FLP Recognition Target Sites Research Articles

The FlpTRAP system for purification of specific, endogenous chromatin regions

The repressor element 1-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) binds to repressor element 1/neuron-restrictive silencer element (RE1/NRSE) sites in the genome and recruits effector proteins to repress its target genes. Here, we developed the FlpTRAP system to isolate endogenously assembled DNA-protein complexes such as the REST/NRSF complex. In the FlpTRAP system, we take advantage of the step-arrest variant of the Flp recombinase, FlpH305L, which, in the presence of Flp recognition target (FRT) DNA, accumulates as FRT DNA-protein adduct. The FlpTRAP system consists of three elements: (i) FlpH305L-containing cell extracts or isolates, (ii) a cell line engineered to harbor the DNA motif of interest flanked by FRT sites, and (iii) affinity selection steps to isolate the target chromatin. Specifically, 3×FLAG-tagged FlpH305L was expressed in insect cell cultures infected with baculovirus, and cell lysates were prepared. The lysate was used to capture the FRT-SNAP25 RE1/NRSE-FRT chromatin from a human medulloblastoma cell line, and the target RE1/NRSE chromatin was isolated by anti-FLAG immunoaffinity chromatography. Using electrophoretic mobility shift assays (EMSAs) and chromatin immunopurification (ChIP), we show that FlpH305L recognized and bound to the FRT sites. Overall, we suggest the FlpTRAP system as a tool to purify endogenous, specific chromatin loci from eukaryotic cells.

Expression of Flp Protein in a Baculovirus/Insect Cell System for Biotechnological Applications.

The Saccharomyces cerevisiae Flp protein is a site-specific recombinase that recognizes and binds to the Flp recognition target (FRT) site, a specific sequence comprised of at least two inverted repeats separated by a spacer. Binding of four monomers of Flp is required to mediate recombination between two FRT sites. Because of its site-specific cleavage characteristics, Flp has been established as a genome engineering tool. Amongst others, Flp is used to direct insertion of genes of interest into eukaryotic cells based on single and double FRT sites. A Flp-encoding plasmid is thereby typically cotransfected with an FRT-harboring donor plasmid. Moreover, Flp can be used to excise DNA sequences that are flanked by FRT sites. Therefore, the aim of this study was to determine whether Flp protein and its step-arrest mutant, FlpH305L, recombinantly expressed in insect cells, can be used for biotechnological applications. Using a baculovirus system, the proteins were expressed as C-terminally 3 × FLAG-tagged proteins and were purified by anti-FLAG affinity selection. As demonstrated by electrophoretic mobility shift assays (EMSAs), purified Flp and FlpH305L bind to FRT-containing DNA. Furthermore, using a cell assay, purified Flp was shown to be active in recombination and to mediate efficient insertion of a donor plasmid into the genome of target cells. Thus, these proteins can be used for applications such as DNA-binding assays, in vitro recombination, or genome engineering.

Gene disruption in Salmonella typhimurim by modified λ Red disruption system.

There are many techniques to knock out directed genes in bacteria, some of which have been described in Salmonella species. In this study, a combination of SOEing PCR method and the λ Red disruption system were used to disrupt phoP gene in wild type and standard strains of Salmonella typhimurium. Three standards PCR and one fusion PCR reactions were performed to construct a linear DNA including upstream and downstream of phoP gene and Kanamycin cassette. As a template plasmid, we used pKD4 which carries kanamycin gene flanked by FRT (FLP recognition target) sites. The resulting construct was electroporated into prepared competent cells of S. typhimurium. The transformants colonies related to the standard strain appeared on the LB-Km-agar plates after incubation, but there was no colony on LB-Km-agar plates corresponding to the wild type strain. The failure in transformation of the wild type strain may be because of inflexibility of the λ Red disruption system in this strain or its unique restriction-modification system. However, by this construct we are able to generate phoP mutant in many of the Salmonella species due to high homology of the phoP gene which exists in different species.

