elements in yeast and in heterologous system (Escherichia coli, Drosophila, mosquito and cultured mammalian cells). In this report, it is shown that transient FLP recombinase expression can recombine and activate an extrachromosomal silent reporter gene following coinjection into fertilized one-cell mouse eggs. Furthermore, it is demonstrated that introduction of a FLP-recombinase expression vector into transgenic one-cell fertilized mouse eggs induces a recombination event at a chromosomal FRT target locus. The resulting event occured at the one-cell stage and deleted a chromosomal tandem array of a FRT containinglacZ expression cassette down to one or two copies. These results demonstrate that the FLP recombinase can be utilized to manipulate the genome of transgenic animals and suggest that FLP recombinase-mediated plasmid-to-chromosome targeting is feasible in microinjected eggs.     

DNA重组酶FLP原核表达与多步法纯化及其活性检测

DNA重组酶FLP存在于酵母2μ质粒上,能识别34bp的FRT位点,并根据2个FRT位点的相对方向完成位点间DNA序列的交换、重组、删除与逆转,在现代分子生物学理论研究与基因工程技术开发中具有广泛应用。构建了在原核大肠杆菌中高效表达FLP重组酶的表达载体pQE32-flpe并建立起相应的原核高效表达体系,在原核细菌大肠杆菌M15菌株中实现FLP酶蛋白的高效表达,同时建立了相应的纯化方法。纯化时先用硫酸铵沉淀法富集FLP酶蛋白,经透析脱盐后再用镍离子鳌合微柱(0.5~1.0mL)亲合层析梯度洗脱的方法获得纯化的FLP酶蛋白。通过构建含有2个方向相同的FRT序列位点的质粒pUC18-FRT-gfp-FRT和含有1个FRT位点的表达载体pET30a-FRT,并分别以其为底物来检测FLP重组酶的删除、交换与重组功能的活性。结果表明,该方法不仅能有效表达FLP酶蛋白,并能行之有效地纯化FLP酶蛋白,以及检测纯化的FLP酶蛋白对DNA序列的删除、重组与交换功能。该方法简单易行并能获得有活性的FLP酶蛋白,为深入研究其机理以及研发相应的DNA重组技术提供重要参考。     

Activity of the c-myc Replicator at an Ectopic Chromosomal Location

DNA replication starts at multiple discrete sites across the human chromosomal c-myc region, including two or more sites within 2.4 kb upstream of the c-myc gene. The corresponding 2.4-kb c-myc origin fragment confers autonomously replicating sequence (ARS) activity on plasmids, which specifically initiate replication in the origin fragment in vitro and in vivo. To test whether the region that displays plasmid replicator activity also acts as a chromosomal replicator, HeLa cell sublines that each contain a single copy of the Saccharomyces cerevisiae FLP recombinase target (FRT) sequence flanked by selectable markers were constructed. A clonal line containing a single unrearranged copy of the transduced c-myc origin was produced by cotransfecting a donor plasmid containing the 2.4-kb c-myc origin fragment and FRT, along with a plasmid expressing the yeast FLP recombinase, into cells containing a chromosomal FRT acceptor site. The amount of short nascent DNA strands at the chromosomal acceptor site was quantitated before and after targeted integration of the origin fragment. Competitive PCR quantitation showed that the c-myc origin construct substantially increased the amount of nascent DNA relative to that at the unoccupied acceptor site and to that after the insertion of non-myc DNA. The abundance of nascent strands was greatest close to the c-myc insert of the integrated donor plasmid, and significant increases in nascent strand abundance were observed at sites flanking the insertion. These results provide biochemical and genetic evidence for the existence of chromosomal replicators in metazoan cells and are consistent with the presence of chromosomal replicator activity in the 2.4-kb region of c-myc origin DNA.     

FLP recombinase of the 2 microns circle plasmid of Saccharomyces cerevisiae bends its DNA target. Isolation of FLP mutants defective in DNA bending

The FLP recombinase is encoded by the yeast plasmid 2 microns circle and catalyses a site-specific recombination reaction that results in inversion of a segment of the 2 micron plasmid. We describe a method for the isolation of inactivating mutations in the FLP gene. The analysis of the recombination and binding activity of defective FLP proteins in vitro resulted in the identification of two classes of mutations: those that completely abolish FLP function by interfering with DNA binding and others that block recombination after the binding step. We have shown that FLP-mediated recombination is accompanied by bending of the DNA target and that mutations in the FLP recombinase that block bending also eliminate recombination.     

The FLP protein contacts both major and minor grooves of its recognition target sequence.

The FLP protein of the 2 microns plasmid of Saccharomyces cerevisiae promotes conservative site-specific recombination between DNA sequences that contain the FLP recognition target (FRT). FLP binds to each of the three 13 base pair symmetry elements in the FRT site in a site-specific manner. We have probed both major and minor groove contacts of FLP using dimethyl sulphate, monoacetyl-4-hydroxyaminoquinoline 1-oxide and potassium permanganate and find that the protein displays extensive interactions with residues of both the major and minor grooves of 10 base pairs of each symmetry element. We find no evidence that the FRT site assumes a single-stranded conformation upon FLP binding.     