Improved bioethanol production from metabolic engineering of Enterobacter aerogenes ATCC 29007

This study investigates the enhancement of bioethanol production using a genetic engineering approach. The bioethanol-producing strain, E. aerogenes ATCC 29007, was engineered by deleting the D-lactate dehydrogenase (ldhA) gene to block the production of lactic acid. The Open-reading frame coding region of ldhA gene was replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one-step method to inactivate chromosomal genes and primers designed to create in-frame deletions upon excision of the resistance cassette. The colony PCR was used to confirm the deleted gene. Glycerol, a useful byproduct in the biodiesel industry, was employed to convert into bioethanol, using engineered E. aerogenes SUMI014. Under optimal conditions of fermentation (34°C, pH 7.5, 78h), bioethanol production by the mutant strain was 34.54g/L, 1.5 times greater than that produced by its wild type (13.09g/L). Subsequent overexpression of alcohol dehydrogenase (adhE) gene in the mutant strain; increased the production of bioethanol up to 38.32g/L. By the combination of gene deletion and overexpression, the bioethanol yield was 0.48g/g when employing 80g/L glycerol. Hence, a significant enhancement in ethanol production was observed. These results may provide valuable guidelines for further engineering bioethanol producers.

Modified Lentiviral LTRs Allow Flp Recombinase–mediated Cassette Exchange and In Vivo Tracing of “Factor-free” Induced Pluripotent Stem Cells

Methods for generating induced pluripotent stem cells (iPSCs) for disease modeling and cell therapies have progressed from integrating vectors to transient delivery of reprogramming factors, avoiding permanent genomic modification. A major limitation of unmodified iPSCs is the assessment of their distribution and contribution to adverse reactions in autologous cell therapy. Here, we report that polycistronic lentiviral vectors with single Flp recombinase (Flp) recognition target (FRT) sites can be used to generate murine iPSCs that are devoid of the reprogramming cassette but carry an intergenic 300-bp long terminal repeat sequence. Performing quantitative polymerase chain reaction on this marker, we could determine genetic identity and tissue contribution of iPSC-derived teratomas in mice. Moreover, we generated iPSCs carrying heterospecific FRT twin sites, enabling excision and recombinase-mediated cassette exchange (RMCE) of the reprogramming cassette for another expression unit of choice. Following screening of iPSCs for "safe harbor" integration sites, expression cassettes were introduced by RMCE into various previously silenced loci of selected single-copy iPSCs. Analysis of DNA methylation showed that RMCE reverted the local epigenetic signature, which allowed transgene expression in undifferentiated iPSCs and in differentiated progeny. These findings support the concept of creating clonotypically defined exchangeable and traceable pluripotent stem cells for disease research and cell therapy.

Toward Position-independent Retroviral Vector Expression in Pluripotent Stem Cells

Toward Position-independent Retroviral Vector Expression in Pluripotent Stem Cells

LoxP-FRT Trap (LOFT): a simple and flexible system for conventional and reversible gene targeting

BackgroundConditional gene knockout (cKO) mediated by the Cre/LoxP system is indispensable for exploring gene functions in mice. However, a major limitation of this method is that gene KO is not reversible. A number of methods have been developed to overcome this, but each method has its own limitations.ResultsWe describe a simple method we have named LOFT [LoxP-flippase (FLP) recognition target (FRT) Trap], which is capable of reversible cKO and free of the limitations associated with existing techniques. This method involves two alleles of a target gene: a standard floxed allele, and a multi-functional allele bearing an FRT-flanked gene-trap cassette, which inactivates the target gene while reporting its expression with green fluorescent protein (GFP); the trapped allele is thus a null and GFP reporter by default, but is convertible into a wild-type allele. The floxed and trapped alleles can typically be generated using a single construct bearing a gene-trap cassette doubly flanked by LoxP and FRT sites, and can be used independently to achieve conditional and constitutive gene KO, respectively. More importantly, in mice bearing both alleles and also expressing the Cre and FLP recombinases, sequential function of the two enzymes should lead to deletion of the target gene, followed by restoration of its expression, thus achieving reversible cKO. LOFT should be generally applicable to mouse genes, including the growing numbers of genes already floxed; in the latter case, only the trapped alleles need to be generated to confer reversibility to the pre-existing cKO models. LOFT has other applications, including the creation and reversal of hypomorphic mutations. In this study we proved the principle of LOFT in the context of T-cell development, at a hypomorphic allele of Baf57/Smarce1 encoding a subunit of the chromatin-remodeling Brg/Brahma-associated factor (BAF) complex. Interestingly, the FLP used in the current work caused efficient reversal in peripheral T cells but not thymocytes, which is advantageous for studying developmental epigenetic programming of T-cell functions, a fundamental issue in immunology.ConclusionsLOFT combines well-established basic genetic methods into a simple and reliable method for reversible gene targeting, with the flexibility of achieving traditional constitutive and conditional KO.

Mutant HFE H63D Protein Is Associated with Prolonged Endoplasmic Reticulum Stress and Increased Neuronal Vulnerability

A specific polymorphism in the hemochromatosis (HFE) gene, H63D, is over-represented in neurodegenerative disorders such as amyotrophic lateral sclerosis and Alzheimer disease. Mutations of HFE are best known as being associated with cellular iron overload, but the mechanism by which HFE H63D might increase the risk of neuron degeneration is unclear. Here, using an inducible expression cell model developed from a human neuronal cell line SH-SY5Y, we reported that the presence of the HFE H63D protein activated the unfolded protein response (UPR). This response was followed by a persistent endoplasmic reticulum (ER) stress, as the signals of UPR sensors attenuated and followed by up-regulation of caspase-3 cleavage and activity. Our in vitro findings were recapitulated in a transgenic mouse model carrying Hfe H67D, the mouse equivalent of the human H63D mutation. In this model, UPR activation was detected in the lumbar spinal cord at 6 months then declined at 12 months in association with increased caspase-3 cleavage. Moreover, upon the prolonged ER stress, the number of cells expressing HFE H63D in early apoptosis was increased moderately. Cell proliferation was decreased without increased cell death. Additionally, despite increased iron level in cells carrying HFE H63D, it appeared that ER stress was not responsive to the change of cellular iron status. Overall, our studies indicate that the HFE H63D mutant protein is associated with prolonged ER stress and chronically increased neuronal vulnerability.

Elimination of the Plasmid Bacterial Backbone in Site-Directed Transgenesis

For cellular and animal transgenesis, FLP- and Cre-recombinase gene capture systems are highly effective to provide stable integration of a donor plasmid carrying the transgene cassette of interest into an engineered genomic locus in a given cell line. However, in many protocols, the entire plasmid bacterial backbone is integrated along with the transgene cassette. Here, we present a very simple yet highly efficient method for excluding plasmid bacterial backbone integration. The transgene cassette, including a single FLP recognition target site, is specifically amplified by PCR, and the resulting DNA ligated into minicircles can serve as donor DNA in FLP-mediated recombination. Interestingly, the elimination of the bacterial backbone increased expression of the inserted transgene. The presented method is simple and efficient for generating transgene cassette insertions devoid of the bacterial backbone.

Construction of genetically defined aroA mutant of a native E. coli O78:K80 isolated from avian colibacillosis, in Iran

A precisely defined, single gene deletion of aroA was made systematically by Datsenko and Wanner based approach in native Escherichia coli O78: K80, and its nature was examined. The aroA gene encodes 5-enolpyrovylshikimate3-phosphate synthase and participates in the aromatic amino acids and folic acid, which are universal metabolic pathways of bacteria. For construction of mutant strain, the gene was replaced by recombination with a chloramphenicol cassette, flanked by FLP recognition target site. Primers designed to create in-frame deletion upon excision of the resistance cassette. The aroA deletion was confirmed by both polymerase chain reaction and failure of the mutant to replicate and grow on minimum medium lacking aromatic amino acids. On the other hand, the wild-type parent strain grows well in this medium. The sequence of the aroA gene in native strain was different, and knowing this subject was important to produce a mutant strain with deletion-based methods of mutation. Furthermore, use of antibiotic cassette replacement facilitates mutant construction in a more cost-effective manner in comparison with other techniques such as use of suicide vectors.

In-Frame Deletion of Escherichia Coli Essential Genes in Complex Regulon

A conditional knockout-rescue system was developed to construct an in-frame deletion strain ofEscherichia coli essential genes. The target was flanked with marker genes and FRT (FLP recognition target) sites, and a plasmid containing arabinose-induced FLP recombinase was transformed. After arabinose induction, cells could survive only when target protein activity was provided in trans. We selected three essential genes as targets, yaeT, fabZ, and dnaE, which are components of the complex eight-gene regulon yaeT-hlpA-lpxD-fabZ-lpxA-1pxB-rnhB-dnaE. Deletion of these three genes exhibit no polar effects on their adjacent genes in terms of cell viability, meaning that this system not only allows for the simplified study of protein interactions and homolog screening in other organisms, but also facilitates the null mutant construction of essential genes.