FLP-mediated recombination for use in hybrid plant production

We have studied the feasibility in Arabidopsis of using a site-specific recombination system FLP/FRT, from the 2 microm plasmid of yeast, for making plant hybrids. Initially, Arabidopsis plants expressing the FLP site-specific recombinase were crossed with plants transformed with a vector containing kanamycin-resistance gene (npt) flanked by FRT sites, which also served to separate the CaMV35S promoter from a promoterless gusA. Hybrid progeny were tested for excision of the npt gene and the positioning of 35S promoter proximal to gusA. GUS activity was observed in the progeny of all crosses, but not in the progeny derived from the self-pollinated homozygous parents. We then induced male sterility in Arabidopsis plants using the antisense expression of a pollen- and tapetum-specific gene, bcp1, flanked by FRT sites. Upon cross-pollination of flowers on the same male-sterile plants with pollen from FLP-containing plants, viable seeds were produced and the progeny hybrid plants developed normally. Molecular analyses revealed that the antisense expression cassette of bcp1 had been excised in these plants. These results show for the first time that a site-specific recombinase can be used to restore fertility in male-sterile plants, providing an alternative method for the production of hybrid seeds and plants.     

FLP protein of 2 mu circle plasmid of yeast induces multiple bends in the FLP recognition target site

The FLP recombinase of the 2 mu plasmid of Saccharomyces cerevisiae binds to a target containing three 13 base-pair symmetry elements called a, b and c. The symmetry elements b and c are in direct orientation while the a element is in inverted orientation with respect to b and c on the opposite side of an eight base-pair core region. Each symmetry element acts as a binding site for the FLP protein. The FLP protein can form three different complexes with the FLP recognition target (FRT site) according to the number of elements within the site that are occupied by the FLP protein. Binding of FLP to the FRT site induces DNA bending. We have measured the angles of bends caused by the binding of the FLP protein to full and partial FRT sites. We find that FLP induces three types of bend in the FRT-containing DNA. The type I bend is approximately 60 degrees and results from a molecule of FLP bound to one symmetry element. The type II bend is greater than 144 degrees and results from FLP molecules bound to symmetry elements a and b. The type III bend is approximately 65 degrees and results from FLP proteins bound to symmetry elements b and c. Certain FLP proteins that are defective in recombination can generate the type I and type III bends but are impaired in their ability to induce the type II bend. We discuss the role of bending in FLP-mediated recombination.     

FLP-mediated recombination of FRT sites in the maize genome.

Molecular evidence is provided for genomic recombinations in maize cells induced by the yeast FLP/FRT site-specific recombination system. The FLP protein recombined FRT sites previously integrated into the maize genome leading to excision of a selectable marker, the neo gene. NPTII activity was not observed after the successful recombination process; instead, the gusA gene was activated by the removal of the blocking DNA fragment. Genomic sequencing in the region of the FRT site (following the recombination reaction) indicated that a precise rearrangement of genomic DNA sequences had taken place. The functional FLP gene could be either expressed transiently or after stable integration into the maize genome. The efficiency of genomic recombinations was high enough that a selection for recombination products, or for FLP expression, was not required. The results presented here establish the FLP/FRT site-specific recombination system as an important tool for controlled modifications of maize genomic DNA.     

Use of green fluorescent protein/Flp recombinase fusion protein and flow cytometric sorting to enrich for cells undergoing Flp-mediated recombination

Flp recombinase has been used extensively for in vivo manipulation of eukaryotic DNA at specific sequences designated as FRT sites. We developed a method to use Flp-mediated recombination without the need for drug resistance or metabolic selection of cells in which recombination has occurred. We generated expression plasmids directing expression of fusion proteins consisting of Flp recombinase and green fluorescent protein (GFP) coding sequences. When the plasmids were introduced into K562 cells containing Flp recombinase substrates and transfected cells were selected for by flow cytometric sorting, GFP-positive cells were enriched 5- to 30-fold for Flp-mediated recombination events compared with unsorted cells. These studies demonstrate the usefulness of GFP/Flp recombinase fusion proteins to manipulate chromosomal DNA in vivo without requiring drug resistance or metabolic marker genes.     