Construction of Escherichia coli K‐12 in‐frame, single‐gene knockout mutants: the Keio collection

We have systematically made a set of precisely defined, single-gene deletions of all nonessential genes in Escherichia coli K-12. Open-reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one-step method for inactivation of chromosomal genes and primers designed to create in-frame deletions upon excision of the resistance cassette. Of 4288 genes targeted, mutants were obtained for 3985. To alleviate problems encountered in high-throughput studies, two independent mutants were saved for every deleted gene. These mutants—the ‘Keio collection'—provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome-wide testing of mutational effects in a common strain background, E. coli K-12 BW25113. We were unable to disrupt 303 genes, including 37 of unknown function, which are candidates for essential genes. Distribution is being handled via GenoBase (http://ecoli.aist-nara.ac.jp/).

Tagging genes with cassette-exchange sites

In an effort to make transgenesis more flexible and reproducible, we developed a system based on novel 5′ and 3′ ‘gene trap’ vectors containing heterospecific Flp recognition target sites and the corresponding ‘exchange’ vectors allowing the insertion of any DNA sequence of interest into the trapped locus. Flp-recombinase-mediated cassette exchange was demonstrated to be highly efficient in our system, even in the absence of locus-specific selection. The feasibility of constructing a library of ES cell clones using our gene trap vectors was tested and a thousand insertion sites were characterized, following electroporation in ES cells, by RACE–PCR and sequencing. We validated the system in vivo for two trapped loci in transgenic mice and demonstrated that the reporter transgenes inserted into the trapped loci have an expression pattern identical to the endogenous genes. We believe that this system will facilitate in vivo studies of gene function and large-scale generation of mouse models of human diseases, caused by not only loss but also gain of function alleles.

Construction of targeted single copy lac fusions using λ Red and FLP-mediated site-specific recombination in bacteria

A simple method for the construction of targeted transcriptional and translational fusions to the lac operon using FLP mediated site-specific recombination is described. Conditional plasmids containing promoterless lacZY genes and the FLP recognition target (FRT) site in both orientations were constructed for generating transcriptional fusions. Similarly, a plasmid used to create translational fusions was constructed in which the endogenous translational start of lacZ has been removed. These plasmids can be transformed into strains containing a single FRT site, which was previously integrated downstream of the promoter of interest using the λ Red recombination method. The FLP protein produced from a helper plasmid that contains a conditional origin of replication promotes site-specific recombination between the FRT sites, resulting in an integrated lac fusion to the gene of interest. Transcriptional fusions to the Salmonella typhimurium genes sodCII and sitA were constructed using this method and shown to respond appropriately to mutations in the respective regulatory genes, rpoS and fur. Translational fusions were also constructed using this method. In this case, expression of β-galactosidase was dependent on translation of the target protein. Given that the FLP recombinase does not require host factors for function and that this method requires no molecular cloning, this method should be applicable for the analysis of gene expression in a variety of organisms.

One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.

We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s). In this procedure, recombination requires the phage lambda Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid. To demonstrate the utility of this approach, we generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the respective PCR products, we made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, especially in genome analysis of E. coli and other bacteria because the procedure can be done in wild-type cells.

DNA sequence determinant for FIp-induced DNA bending.

The Flp site-specific recombinase from Saccharomyces cerevisiae induces DNA bending upon interaction with the Flp recognition target (FRT) site. The minimal FRT site comprises the inverted a and b binding elements, which flank a central 8 bp core region. The DNA bend in a complex of two Flp monomers bound to the FRT site is located in the middle of the core region. When the central AT basepair was replaced with a CG, the DNA bend was positioned at the outside end of the core region adjacent to the a binding element. The other basepairs surrounding the central AT basepair were not important to the position of Flp-induced bends. The change also decreased Flp-mediated cleavage of the top strand of the FRT site and increased Flp-mediated cleavage of the bottom strand. The overall recombination proficiency of the site was impaired. We conclude that the central AT basepair provides a point of flexure in the FRT site, which Flp uses to position the bend in dimeric Flp-DNA complexes, and that the structure of the core DNA influences the functionality of the site.

DNA cassette exchange in ES cells mediated by Flp recombinase: an efficient strategy for repeated modification of tagged loci by marker-free constructs.