Excision of selectable marker gene from transgenic tobacco using the GM-gene-deletor system regulated by a heat-inducible promoter

To excise a selectable marker gene from transgenic plants, a new binary expression vector based on the 'genetically modified (GM)-gene-deletor' system was constructed. In this vector, the gene coding for FLP site-specific recombinase under the control of a heat shock-inducible promoter HSP18.2 from Arabidopsis thaliana and isopentenyltransferase gene (ipt), as a selectable marker gene under the control of the cauliflower mosaic virus 35S (CaMV 35S) promoter, were flanked by two loxP/FRT fusion sequences as recombination sites in direct orientation. Histochemical staining for GUS activity showed that, upon induction by heat shock, all exogenous DNA, including the selectable marker gene ipt, beta-glucuronidase (gusA) gene and the FLP recombinase gene, between two loxP/FRT sites was eliminated efficiently from primary transgenic tobacco plants. Molecular analysis further confirmed that excision of the marker gene (ipt) was heritable and stable. Our approach provides a reliable strategy for auto-excising a selectable marker gene from calli, shoots or other tissues of transgenic plants after transformation and producing marker-free transgenic plants.     

Mutagenesis of a conserved region of the gene encoding the FLP recombinase of Saccharomyces cerevisiae. A role for arginine 191 in binding and ligation.

The FLP recombinase from the 2 microns plasmid of Saccharomyces cerevisiae contains a region from amino acid 185 to 203 that is conserved among several FLP-like proteins from different yeasts. Using site-directed mutagenesis, we have made mutations in this region of the FLP gene. Five of twelve mutations in the region yielded proteins that were unable to bind to the FLP recombination target (FRT) site. A change of arginine at position 191 to lysine resulted in a protein (FLP-R191K) that could bind to the FRT site but could not catalyze recombination. This mutant protein accumulated as a stable protein-DNA complex in which one of the two bound FLP proteins was covalently attached to the DNA. FLP-R191K was defective in strand exchange and ligation and was unable to promote protein-protein interaction with half-FRT sites. The conservation of three residues in all members of the integrase family of site-specific recombinases (His305, Arg308, Tyr343 in FLP) implies a common mechanism of recombination. The conservation of arginine 191 and the properties of the FLP-R191K mutant protein suggest that this arginine also plays an important role in the mechanism of FLP-mediated site-specific recombination.     

High efficiency, site-specific excision of a marker gene by the phage P1 cre-loxP system in the yellow fever mosquito, Aedes aegypti

The excision of specific DNA sequences from integrated transgenes in insects permits the dissection in situ of structural elements that may be important in controlling gene expression. Furthermore, manipulation of potential control elements in the context of a single integration site mitigates against insertion site influences of the surrounding genome. The cre-loxP site-specific recombination system has been used successfully to remove a marker gene from transgenic yellow fever mosquitoes, Aedes aegypti. A total of 33.3% of all fertile families resulting from excision protocols showed evidence of cre-loxP-mediated site-specific excision. Excision frequencies were as high as 99.4% within individual families. The cre recombinase was shown to precisely recognize loxP sites in the mosquito genome and catalyze excision. Similar experiments with the FLP/FRT site-specific recombination system failed to demonstrate excision of the marker gene from the mosquito chromosomes.     

Positive selection of FLP-mediated unequal sister chromatid exchange products in mammalian cells.

Site-specific recombination provides a powerful tool for studying gene function at predetermined chromosomal sites. Here we describe the use of a blasticidin resistance system to select for recombination in mammalian cells using the yeast enzyme FLP. The vector is designed so that site-specific recombination reconstructs the antibiotic resistance marker within the sequences flanked by the FLP target sites. This approach allows the detection of DNA excised by FLP-mediated recombination and facilitates the recovery of recombination products that would not be detected by available screening strategies. We used this system to show that the molecules excised by intrachromosomal recombination between tandem FLP recombinase target sites do not reintegrate into the host genome at detectable frequencies. We further applied the direct selection approach to recover a rare FLP-mediated recombination event displaying the characteristics of an unequal sister chromatid exchange between FLP target sites. Implications of this approach for the generation of duplications to assess their effect on gene dosage and chromosome stability are discussed.     

The minimal duplex DNA sequence required for site-specific recombination promoted by the FLP protein of yeast in vitro.

The 2-micron plasmid of the yeast Saccharomyces cerevisiae codes for a site-specific recombinase ('FLP') that efficiently catalyses recombination across the plasmid's two 599 bp repeats both in vivo and in vitro. We have used the partially purified FLP protein to define the minimal duplex DNA sequence required for intra- and intermolecular recombination in vitro. Previous DNase footprinting experiments had shown that FLP protected 50 bp of DNA around the recombination site. We made BAL31 deletions and synthetic FLP sites to show that the minimal length of the site that was able to recombine with a wild-type site was 22 bp. The site consists of two 7 bp inverted repeats surrounding an 8 bp core region. We also showed that the deleted sites recombined with themselves and that one of three 13 bp repeated elements within the FLP target sequence was not necessary for efficient recombination in vitro. Mutants lacking this redundant 13 bp element required a lower amount of FLP recombinase to achieve maximal yield of recombination than the wild type site. Finally, we discuss the structure of the FLP site in relation to the proposed function of FLP recombination in copy number amplification of the 2-micron plasmid in vivo.