The repeated modification of a genomic locus is a technically demanding but powerful strategy to analyze the function of a particular gene product or the role of cis-regulatory DNA elements in mammalian cells. The initial step is "tagging" a site with a selectable marker which is done by homologous recombination (HR) to modify a known locus or by random integration to study cis-regulatory elements at a reproducibly accessible genomic location. The tag is then used to target the construct of choice during an exchange step. Presented here is a novel technique in which the exchange is independent of HR and does not introduce vector sequences. It relies on our previous studies on the replacement of DNA cassettes by FLP-recombinase, whereby some common limitations can be overcome. To this end, the tag, a hygtk positive/negative selection marker, is integrated into the genome of embryonic stem (ES) cells. This marker is flanked by a wild-type Flp-recognition target (FRT) site on one end and by a modified heterospecific FRT site on the other. Successful Flp-mediated replacement of the hygtk cassette is enriched by ganciclovir (GANC) selection for cells that lack the encoded fusion protein. Thereby, the hygtk gene can be exchanged for virtually any sequence in a single efficient step without the need of introducing a positive selectable marker. The system can hence be used to analyze the function of either a gene product or regulatory sequences in ES cells or the transgenic mice derived thereof.

Determinants of the position of a Flp-induced DNA bend.

The Flp site-specific recombinase from Saccharomyces cerevisiae induces DNA bending upon interaction with the Flp recognition target (FRT) site. The minimal FRT site is comprised of two inverted binding elements which flank a central core region. Binding of a single monomer of Flp to DNA induces a DNA bend of 60 degrees. The position of this bend differed depending on whether the substrate contained a single binding element or a two-element FRT site. In the present work we tested and disproved a model in which a single Flp monomer interacts with both symmetry elements of a single FRT site. Likewise, we showed that a model in which a Flp monomer dissociates from a singly occupied FRT site and reassociates with the unbound element of another singly occupied FRT site during electrophoresis, does not account for the apparent shift in the position of the bend centre. It seems that the movement of a Flp monomer between the a and b elements of one FRT site during electrophoresis accounts for this anomaly. The position of the DNA bend resulting from the association of a Flp monomer with the FRT site is also influenced by the DNA sequences flanking the site. We conclude that attempts to measure the bend centre of a complex of one Flp molecule bound to a DNA containing two binding elements give misleading results. The position of the bend is more accurately measured in the presence of a single binding element.

The Role of Single-stranded DNA in Flp-mediated Strand Exchange

The Flp recognition target site contains two inverted 13-base pair (bp) Flp binding sequences that surround an 8-bp core region. Flp recombinase has been shown to carry out strand ligation independently of its ability to execute strand cleavage. Using a synthetic activated DNA substrate bearing a 3'-phosphotyrosine group, we have developed an assay to measure strand exchange by Flp proteins. We have shown that wild-type Flp protein was able to catalyze strand exchange using DNA substrates containing 8-bp duplex core sequences. Mutant Flp proteins that are defective in either DNA bending or DNA cleavage were also impaired in their abilities to carry out strand exchange. The inability of these mutant proteins to execute strand exchange could be overcome by providing a DNA substrate containing a single-stranded core sequence. This single-stranded core sequence could be as small as 3 nucleotides. Full activity of mutant Flp proteins in strand exchange was observed when both partner DNAs contained an 8-nucleotide single-stranded core region. Using suicide substrates, we showed that single-stranded DNA is also important for strand exchange reactions where Flp-mediated strand cleavage is required. These results suggest that the ability of Flp to induce DNA bending and strand cleavage may be crucial for strand exchange. We propose that both DNA bending and strand cleavage may be required to separate the strands of the core region and that single-stranded DNA in the core region might be an intermediate in Flp-mediated DNA recombination.

Molecular engineering with the FRT sequence of the yeast 2 μm plasmid: [ cir°] segregant enrichment by counterselection for 2 μm site-specific recombination

Site-specific recombination systems from bacteriophage and yeasts are becoming precious tools for manipulating DNA both in vitro and in living organisms. In this work we describe the isolation of yeast Saccharomyces cerevisiae segregants which have lost the highly stable 2 μm DNA plasmid, exploiting the site-specific recombination system of 2 μm itself. We efficiently isolated [ cir o] segregants from two haploid yeast strains and also a diploid. Moreover, the effect of mutations in the core region of the FRT (Flp Recognition Target) sequence was investigated in vivo, studying the result of the recombination event between several mutated and wild-type FRT sequences. From our result it seems that the identity between the core regions of two FRT sites is necessary but not sufficient, indicating that the core sequence itself has a relevant function in the recombination mechanism in vivo.